Marlene Rabinovitch

All Publications

• Pathology and pathobiology of pulmonary hypertension: current insights and future directions. The European respiratory journal Guignabert, C., Aman, J., Bonnet, S., Dorfmüller, P., Olschewski, A. J., Pullamsetti, S., Rabinovitch, M., Schermuly, R. T., Humbert, M., Stenmark, K. R. 2024

  Abstract

  In recent years, major advances have been made in the understanding of the cellular and molecular mechanisms driving pulmonary vascular remodelling in various forms of pulmonary hypertension, including pulmonary arterial hypertension, pulmonary hypertension associated with left heart disease, pulmonary hypertension associated with chronic lung disease and hypoxia, and chronic thromboembolic pulmonary hypertension. However, the survival rates for these different forms of pulmonary hypertension remain unsatisfactory, underscoring the crucial need to more effectively translate innovative scientific knowledge into healthcare interventions. In these proceedings of the 7th World Symposium on Pulmonary Hypertension, we delve into recent developments in the field of pathology and pathophysiology, prioritising them while questioning their relevance to different subsets of pulmonary hypertension. In addition, we explore how the latest omics and other technological advances can help us better and more rapidly understand the myriad basic mechanisms contributing to the initiation and progression of pulmonary vascular remodelling. Finally, we discuss strategies aimed at improving patient care, optimising drug development, and providing essential support to advance research in this field.

  View details for DOI 10.1183/13993003.01095-2024

  View details for PubMedID 39209474

• Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity. Cell genomics Tervi, A., Ramste, M., Abner, E., Cheng, P., Lane, J. M., Maher, M., Valliere, J., Lammi, V., Strausz, S., Riikonen, J., Nguyen, T., Martyn, G. E., Sheth, M. U., Xia, F., Docampo, M. L., Gu, W., Esko, T., Saxena, R., Pirinen, M., Palotie, A., Ripatti, S., Sinnott-Armstrong, N., Daly, M., Engreitz, J. M., Rabinovitch, M., Heckman, C. A., Quertermous, T., Jones, S. E., Ollila, H. M. 2024: 100630

  Abstract

  Raynaud's syndrome is a dysautonomia where exposure to cold causes vasoconstriction and hypoxia, particularly in the extremities. We performed meta-analysis in four cohorts and discovered eight loci (ADRA2A, IRX1, NOS3, ACVR2A, TMEM51, PCDH10-DT, HLA, and RAB6C) where ADRA2A, ACVR2A, NOS3, TMEM51, and IRX1 co-localized with expression quantitative trait loci (eQTLs), particularly in distal arteries. CRISPR gene editing further showed that ADRA2A and NOS3 loci modified gene expression and in situ RNAscope clarified the specificity of ADRA2A in small vessels and IRX1 around small capillaries in the skin. A functional contraction assay in the cold showed lower contraction in ADRA2A-deficient and higher contraction in ADRA2A-overexpressing smooth muscle cells. Overall, our study highlights the power of genome-wide association testing with functional follow-up as a method to understand complex diseases. The results indicate temperature-dependent adrenergic signaling through ADRA2A, effects at the microvasculature by IRX1, endothelial signaling by NOS3, and immune mechanisms by the HLA locus in Raynaud's syndrome.

  View details for DOI 10.1016/j.xgen.2024.100630

  View details for PubMedID 39142284

• Life-saving effect of pulmonary surfactant in premature babies. The Journal of clinical investigation Raj, J. U., Bland, R. D., Bhattacharya, J., Rabinovitch, M., Matthay, M. A. 2024; 134 (9)

  Abstract

  The discovery and replacement of lung surfactant have helped increase survival rates for neonatal respiratory distress syndrome in extremely premature infants.

  View details for DOI 10.1172/JCI179948

  View details for PubMedID 38690742

• Reduced FOXF1 links unrepaired DNA damage to pulmonary arterial hypertension. Nature communications Isobe, S., Nair, R. V., Kang, H. Y., Wang, L., Moonen, J. R., Shinohara, T., Cao, A., Taylor, S., Otsuki, S., Marciano, D. P., Harper, R. L., Adil, M. S., Zhang, C., Lago-Docampo, M., Körbelin, J., Engreitz, J. M., Snyder, M. P., Rabinovitch, M. 2023; 14 (1): 7578

  Abstract

  Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension.

  View details for DOI 10.1038/s41467-023-43039-y

  View details for PubMedID 37989727

  View details for PubMedCentralID 4737700

• Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy. American journal of respiratory and critical care medicine Ferrian, S., Cao, A., McCaffrey, E. F., Saito, T., Greenwald, N. F., Nicolls, M. R., Bruce, T., Zamanian, R. T., Del Rosario, P., Rabinovitch, M., Angelo, M. 2023

  Abstract

  Rationale: Elucidating the immune landscape within and surrounding pulmonary arteries (PAs) is critical in understanding immune-driven vascular pathology in pulmonary arterial hypertension (PAH). Although more severe vascular pathology is often observed in hereditary (H)PAH patients with BMPR2 mutations, the involvement of specific immune cell subsets remains unclear. Methods: We used cutting-edge multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to compare PAs and adjacent tissue in PAH lungs (idiopathic (I)PAH and HPAH) with unused donor lungs. Measurements: We quantified immune cells' proximity and abundance, focusing on those linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells (mo-DCs) within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In HPAH patients, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving TIM-3+ T cells. This correlated with an expanded DC subset expressing IDO-1, TIM-3, and SAMHD1, alongside increased neutrophils, SMCs, and α-SMA+ECs, reinforcing the severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared to IPAH. Our findings emphasize the therapeutic potential of targeting mo-DCs, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

  View details for DOI 10.1164/rccm.202209-1761OC

  View details for PubMedID 37934691

• A computational growth and remodeling framework for adaptive and maladaptive pulmonary arterial hemodynamics. Biomechanics and modeling in mechanobiology Szafron, J. M., Yang, W., Feinstein, J. A., Rabinovitch, M., Marsden, A. L. 2023

  Abstract

  Hemodynamic loading is known to contribute to the development and progression of pulmonary arterial hypertension (PAH). This loading drives changes in mechanobiological stimuli that affect cellular phenotypes and lead to pulmonary vascular remodeling. Computational models have been used to simulate mechanobiological metrics of interest, such as wall shear stress, at single time points for PAH patients. However, there is a need for new approaches that simulate disease evolution to allow for prediction of long-term outcomes. In this work, we develop a framework that models the pulmonary arterial tree through adaptive and maladaptive responses to mechanical and biological perturbations. We coupled a constrained mixture theory-based growth and remodeling framework for the vessel wall with a morphometric tree representation of the pulmonary arterial vasculature. We show that non-uniform mechanical behavior is important to establish the homeostatic state of the pulmonary arterial tree, and that hemodynamic feedback is essential for simulating disease time courses. We also employed a series of maladaptive constitutive models, such as smooth muscle hyperproliferation and stiffening, to identify critical contributors to development of PAH phenotypes. Together, these simulations demonstrate an important step toward predicting changes in metrics of clinical interest for PAH patients and simulating potential treatment approaches.

  View details for DOI 10.1007/s10237-023-01744-z

  View details for PubMedID 37658985

• Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function. Vascular pharmacology Culley, M. K., Rao, R. J., Mehta, M., Zhao, J., El Khoury, W., Harvey, L. D., Perk, D., Tai, Y. Y., Tang, Y., Shiva, S., Rabinovitch, M., Gu, M., Bertero, T., Chan, S. Y. 2023: 107181

  Abstract

  Deficiency of iron‑sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

  View details for DOI 10.1016/j.vph.2023.107181

  View details for PubMedID 37164245

• A Computational Growth and Remodeling Framework for Adaptive and Maladaptive Pulmonary Arterial Hemodynamics. bioRxiv : the preprint server for biology Szafron, J. M., Yang, W., Feinstein, J. A., Rabinovitch, M., Marsden, A. L. 2023

  Abstract

  Hemodynamic loading is known to contribute to the development and progression of pulmonary arterial hypertension (PAH). This loading drives changes in mechanobiological stimuli that affect cellular phenotypes and lead to pulmonary vascular remodeling. Computational models have been used to simulate mechanobiological metrics of interest, such as wall shear stress, at single time points for PAH patients. However, there is a need for new approaches that simulate disease evolution to allow for prediction of long-term outcomes. In this work, we develop a framework that models the pulmonary arterial tree through adaptive and maladaptive responses to mechanical and biological perturbations. We coupled a constrained mixture theory-based growth and remodeling framework for the vessel wall with a morphometric tree representation of the pulmonary arterial vasculature. We show that non-uniform mechanical behavior is important to establish the homeostatic state of the pulmonary arterial tree, and that hemodynamic feedback is essential for simulating disease time courses. We also employed a series of maladaptive constitutive models, such as smooth muscle hyperproliferation and stiffening, to identify critical contributors to development of PAH phenotypes. Together, these simulations demonstrate an important step towards predicting changes in metrics of clinical interest for PAH patients and simulating potential treatment approaches.

  View details for DOI 10.1101/2023.04.20.537714

  View details for PubMedID 37131683

  View details for PubMedCentralID PMC10153237

• Are Senolytic Agents Guilty of Overkill or Inappropriate Age Discrimination? Circulation Rabinovitch, M. 2023; 147 (8): 667-668

  View details for DOI 10.1161/CIRCULATIONAHA.122.060247

  View details for PubMedID 36802881

• Dysregulated Smooth Muscle Cell BMPR2-ARRB2 Axis Causes Pulmonary Hypertension. Circulation research Wang, L., Moonen, J. R., Cao, A., Isobe, S., Li, C. G., Tojais, N. F., Taylor, S., Marciano, D. P., Chen, P., Gu, M., Li, D., Harper, R. L., El-Bizri, N., Kim, Y., Stankunas, K., Rabinovitch, M. 2023

  Abstract

  OBJECTIVE: Mutations in bone morphogenetic protein receptor 2 (BMPR2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH.METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism.RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary artery. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of phosphorylated extracellular signal related kinase 1/2-pP38-pSMAD2/3 mediating elevation in ARRB2 (beta-arrestin2), phosphorylated protein kinase B, and CTNNB1 (beta-catenin) and reduction in RHOA and RAC1. Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension.CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

  View details for DOI 10.1161/CIRCRESAHA.121.320541

  View details for PubMedID 36744494

• Respiratory viruses and postoperative hemodynamics in patients with unrestrictive congenital cardiac communications: a prospective cohort study. European journal of medical research Abud, K. C., Machado, C. M., Vilas Boas, L. S., Maeda, N. Y., Carvalho, E. S., Souza, M. F., Gaiolla, P. V., Castro, C. R., Pereira, J., Rabinovitch, M., Lopes, A. A. 2023; 28 (1): 38

  Abstract

  Pulmonary vascular abnormalities pose a risk for severe life-threatening hemodynamic disturbances following surgical repair of congenital cardiac communications (CCCs). In the distal lung, small airways and vessels share a common microenvironment, where biological crosstalks take place. Because respiratory cells infected by viruses express a number of molecules with potential impact on airway and vascular remodeling, we decided to test the hypothesis that CCC patients carrying viral genomes in the airways might be at a higher risk for pulmonary (and systemic) hemodynamic disturbances postoperatively.Sixty patients were prospectively enrolled (age 11 [7-16] months, median with interquartile range). Preoperative pulmonary/systemic mean arterial pressure ratio (PAP/SAP) was 0.78 (0.63-0.88). The presence or absence of genetic material for respiratory viruses in nasopharyngeal and tracheal aspirates was investigated preoperatively in the absence of respiratory symptoms using real-time polymerase chain reaction (kit for detection of 19 pathogens). Post-cardiopulmonary bypass (CPB) inflammatory reaction was analyzed by measuring serum levels of 36 inflammatory proteins (immunoblotting) 4 h after its termination. Postoperative hemodynamics was assessed using continuous recording of PAP and SAP with calculation of PAP/SAP ratio.Viral genomes were detected in nasopharynx and the trachea in 64% and 38% of patients, respectively. Rhinovirus was the most prevalent agent. The presence of viral genomes in the trachea was associated with an upward shift of postoperative PAP curve (p = 0.011) with a PAP/SAP of 0.44 (0.36-0.50) in patients who were positive versus 0.34 (0.30-0.45) in those who were negative (p = 0.008). The presence or absence of viral genomes in nasopharynx did not help predict postoperative hemodynamics. Postoperative PAP/SAP was positively correlated with post-CPB levels of interleukin-1 receptor antagonist (p = 0.026), macrophage migration inhibitory factor (p = 0.019) and monocyte chemoattractant protein-1 (p = 0.031), particularly in patients with virus-positive tracheal aspirates.Patients with CCCs carrying respiratory viral genomes in lower airways are at a higher risk for postoperative pulmonary hypertension, thus deserving special attention and care. Preoperative exposure to respiratory viruses and post-CPB inflammatory reaction seem to play a combined role in determining the postoperative behavior of the pulmonary circulation.

  View details for DOI 10.1186/s40001-023-01003-y

  View details for PubMedID 36670454

• KLF4 recruits SWI/SNF to increase chromatin accessibility and reprogram the endothelial enhancer landscape under laminar shear stress. Nature communications Moonen, J. R., Chappell, J., Shi, M., Shinohara, T., Li, D., Mumbach, M. R., Zhang, F., Nair, R. V., Nasser, J., Mai, D. H., Taylor, S., Wang, L., Metzger, R. J., Chang, H. Y., Engreitz, J. M., Snyder, M. P., Rabinovitch, M. 2022; 13 (1): 4941

  Abstract

  Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease.

  View details for DOI 10.1038/s41467-022-32566-9

  View details for PubMedID 35999210

• KMT2D-NOTCH Mediates Coronary Abnormalities in Hypoplastic Left Heart Syndrome. Circulation research Yu, Z., Zhou, X., Liu, Z., Pastrana-Gomez, V., Liu, Y., Guo, M., Tian, L., Nelson, T. J., Wang, N., Mital, S., Chitayat, D., Wu, J. C., Rabinovitch, M., Wu, S. M., Snyder, M. P., Miao, Y., Gu, M. 2022: 101161CIRCRESAHA122320783

  View details for DOI 10.1161/CIRCRESAHA.122.320783

  View details for PubMedID 35762338

• Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension. American journal of respiratory and critical care medicine Taylor, S., Isobe, S., Cao, A., Contrepois, K., Benayoun, B. A., Jiang, L., Wang, L., Melemenidis, S., Ozen, M. O., Otsuki, S., Shinohara, T., Sweatt, A. J., Kaplan, J., Moonen, J., Marciano, D. P., Gu, M., Miyagawa, K., Hayes, B., Sierra, R. G., Kupitz, C. J., Del Rosario, P. A., Hsi, A., Thompson, A. A., Ariza, M. E., Demirci, U., Zamanian, R. T., Haddad, F., Nicolls, M. R., Snyder, M. P., Rabinovitch, M. 2022

  Abstract

  RATIONALE: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear.OBJECTIVES: Relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling.METHODS: Production of elastase, release of extracellular traps, adhesion and migration were assessed in neutrophils from pulmonary arterial hypertension patients and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension and we determined whether they produce pulmonary hypertension in mice.MEASUREMENTS AND MAIN RESULTS: Neutrophils from pulmonary arterial hypertension patients produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated beta1integrin and vinculin identified on proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic interferon signature that we related to an increase in human endogenous retrovirus k envelope protein. Transfection of human endogenous retrovirus k envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and interferon genes, whereas vinculin is increased by human endogenous retrovirus k dUTPase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus k envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor.CONCLUSIONS: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

  View details for DOI 10.1164/rccm.202102-0446OC

  View details for PubMedID 35696338

• Role of endothelial cells in pulmonary fibrosis via SREBP2 activation. JCI insight Martin, M., Zhang, J., Miao, Y., He, M., Kang, J., Huang, H., Chou, C., Huang, T., Hong, H., Su, S., Wong, S. S., Harper, R. L., Wang, L., Bhattacharjee, R., Huang, H., Chen, Z. B., Malhotra, A., Rabinovitch, M., Hagood, J. S., Shyy, J. Y. 2021; 6 (22)

  Abstract

  Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), alpha-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.

  View details for DOI 10.1172/jci.insight.125635

  View details for PubMedID 34806652

• Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19. The Journal of experimental medicine Wilk, A. J., Lee, M. J., Wei, B., Parks, B., Pi, R., Martinez-Colon, G. J., Ranganath, T., Zhao, N. Q., Taylor, S., Becker, W., Stanford COVID-19 Biobank, Jimenez-Morales, D., Blomkalns, A. L., O'Hara, R., Ashley, E. A., Nadeau, K. C., Yang, S., Holmes, S., Rabinovitch, M., Rogers, A. J., Greenleaf, W. J., Blish, C. A. 2021; 218 (8)

  Abstract

  Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-kappaB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.

  View details for DOI 10.1084/jem.20210582

  View details for PubMedID 34128959

• Monocyte released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition. JCI insight Otsuki, S., Saito, T., Taylor, S., Li, D., Moonen, J., Marciano, D. P., Harper, R. L., Cao, A., Wang, L., Ariza, M. E., Rabinovitch, M. 2021

  Abstract

  Previously we reported heightened expression of human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of PA hypertension (PAH) patients. Furthermore, recombinant HERV-K dUTPase increased IL6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG, and proinflammatory molecules IL6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response (MYD)-88 to increase IL6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-kappaB in addition to pJNK-pSMAD3 required for SNAIL, and pSTAT1 for IL6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-kappaB in addition to pERK1/2-activating transcription factor (ATF)-2 to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated pro-inflammatory molecules.

  View details for DOI 10.1172/jci.insight.146416

  View details for PubMedID 34185707

• Frataxin deficiency promotes endothelial senescence in pulmonary hypertension JOURNAL OF CLINICAL INVESTIGATION Culley, M. K., Zhao, J., Tai, Y., Tang, Y., Perk, D., Negi, V., Yu, Q., Woodcock, C. C., Handen, A., Speyer, G., Kim, S., Lai, Y., Satoh, T., Watson, A. M., Al Aaraj, Y., Sembrat, J., Rojas, M., Goncharov, D., Goncharova, E. A., Khan, O. F., Anderson, D. G., Dahlman, J. E., Gurkar, A. U., Lafyatis, R., Fayyaz, A. U., Redfield, M. M., Gladwin, M. T., Rabinovitch, M., Gu, M., Bertero, T., Chan, S. Y. 2021; 131 (11)

  View details for DOI 10.1172/JCI136459.

  View details for Web of Science ID 000656988800004

• iPSC-endothelial cell phenotypic drug screening and in silico analyses identify tyrphostin-AG1296 for pulmonary arterial hypertension. Science translational medicine Gu, M., Donato, M., Guo, M., Wary, N., Miao, Y., Mao, S., Saito, T., Otsuki, S., Wang, L., Harper, R. L., Sa, S., Khatri, P., Rabinovitch, M. 2021; 13 (592)

  Abstract

  Pulmonary arterial hypertension (PAH) is a progressive disorder leading to occlusive vascular remodeling. Current PAH therapies improve quality of life but do not reverse structural abnormalities in the pulmonary vasculature. Here, we used high-throughput drug screening combined with in silico analyses of existing transcriptomic datasets to identify a promising lead compound to reverse PAH. Induced pluripotent stem cell-derived endothelial cells generated from six patients with PAH were exposed to 4500 compounds and assayed for improved cell survival after serum withdrawal using a chemiluminescent caspase assay. Subsequent validation of caspase activity and improved angiogenesis combined with data analyses using the Gene Expression Omnibus and Library of Integrated Network-Based Cellular Signatures databases revealed that the lead compound AG1296 was positively associated with an anti-PAH gene signature. AG1296 increased abundance of bone morphogenetic protein receptors, downstream signaling, and gene expression and suppressed PAH smooth muscle cell proliferation. AG1296 induced regression of PA neointimal lesions in lung organ culture and PA occlusive changes in the Sugen/hypoxia rat model and reduced right ventricular systolic pressure. Moreover, AG1296 improved vascular function and BMPR2 signaling and showed better correlation with the anti-PAH gene signature than other tyrosine kinase inhibitors. Specifically, AG1296 up-regulated small mothers against decapentaplegic (SMAD) 1/5 coactivators, cAMP response element-binding protein 3 (CREB3), and CREB5: CREB3 induced inhibitor of DNA binding 1 and downstream genes that improved vascular function. Thus, drug discovery for PAH can be accelerated by combining phenotypic screening with in silico analyses of publicly available datasets.

  View details for DOI 10.1126/scitranslmed.aba6480

  View details for PubMedID 33952674

• Frataxin deficiency promotes endothelial senescence in pulmonary hypertension. The Journal of clinical investigation Culley, M. K., Zhao, J., Tai, Y. Y., Tang, Y., Perk, D., Negi, V., Yu, Q., Woodcock, C. C., Handen, A., Speyer, G., Kim, S., Lai, Y., Satoh, T., Watson, A. M., Al Aaraj, Y., Sembrat, J., Rojas, M., Goncharov, D., Goncharova, E. A., Khan, O. F., Anderson, D. G., Dahlman, J. E., Gurkar, A. U., Lafyatis, R., Fayyaz, A. U., Redfield, M. M., Gladwin, M. T., Rabinovitch, M., Gu, M., Bertero, T., Chan, S. Y. 2021

  Abstract

  The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S-containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell-derived endothelial cells from Friedreich's ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency-dependent senescence drove vessel inflammation, remodeling, and PH, while pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.

  View details for DOI 10.1172/JCI136459

  View details for PubMedID 33905372

• ALDH1A3 Coordinates Metabolism with Gene Regulation in Pulmonary Arterial Hypertension. Circulation Li, D., Shao, N., Moonen, J., Zhao, Z., Shi, M., Otsuki, S., Wang, L., Nguyen, T., Yan, E., Marciano, D. P., Contrepois, K., Li, C. G., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2021

  Abstract

  Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. Methods: RNA Sequencing was applied to pulmonary arterial (PA) SMC from PAH patients with and without a BMPR2 mutation vs. control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. Results: ALDH1A3 mRNA and protein were increased in PAH vs control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced PA muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl-CoA to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at nuclear factor Y (NFY)A binding sites via the acetyltransferase KAT2B and permitted NFY mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was beta-catenin dependent. Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not EC, is an important therapeutic consideration.

  View details for DOI 10.1161/CIRCULATIONAHA.120.048845

  View details for PubMedID 33764154

• Computational simulation-derived hemodynamic and biomechanical properties of the pulmonary arterial tree early in the course of ventricular septal defects. Biomechanics and modeling in mechanobiology Dong, M. L., Lan, I. S., Yang, W., Rabinovitch, M., Feinstein, J. A., Marsden, A. L. 2021

  Abstract

  Untreated ventricular septal defects (VSDs) can lead to pulmonary arterial hypertension (PAH) characterized by elevated pulmonary artery (PA) pressure and vascular remodeling, known as PAH associated with congenital heart disease (PAH-CHD). Though previous studies have investigated hemodynamic effects on vascular mechanobiology in late-stage PAH, hemodynamics leading to PAH-CHD initiation have not been fully quantified. We hypothesize that abnormal hemodynamics from left-to-right shunting in early stage VSDs affects PA biomechanical properties leading to PAH initiation. To model PA hemodynamics in healthy, small, moderate, and large VSD conditions prior to the onset of vascular remodeling, computational fluid dynamics simulations were performed using a 3D finite element model of a healthy 1-year-old's proximal PAs and a body-surface-area-scaled 0D distal PA tree. VSD conditions were modeled with increased pulmonary blood flow to represent degrees of left-to-right shunting. In the proximal PAs, pressure, flow, strain, and wall shear stress (WSS) increased with increasing VSD size; oscillatory shear index decreased with increasing VSD size in the larger PA vessels. WSS was higher in smaller diameter vessels and increased with VSD size, with the large VSD condition exhibiting WSS >100 dyn/cm[Formula: see text], well above values typically used to study dysfunctional mechanotransduction pathways in PAH. This study is the first to estimate hemodynamic and biomechanical metrics in the entire pediatric PA tree with VSD severity at the stage leading to PAH initiation and has implications for future studies assessing effects of abnormal mechanical stimuli on endothelial cells and vascular wall mechanics that occur during PAH-CHD initiation and progression.

  View details for DOI 10.1007/s10237-021-01519-4

  View details for PubMedID 34585299

• Severe Pulmonary Arterial Hypertension is Characterized by Increased Neutrophil Elastase and Relative Elafin Deficiency. Chest Sweatt, A. J., Miyagawa, K., Rhodes, C. J., Taylor, S., Del Rosario, P. A., Hsi, A., Haddad, F., Spiekerkoetter, E., Bental-Roof, M., Bland, R. D., Swietlik, E. M., Gräf, S., Wilkins, M. R., Morrell, N. W., Nicolls, M. R., Rabinovitch, M., Zamanian, R. T. 2021

  Abstract

  Preclinical evidence implicates neutrophil elastase (NE) in PAH pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation.Are circulating NE and elafin levels abnormal in PAH and associated with clinical severity?. In an observational Stanford University PAH cohort (N=249), plasma NE and elafin were measured in comparison to healthy controls (N=106) then related to clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (N=75, N=357). Mixed effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE/elafin balance on pulmonary artery endothelial cells (PAECs) from PAH patients.Relative to controls, patients had increased NE (205.1 [123.6-387.3] vs. 97.6 [74.4-126.6] ng/mL, P<0.0001) and decreased elafin (32.0 [15.3-59.1] vs. 45.5 [28.1-92.8] ng/mL, P<0.0001) independent of PAH subtype, illness duration, and therapies. Higher NE associated with worse symptom severity, shorter six-minute walk distance, higher NT-proBNP, greater right ventricular dysfunction, worse hemodynamics, increased circulating neutrophils, elevated cytokine levels, and lower blood BMPR2 expression. In Stanford patients, NE>168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (HR 2.52, CI 1.36-4.65, P=0.003) or prognostic cytokines (HR 2.63, CI 1.42-4.87, P=0.001), and NE added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH-PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients.NE is increased and elafin deficient across PAH subtypes. NE associates with disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin.

  View details for DOI 10.1016/j.chest.2021.06.028

  View details for PubMedID 34181952

• Patient-Specific Induced Pluripotent Stem Cells Implicate Intrinsic Impaired Contractility in Hypoplastic Left Heart Syndrome. Circulation Paige, S. L., Galdos, F. X., Lee, S., Chin, E. T., Ranjbarvaziri, S., Feyen, D. A., Darsha, A. K., Xu, S., Ryan, J. A., Beck, A. L., Qureshi, M. Y., Miao, Y., Gu, M., Bernstein, D., Nelson, T. J., Mercola, M., Rabinovitch, M., Ashley, E. A., Parikh, V. N., Wu, S. M. 2020; 142 (16): 1605–8

  View details for DOI 10.1161/CIRCULATIONAHA.119.045317

  View details for PubMedID 33074758

• Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome. Cell stem cell Miao, Y., Tian, L., Martin, M., Paige, S. L., Galdos, F. X., Li, J., Klein, A., Zhang, H., Ma, N., Wei, Y., Stewart, M., Lee, S., Moonen, J., Zhang, B., Grossfeld, P., Mital, S., Chitayat, D., Wu, J. C., Rabinovitch, M., Nelson, T. J., Nie, S., Wu, S. M., Gu, M. 2020

  Abstract

  Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

  View details for DOI 10.1016/j.stem.2020.07.015

  View details for PubMedID 32810435

• Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy. Science translational medicine Sayed, N., Liu, C., Ameen, M., Himmati, F., Zhang, J. Z., Khanamiri, S., Moonen, J., Wnorowski, A., Cheng, L., Rhee, J., Gaddam, S., Wang, K. C., Sallam, K., Boyd, J. H., Woo, Y. J., Rabinovitch, M., Wu, J. C. 2020; 12 (554)

  Abstract

  Mutations in LMNA, the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in LMNA Human induced pluripotent stem cell (iPSC)-derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized. Patients with LMNA-related DCM exhibited clinical endothelial dysfunction, and their iPSC-ECs showed decreased functionality as seen by impaired angiogenesis and nitric oxide (NO) production. Moreover, genome-edited isogenic iPSC lines recapitulated the EC disease phenotype in which LMNA-corrected iPSC-ECs showed restoration of EC function. Simultaneous profiling of chromatin accessibility and gene expression dynamics by combining assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) as well as loss-of-function studies identified Kruppel-like factor 2 (KLF2) as a potential transcription factor responsible for the EC dysfunction. Gain-of-function studies showed that treatment of LMNA iPSC-ECs with KLF2 agonists, including lovastatin, rescued the EC dysfunction. Patients with LMNA-related DCM treated with lovastatin showed improvements in clinical endothelial dysfunction as indicated by increased reactive hyperemia index. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from patients exhibiting the DCM phenotype showed improvement in CM function when cocultured with iPSC-ECs and lovastatin. These results suggest that impaired cross-talk between ECs and CMs can contribute to the pathogenesis of LMNA-related DCM, and statin may be an effective therapy for vascular dysfunction in patients with cardiolaminopathy.

  View details for DOI 10.1126/scitranslmed.aax9276

  View details for PubMedID 32727917

• Cellular senescence impairs the reversibility of pulmonary arterial hypertension. Science translational medicine van der Feen, D. E., Bossers, G. P., Hagdorn, Q. A., Moonen, J., Kurakula, K., Szulcek, R., Chappell, J., Vallania, F., Donato, M., Kok, K., Kohli, J. S., Petersen, A. H., van Leusden, T., Demaria, M., Goumans, M. T., De Boer, R. A., Khatri, P., Rabinovitch, M., Berger, R. M., Bartelds, B. 2020; 12 (554)

  Abstract

  Pulmonary arterial hypertension (PAH) in congenital cardiac shunts can be reversed by hemodynamic unloading (HU) through shunt closure. However, this reversibility potential is lost beyond a certain point in time. The reason why PAH becomes irreversible is unknown. In this study, we used MCT+shunt-induced PAH in rats to identify a dichotomous reversibility response to HU, similar to the human situation. We compared vascular profiles of reversible and irreversible PAH using RNA sequencing. Cumulatively, we report that loss of reversibility is associated with a switch from a proliferative to a senescent vascular phenotype and confirmed markers of senescence in human PAH-CHD tissue. In vitro, we showed that human pulmonary endothelial cells of patients with PAH are more vulnerable to senescence than controls in response to shear stress and confirmed that the senolytic ABT263 induces apoptosis in senescent, but not in normal, endothelial cells. To support the concept that vascular cell senescence is causal to the irreversible nature of end-stage PAH, we targeted senescence using ABT263 and induced reversal of the hemodynamic and structural changes associated with severe PAH refractory to HU. The factors that drive the transition from a reversible to irreversible pulmonary vascular phenotype could also explain the irreversible nature of other PAH etiologies and provide new leads for pharmacological reversal of end-stage PAH.

  View details for DOI 10.1126/scitranslmed.aaw4974

  View details for PubMedID 32727916

• Proceedings From the 2019 Stanford Single Ventricle Scientific Summit: Advancing Science for Single Ventricle Patients: From Discovery to Clinical Applications. Journal of the American Heart Association Reddy, S., Handler, S. S., Wu, S., Rabinovitch, M., Wright, G. 2020; 9 (7): e015871

  Abstract

  Abstracts Because of remarkable advances in survival over the past 40years, the worldwide population of individuals with single ventricle heart disease living with Fontan circulation has grown to 70000, with nearly half aged >18years. Survival to at least 30years of age is now achievable for 75% of Fontan patients. On the other hand, single ventricle patients account for the largest group of the 6000 to 8000 children hospitalized with circulation failure, with or without heart failure annually in the United States, with the highest in-hospital mortality. Because there is little understanding of the underlying mechanisms of heart failure, arrhythmias, pulmonary and lymphatic vascular abnormalities, and other morbidities, there are no specific treatments to maintain long-term myocardial performance or to optimize overall patient outcomes.

  View details for DOI 10.1161/JAHA.119.015871

  View details for PubMedID 32188306

• Image-based scaling laws for somatic growth and pulmonary artery morphometry from infant- to adulthood. American journal of physiology. Heart and circulatory physiology Dong, M. L., Yang, W. n., Tamaresis, J. S., Chan, F. P., Zucker, E. J., Kumar, S. n., Rabinovitch, M. n., Marsden, A. L., Feinstein, J. A. 2020

  Abstract

  Pulmonary artery (PA) morphometry has been extensively explored in adults, with particular focus on intra-acinar arteries. However, scaling law relationships for length and diameter of extensive pre-acinar PAs by age have not been previously reported for in vivo human data. To understand pre-acinar PA growth spanning children to adults, we performed morphometric analyses of all PAs visible in the computed tomography (CT) and magnetic resonance (MR) images from a healthy subject cohort (n=16; age: 1-51 years; body surface area, BSA: 0.49-2.01 m2). Subject-specific anatomic PA models were constructed from CT and MR images, and morphometric information - diameter, length, tortuosity, bifurcation angle, and connectivity - was extracted and sorted into diameter-defined Strahler orders. Validation of Murray's law, describing optimal scaling exponents of radii for branching vessels, was performed to determine how closely PAs conform to this classical relationship. Using regression analyses of vessel diameters and lengths against orders and patient metrics (BSA, age, height), we found that diameters increased exponentially with order and allometrically with patient metrics, and length increased allometrically with patient metrics, albeit weakly. The average tortuosity index of all vessels was 0.026 ± 0.024, average bifurcation angle was 28.2º ± 15.1º, and average Murray's law exponent was 2.92 ± 1.07. We report a set of scaling laws for vessel diameter and length, along with other morphometric information. These provide an initial understanding of healthy structural pre-acinar PA development with age, which can be used for computational modeling studies and comparison with diseased PA anatomy.

  View details for DOI 10.1152/ajpheart.00123.2020

  View details for PubMedID 32618514

• Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program. Stem cell research Kanchan, K. n., Iyer, K. n., Yanek, L. R., Carcamo-Orive, I. n., Taub, M. A., Malley, C. n., Baldwin, K. n., Becker, L. C., Broeckel, U. n., Cheng, L. n., Cowan, C. n., D'Antonio, M. n., Frazer, K. A., Quertermous, T. n., Mostoslavsky, G. n., Murphy, G. n., Rabinovitch, M. n., Rader, D. J., Steinberg, M. H., Topol, E. n., Yang, W. n., Knowles, J. W., Jaquish, C. E., Ruczinski, I. n., Mathias, R. A. 2020; 46: 101803

  Abstract

  Human induced pluripotent stem cell (hiPSC) lines have previously been generated through the NHLBI sponsored NextGen program at nine individual study sites. Here, we examined the structural integrity of 506 hiPSC lines as determined by copy number variations (CNVs). We observed that 149 hiPSC lines acquired 258 CNVs relative to donor DNA. We identified six recurrent regions of CNVs on chromosomes 1, 2, 3, 16 and 20 that overlapped with cancer associated genes. Furthermore, the genes mapping to regions of acquired CNVs show an enrichment in cancer related biological processes (IL6 production) and signaling cascades (JNK cascade & NFκB cascade). The genomic region of instability on chr20 (chr20q11.2) includes transcriptomic signatures for cancer associated genes such as ID1, BCL2L1, TPX2, PDRG1 and HCK. Of these HCK shows statistically significant differential expression between carrier and non-carrier hiPSC lines. Overall, while a low level of genomic instability was observed in the NextGen generated hiPSC lines, the observation of structural instability in regions with known cancer associated genes substantiates the importance of systematic evaluation of genetic variations in hiPSCs before using them as disease/research models.

  View details for DOI 10.1016/j.scr.2020.101803

  View details for PubMedID 32442913

• MicroRNA-483 amelioration of experimental pulmonary hypertension. EMBO molecular medicine Zhang, J. n., He, Y. n., Yan, X. n., Chen, S. n., He, M. n., Lei, Y. n., Zhang, J. n., Gongol, B. n., Gu, M. n., Miao, Y. n., Bai, L. n., Cui, X. n., Wang, X. n., Zhang, Y. n., Fan, F. n., Li, Z. n., Shen, Y. n., Chou, C. H., Huang, H. D., Malhotra, A. n., Rabinovitch, M. n., Jing, Z. C., Shyy, J. Y. 2020: e11303

  Abstract

  Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-β (TGF-β), TGF-β receptor 2 (TGFBR2), β-catenin, connective tissue growth factor (CTGF), interleukin-1β (IL-1β), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses.

  View details for DOI 10.15252/emmm.201911303

  View details for PubMedID 32324970

• Remodeling of active endothelial enhancers is associated with aberrant gene-regulatory networks in pulmonary arterial hypertension. Nature communications Reyes-Palomares, A. n., Gu, M. n., Grubert, F. n., Berest, I. n., Sa, S. n., Kasowski, M. n., Arnold, C. n., Shuai, M. n., Srivas, R. n., Miao, S. n., Li, D. n., Snyder, M. P., Rabinovitch, M. n., Zaugg, J. B. 2020; 11 (1): 1673

  Abstract

  Environmental and epigenetic factors often play an important role in polygenic disorders. However, how such factors affect disease-specific tissues at the molecular level remains to be understood. Here, we address this in pulmonary arterial hypertension (PAH). We obtain pulmonary arterial endothelial cells (PAECs) from lungs of patients and controls (n = 19), and perform chromatin, transcriptomic and interaction profiling. Overall, we observe extensive remodeling at active enhancers in PAH PAECs and identify hundreds of differentially active TFs, yet find very little transcriptomic changes in steady-state. We devise a disease-specific enhancer-gene regulatory network and predict that primed enhancers in PAH PAECs are activated by the differentially active TFs, resulting in an aberrant response to endothelial signals, which could lead to disturbed angiogenesis and endothelial-to-mesenchymal-transition. We validate these predictions for a selection of target genes in PAECs stimulated with TGF-β, VEGF or serotonin. Our study highlights the role of chromatin state and enhancers in disease-relevant cell types of PAH.

  View details for DOI 10.1038/s41467-020-15463-x

  View details for PubMedID 32245974

• Landscape of cohesin-mediated chromatin loops in the human genome. Nature Grubert, F. n., Srivas, R. n., Spacek, D. V., Kasowski, M. n., Ruiz-Velasco, M. n., Sinnott-Armstrong, N. n., Greenside, P. n., Narasimha, A. n., Liu, Q. n., Geller, B. n., Sanghi, A. n., Kulik, M. n., Sa, S. n., Rabinovitch, M. n., Kundaje, A. n., Dalton, S. n., Zaugg, J. B., Snyder, M. n. 2020; 583 (7818): 737–43

  Abstract

  Physical interactions between distal regulatory elements have a key role in regulating gene expression, but the extent to which these interactions vary between cell types and contribute to cell-type-specific gene expression remains unclear. Here, to address these questions as part of phase III of the Encyclopedia of DNA Elements (ENCODE), we mapped cohesin-mediated chromatin loops, using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), and analysed gene expression in 24 diverse human cell types, including core ENCODE cell lines. Twenty-eight per cent of all chromatin loops vary across cell types; these variations modestly correlate with changes in gene expression and are effective at grouping cell types according to their tissue of origin. The connectivity of genes corresponds to different functional classes, with housekeeping genes having few contacts, and dosage-sensitive genes being more connected to enhancer elements. This atlas of chromatin loops complements the diverse maps of regulatory architecture that comprise the ENCODE Encyclopedia, and will help to support emerging analyses of genome structure and function.

  View details for DOI 10.1038/s41586-020-2151-x

  View details for PubMedID 32728247

• PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension. Circulation research Hennigs, J. K., Cao, A. n., Li, C. G., Shi, M. n., Mienert, J. n., Miyagawa, K. n., Körbelin, J. n., Marciano, D. P., Chen, P. I., Roughley, M. n., Elliott, M. V., Harper, R. L., Bill, M. A., Chappell, J. n., Moonen, J. R., Diebold, I. n., Wang, L. n., Snyder, M. P., Rabinovitch, M. n. 2020

  Abstract

  Rationale: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of bone morphogenetic protein type 2 receptor (BMPR2) via two downstream transcription factors, PPARγ and p53. Objective: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ- p53 transcription factor complex in the pulmonary endothelium. Methods and Results: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial (PA) and lung microvascular (MV) endothelial cells (EC) in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation (ChIP) sequencing (seq) and RNA-seq established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung EC survival, angiogenesis and DNA repair including, EPHA2, FHL2, JAG1, SULF2 and TIGAR. Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in PAEC subjected to oxidative stress. In EC-Bmpr2-knockout mice unable to stabilize p53 in ECs under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAEC from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. Conclusions: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAEC downstream of dysfunctional BMPR2 to rehabilitate PAH PAEC, regenerate pulmonary microvessels and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.

  View details for DOI 10.1161/CIRCRESAHA.119.316339

  View details for PubMedID 33322916

• Targeted Proteomics of Right Heart Adaptation to Pulmonary Arterial Hypertension. The European respiratory journal Amsallem, M. n., Sweatt, A. J., Arthur Ataam, J. n., Guihaire, J. n., Lecerf, F. n., Lambert, M. n., Ghigna, M. R., Ali, M. K., Mao, Y. n., Fadel, E. n., Rabinovitch, M. n., de Jesus Perez, V. n., Spiekerkoetter, E. n., Mercier, O. n., Haddad, F. n., Zamanian, R. T. 2020

  Abstract

  No prior proteomic screening study has centered on the right ventricle (RV) in pulmonary arterial hypertension (PAH). This study investigates the circulating proteomic profile associated with right heart maladaptive phenotype (RHMP) in PAH.Plasma proteomic profiling was performed using multiplex immunoassay in 121 PAH patients (discovery cohort) and 76 patients (validation cohort). The association between proteomic markers and RHMP (defined by the Mayo right heart score [combining RV strain, New York Heart Association NYHA class and NT-proBNP] and Stanford score [RV end-systolic remodelling index, NYHA and NT-proBNP]) was assessed by partial least squares regression. Biomarkers expressions were measured in RV samples from PAH patients and controls, and pulmonary artery banding (PAB) mice.High levels of hepatic growth factor (HGF), stem cell growth factor beta, nerve growth factor and stromal derived factor-1 were associated with worse Mayo and Stanford scores independently from pulmonary resistance or pressure in both cohorts (the validation cohort had more severe disease features: lower cardiac index and higher NT-proBNP). In both cohorts, HGF added value to the REVEAL score in the prediction of death, transplant, or hospitalisation at 3 years. RV expression levels of HGF and its receptor c-Met were higher in end-stage PAH patients than controls, and in PAB mice than shams.High plasma HGF levels are associated with RHMP and predictive of 3-year clinical worsening. Both HGF and c-Met RV expression levels are increased in PAH. Assessing plasma HGF levels might identify patients at risk for heart failure who warrant closer follow-up and intensified therapy.

  View details for DOI 10.1183/13993003.02428-2020

  View details for PubMedID 33334941

• Parameters associated with outcome in pediatric patients with congenital heart disease and pulmonary hypertension subjected to combined vasodilator and surgical treatments PULMONARY CIRCULATION Thomaz, A., Kajita, L. J., Aiello, V. D., Zorzanelli, L., Galas, F. G., Machado, C. G., Barbero-Marcial, M., Jatene, M. B., Rabinovitch, M., Lopes, A. 2019; 9 (3)

  View details for DOI 10.1177/2045894019837885

  View details for Web of Science ID 000480238100001

• Evolution of hemodynamic forces in the pulmonary tree with progressively worsening pulmonary arterial hypertension in pediatric patients BIOMECHANICS AND MODELING IN MECHANOBIOLOGY Yang, W., Dong, M., Rabinovitch, M., Chan, F. P., Marsden, A. L., Feinstein, J. A. 2019; 18 (3): 779–96

  View details for DOI 10.1007/s10237-018-01114-0

  View details for Web of Science ID 000467394100015

• Discovery of Distinct Immune Phenotypes Using Machine Learning in Pulmonary Arterial Hypertension CIRCULATION RESEARCH Sweatt, A. J., Hedlin, H. K., Balasubramanian, V., Hsi, A., Blum, L. K., Robinson, W. H., Haddad, F., Hickey, P. M., Condliffe, R., Lawrie, A., Nicolls, M. R., Rabinovitch, M., Khatri, P., Zamanian, R. T. 2019; 124 (6): 904–19

  View details for DOI 10.1161/CIRCRESAHA.118.313911

  View details for Web of Science ID 000469341600015

• EXPRESS: Parameters associated with outcome in pediatric patients with congenital heart disease and pulmonary hypertension subjected to combined vasodilator and surgical treatments. Pulmonary circulation Thomaz, A. M., Kajita, L. J., Aiello, V. D., Zorzanelli, L., Galas, F. R., Machado, C. G., Barbero-Marcial, M., Jatene, M. B., Rabinovitch, M., Lopes, A. A. 2019: 2045894019837885

  View details for PubMedID 30806154

• PPARgamma Interaction with UBR5/ATMIN Promotes DNA Repair to Maintain Endothelial Homeostasis. Cell reports Li, C. G., Mahon, C., Sweeney, N. M., Verschueren, E., Kantamani, V., Li, D., Hennigs, J. K., Marciano, D. P., Diebold, I., Abu-Halawa, O., Elliott, M., Sa, S., Guo, F., Wang, L., Cao, A., Guignabert, C., Sollier, J., Nickel, N. P., Kaschwich, M., Cimprich, K. A., Rabinovitch, M. 2019; 26 (5): 1333

  Abstract

  Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor gamma (PPARgamma) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARgamma promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARgamma depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARgamma DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARgamma signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARgamma-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARgamma DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.

  View details for DOI 10.1016/j.celrep.2019.01.013

  View details for PubMedID 30699358

• Smooth Muscle Contact Drives Endothelial Regeneration by BMPR2-Notch1-Mediated Metabolic and Epigenetic Changes CIRCULATION RESEARCH Miyagawa, K., Shi, M., Chen, P., Hennigs, J. K., Zhao, Z., Wang, M., Li, C. G., Saito, T., Taylor, S., Sa, S., Cao, A., Wang, L., Snyder, M. P., Rabinovitch, M. 2019; 124 (2): 211–24

  View details for DOI 10.1161/CIRCRESAHA.118.313374

  View details for Web of Science ID 000469341100015

• Evolution of hemodynamic forces in the pulmonary tree with progressively worsening pulmonary arterial hypertension in pediatric patients. Biomechanics and modeling in mechanobiology Yang, W., Dong, M., Rabinovitch, M., Chan, F. P., Marsden, A. L., Feinstein, J. A. 2019

  Abstract

  Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling resulting in right ventricular (RV) dysfunction and ultimately RV failure. Mechanical stimuli acting on the vessel walls of the full pulmonary tree have not previously been comprehensively characterized. The goal of this study is to characterize wall shear stress (WSS) and strain in pediatric PAH patients at different stages of disease severity using computational patient-specific modeling. Computed tomography, magnetic resonance imaging and right heart catheterization data were collected and assimilated into pulmonary artery (PA) models for patients with and without PAH. Patients were grouped in three disease severity groups (control, moderate and severe) based on clinical evaluations. A finite element solver was employed to quantify hemodynamics and wall strains. To estimate WSS in the distal small PAs with diameters ranging from 50 to 500[Formula: see text], a morphometric tree model was created, with inputs coming from outlets of the 3D model. WSS in the proximal PAs decreased with disease severity (control 20.5 vs. moderate 15.8 vs. severe 6.3[Formula: see text], [Formula: see text]). Oscillatory shear index increased in the main pulmonary artery (MPA) with disease severity (0.13 vs. 0.13 vs. 0.2, [Formula: see text]). Wall strains measured by the first invariant of Green strain tensor decreased with disease severity (0.16 vs. 0.12 vs. 0.11, [Formula: see text]). Mean WSS for the distal PAs between 100 and 500[Formula: see text] significantly increased with disease severity (20 vs. 52 vs. 116 [Formula: see text], [Formula: see text]). In conclusion, 3D flow simulations showed that WSS is significantly decreased in the MPA with disease while the mathematical morphometric model suggested increased WSS in the distal small vessels. Computational models can reveal mechanical stimuli acting on vessel walls that may inform patient risk stratification and flow shear experiments.

  View details for PubMedID 30635853

• Hot topics in the mechanisms of pulmonary arterial hypertension disease: cancer-like pathobiology, the role of the adventitia, systemic involvement, and right ventricular failure. Pulmonary circulation Spiekerkoetter, E. n., Goncharova, E. A., Guignabert, C. n., Stenmark, K. n., Kwapiszewska, G. n., Rabinovitch, M. n., Voelkel, N. n., Bogaard, H. J., Graham, B. n., Pullamsetti, S. S., Kuebler, W. M. 2019; 9 (4): 2045894019889775

  Abstract

  In order to intervene appropriately and develop disease-modifying therapeutics for pulmonary arterial hypertension, it is crucial to understand the mechanisms of disease pathogenesis and progression. We herein discuss four topics of disease mechanisms that are currently highly debated, yet still unsolved, in the field of pulmonary arterial hypertension. Is pulmonary arterial hypertension a cancer-like disease? Does the adventitia play an important role in the initiation of pulmonary vascular remodeling? Is pulmonary arterial hypertension a systemic disease? Does capillary loss drive right ventricular failure? While pulmonary arterial hypertension does not replicate all features of cancer, anti-proliferative cancer therapeutics might still be beneficial in pulmonary arterial hypertension if monitored for safety and tolerability. It was recognized that the adventitia as a cell-rich compartment is important in the disease pathogenesis of pulmonary arterial hypertension and should be a therapeutic target, albeit the data are inconclusive as to whether the adventitia is involved in the initiation of neointima formation. There was agreement that systemic diseases can lead to pulmonary arterial hypertension and that pulmonary arterial hypertension can have systemic effects related to the advanced lung pathology, yet there was less agreement on whether idiopathic pulmonary arterial hypertension is a systemic disease per se. Despite acknowledging the limitations of exactly assessing vascular density in the right ventricle, it was recognized that the failing right ventricle may show inadequate vascular adaptation resulting in inadequate delivery of oxygen and other metabolites. Although the debate was not meant to result in a definite resolution of the specific arguments, it sparked ideas about how we might resolve the discrepancies by improving our disease modeling (rodent models, large-animal studies, studies of human cells, tissues, and organs) as well as standardization of the models. Novel experimental approaches, such as lineage tracing and better three-dimensional imaging of experimental as well as human lung and heart tissues, might unravel how different cells contribute to the disease pathology.

  View details for DOI 10.1177/2045894019889775

  View details for PubMedID 31798835

  View details for PubMedCentralID PMC6868582

• Phenotypically-Silent Bone Morphogenetic Protein Receptor 2 (Bmpr2) Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing The Risk for Neointimal Transformation. Circulation Tian, W. n., Jiang, X. n., Sung, Y. K., Shuffle, E. n., Wu, T. H., Kao, P. N., Tu, A. B., Dorfmüller, P. n., Cao, A. n., Wang, L. n., Peng, G. n., Kim, Y. n., Zhang, P. n., Chappell, J. n., Pasupneti, S. n., Dahms, P. n., Maguire, P. n., Chaib, H. n., Zamanian, R. n., Peters-Golden, M. n., Snyder, M. P., Voelkel, N. F., Humbert, M. n., Rabinovitch, M. n., Nicolls, M. R. 2019

  Abstract

  Bmpr2 mutations are critical risk factors for hereditary pulmonary arterial hypertension (hPAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-lipoxygenase (5-LO) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4 (LTB4), are candidates for the 'second hit'. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically-silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats.Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to one year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus (AdAlox5), monocrotaline, SU5416, SU5416 with chronic hypoxia or chronic hypoxia alone. Bmpr2-mutant hPAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells (PAECs) with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes.Lung inflammation, induced by intratracheal delivery of AdAlox5, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated LTB4 biosynthesis. Bmpr2-mutant hPAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant, and proliferative PAECs with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced LTB4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated NF-κB, IL-6, and TGF-β signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation.In a new 'two-hit' model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced LTB4 production. This study offers one explanation of how an environmental injury unleashes the destructive potential of an otherwise-silent genetic mutation.

  View details for DOI 10.1161/CIRCULATIONAHA.119.040629

  View details for PubMedID 31462075

• Discovery of Distinct Immune Phenotypes Using Machine Learning in Pulmonary Arterial Hypertension. Circulation research Sweatt, A. J., Hedlin, H. K., Balasubramanian, V. n., Hsi, A. n., Blum, L. K., Robinson, W. H., Haddad, F. n., Hickey, P. M., Condliffe, R. A., Lawrie, A. n., Nicolls, M. R., Rabinovitch, M. n., Khatri, P. n., Zamanian, R. T. 2019

  Abstract

  Accumulating evidence implicates inflammation in pulmonary arterial hypertension (PAH) and therapies targeting immunity are under investigation, though it remains unknown if distinct immune phenotypes exist.Identify PAH immune phenotypes based on unsupervised analysis of blood proteomic profiles.In a prospective observational study of Group 1 PAH patients evaluated at Stanford University (discovery cohort, n=281) and University of Sheffield (validation cohort, n=104) between 2008-2014, we measured a circulating proteomic panel of 48 cytokines, chemokines, and factors using multiplex immunoassay. Unsupervised machine learning (consensus clustering) was applied in both cohorts independently to classify patients into proteomic immune clusters, without guidance from clinical features. To identify central proteins in each cluster, we performed partial correlation network analysis. Clinical characteristics and outcomes were subsequently compared across clusters. Four PAH clusters with distinct proteomic immune profiles were identified in the discovery cohort. Cluster 2 (n=109) had low cytokine levels similar to controls. Other clusters had unique sets of upregulated proteins central to immune networks- cluster 1 (n=58)(TRAIL, CCL5, CCL7, CCL4, MIF), cluster 3 (n=77)(IL-12, IL-17, IL-10, IL-7, VEGF), and cluster 4 (n=37)(IL-8, IL-4, PDGF-β, IL-6, CCL11). Demographics, PAH etiologies, comorbidities, and medications were similar across clusters. Non-invasive and hemodynamic surrogates of clinical risk identified cluster 1 as high-risk and cluster 3 as low-risk groups. Five-year transplant-free survival rates were unfavorable for cluster 1 (47.6%, CI 35.4-64.1%) and favorable for cluster 3 (82.4%, CI 72.0-94.3%)(across-cluster p<0.001). Findings were replicated in the validation cohort, where machine learning classified four immune clusters with comparable proteomic, clinical, and prognostic features.Blood cytokine profiles distinguish PAH immune phenotypes with differing clinical risk that are independent of World Health Organization Group 1 subtypes. These phenotypes could inform mechanistic studies of disease pathobiology and provide a framework to examine patient responses to emerging therapies targeting immunity.

  View details for PubMedID 30661465

• Inflammatory Basis of Pulmonary Arterial Hypertension: Implications for Perioperative and Critical Care Medicine. Anesthesiology Goldenberg, N. M., Rabinovitch, M. n., Steinberg, B. E. 2019

  View details for DOI 10.1097/ALN.0000000000002740

  View details for PubMedID 31094755

• High-throughput Drug Screen to Reverse Phenotype of Pulmonary Artery Hypertension Ipsc Derived Vascular Cells Combined with Bioinformatics Uncovers Promising Therapies Gu, M., Donate, M., Miao, Y., Mao, S., Saito, T., Otsuki, S., Wang, L., Harper, R., Sa, S., Khatri, P., Rabinovitch, M. LIPPINCOTT WILLIAMS & WILKINS. 2018: E70

  View details for Web of Science ID 000454198400020

• Smooth Muscle Contact Drives Endothelial Regeneration by BMPR2-Notch1 Mediated Metabolic and Epigenetic Changes. Circulation research Miyagawa, K., Shi, M., Chen, P., Hennigs, J. K., Zhao, Z., Wang, M., Li, C. G., Saito, T., Taylor, S., Sa, S., Cao, A., Wang, L., Snyder, M. P., Rabinovitch, M. 2018

  Abstract

  RATIONALE: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension.OBJECTIVE: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity.METHODS AND RESULTS: We found that in SMC and EC contact co-cultures, bone morphogenetic protein receptor 2 (BMPR2) is required by both cell types to produce collagen IV to activate integrin-linked kinase. This enzyme directs phospho c-Jun N-terminal kinase (p-JNK) to the EC membrane, where it stabilizes presenilin1 and releases Notch1 intracellular domain (N1ICD) to promote EC proliferation. This response is necessary for EC regeneration following carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of pre-capillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3. ChIP-seq analyses showed that PFKFB3 is required for citrate-dependent histone acetylation (H3K27) at enhancer sites of genes regulated by the acetyl transferase p300, and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis.CONCLUSIONS: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, to maintain monolayer integrity and vascular homeostasis in response to injury.

  View details for PubMedID 30582451

• Elafin Treatment Rescues EGFR-Klf4 Signaling and Lung Cell Survival in Ventilated Newborn Mice. American journal of respiratory cell and molecular biology Alejandre Alcazar, M. A., Kaschwich, M., Ertsey, R., Preuss, S., Milla, C., Mujahid, S., Masumi, J., Khan, S., Mokres, L. M., Tian, L., Mohr, J., Hirani, D. V., Rabinovitch, M., Bland, R. D. 2018; 59 (5): 623–34

  Abstract

  Mechanical ventilation with O2-rich gas (MV-O2) inhibits alveologenesis and lung growth. We previously showed that MV-O2 increased elastase activity and apoptosis in lungs of newborn mice, whereas elastase inhibition by elafin suppressed apoptosis and enabled lung growth. Pilot studies suggested that MV-O2 reduces lung expression of prosurvival factors phosphorylated epidermal growth factor receptor (pEGFR) and Kruppel-like factor 4 (Klf4). Here, we sought to determine whether apoptosis and lung growth arrest evoked by MV-O2 reflect disrupted pEGFR-Klf4 signaling, which elafin treatment preserves, and to assess potential biomarkers of bronchopulmonary dysplasia (BPD). Five-day-old mice underwent MV with air or 40% O2 for 8-24 hours with or without elafin treatment. Unventilated pups served as controls. Immunoblots were used to assess lung pEGFR and Klf4 proteins. Cultured MLE-12 cells were exposed to AG1478 (EGFR inhibitor), Klf4 siRNA, or vehicle to assess effects on proliferation, apoptosis, and EGFR regulation of Klf4. Plasma elastase and elafin levels were measured in extremely premature infants. In newborn mice, MV with air or 40% O2 inhibited EGFR phosphorylation and suppressed Klf4 protein content in lungs (vs. unventilated controls), yielding increased apoptosis. Elafin treatment inhibited elastase, preserved lung pEGFR and Klf4, and attenuated the apoptosis observed in lungs of vehicle-treated mice. In MLE-12 studies, pharmacological inhibition of EGFR and siRNA suppression of Klf4 increased apoptosis and reduced proliferation, and EGFR inhibition decreased Klf4. Plasma elastase levels were more than twofold higher, without a compensating increase of plasma elafin, in infants with BPD, compared to infants without BPD. These findings indicate that pEGFR-Klf4 is a novel prosurvival signaling pathway in lung epithelium that MV disrupts. Elafin preserves pEGFR-Klf4 signaling and inhibits apoptosis, thereby enabling lung growth during MV. Together, our animal and human data raise the question: would elastase inhibition prevent BPD in high-risk infants exposed to MV-O2?

  View details for PubMedID 29894205

• The Role of Neutrophils and Neutrophil Elastase in Pulmonary Arterial Hypertension FRONTIERS IN MEDICINE Taylor, S., Dirir, O., Zamanian, R. T., Rabinovitch, M., Thompson, A. 2018; 5

  View details for DOI 10.3389.fmed.2018.00217

  View details for Web of Science ID 000440761000001

• The Role of Neutrophils and Neutrophil Elastase in Pulmonary Arterial Hypertension. Frontiers in medicine Taylor, S., Dirir, O., Zamanian, R. T., Rabinovitch, M., Thompson, A. A. 2018; 5: 217

  Abstract

  Pulmonary arterial hypertension (PAH) is a severe vasculopathy characterized by the presence of fibrotic lesions in the arterial wall and the loss of small distal pulmonary arteries. The vasculopathy is accompanied by perivascular inflammation and increased protease levels, with neutrophil elastase notably implicated in aberrant vascular remodeling. However, the source of elevated elastase levels in PAH remains unclear. A major source of neutrophil elastase is the neutrophil, an understudied cell population in PAH. The principal function of neutrophils is to destroy invading pathogens by means of phagocytosis and NET formation, but proteases, chemokines, and cytokines implicated in PAH can be released by and/or prime and activate neutrophils. This review focuses on the contribution of inflammation to the development and progression of the disease, highlighting studies implicating neutrophils, neutrophil elastase, and other neutrophil proteases in PAH. The roles of cytokines, chemokines, and neutrophil elastase in the disease are discussed and we describe new insight into the role neutrophils potentially play in the pathogenesis of PAH.

  View details for DOI 10.3389/fmed.2018.00217

  View details for PubMedID 30131961

  View details for PubMedCentralID PMC6090899

• Right Ventricular Stroke Work Correlates with Outcomes in Pediatric Pulmonary Arterial Hypertension. Pulmonary circulation Yang, W., Marsden, A. L., Ogawa, M. T., Sakarovitch, C., Hall, K. K., Rabinovitch, M., Feinstein, J. A. 2018: 2045894018780534

  View details for PubMedID 29767574

• Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives. The European respiratory journal Humbert, M. n., Guignabert, C. n., Bonnet, S. n., Dorfmüller, P. n., Klinger, J. R., Nicolls, M. R., Olschewski, A. J., Pullamsetti, S. S., Schermuly, R. T., Stenmark, K. R., Rabinovitch, M. n. 2018

  Abstract

  Clinical and translational research has played a major role in advancing our understanding of pulmonary hypertension (PH), including pulmonary arterial hypertension and other forms of PH with severe vascular remodelling (e.g. chronic thromboembolic PH and pulmonary veno-occlusive disease). However, PH remains an incurable condition with a high mortality rate, underscoring the need for a better transfer of novel scientific knowledge into healthcare interventions. Herein, we review recent findings in pathology (with the questioning of the strict morphological categorisation of various forms of PH into pre- or post-capillary involvement of pulmonary vessels) and cellular mechanisms contributing to the onset and progression of pulmonary vascular remodelling associated with various forms of PH. We also discuss ways to improve management and to support and optimise drug development in this research field.

  View details for PubMedID 30545970

• A Pro - Con debate: Current Controversies in PAH Pathogenesis at the American Thoracic Society International Meeting in 2017. American journal of physiology. Lung cellular and molecular physiology Kuebler, W. M., Nicolls, M. R., Olschewski, A. n., Abe, K. n., Rabinovitch, M. n., Stewart, D. J., Chan, S. Y., Morrell, N. W., Archer, S. L., Spiekerkoetter, E. n. 2018

  Abstract

  The following review summarizes the pro-con debate about current controversies regarding the pathogenesis of pulmonary arterial hypertension (PAH) that took place at the American Thoracic Society Conference in May 2017. Leaders in the field of PAH research discussed the importance of the immune system, the role of hemodynamic stress and endothelial apoptosis as well as bone morphogenetic protein receptor 2 (BMPR2) signaling in PAH pathogenesis. While this summary does not intend to resolve obvious conflicts in opinion, we hope that the presented arguments entice further discussions and draw a new generation of enthusiastic researchers into this vibrant field of science to bridge existing gaps for a better understanding and therapy of this fatal disease.

  View details for PubMedID 29877097

• A Dominant Role for Regulatory T Cells in Protecting Females Against Pulmonary Hypertension. Circulation research Tamosiuniene, R. n., Manouvakhova, O. n., Mesange, P. n., Saito, T. n., Qian, J. n., Sanyal, M. n., Lin, Y. C., Nguyen, L. P., Luria, A. n., Tu, A. B., Sante, J. M., Rabinovitch, M. n., Fitzgerald, D. J., Graham, B. B., Habtezion, A. n., Voelkel, N. F., Aurelian, L. n., Nicolls, M. R. 2018

  Abstract

  Rationale: Pulmonary arterial hypertension (PH) is a life-threatening condition associated with immune dysregulation and abnormal regulatory T cell (Treg) activity, but it is currently unknown whether and how abnormal Treg function differentially affects males and females. Objective: To evaluate whether and how Treg-deficiency differentially affects male and female rats in experimental PH. Methods and Results: Male and female athymicrnu/rnurats, lacking Tregs, were treated with the vascular endothelial growth factor receptor-2 (VEGFR2) inhibitor SU5416 or chronic hypoxia and evaluated for PH; some animals underwent Treg immune reconstitution (IR) before SU5416 administration. Plasma prostacyclin (PGI2) levels were measured. Lung and right ventricles (RVs) were assessed for the expression of the vasoprotective proteins cyclooxygenase-2 (COX-2), prostacyclin synthase (PTGIS), programmed death ligand-1 (PDL-1), and heme oxygenase-1 (HO-1). Inhibitors of these pathways were administered to athymic rats undergoing Treg IR. Finally, human cardiac microvascular endothelial cells co-cultured with Tregs were evaluated for COX-2, PDL-1, HO-1, and estrogen receptor (ER) expression, and culture supernatants were assayed for PGI2 and IL-10. SU5416-treatment and chronic hypoxia produced more severe PH in female than male athymic rats. Females were distinguished by greater pulmonary inflammation, augmented RV fibrosis, lower plasma PGI2 levels, decreased lung COX-2, PTGIS, HO-1 and PDL-1 expression and reduced RV PDL-1 levels. In both sexes, Treg IR protected against PH development and raised levels of plasma PGI2 and cardiopulmonary COX-2, PTGIS, PDL-1, and HO-1. Inhibiting COX-2, HO-1, and programmed death-1 (PD1)/PDL1 pathways abrogated Treg protection. In vitro, human Tregs directly upregulated endothelial COX-2, PDL1, HO-1, ERs and increased supernatant levels of PGI2 and IL-10. Conclusions: In two animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

  View details for PubMedID 29545367

• Right Heart End-Systolic Remodeling Index Strongly Predicts Outcomes in Pulmonary Arterial Hypertension: Comparison With Validated Models. Circulation. Cardiovascular imaging Amsallem, M., Sweatt, A. J., Aymami, M. C., Kuznetsova, T., Selej, M., Lu, H., Mercier, O., Fadel, E., Schnittger, I., McConnell, M. V., Rabinovitch, M., Zamanian, R. T., Haddad, F. 2017; 10 (6)

  Abstract

  Right ventricular (RV) end-systolic dimensions provide information on both size and function. We investigated whether an internally scaled index of end-systolic dimension is incremental to well-validated prognostic scores in pulmonary arterial hypertension.From 2005 to 2014, 228 patients with pulmonary arterial hypertension were prospectively enrolled. RV end-systolic remodeling index (RVESRI) was defined by lateral length divided by septal height. The incremental values of RV free wall longitudinal strain and RVESRI to risk scores were determined. Mean age was 49±14 years, 78% were female, 33% had connective tissue disease, 52% were in New York Heart Association class ≥III, and mean pulmonary vascular resistance was 11.2±6.4 WU. RVESRI and right atrial area were strongly connected to the other right heart metrics. Three zones of adaptation (adapted, maladapted, and severely maladapted) were identified based on the RVESRI to RV systolic pressure relationship. During a mean follow-up of 3.9±2.4 years, the primary end point of death, transplant, or admission for heart failure was reached in 88 patients. RVESRI was incremental to risk prediction scores in pulmonary arterial hypertension, including the Registry to Evaluate Early and Long-Term PAH Disease Management score, the Pulmonary Hypertension Connection equation, and the Mayo Clinic model. Using multivariable analysis, New York Heart Association class III/IV, RVESRI, and log NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) were retained (χ(2), 62.2; P<0.0001). Changes in RVESRI at 1 year (n=203) were predictive of outcome; patients initiated on prostanoid therapy showed the greatest improvement in RVESRI. Among right heart metrics, RVESRI demonstrated the best test-retest characteristics.RVESRI is a simple reproducible prognostic marker in patients with pulmonary arterial hypertension.

  View details for DOI 10.1161/CIRCIMAGING.116.005771

  View details for PubMedID 28592589

• Enhancing Insights into Pulmonary Vascular Disease (PVD) Through a Precision Medicine Approach. A Joint NHLBI-CMREF Workshop Report. American journal of respiratory and critical care medicine Newman, J. H., Rich, S., Abman, S. H., Alexander, J. H., Barnard, J., Beck, G. J., Benza, R. L., Bull, T. M., Chan, S. Y., Chun, H. J., Doogan, D., Dupuis, J., Erzurum, S. C., Frantz, R. P., Geraci, M., Gillies, H., Gladwin, M., Gray, M. P., Hemnes, A. R., Herbst, R. S., Hernandez, A. F., Hill, N. S., Horn, E. M., Hunter, K., Jing, Z., Johns, R., Kaul, S., Kawut, S. M., Lahm, T., Leopold, J. A., Lewis, G. D., Mathai, S. C., McLaughlin, V. V., Michelakis, E. D., Nathan, S. D., Nichols, W., Page, G., Rabinovitch, M., Rich, J., Rischard, F., Rounds, S., Shah, S. J., Tapson, V. F., Lowy, N., Stockbridge, N., Weinmann, G., Xiao, L. 2017

  Abstract

  The Division of Lung Diseases of the NHLBI and the Cardiovascular Medical Education and Research Fund held a workshop to discuss how to leverage the anticipated scientific output from the recently launched "Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics" (PVDOMICS) program to develop newer approaches to pulmonary vascular disease. PVDOMICS is a collaborative, protocol-driven network to analyze all patient populations with pulmonary hypertension to define novel pulmonary vascular disease (PVD) phenotypes. Stakeholders, including basic, translational, and clinical investigators; clinicians; patient advocacy organizations; regulatory agencies; and pharmaceutical industry experts, joined to discuss the application of precision medicine to PVD clinical trials. Recommendations were generated for discussion of research priorities in line with NHLBI Strategic Vision Goals that include: (1) A national effort, involving all the stakeholders, should seek to coordinate biosamples and biodata from all funded programs to a web-based repository so that information can be shared and correlated with other research projects. Example programs sponsored by NHLBI include PVDOMICS, Pulmonary Hypertension Breakthrough Initiative, the National Biological Sample and Data Repository for PAH, and the National Precision Medicine Initiative. (2) A task force to develop a master clinical trials protocol for PVD to apply precision medicine principles to future clinical trials. Specific features include: (a) adoption of smaller clinical trials that incorporate biomarker-guided enrichment strategies, using adaptive and innovative statistical designs; and (b) development of newer endpoints that reflect well-defined and clinically meaningful changes. (3) Development of updated and systematic variables in imaging, hemodynamic, cellular, genomic, and metabolic tests that will help precisely identify individual and shared features of PVD and serve as the basis of novel phenotypes for therapeutic interventions.

  View details for DOI 10.1164/rccm.201701-0150WS

  View details for PubMedID 28430547

• Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers CELL STEM CELL Gu, M., Shao, N., Sa, S., Li, D., Termglinchan, V., Ameen, M., Karakikes, I., Sosa, G., Grubert, F., Lee, J., Cao, A., Taylor, S., Ma, Y., Zhao, Z., Chappell, J., Hamid, R., Austin, E. D., Gold, J. D., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2017; 20 (4): 490-?

  View details for DOI 10.1016/j.stem.2016.08.019

  View details for Web of Science ID 000398350800013

• Long-term miR-29b suppression reduces aneurysm formation in a Marfan mouse model. Physiological reports Okamura, H., Emrich, F., Trojan, J., Chiu, P., Dalal, A. R., Arakawa, M., Sato, T., Penov, K., Koyano, T., Pedroza, A., Connolly, A. J., Rabinovitch, M., Alvira, C., Fischbein, M. P. 2017; 5 (8)

  Abstract

  Aortic root aneurysm formation and subsequent dissection and/or rupture remain the leading cause of death in patients with Marfan syndrome. Our laboratory has reported that miR-29b participates in aortic root/ascending aorta extracellular matrix remodeling during early aneurysm formation in Fbn1(C1039G/+) Marfan mice. Herein, we sought to determine whether miR-29b suppression can reduce aneurysm formation long-term. Fbn1(C1039G/+) Marfan mice were treated with retro-orbital LNA-anti-miR-29b inhibitor or scrambled-control-miR before aneurysms develop either (1) a single dose prenatally (pregnant Fbn1(C1039G/+) mice at 14.5 days post-coitum) (n = 8-10, each group) or (2) postnatally every other week, from 2 to 22 weeks of age, and sacrificed at 24 weeks (n = 8-10, each group). To determine if miR-29b blockade was beneficial even after aneurysms develop, a third group of animals were treated every other week, starting at 8 weeks of age, until sacrificed (n = 4-6, each group). miR-29b inhibition resulted in aneurysm reduction, increased elastogenesis, decreased matrix metalloproteinase activity and decreased elastin breakdown. Prenatal LNA-anti-miR-29b inhibitor treatment decreased aneurysm formation up to age 32 weeks, whereas postnatal treatment was effective up to 16 weeks. miR-29b blockade did not slow aortic growth once aneurysms already developed. Systemic miR-29b inhibition significantly reduces aneurysm development long-term in a Marfan mouse model. Drug administration during aortic wall embryologic development appears fundamental. miR-29b suppression could be a potential therapeutic target for reducing aneurysm formation in Marfan syndrome patients.

  View details for DOI 10.14814/phy2.13257

  View details for PubMedID 28455451

• Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Sa, S., Gu, M., Chappe, J., Shao, N., Ameen, M., Elliott, K. A., Li, D., Grubert, F., Li, C. G., Taylor, S., Cao, A., Ma, Y., Fong, R., Nguyen, L., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2017; 195 (7): 930-941

  View details for DOI 10.1164/rccm.201606-1200OC

  View details for Web of Science ID 000398017200016

• Translating Research into Improved Patient Care in Pulmonary Arterial Hypertension AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Bonnet, S., Provencher, S., Guignabert, C., Perros, F., Boucherat, O., Schermuly, R. T., Hassoun, P. M., Rabinovitch, M., Nicolls, M. R., Humbert, M. 2017; 195 (5): 583-595

  Abstract

  Despite important advances in its therapeutic management, pulmonary arterial hypertension (PAH) remains an incurable disease. Although numerous drugs exhibited beneficial effects in preclinical settings, only few have reached clinical trial phases, highlighting the challenges of translating preclinical investigations into clinical trials. Potential reasons for delayed PAH drug development may include the inherent limitations of the currently available animal and in vitro models, potential lack of appropriate standardization of the experimental design, regulatory agencies requirements, competing clinical trials and insufficient funding. Although this is not unique to PAH, there is urgency for reducing the number of false positive signals in preclinical studies and optimizing the development of innovative therapeutic targets through performance of clinical trials based on more robust experimental data. The current review discusses the challenges and opportunities in preclinical research to foster drug development in PAH.

  View details for DOI 10.1164/rccm.201607-1515PP

  View details for Web of Science ID 000395357400008

• Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension. JCI insight Chen, P., Cao, A., Miyagawa, K., Tojais, N. F., Hennigs, J. K., Li, C. G., Sweeney, N. M., Inglis, A. S., Wang, L., Li, D., Ye, M., Feldman, B. J., Rabinovitch, M. 2017; 2 (2)

  Abstract

  Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.

  View details for DOI 10.1172/jci.insight.90427

  View details for PubMedID 28138562

• Relation of Cytokine Profile to Clinical and Hemodynamic Features in Young Patients With Congenital Heart Disease and Pulmonary Hypertension AMERICAN JOURNAL OF CARDIOLOGY Zorzanelli, L., Maeda, N., Clave, M., Thomaz, A., Galas, F., Rabinovitch, M., Lopes, A. 2017; 119 (1): 119-125

  Abstract

  In congenital heart disease, severity of pulmonary hypertension and operability is defined by noninvasive parameters (clinical history, physical examination, and echocardiography) and sometimes, cardiac catheterization. We investigated how circulating levels of inflammatory mediators correlate with such parameters in a young pediatric population (age, 2.0 months to 3.1 years) and the effects of preoperative pulmonary vasodilator therapy with sildenafil. Cytokines were analyzed in serum using chemiluminescence signals. In the whole patient group (n = 47), interleukin 17E, a Th2 immune response mediator increased with increasing age, considered as a parameter of disease severity (R(2) = 0.24, p <0.001), whereas the angiogenic chemokine growth-regulated oncogene alpha decreased (R(2) = 0.21, p = 0.001). Macrophage migration inhibitory factor chemokine was greater in subjects with elevated pulmonary vascular resistance (n = 16, p = 0.022), whereas regulated on activation, normal T cell expressed and secreted chemokine was greater in subjects with pulmonary congestion due to increased pulmonary blood flow (n = 31, p = 0.037). The observations were the same for the specific subpopulation of patients with Down syndrome (p = 0.009 and p = 0.012 for migration inhibitory factor and regulated on activation, normal T cell expressed and secreted in the respective subgroups). Sildenafil administration to patients with elevated pulmonary vascular resistance resulted in improvement of pulmonary blood flow (p = 0.012) and systemic oxygen saturation (p = 0.010), with a decrease in serum interleukin 6 (p = 0.027) and soluble ICAM-1 (p = 0.011). In conclusion, levels of circulating inflammatory molecules seem to correlate with disease severity in this population, with potential pathophysiological and therapeutic implications.

  View details for DOI 10.1016/j.amjcard.2016.09.020

  View details for Web of Science ID 000391246900019

  View details for PubMedID 28247848

• Codependence of Bone Morphogenetic Protein Receptor 2 and Transforming Growth Factor-β in Elastic Fiber Assembly and Its Perturbation in Pulmonary Arterial Hypertension. Arteriosclerosis, thrombosis, and vascular biology Tojais, N. F., Cao, A. n., Lai, Y. J., Wang, L. n., Chen, P. I., Alcazar, M. A., de Jesus Perez, V. n., Hopper, R. K., Rhodes, C. J., Bill, M. A., Sakai, L. Y., Rabinovitch, M. n. 2017

  Abstract

  We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly.Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cell and adventitial fibroblasts, TGFβ1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFβ1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFβ1-stimulated elastic fiber assemblies were impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFβ1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblast stimulated with BMP4 or TGFβ1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension.Disrupting BMPR2 impairs TGFβ1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

  View details for PubMedID 28619995

• Features and Outcomes of Methamphetamine Associated Pulmonary Arterial Hypertension. American journal of respiratory and critical care medicine Zamanian, R. T., Hedlin, H. n., Greuenwald, P. n., Wilson, D. M., Segal, J. I., Jorden, M. n., Kudelko, K. n., Liu, J. n., Hsi, A. n., Rupp, A. n., Sweatt, A. J., Tuder, R. n., Berry, G. J., Rabinovitch, M. n., Doyle, R. L., De Jesus Perez, V. n., Kawut, S. M. 2017

  Abstract

  While amphetamines are recognized as "likely" agents to cause drugs and toxins associated pulmonary arterial hypertension (PAH), (meth)amphetamine associated PAH (Meth-APAH) has not been well described.To prospectively characterize the clinical presentation, histopathology, and outcomes of Meth-APAH compared to those of idiopathic PAH (iPAH).We performed a prospective cohort study of Meth-APAH and iPAH patients presenting to the Stanford University Pulmonary Hypertension Program between 2003-2015. Clinical, pulmonary angiography, histopathology, and outcomes data were compared. We used data from the Healthcare Cost and Utilization Project to estimate the epidemiology of PAH in (meth)amphetamine abusers hospitalized in California.The study sample included 90 Meth-APAH and 97 iPAH patients. Meth-APAH patients were less likely to be female, but similar in age, body mass index, and six minute walk distance to iPAH patients. Meth-PAH patients reported more advanced heart failure symptoms, had significantly higher right atrial pressure (12.7±6.8 vs. 9.8±5.1 mmHg, p=0.001), and lower stroke volume index (22.2±7.1 vs 25.5±8.7 mL/m2, p=0.01). Event-free survival in Meth-APAH was 64.2%, 47.2%, and 25% at 2.5, 5, and 10 years respectively, representing more than double the risk of clinical worsening or death compared to iPAH (HR 2.04, 95% CI 1.28-3.25, p=0.003) independent of confounders. California data demonstrated a 2.6 fold increase in risk of PAH diagnosis in hospitalized (meth)amphetamine users.Meth-APAH is a severe and progressive form of PAH with poor outcomes. Future studies should focus on mechanisms of disease and potential therapeutic considerations.

  View details for PubMedID 28934596

• Upregulation of HERV-K is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension. Circulation Saito, T. n., Miyagawa, K. n., Chen, S. Y., Tamosiuniene, R. n., Wang, L. n., Sharp, O. n., Samayoa, E. n., Harada, D. n., Moonen, J. A., Cao, A. n., Chen, P. I., Hennigs, J. K., Gu, M. n., Li, C. G., Leib, R. D., Li, D. n., Adams, C. M., Del Rosario, P. A., Bill, M. A., Haddad, F. n., Montoya, J. G., Robinson, W. n., Fantl, W. J., Nolan, G. P., Zamanian, R. T., Nicolls, M. R., Chiu, C. Y., Ariza, M. E., Rabinovitch, M. n. 2017

  Abstract

  Background -Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue and elevated cytokines have been related to PAH pathogenesis but without clear understanding of how these abnormalities are initiated, perpetuated and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Methods -Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry (LCMS), confirmed by ELISA, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next generation sequencing, functional studies in cultured monocytes and endothelial cells (EC) and hemodynamic and lung studies in a rat. Results -SAM domain and HD1 domain-containing protein (SAMHD1), an innate immune factor that suppresses HIV replication was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH vs. 12 control lungs. Elevated SAMHD1 was localized to endothelial cells (EC), perivascular dendritic cells and macrophages and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH vs. control lungs (n=4 each). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) mRNAs were elevated in PAH vs. control lungs (n=10) and proteins were localized to macrophages. HERV-K dUTPase induced SAMHD1 and pro-inflammatory cytokines (e.g., IL6, IL1β and TNFα) in circulating monocytes and pulmonary arterial (PA) EC, and activated B cells. Vulnerability of PAEC to apoptosis was increased by HERV-K dUTPase in an IL6 independent manner. Furthermore, three weekly injections of HERV-K dUTPase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8), and elevated IL6. Conclusions -Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

  View details for PubMedID 28935667

• NETs Activate Pulmonary Arterial Endothelial Cells. Arteriosclerosis, thrombosis, and vascular biology Rabinovitch, M. 2016; 36 (10): 2035-7

  View details for DOI 10.1161/ATVBAHA.116.308206

  View details for PubMedID 27655779

• In Pulmonary Arterial Hypertension, Reduced BMPR2 Promotes Endothelial-to-Mesenchymal Transition via HMGA1 and Its Target Slug CIRCULATION Hopper, R. K., Moonen, J. A., Diebold, I., Cao, A., Rhodes, C. J., Tojais, N. F., Hennigs, J. K., Gu, M., Wang, L., Rabinovitch, M. 2016; 133 (18): 1783-?

  Abstract

  -We previously reported high-throughput RNA sequencing analyses that identified heightened expression of the chromatin architectural factor High Mobility Group AT-hook 1 (HMGA1) in pulmonary arterial (PA) endothelial cells (ECs) from idiopathic PA hypertension (IPAH) patients compared to controls. Since HMGA1 promotes epithelial to mesenchymal transition in cancer, we hypothesized that increased HMGA1 could induce transition of PAECs to a smooth muscle (SM)-like mesenchymal phenotype (EndMT), explaining both dysregulation of PAEC function and possible cellular contribution to the occlusive remodeling that characterizes advanced IPAH.-We documented increased HMGA1 in PAECs cultured from IPAH vs. donor control lungs. Confocal microscopy of lung explants localized the increase in HMGA1 consistently to PA endothelium, and identified many cells double-positive for HMGA1 and smooth muscle 22 alpha (SM22α) in occlusive and plexogenic lesions. Since decreased expression and function of bone morphogenetic protein receptor (BMPR)2 is observed in PAH, we reduced BMPR2 by siRNA in control PAECs and documented an increase in HMGA1 protein. Consistent with transition of PAECs by HMGA1, we detected reduced PECAM-1 (CD31) and increased EndMT markers, αSMA, SM22α, calponin, phospho-vimentin and Slug. The transition was associated with spindle SM-like morphology, and the increase in αSMA was largely reversed by joint knockdown of BMPR2 and HMGA1 or Slug. Pulmonary ECs from mice with EC-specific loss of BMPR2 showed similar gene and protein changes.-Increased HMGA1 in PAECs resulting from dysfunctional BMPR2 signaling can transition endothelium to SM-like cells associated with PAH.

  View details for DOI 10.1161/CIRCULATIONAHA.115.020617

  View details for Web of Science ID 000375604400008

  View details for PubMedID 27045138

• Loss of PPAR gamma in endothelial cells leads to impaired angiogenesis JOURNAL OF CELL SCIENCE Vattulainen-Collanus, S., Akinrinade, O., Li, M., Koskenvuo, M., Li, C. G., Rao, S. P., Perez, V. D., Yuan, K., Sawada, H., Koskenvuo, J. W., Alvira, C., Rabinovitch, M., Alastalo, T. 2016; 129 (4): 693-705

  Abstract

  Tie2 promoter-mediated loss of peroxisome proliferator-activated receptor gamma (PPARγ) in mice leads to osteopetrosis and pulmonary arterial hypertension. Vascular disease is associated with loss of PPARγ in pulmonary microvascular endothelial cells (PMVEC), we evaluated the role of PPARγ in PMVEC functions, such as angiogenesis and migration. The role of PPARγ in angiogenesis was evaluated in Tie2CrePPARγ(flox/flox) and wild type (WT) mice, and in mouse and human PMVECs. RNA-sequencing and bioinformatic approaches were utilized to reveal angiogenesis-associated targets for PPARγ. Tie2CrePPARγ(flox/flox) mice showed an impaired angiogenic capacity. Analysis of endothelial progenitor-like cells using bone marrow transplantation combined with evaluation of isolated PMVECs revealed that loss of PPARγ attenuates the migration and angiogenic capacity of mature PMVECs. PPARγ-deficient human PMVECs showed a similar migration defect in culture. Bioinformatic and experimental analyses revealed E2F1 as a novel target of PPARγ in the regulation of PMVEC migration. Disruption of the PPARγ-E2F1 axis was associated with a dysregulated Wnt pathway related to the GSK3β interaction protein. In conclusion, PPARγ plays an important role in sustaining angiogenic potential in mature PMVECs through E2F1-mediated gene regulation.

  View details for DOI 10.1242/jcs.169011

  View details for Web of Science ID 000370240900006

• Serum Cytokines in Young Pediatric Patients with Congenital Cardiac Shunts and Altered Pulmonary Hemodynamics MEDIATORS OF INFLAMMATION Zorzanelli, L., Maeda, N. Y., Clave, M. M., Aiello, V. D., Rabinovitch, M., Lopes, A. A. 2016

  Abstract

  Background and Objective. Inflammation is central in the pathogenesis of pulmonary hypertension. We investigated how serum cytokines correlate with clinical features, hemodynamics, and lung histology in young patients with pulmonary hypertension associated with congenital cardiac shunts. Design. Prospective, observational study. Methods and Results. Patients (n = 44) were aged 2.6 to 37.6 months. Group I patients (n = 31) were characterized by pulmonary congestion and higher pulmonary blood flow compared to group II (p = 0.022), with no need for preoperative cardiac catheterization. Group II patients (n = 13) had no congestive features. At catheterization, they had elevated pulmonary vascular resistance (5.7 [4.4-7.4] Wood units·m(2), geometric mean with 95% CI). Cytokines were measured by chemiluminescence. Macrophage migration inhibitory factor (MIF) was found to be inversely related to pulmonary blood flow (r = -0.33, p = 0.026) and was higher in group II (high pulmonary vascular resistance) compared to group I (high pulmonary blood flow) (p = 0.017). In contrast, RANTES chemokine (regulated on activation, normal T cell expressed and secreted) was characteristically elevated in Group I (p = 0.022). Interleukin 16 was also negatively related to pulmonary blood flow (rS = -0.33, p = 0.029) and was higher in patients with obstructive vasculopathy at intraoperative lung biopsy (p = 0.021). Conclusion. Cytokines seem to be important and differentially regulated in subpopulations of young patients with cardiac shunts.

  View details for DOI 10.1155/2016/7672048

  View details for Web of Science ID 000383105800001

  View details for PubMedID 27656048

• Leukotriene B-4 Activates Pulmonary Artery Adventitial Fibroblasts in Pulmonary Hypertension HYPERTENSION Qian, J., Tian, W., Jiang, X., Tamosiuniene, R., Sung, Y. K., Shuffle, E. M., Tu, A. B., Valenzuela, A., Jiang, S., Zamanian, R. T., Fiorentino, D. F., Voelkel, N. F., Peters-Golden, M., Stenmark, K. R., Chung, L., Rabinovitch, M., Nicolls, M. R. 2015; 66 (6): 1227-1239

  Abstract

  A recent study demonstrated a significant role for leukotriene B4 (LTB4) causing pulmonary vascular remodeling in pulmonary arterial hypertension. LTB4 was found to directly injure luminal endothelial cells and promote growth of the smooth muscle cell layer of pulmonary arterioles. The purpose of this study was to determine the effects of LTB4 on the pulmonary adventitial layer, largely composed of fibroblasts. Here, we demonstrate that LTB4 enhanced human pulmonary artery adventitial fibroblast proliferation, migration, and differentiation in a dose-dependent manner through its cognate G-protein-coupled receptor, BLT1. LTB4 activated human pulmonary artery adventitial fibroblast by upregulating p38 mitogen-activated protein kinase as well as Nox4-signaling pathways. In an autoimmune model of pulmonary hypertension, inhibition of these pathways blocked perivascular inflammation, decreased Nox4 expression, reduced reactive oxygen species production, reversed arteriolar adventitial fibroblast activation, and attenuated pulmonary hypertension development. This study uncovers a novel mechanism by which LTB4 further promotes pulmonary arterial hypertension pathogenesis, beyond its established effects on endothelial and smooth muscle cells, by activating adventitial fibroblasts.

  View details for DOI 10.1161/HYPERTENSIONAHA.115.06370

  View details for PubMedID 26558820

• RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Rhodes, C. J., Im, H., Cao, A., Hennigs, J. K., Wang, L., Sa, S., Chen, P., Nickel, N. P., Miyagawa, K., Hopper, R. K., Tojais, N. F., Li, C. G., Gu, M., Spiekerkoetter, E., Xian, Z., Chen, R., Zhao, M., Kaschwich, M., del Rosario, P. A., Bernstein, D., Zamanian, R. T., Wu, J. C., Snyder, M. P., Rabinovitch, M. 2015; 192 (3): 356-366

  Abstract

  Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.RNA sequencing was utilized to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension vs. controls and to assess the functional significance of major differentially expressed transcripts.The endothelial transcriptomes from seven control and six idiopathic pulmonary arterial hypertension patients' lungs were analyzed. Differentially expressed genes were related to BMPR2 signaling. Those downregulated were assessed for function in cultured cells, and in a transgenic mouse.Fold-differences in ten genes were significant (p<0.05), four increased and six decreased in patients vs.No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated six/ten patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2 regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2 and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration and tube formation. In mice null for the EFNA1 receptor, EphA2, vs. controls, VEGF receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy and loss of small arteries.The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.

  View details for DOI 10.1164/rccm.201408-1528OC

  View details for PubMedID 26030479

• Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension. American journal of respiratory and critical care medicine Spiekerkoetter, E., Sung, Y. K., Sudheendra, D., Bill, M., Aldred, M. A., van de Veerdonk, M. C., Vonk Noordegraaf, A., Long-Boyle, J., Dash, R., Yang, P. C., Lawrie, A., Swift, A. J., Rabinovitch, M., Zamanian, R. T. 2015; 192 (2): 254-257

  View details for DOI 10.1164/rccm.201411-2061LE

  View details for PubMedID 26177174

• Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor, Elafin, to Maintain Graft Perfusion During Acute Rejection AMERICAN JOURNAL OF TRANSPLANTATION Jiang, X., Nguyen, T. T., Tian, W., Sung, Y. K., Yuan, K., Qian, J., Rajadas, J., Sallenave, J., Nickel, N. P., Perez, V. d., RABINOVITCH, M., Nicolls, M. R. 2015; 15 (7): 1768-1781

  Abstract

  The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.

  View details for DOI 10.1111/ajt.13189

  View details for Web of Science ID 000356494300013

  View details for PubMedID 25727073

• Elafin Reverses Pulmonary Hypertension via Caveolin-1-Dependent Bone Morphogenetic Protein Signaling AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Nickel, N. P., Spiekerkoetter, E., Gu, M., Li, C. G., Li, H., Kaschwich, M., Diebold, I., Hennigs, J. K., Kim, K., Miyagawa, K., Wang, L., Cao, A., Sa, S., Jiang, X., Stockstill, R. W., Nicolls, M. R., Zamanian, R. T., Bland, R. D., Rabinovitch, M. 2015; 191 (11): 1273-1286

  Abstract

  Pulmonary arterial hypertension is characterized by endothelial cell dysfunction, impaired BMPR2 signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates the development of hypoxic pulmonary hypertension in mice, but its potential to improve endothelial cell function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown.To assess elafin-mediated regression of pulmonary vascular pathology in rats with pulmonary hypertension induced by VEGF receptor blockade and hypoxia (Sugen/Hypoxia), and in lung explants from pulmonary hypertension patients. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells from controls and patients, and to elucidate the underlying mechanism. Methods, Measurements and Main Results: In Sugen/Hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a product of BMPR2 signaling. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and in lung organ culture elafin decreased neointimal lesions. In normal and patient pulmonary artery endothelial cells, elafin enhanced survival and promoted angiogenesis by increasing pSMAD dependent and independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of caveolin-1 on endothelial surfaces.Elafin reverses obliterative changes in rat and human pulmonary arteries via elastase inhibition and caveolin-1 dependent amplification of BMPR2 signaling.

  View details for DOI 10.1164/rccm.201412-2291OC

  View details for Web of Science ID 000356105000014

  View details for PubMedID 25853696

• BMPR2 Preserves Mitochondrial Function and DNA during Reoxygenation to Promote Endothelial Cell Survival and Reverse Pulmonary Hypertension CELL METABOLISM Diebold, I., Hennigs, J. K., Miyagawa, K., Li, C. G., Nickel, N. P., Kaschwich, M., Cao, A., Wang, L., Reddy, S., Chen, P., Nakahira, K., Alcazar, M. A., Hopper, R. K., Ji, L., Feldman, B. J., Rabinovitch, M. 2015; 21 (4): 596-608

  Abstract

  Mitochondrial dysfunction, inflammation, and mutant bone morphogenetic protein receptor 2 (BMPR2) are associated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels, and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1α and TFAM, regulators of mitochondrial biogenesis, and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1α, NRF2, TFAM, mitochondrial membrane potential, and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1α, TFAM, mitochondrial membrane potential, ATP production, and glycolysis, and induces mitochondrial fission and a pro-inflammatory state. These features are recapitulated in PAECs from PAH patients with mutant BMPR2.

  View details for DOI 10.1016/j.cmet.2015.03.010

  View details for Web of Science ID 000352500800014

  View details for PubMedID 25863249

• Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice. American journal of physiology. Lung cellular and molecular physiology Hilgendorff, A., Parai, K., Ertsey, R., Navarro, E., Jain, N., Carandang, F., Peterson, J., Mokres, L., Milla, C., Preuss, S., Alcazar, M. A., Khan, S., Masumi, J., Ferreira-Tojais, N., Mujahid, S., Starcher, B., Rabinovitch, M., Bland, R. 2015; 308 (5): L464-78

  Abstract

  Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice (Eln(+/-)) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type (Eln(+/+)) and Eln(+/-) littermates at baseline and after MV with air for 8-24 h. Lungs of unventilated Eln(+/-) mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln(+/+) pups. Eln(+/-) lungs contained fewer capillaries than Eln(+/+) lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln(+/+) neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln(+/-) mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln(+/-) than in Eln(+/+) pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln(+/-) compared with Eln(+/+) mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln(+/+) and Eln(+/-) mice. Paucity of lung capillaries in Eln(+/-) newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln(+/-) mice.

  View details for DOI 10.1152/ajplung.00278.2014

  View details for PubMedID 25539853

  View details for PubMedCentralID PMC4346771

• Enhanced Caspase Activity Contributes to Aortic Wall Remodeling and Early Aneurysm Development in a Murine Model of Marfan Syndrome ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Emrich, F. C., Okamura, H., Dalal, A. R., Penov, K., Merk, D. R., Raaz, U., Hennigs, J. K., Chin, J. T., Miller, M. O., Pedroza, A. J., Craig, J. K., Koyano, T. K., Blankenberg, F. G., Connolly, A. J., Mohr, F. W., Alvira, C. M., Rabinovitch, M., Fischbein, M. P. 2015; 35 (1): 146-154

  Abstract

  Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome.Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs.Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.

  View details for DOI 10.1161/ATVBAHA.114.304364

  View details for PubMedID 25359856

• Targeting the Wnt signaling pathways in pulmonary arterial hypertension. Drug discovery today de Jesus Perez, V., Yuan, K., Alastalo, T., Spiekerkoetter, E., Rabinovitch, M. 2014; 19 (8): 1270-1276

  Abstract

  Pulmonary arterial hypertension (PAH) is a life-threatening disorder that is associated with elevated pulmonary pressures and right heart failure resulting from progressive loss and thickening of small pulmonary arteries. Despite their ability to improve symptoms, current therapies fail to prevent disease progression, leaving lung transplantation as the only therapy in end-stage PAH. To overcome the limitations of current therapies, there is an active search for disease-modifying agents capable of altering the natural history of, and improving clinical outcomes in, PAH. The Wnt signaling pathways have emerged as attractive treatment targets in PAH given their role in the preservation of pulmonary vascular homeostasis and the recent development of Wnt-specific compounds and biological therapies capable of modulating pathway activity. In this review, we summarize the literature describing the role of Wnt signaling in the pulmonary circulation and discuss promising advances in the field of Wnt therapeutics that could lead to novel clinical therapies capable of preventing and/or reversing pulmonary vascular pathology in patients with this devastating disease.

  View details for DOI 10.1016/j.drudis.2014.06.014

  View details for PubMedID 24955837

• Inflammation and Immunity in the Pathogenesis of Pulmonary Arterial Hypertension CIRCULATION RESEARCH Rabinovitch, M., Guignabert, C., Humbert, M., Nicolls, M. R. 2014; 115 (1): 165-175

  Abstract

  This review summarizes an expanding body of knowledge indicating that failure to resolve inflammation and altered immune processes underlie the development of pulmonary arterial hypertension. The chemokines and cytokines implicated in pulmonary arterial hypertension that could form a biomarker platform are discussed. Pre-clinical studies that provide the basis for dysregulated immunity in animal models of the disease are reviewed. In addition, we present therapies that target inflammatory/immune mechanisms that are currently enrolling patients, and discuss others in development. We show how genetic and metabolic abnormalities are inextricably linked to dysregulated immunity and adverse remodeling in the pulmonary arteries.

  View details for DOI 10.1161/CIRCRESAHA.113.301141

  View details for Web of Science ID 000337738900018

  View details for PubMedID 24951765

• Repair of congenital heart disease with associated pulmonary hypertension in children: what are the minimal investigative procedures? Consensus statement from the Congenital Heart Disease and Pediatric Task Forces, Pulmonary Vascular Research Institute (PVRI). Pulmonary circulation Lopes, A. A., Barst, R. J., Haworth, S. G., Rabinovitch, M., Al Dabbagh, M., Del Cerro, M. J., Ivy, D., Kashour, T., Kumar, K., Harikrishnan, S., D'Alto, M., Thomaz, A. M., Zorzanelli, L., Aiello, V. D., Mocumbi, A. O., Santana, M. V., Galal, A. N., Banjar, H., Tamimi, O., Heath, A., Flores, P. C., Diaz, G., Sandoval, J., Kothari, S., Moledina, S., Gonçalves, R. C., Barreto, A. C., Binotto, M. A., Maia, M., Al Habshan, F., Adatia, I. 2014; 4 (2): 330-341

  Abstract

  Standardization of the diagnostic routine for children with congenital heart disease associated with pulmonary arterial hypertension (PAH-CHD) is crucial, in particular since inappropriate assignment to repair of the cardiac lesions (e.g., surgical repair in patients with elevated pulmonary vascular resistance) may be detrimental and associated with poor outcomes. Thus, members of the Congenital Heart Disease and Pediatric Task Forces of the Pulmonary Vascular Research Institute decided to conduct a survey aimed at collecting expert opinion from different institutions in several countries, covering many aspects of the management of PAH-CHD, from clinical recognition to noninvasive and invasive diagnostic procedures and immediate postoperative support. In privileged communities, the vast majority of children with congenital cardiac shunts are now treated early in life, on the basis of noninvasive diagnostic evaluation, and have an uneventful postoperative course, with no residual PAH. However, a small percentage of patients (older at presentation, with extracardiac syndromes or absence of clinical features of increased pulmonary blood flow, thus suggesting elevated pulmonary vascular resistance) remain at a higher risk of complications and unfavorable outcomes. These patients need a more sophisticated diagnostic approach, including invasive procedures. The authors emphasize that decision making regarding operability is based not only on cardiac catheterization data but also on the complete diagnostic picture, which includes the clinical history, physical examination, and all aspects of noninvasive evaluation.

  View details for DOI 10.1086/675995

  View details for PubMedID 25006452

  View details for PubMedCentralID PMC4070778

• Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension. journal of experimental medicine Sawada, H., Saito, T., Nickel, N. P., Alastalo, T., Glotzbach, J. P., Chan, R., Haghighat, L., Fuchs, G., Januszyk, M., Cao, A., Lai, Y., Perez, V. d., Kim, Y., Wang, L., Chen, P., Spiekerkoetter, E., Mitani, Y., Gurtner, G. C., Sarnow, P., Rabinovitch, M. 2014; 211 (2): 263-280

  Abstract

  Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

  View details for DOI 10.1084/jem.20111741

  View details for PubMedID 24446489

• Relevant issues in the pathology and pathobiology of pulmonary hypertension. Journal of the American College of Cardiology Tuder, R. M., Archer, S. L., Dorfmüller, P., Erzurum, S. C., Guignabert, C., Michelakis, E., Rabinovitch, M., Schermuly, R., Stenmark, K. R., Morrell, N. W. 2013; 62 (25): D4-12

  Abstract

  Knowledge of the pathobiology of pulmonary hypertension (PH) continues to accelerate. However, fundamental gaps remain in our understanding of the underlying pathological changes in pulmonary arteries and veins in the different forms of this syndrome. Although PH primarily affects the arteries, venous disease is increasingly recognized as an important entity. Moreover, prognosis in PH is determined largely by the status of the right ventricle, rather than the levels of pulmonary artery pressures. It is increasingly clear that although vasospasm plays a role, PH is an obstructive lung panvasculopathy. Disordered metabolism and mitochondrial structure, inflammation, and dysregulation of growth factors lead to a proliferative, apoptosis-resistant state. These abnormalities may be acquired, genetically mediated as a result of mutations in bone morphogenetic protein receptor-2 or activin-like kinase-1, or epigenetically inherited (as a result of epigenetic silencing of genes such as superoxide dismutase-2). There is a pressing need to better understand how the pathobiology leads to severe disease in some patients versus mild PH in others. Recent recognition of a potential role of acquired abnormalities of mitochondrial metabolism in the right ventricular myocytes and pulmonary vascular cells suggests new therapeutic approaches, diagnostic modalities, and biomarkers. Finally, dissection of the role of pulmonary inflammation in the initiation and promotion of PH has revealed a complex yet fascinating interplay with pulmonary vascular remodeling, promising to lead to novel therapeutics and diagnostics. Emerging concepts are also relevant to the pathobiology of PH, including a role for bone marrow and circulating progenitor cells and microribonucleic acids. Continued interest in the interface of the genetic basis of PH and cellular and molecular pathogenetic links should further expand our understanding of the disease.

  View details for DOI 10.1016/j.jacc.2013.10.025

  View details for PubMedID 24355640

  View details for PubMedCentralID PMC3970402

• Blocking Macrophage Leukotriene B-4 Prevents Endothelial Injury and Reverses Pulmonary Hypertension SCIENCE TRANSLATIONAL MEDICINE Tian, W., Jiang, X., Tamosiuniene, R., Sung, Y. K., Qian, J., Dhillon, G., Gera, L., Farkas, L., Rabinovitch, M., Zamanian, R. T., Inayathullah, M., Fridlib, M., Rajadas, J., Peters-Golden, M., Voelkel, N. F., Nicolls, M. R. 2013; 5 (200)

  Abstract

  Pulmonary hypertension (PH) is a serious condition that affects mainly young and middle-aged women, and its etiology is poorly understood. A prominent pathological feature of PH is accumulation of macrophages near the arterioles of the lung. In both clinical tissue and the SU5416 (SU)/athymic rat model of severe PH, we found that the accumulated macrophages expressed high levels of leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme for leukotriene B4 (LTB4). Moreover, macrophage-derived LTB4 directly induced apoptosis in pulmonary artery endothelial cells (PAECs). Further, LTB4 induced proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We found that LTB4 acted through its receptor, BLT1, to induce PAEC apoptosis by inhibiting the protective endothelial sphingosine kinase 1 (Sphk1)-endothelial nitric oxide synthase (eNOS) pathway. Blocking LTA4H decreased in vivo LTB4 levels, prevented PAEC apoptosis, restored Sphk1-eNOS signaling, and reversed fulminant PH in the SU/athymic rat model of PH. Antagonizing BLT1 similarly reversed established PH. Inhibition of LTB4 biosynthesis or signal transduction in SU-treated athymic rats with established disease also improved cardiac function and reopened obstructed arterioles; this approach was also effective in the monocrotaline model of severe PH. Human plexiform lesions, one hallmark of PH, showed increased numbers of macrophages, which expressed LTA4H, and patients with connective tissue disease-associated pulmonary arterial hypertension exhibited significantly higher LTB4 concentrations in the systemic circulation than did healthy subjects. These results uncover a possible role for macrophage-derived LTB4 in PH pathogenesis and identify a pathway that may be amenable to therapeutic targeting.

  View details for Web of Science ID 000323705100010

• FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. journal of clinical investigation Spiekerkoetter, E., Tian, X., Cai, J., Hopper, R. K., Sudheendra, D., Li, C. G., El-Bizri, N., Sawada, H., Haghighat, R., Chan, R., Haghighat, L., de Jesus Perez, V., Wang, L., Reddy, S., Zhao, M., Bernstein, D., Solow-Cordero, D. E., Beachy, P. A., Wandless, T. J., ten Dijke, P., Rabinovitch, M. 2013; 123 (8): 3600-3613

  Abstract

  Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

  View details for DOI 10.1172/JCI65592

  View details for PubMedID 23867624

• Molecular determinants of lung development. Annals of the American Thoracic Society Morrisey, E. E., Cardoso, W. V., Lane, R. H., Rabinovitch, M., Abman, S. H., Ai, X., Albertine, K. H., Bland, R. D., Chapman, H. A., Checkley, W., Epstein, J. A., Kintner, C. R., Kumar, M., Minoo, P., Mariani, T. J., McDonald, D. M., Mukouyama, Y., Prince, L. S., Reese, J., Rossant, J., Shi, W., Sun, X., Werb, Z., Whitsett, J. A., Gail, D., Blaisdell, C. J., Lin, Q. S. 2013; 10 (2): S12-6

  Abstract

  Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

  View details for DOI 10.1513/AnnalsATS.201207-036OT

  View details for PubMedID 23607856

• Combining induced pluripotent stem cell with next generation sequencing technology to gain new insights into pathobiology and treatment of pulmonary arterial hypertension. Pulmonary circulation Rabinovitch, M. 2013; 3 (1): 153-155

  View details for DOI 10.4103/2045-8932.109963

  View details for PubMedID 23662192

  View details for PubMedCentralID PMC3641723

• Anticipated classes of new medications and molecular targets for pulmonary arterial hypertension PULMONARY CIRCULATION Morrell, N. W., Archer, S. L., DeFelice, A., Evans, S., Fiszman, M., Martin, T., Saulnier, M., Rabinovitch, M., Schermuly, R., Stewart, D., Truebel, H., Walker, G., Stenmark, K. R. 2013; 3 (1): 226-244

  View details for DOI 10.4103/2045-8932.109940

  View details for Web of Science ID 000209981400033

• Patchy deletion of Bmpr1a potentiates proximal pulmonary artery remodeling in mice exposed to chronic hypoxia BIOMECHANICS AND MODELING IN MECHANOBIOLOGY Vanderpool, R. R., El-Bizri, N., Rabinovitch, M., Chesler, N. C. 2013; 12 (1): 33-42

  Abstract

  Reduced vascular expression of bone morphogenetic protein type IA receptor (Bmpr1a) has been found in patients with pulmonary arterial hypertension. Our previous studies in mice with patchy deletion of Bmpr1a in vascular smooth muscle cells and cardiac myocytes showed decreased distal vascular remodeling despite a similar severity of hypoxic pulmonary hypertension (HPH). We speculate increased stiffness from ectopic deposition of collagen in proximal pulmonary arteries might account for HPH. Pulsatile pressure-flow relationships were measured in isolated, ventilated, perfused lungs of SM22α;TRE-Cre;R26R;Bmpr1a(flox/flox) (KO) mice and wild-type littermates, following 21 days (hypoxia) and 0 days (control) of chronic hypoxia. Pulmonary vascular impedance, which yields insight into proximal and distal arterial remodeling, was calculated. Reduced Bmpr1a expression had no effect on input impedance Z(0) (P = 0.52) or characteristic impedance Z(C) (P = 0.18) under control conditions; it also had no effect on the decrease in Z(0) via acute rho kinase inhibition. However, following chronic hypoxia, reduced Bmpr1a expression increased Z(C) (P < 0.001) without affecting Z(0) (P = 0.72). These results demonstrate that Bmpr1a deficiency does not significantly alter the hemodynamic function of the distal vasculature or its response to chronic hypoxia but larger, more proximal arteries are affected. In particular, reduced Bmpr1a expression likely decreased dilatation and increased stiffening in response to hypoxia, probably by collagen accumulation. Increased PA stiffness can have a significant impact on right ventricular function. This study illustrates for the first time how proximal pulmonary artery changes in the absence of distal pulmonary artery changes contribute to pulmonary arterial hypertension.

  View details for DOI 10.1007/s10237-012-0379-6

  View details for Web of Science ID 000313480100004

  View details for PubMedID 22314711

• Chronic effects of pulmonary artery stenosis on hemodynamic and structural development of the lungs. American journal of physiology. Lung cellular and molecular physiology Razavi, H., Stewart, S. E., Xu, C., Sawada, H., Zarafshar, S. Y., Taylor, C. A., Rabinovitch, M., Feinstein, J. A. 2013; 304 (1): L17-28

  Abstract

  Pulmonary artery (PA) stenosis is a difficult obstructive defect to manage since clinicians cannot know a priori which obstructions to treat and when. Prognosis of PA stenosis and its chronic effects on lung development are poorly understood. This study aimed to characterize the hemodynamic and structural effects of PA stenosis during development. Fourteen male Sprague-Dawley rats underwent left PA (LPA) banding at age 21 days, and 13 underwent sham operation. Hemodynamic and structural impacts were studied longitudinally at 20, 36, 52, 100, and 160 days. Chronic LPA banding resulted in a significant reduction in LPA flow (P < 0.0001) and size of both proximal LPA (P < 0.0001) and distal LPA (P < 0.01), as well as a significant increase in flow and size of the right PA (P < 0.05) throughout development. Flows and sizes adapted such that normal levels of wall shear were restored after banding. At 160 days, LPA banding resulted in a significant decrease in left lung volume and an increase in right lung volume but no significant differences in total lung volume. There was an elevation of proximal LPA pressure as well as right ventricular hypertrophy in the banded animals. The banded lung exhibited arterial disorganization, loss of vessels, and enlargement of its bronchial arteries, whereas the contralateral lung showed signs of vascular pathology. There are consequences on development of both lungs in the presence of an LPA stenosis at young age. These results suggest that early intervention may be necessary to optimize left lung growth and minimize right lung vascular pathology.

  View details for DOI 10.1152/ajplung.00412.2011

  View details for PubMedID 23043077

• Loss of adenomatous poliposis coli-a3 integrin interaction promotes endothelial apoptosis in mice and humans. Circulation research de Jesus Perez, V. A., Yuan, K., Orcholski, M. E., Sawada, H., Zhao, M., Li, C. G., Tojais, N. F., Nickel, N., Rajagopalan, V., Spiekerkoetter, E., Wang, L., Dutta, R., Bernstein, D., Rabinovitch, M. 2012; 111 (12): 1551-1564

  Abstract

  Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/β-catenin pathway, we proposed that β-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH.This study aims to establish the role of β-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury.To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive β-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.

  View details for DOI 10.1161/CIRCRESAHA.112.267849

  View details for PubMedID 23011394

  View details for PubMedCentralID PMC3821702

• Molecular pathogenesis of pulmonary arterial hypertension JOURNAL OF CLINICAL INVESTIGATION Rabinovitch, M. 2012; 122 (12): 4306-4313

  Abstract

  Recent clinical and experimental studies are redefining the cellular and molecular bases of pulmonary arterial hypertension (PAH). The genetic abnormalities first identified in association with the idiopathic form of PAH--together with a vast increase in our understanding of cell signaling, cell transformation, and cell-cell interactions; gene expression; microRNA processing; and mitochondrial and ion channel function--have helped explain the abnormal response of vascular cells to injury. Experimental and clinical studies now converge on the intersection and interactions between a genetic predisposition involving the BMPR2 signaling pathway and an impaired metabolic and chronic inflammatory state in the vessel wall. These deranged processes culminate in an exuberant proliferative response that occludes the pulmonary arterial (PA) lumen and obliterates the most distal intraacinar vessels. Here, we describe emerging therapies based on preclinical studies that address these converging pathways.

  View details for DOI 10.1172/JCI60658

  View details for Web of Science ID 000311926200003

  View details for PubMedID 23202738

  View details for PubMedCentralID PMC3533531

• Neonatal mice genetically modified to express the elastase inhibitor elafin are protected against the adverse effects of mechanical ventilation on lung growth. American journal of physiology. Lung cellular and molecular physiology Hilgendorff, A., Parai, K., Ertsey, R., Juliana Rey-Parra, G., Thébaud, B., Tamosiuniene, R., Jain, N., Navarro, E. F., Starcher, B. C., Nicolls, M. R., Rabinovitch, M., Bland, R. D. 2012; 303 (3): L215-27

  Abstract

  Mechanical ventilation (MV) with O(2)-rich gas (MV-O(2)) offers life-saving treatment for newborn infants with respiratory failure, but it also can promote lung injury, which in neonates translates to defective alveolar formation and disordered lung elastin, a key determinant of lung growth and repair. Prior studies in preterm sheep and neonatal mice showed that MV-O(2) stimulated lung elastase activity, causing degradation and remodeling of matrix elastin. These changes yielded an inflammatory response, with TGF-β activation, scattered elastic fibers, and increased apoptosis, culminating in defective alveolar septation and arrested lung growth. To see whether sustained inhibition of elastase activity would prevent these adverse pulmonary effects of MV-O(2), we did studies comparing wild-type (WT) and mutant neonatal mice genetically modified to express in their vascular endothelium the human serine elastase inhibitor elafin (Eexp). Five-day-old WT and Eexp mice received MV with 40% O(2) (MV-O(2)) for 24-36 h. WT and Eexp controls breathed 40% O(2) without MV. MV-O(2) increased lung elastase and MMP-9 activity, resulting in elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 increased 6-fold), apoptosis (cleaved-caspase-3 increased 10-fold), and inflammation (NF-κB activation, influx of neutrophils and monocytes) in lungs of WT vs. unventilated controls. These changes were blocked or blunted during MV-O(2) of Eexp mice. Scattered lung elastin and emphysematous alveoli observed in WT mice after 36 h of MV-O(2) were attenuated in Eexp mice. Both WT and Eexp mice showed defective VEGF signaling (decreased lung VEGF-R2 protein) and loss of pulmonary microvessels after lengthy MV-O(2), suggesting that elafin's beneficial effects during MV-O(2) derived primarily from preserving matrix elastin and suppressing lung inflammation, thereby enabling alveolar formation during MV-O(2). These results suggest that degradation and remodeling of lung elastin can contribute to defective lung growth in response to MV-O(2) and might be targeted therapeutically to prevent ventilator-induced neonatal lung injury.

  View details for DOI 10.1152/ajplung.00405.2011

  View details for PubMedID 22683569

  View details for PubMedCentralID PMC3423862

• Development of pulmonary arterial hypertension in mice over-expressing S100A4/Mts1 is specific to females RESPIRATORY RESEARCH Dempsie, Y., Nilsen, M., White, K., Mair, K. M., Loughlin, L., Ambartsumian, N., Rabinovitch, M., MacLean, M. R. 2011; 12

  Abstract

  Idiopathic and familial forms of pulmonary arterial hypertension (PAH) occur more frequently in women than men. However, the reason for this remains unknown. Both the calcium binding protein S100A4/Mts1 (Mts1) and its endogenous receptor (receptor for advanced glycosylation end products; RAGE) have been implicated in the development of PAH. We wished to investigate if the Mts1/RAGE pathway may play a role in the gender bias associated with PAH.We investigated the effects of gender on development of PAH in mice over-expressing Mts1 (Mts1+ mice) via measurement of pulmonary arterial remodeling, systolic right ventricular pressure (sRVP) and right ventricular hypertrophy (RVH). Gender differences in pulmonary arterial Mts1 and RAGE expression were assessed by qRT-PCR and immunohistochemistry. Western blotting and cell counts were used to investigate interactions between 17β-estradiol, Mts1 and RAGE on proliferation of human pulmonary artery smooth muscle cells (hPASMCs). Statistical analysis was by one-way analysis of variance with Dunnetts post test or two-way analysis of variance with Bonferronis post test, as appropriate.Female Mts1+ mice developed increased sRVP and pulmonary vascular remodeling, whereas male Mts1+ mice remained unaffected. The development of plexiform-like lesions in Mts1+ mice was specific to females. These lesions stained positive for both Mts1 and RAGE in the endothelial and adventitial layers. Expression of pulmonary arterial Mts1 was greater in female than male Mts1+ mice, and was localised to the medial and adventitial layers in non plexiform-like pulmonary arteries. RAGE gene expression and immunoreactivity were similar between male and female Mts1+ mice and RAGE staining was localised to the endothelial layer in non plexiform-like pulmonary arteries adjacent to airways. In non-plexiform like pulmonary arteries not associated with airways RAGE staining was present in the medial and adventitial layers. Physiological concentrations of 17β-estradiol increased Mts1 expression in hPASMCs. 17β-estradiol-induced hPASMC proliferation was inhibited by soluble RAGE, which antagonises the membrane bound form of RAGE.Mts1 over-expression combined with female gender is permissive to the development of experimental PAH in mice. Up-regulation of Mts1 and subsequent activation of RAGE may contribute to 17β-estradiol-induced proliferation of hPASMCs.

  View details for DOI 10.1186/1465-9921-12-159

  View details for Web of Science ID 000300491900001

  View details for PubMedID 22185646

• Network Analysis Identifies the Orphan Receptor Tyrosine Kinase Ros1 as a Determinant of Glutathione Peroxidase-1 Mediated Vascular Remodeling Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Ali, Z. A., Perez, V. D., Raiesdana, A., Leeper, N. J., Pan, S., Qu, X., Ali, A., Haghighat, R., Kato, K., Channon, K. M., Rabinovitch, M., Quertermous, T., Ashley, E. A. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738703092

• Regulatory T Cells Limit Vascular Endothelial Injury and Prevent Pulmonary Hypertension CIRCULATION RESEARCH Tamosiuniene, R., Tian, W., Dhillon, G., Wang, L., Sung, Y. K., Gera, L., Patterson, A. J., Agrawal, R., Rabinovitch, M., Ambler, K., Long, C. S., Voelkel, N. F., Nicolls, M. R. 2011; 109 (8): 867-U120

  Abstract

  Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood.To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH.Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4(+)CD25(hi) or CD4(+)CD25(-) T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3(+), IL-10- and TGF-β-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways.PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

  View details for DOI 10.1161/CIRCRESAHA.110.236927

  View details for PubMedID 21868697

• Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions AMERICAN JOURNAL OF PATHOLOGY Kim, Y., Haghighat, L., Spiekerkoetter, E., Sawada, H., Alvira, C. M., Wang, L., Acharya, S., Rodriguez-Colon, G., Orton, A., Zhao, M., Rabinovitch, M. 2011; 179 (3): 1560-1572

  Abstract

  Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.

  View details for DOI 10.1016/j.ajpath.2011.05.051

  View details for PubMedID 21763677

• Inhibiting Lung Elastase Activity Enables Lung Growth in Mechanically Ventilated Newborn Mice AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Hilgendorff, A., Parai, K., Ertsey, R., Jain, N., Navarro, E. F., Peterson, J. L., Tamosiuniene, R., Nicolls, M. R., Starcher, B. C., Rabinovitch, M., Bland, R. D. 2011; 184 (5): 537-546

  Abstract

  Mechanical ventilation with O₂-rich gas (MV-O₂) offers life-saving treatment for respiratory failure, but also promotes lung injury. We previously reported that MV-O2 of newborn mice increased lung elastase activity, causing elastin degradation and redistribution of elastic fibers from septal tips to alveolar walls. These changes were associated with transforming growth factor (TGF)-β activation and increased apoptosis leading to defective alveolarization and lung growth arrest, as seen in neonatal chronic lung disease.To determine if intratracheal treatment of newborn mice with the serine elastase inhibitor elafin would prevent MV-O₂-induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest.Five-day-old mice were treated via tracheotomy with recombinant human elafin or vehicle (lactated-Ringer solution), followed by MV with 40% O₂ for 8-24 hours; control animals breathed 40% O₂ without MV. At study's end, lungs were harvested to assess key variables noted below.MV-O₂ of vehicle-treated pups increased lung elastase and matrix metalloproteinase-9 activity when compared with unventilated control animals, causing elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 tripled), and apoptosis (cleaved-caspase-3 increased 10-fold). Quantitative lung histology showed larger and fewer alveoli, greater inflammation, and scattered elastic fibers. Elafin blocked these MV-O₂-induced changes.Intratracheal elafin, by blocking lung protease activity, prevented MV-O₂-induced elastin degradation, TGF-β activation, apoptosis, and dispersion of matrix elastin, and attenuated lung structural abnormalities noted in vehicle-treated mice after 24 hours of MV-O₂. These findings suggest that elastin breakdown contributes to defective lung growth in response to MV-O₂ and might be targeted therapeutically to prevent MV-O₂-induced lung injury.

  View details for DOI 10.1164/rccm.201012-2010OC

  View details for Web of Science ID 000294478200014

  View details for PubMedID 21562133

  View details for PubMedCentralID PMC3175547

• Disruption of PPAR gamma/beta-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival JOURNAL OF CLINICAL INVESTIGATION Alastalo, T., Li, M., Perez, V. D., Pham, D., Sawada, H., Wang, J. K., Koskenvuo, M., Wang, L., Freeman, B. A., Chang, H. Y., Rabinovitch, M. 2011; 121 (9): 3735-3746

  Abstract

  Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

  View details for DOI 10.1172/JCI43382

  View details for PubMedID 21821917

• Inhibition of Transforming Growth Factor beta Worsens Elastin Degradation in a Murine Model of Kawasaki Disease AMERICAN JOURNAL OF PATHOLOGY Alvira, C. M., Guignabert, C., Kim, Y., Chen, C., Wang, L., Duong, T. T., Yeung, R. S., Li, D. Y., Rabinovitch, M. 2011; 178 (3): 1210-1220

  Abstract

  Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

  View details for DOI 10.1016/j.ajpath.2010.11.054

  View details for PubMedID 21356372

• Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome NATURE IMMUNOLOGY Nakahira, K., Haspel, J. A., Rathinam, V. A., Lee, S., Dolinay, T., Lam, H. C., Englert, J. A., Rabinovitch, M., Cernadas, M., Kim, H. P., Fitzgerald, K. A., Ryter, S. W., Choi, A. M. 2011; 12 (3): 222-U57

  Abstract

  Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

  View details for DOI 10.1038/ni.1980

  View details for Web of Science ID 000287354400009

  View details for PubMedID 21151103

  View details for PubMedCentralID PMC3079381

• Autophagic Protein LC3B Confers Resistance against Hypoxia-Induced Pulmonary Hypertension AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Lee, S., Smith, A., Guo, L., Alastalo, T., Li, M., Sawada, H., Liu, X., Chen, Z., Ifedigbo, E., Lin, Y., Feghali-Bostwick, C., Ryter, S. W., Kim, H. P., Rabinovitch, M., Choi, A. M. 2011; 183 (5): 649-658

  Abstract

  Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B(-/-)), or early growth response-1 (Egr-1(-/-)), were evaluated for vascular morphology and hemodynamics.Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B(-/-) and Egr-1(-/-) mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.

  View details for DOI 10.1164/rccm.201005-0746OC

  View details for Web of Science ID 000288296000018

  View details for PubMedID 20889906

  View details for PubMedCentralID PMC3081281

• BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways JOURNAL OF CELL BIOLOGY Perez, V. A., Ali, Z., Alastalo, T., Ikeno, F., Sawada, H., Lai, Y., Kleisli, T., Spiekerkoetter, E., Qu, X., Rubinos, L. H., Ashley, E., Amieva, M., Dedhar, S., Rabinovitch, M. 2011; 192 (1): 171-188

  Abstract

  We present a novel cell-signaling paradigm in which bone morphogenetic protein 2 (BMP-2) consecutively and interdependently activates the wingless (Wnt)-β-catenin (βC) and Wnt-planar cell polarity (PCP) signaling pathways to facilitate vascular smooth muscle motility while simultaneously suppressing growth. We show that BMP-2, in a phospho-Akt-dependent manner, induces βC transcriptional activity to produce fibronectin, which then activates integrin-linked kinase 1 (ILK-1) via α4-integrins. ILK-1 then induces the Wnt-PCP pathway by binding a proline-rich motif in disheveled (Dvl) and consequently activating RhoA-Rac1-mediated motility. Transfection of a Dvl mutant that binds βC without activating RhoA-Rac1 not only prevents BMP-2-mediated vascular smooth muscle cell motility but promotes proliferation in association with persistent βC activity. Interfering with the Dvl-dependent Wnt-PCP activation in a murine stented aortic graft injury model promotes extensive neointima formation, as shown by optical coherence tomography and histopathology. We speculate that, in response to injury, factors that subvert BMP-2-mediated tandem activation of Wnt-βC and Wnt-PCP pathways contribute to obliterative vascular disease in both the systemic and pulmonary circulations.

  View details for DOI 10.1083/jcb.201008060

  View details for PubMedID 21220513

• Strategic Plan for Lung Vascular Research An NHLBI-ORDR Workshop Report AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Erzurum, S., Rounds, S. I., Stevens, T., Aldred, M., Aliotta, J., Archer, S. L., Asosingh, K., Balaban, R., Bauer, N., Bhattacharya, J., Bogaard, H., Choudhary, G., Dorn, G. W., Dweik, R., Fagan, K., Fallon, M., Finkel, T., Geraci, M., Gladwin, M. T., Hassoun, P. M., Humbert, M., Kaminski, N., Kawut, S. M., Loscalzo, J., McDonald, D., McMurtry, I. F., Newman, J., Nicolls, M., Rabinovitch, M., Shizuru, J., Oka, M., Polgar, P., Rodman, D., Schumacker, P., Stenmark, K., Tuder, R., Voelkel, N., Sullivan, E., Weinshilboum, R., Yoder, M. C., Zhao, Y., Gail, D., Moore, T. M. 2010; 182 (12): 1554-1562

  Abstract

  The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype-phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes.

  View details for DOI 10.1164/rccm.201006-0869WS

  View details for Web of Science ID 000285534600016

  View details for PubMedID 20833821

  View details for PubMedCentralID PMC3029941

• Adverse Pulmonary Effects of Mechanical Ventilation Are Blunted in Transgenic Newborn Mice That Over-Express the Serine Elastase Inhibitor Elafin Hilgendorff, A., Parai, K., Ertsey, R., Starcher, B., Jain, N., Rabinovitch, M., Bland, R. SPRINGER. 2010: 384–85

  View details for Web of Science ID 000274036700037

• Emerging therapies for the treatment of pulmonary hypertension PEDIATRIC CRITICAL CARE MEDICINE Stenmark, K. R., Rabinovitch, M. 2010; 11: S85-S90

  Abstract

  Current treatment of pulmonary arterial hypertension, which includes the use of prostacyclins, endothelin receptor antagonists, and phosphodiesterase type 5 inhibitors, either alone or in combination, often leads to improvements in functional capacity and modest decreases in pulmonary artery pressure. Disappointingly, however, two recent meta-analysis reviewing the controlled trials in pulmonary arterial hypertension, using these three agents, demonstrated little or no increase in survival. Importantly, however, increasing knowledge of the cellular and molecular basis of pulmonary arterial hypertension has led to the development of new agents aimed at either reversing sustained vasoconstriction or stopping/reversing the abnormal cell and extracellular matrix accumulation that, in combination, obstruct pulmonary blood flow and ultimately cause right heart failure. Rho kinase inhibitors, vasodilator peptides (such as vasoactive intestinal peptide and adrenomedullin), and endothelial nitric oxide synthase coupling agents (cicletanine) have been shown sometimes to exert potent pulmonary vasodilatory effects in animal models and in pilot studies in humans. Tyrosine kinase inhibitors (platelet-derived growth factor and epidermal growth factor receptor inhibitors), multikinase inhibitors (tyrosine kinase and serine/threonine kinase), elastase inhibitors, metabolic modulators (e.g., dichloroacetate), survivin inhibitors, and HMG-COA reductase inhibitors have been shown to reverse pulmonary hypertension in rodent models of pulmonary hypertension through inhibition of cell proliferation and induction of apoptosis. Early success in human pulmonary arterial hypertension with tyrosine kinase inhibitors has appeared in case reports. Furthermore, anti-inflammatory/immunomodulatory agents (thiazolidinedinones, rapamycin, cyclosporine, and STAT3 inhibitors) have been demonstrated to be effective at reducing vascular remodeling in animal models. Collectively, these studies are exciting and open potential new avenues for treatment. Caution should be exercised, however, as many agents, which are successful at preventing or reversing pulmonary arterial hypertension in currently used animal models, do not result in similar long-term success in the treatment of human pulmonary arterial hypertension.

  View details for DOI 10.1097/PCC.0b013e3181c76db3

  View details for Web of Science ID 000275411600017

  View details for PubMedID 20216170

• PPAR gamma and the Pathobiology of Pulmonary Arterial Hypertension 2008 Grover Conference on Membrane Receptors, Channels and Transporters in Pulmonary Circulation Rabinovitch, M. SPRINGER-VERLAG BERLIN. 2010: 447–458

  Abstract

  Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that functions as a transcription factor to regulate adipogenesis and metabolism by binding to PPAR response elements (PPAREs) in the promoter region of various target genes. Activation of PPARgamma suppresses smooth muscle cell proliferation and migration. This chapter discusses the potential protective role of PPARgamma and its downstream signaling cascades in the development of pulmonary arterial hypertension. Furthermore, the chapter also provides an overview on the cellular and molecular mechanisms involved in PPARgamma-mediated inhibitory effect on pulmonary vascular remodeling, a major contributor to the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension.

  View details for Web of Science ID 000293426500029

  View details for PubMedID 20204748

• Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Mokres, L. M., Parai, K., Hilgendorff, A., Ertsey, R., Alvira, C. M., Rabinovitch, M., Bland, R. D. 2010; 298 (1): L23-L35

  Abstract

  Defective lung septation and angiogenesis, quintessential features of neonatal chronic lung disease (CLD), typically result from lengthy exposure of developing lungs to mechanical ventilation (MV) and hyperoxia. Previous studies showed fewer alveoli and microvessels, with reduced VEGF and increased transforming growth factor-beta (TGFbeta) signaling, and excess, scattered elastin in lungs of premature infants and lambs with CLD vs. normal controls. MV of newborn mice with 40% O(2) for 24 h yielded similar lung structural abnormalities linked to impaired VEGF signaling, dysregulated elastin production, and increased apoptosis. These studies could not determine the relative importance of cyclic stretch vs. hyperoxia in causing these lung growth abnormalities. We therefore studied the impact of MV for 24 h with air on alveolar septation (quantitative lung histology), angiogenesis [CD31 quantitative-immunohistochemistry (IHC), immunoblots], apoptosis [TdT-mediated dUTP nick end labeling (TUNEL), active caspase-3 assays], VEGF signaling [VEGF-A, VEGF receptor 1 (VEGF-R1), VEGF-R2 immunoblots], TGFbeta activation [phosphorylated Smad2 (pSmad2) quantitative-IHC], and elastin production (tropoelastin immunoblots, quantitative image analysis of Hart's stained sections) in lungs of 6-day-old mice. Compared with unventilated controls, MV caused a 3-fold increase in alveolar area, approximately 50% reduction in alveolar number and endothelial surface area, >5-fold increase in apoptosis, >50% decrease in lung VEGF-R2 protein, 4-fold increase of pSmad2 protein, and >50% increase in lung elastin, which was distributed throughout alveolar walls rather than at septal tips. This study is the first to show that prolonged MV of developing lungs, without associated hyperoxia, can inhibit alveolar septation and angiogenesis and increase apoptosis and lung elastin, findings that could reflect stretch-induced changes in VEGF and TGFbeta signaling, as reported in CLD.

  View details for DOI 10.1152/ajplung.00251.2009

  View details for Web of Science ID 000272827900005

  View details for PubMedID 19854954

  View details for PubMedCentralID PMC2806196

• Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Guignabert, C., Alvira, C. M., Alastalo, T., Sawada, H., Hansmann, G., Zhao, M., Wang, L., El-Bizri, N., Rabinovitch, M. 2009; 297 (6): L1082-L1090

  Abstract

  Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

  View details for DOI 10.1152/ajplung.00199.2009

  View details for Web of Science ID 000272017900009

  View details for PubMedID 19801450

  View details for PubMedCentralID PMC2793182

• Cholinergic Modulation of Angiogenesis: Role of the 7 Nicotinic Acetylcholine Receptor JOURNAL OF CELLULAR BIOCHEMISTRY Wu, J. C., Chruscinski, A., Perez, V. A., Singh, H., Pitsiouni, M., Rabinovitch, M., Utz, P. J., Cooke, J. P. 2009; 108 (2): 433-446

  Abstract

  Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except alpha(2), alpha(4), gamma, and delta in four different types of ECs. Using siRNA methodology, we found that the alpha(7) nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The alpha(9) and alpha(7) nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the alpha(7) nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.

  View details for DOI 10.1002/jcb.22270

  View details for Web of Science ID 000270438000012

  View details for PubMedID 19623583

  View details for PubMedCentralID PMC3140170

• ADVERSE EFFECTS OF MECHANICAL VENTILATION (MV) ARE BLUNTED IN TRANSGENIC NEWBORN MICE THAT OVER-EXPRESS THE SERINE ELASTASE INHIBITOR ELAFIN Hilgendorff, A., Parai, K., Ertsey, R., Mokres, L., Rey-Parra, J., Thebaud, B., Jain, N., Navarro, E., RABINOVITCH, M., Bland, R. WILEY-BLACKWELL. 2009: 27–27

  View details for Web of Science ID 000269862100068

• S100A4 and Bone Morphogenetic Protein-2 Codependently Induce Vascular Smooth Muscle Cell Migration via Phospho-Extracellular Signal-Regulated Kinase and Chloride Intracellular Channel 4 CIRCULATION RESEARCH Spiekerkoetter, E., Guignabert, C., Perez, V. D., Alastalo, T., Powers, J. M., Wang, L., Lawrie, A., Ambartsumian, N., Schmidt, A., Berryman, M., Ashley, R. H., Rabinovitch, M. 2009; 105 (7): 639-U37

  Abstract

  S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products).We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension.Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells.Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

  View details for DOI 10.1161/CIRCRESAHA.109.205120

  View details for Web of Science ID 000270150800006

  View details for PubMedID 19713532

  View details for PubMedCentralID PMC2818124

• Inflammation, Growth Factors, and Pulmonary Vascular Remodeling 4th World Symposium on Pulmonary Hypertension Hassoun, P. M., Mouthon, L., Barbera, J. A., Eddahibi, S., Flores, S. C., Grimminger, F., Jones, P. L., Maitland, M. L., Michelakis, E. D., Morrell, N. W., Newman, J. H., Rabinovitch, M., Schermuly, R., Stenmark, K. R., Voelkel, N. F., Yuan, J. X., Humbert, M. ELSEVIER SCIENCE INC. 2009: S10–S19

  Abstract

  Inflammatory processes are prominent in various types of human and experimental pulmonary hypertension (PH) and are increasingly recognized as major pathogenic components of pulmonary vascular remodeling. Macrophages, T and B lymphocytes, and dendritic cells are present in the vascular lesions of PH, whether in idiopathic pulmonary arterial hypertension (PAH) or PAH related to more classical forms of inflammatory syndromes such as connective tissue diseases, human immunodeficiency virus (HIV), or other viral etiologies. Similarly, the presence of circulating chemokines and cytokines, viral protein components (e.g., HIV-1 Nef), and increased expression of growth (such as vascular endothelial growth factor and platelet-derived growth factor) and transcriptional (e.g., nuclear factor of activated T cells or NFAT) factors in these patients are thought to contribute directly to further recruitment of inflammatory cells and proliferation of smooth muscle and endothelial cells. Other processes, such as mitochondrial and ion channel dysregulation, seem to convey a state of cellular resistance to apoptosis; this has recently emerged as a necessary event in the pathogenesis of pulmonary vascular remodeling. Thus, the recognition of complex inflammatory disturbances in the vascular remodeling process offers potential specific targets for therapy and has recently led to clinical trials investigating, for example, the use of tyrosine kinase inhibitors. This paper provides an overview of specific inflammatory pathways involving cells, chemokines and cytokines, cellular dysfunctions, growth factors, and viral proteins, highlighting their potential role in pulmonary vascular remodeling and the possibility of future targeted therapy.

  View details for DOI 10.1016/j.jacc.2009.04.006

  View details for Web of Science ID 000267547500003

  View details for PubMedID 19555853

• LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor JOURNAL OF CELL SCIENCE Ying, L., Lau, A., Alvira, C. M., West, R., Cann, G. M., Zhou, B., Kinnear, C., Jan, E., Sarnow, P., van de Rijn, M., Rabinovitch, M. 2009; 122 (9): 1441-1451

  Abstract

  Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.

  View details for DOI 10.1242/jcs.025957

  View details for PubMedID 19366727

• Can we start to think about consensus-oriented clinical practices? CARDIOLOGY IN THE YOUNG Lopes, A. A., Rabinovitch, M. 2009; 19: 1-3

  View details for DOI 10.1017/S1047951109003862

  View details for Web of Science ID 000283289400001

  View details for PubMedID 19419597

• Balloon occlusion pulmonary wedge angiography and lung biopsy assessment in the child with a congenital cardiac defect CARDIOLOGY IN THE YOUNG Rabinovitch, M., Haworth, S. G. 2009; 19: 13-15

  View details for DOI 10.1017/S1047951109003898

  View details for Web of Science ID 000283289400004

  View details for PubMedID 19419600

• Insulin resistance in pulmonary arterial hypertension EUROPEAN RESPIRATORY JOURNAL Zamanian, R. T., Hansmann, G., Snook, S., Lilienfeld, D., Rappaport, K. M., Reaven, G. M., RABINOVITCH, M., Doyle, R. L. 2009; 33 (2): 318-324

  Abstract

  Although obesity, dyslipidemia and insulin resistance (IR) are well known risk factors for systemic cardiovascular disease, their impact on pulmonary arterial hypertension (PAH) is unknown. The present authors' previous studies indicate that IR may be a risk factor for PAH. The current study has investigated the prevalence of IR in PAH and explored its relationship with disease severity. Clinical data and fasting blood samples were evaluated in 81 nondiabetic PAH females. In total, 967 National Health and Nutrition Examination Surveys (NHANES) females served as controls. The fasting triglyceride to high-density lipoprotein cholesterol ratio was used as a surrogate of insulin sensitivity. While body mass index was similar in NHANES versus PAH females (28.6 versus 28.7 kg.m(-2)), PAH females were more likely to have IR (45.7 versus 21.5%) and less likely to be insulin sensitive (IS; 43.2 versus 57.8%). PAH females mostly (82.7%) had New York Heart Association (NYHA) class II and III symptoms. Aetiology, NYHA class, 6-min walk-distance and haemodynamics did not differ between IR and IS PAH groups. However, the presence of IR and a higher NYHA class was associated with poorer 6-months event-free survival (58 versus 79%). Insulin resistance appears to be more common in pulmonary arterial hypertension females than in the general population, and may be a novel risk factor or disease modifier that might impact on survival.

  View details for DOI 10.1183/09031936.00000508

  View details for Web of Science ID 000263709300014

  View details for PubMedID 19047320

  View details for PubMedCentralID PMC2785883

• Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-beta-catenin and Wnt-RhoA-Rac1 pathways JOURNAL OF CELL BIOLOGY Perez, V. A., Alastalo, T., Wu, J. C., Axelrod, J. D., Cooke, J. P., Amieva, M., Rabinovitch, M. 2009; 184 (1): 83-99

  Abstract

  Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-beta, induces beta-catenin (beta-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA-Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both beta-C- and Dvl-mediated RhoA-Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2-mediated angiogenesis.

  View details for DOI 10.1083/jcb.200806049

  View details for Web of Science ID 000262867000010

  View details for PubMedID 19139264

  View details for PubMedCentralID PMC2615088

• VEGF blockade inhibits angiogenesis and reepithelialization of endometrium FASEB JOURNAL Fan, X., Krieg, S., Kuo, C. J., Wiegand, S. J., Rabinovitch, M., Druzin, M. L., Brenner, R. M., Giudice, L. C., Nayak, N. R. 2008; 22 (10): 3571-3580

  Abstract

  Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangiogenesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2-neuropilin 1 (VEGFR2-NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endometrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angiogenesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.

  View details for DOI 10.1096/fj.08-111401

  View details for Web of Science ID 000259642600019

  View details for PubMedID 18606863

  View details for PubMedCentralID PMC2537439

• Emerging concepts and translational priorities in pulmonary arterial hypertension CIRCULATION Michelakis, E. D., Wilkins, M. R., Rabinovitch, M. 2008; 118 (14): 1486-1495

  View details for DOI 10.1161/CIRCULATIONAHA.106.673988

  View details for Web of Science ID 000259648100012

  View details for PubMedID 18824655

• SM22 alpha-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis DEVELOPMENT El-Bizri, N., Guignabert, C., Wang, L., Cheng, A., Stankunas, K., Chang, C., Mishina, Y., Rabinovitch, M. 2008; 135 (17): 2981-2991

  Abstract

  Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain.

  View details for DOI 10.1242/dev.017863

  View details for Web of Science ID 000258395500016

  View details for PubMedID 18667463

  View details for PubMedCentralID PMC2653628

• Molecular pathogenesis of pulmonary arterial hypertension JOURNAL OF CLINICAL INVESTIGATION Rabinovitch, M. 2008; 118 (7): 2372-2379

  Abstract

  Recent investigations have suggested that it might be possible to reverse the pathology of pulmonary arterial hypertension (PAH), a disorder that can be rapidly progressive and fatal despite current treatments including i.v. prostacyclin. This review will address the cellular and molecular processes implicated in clinical, genetic, and experimental studies as underlying the pulmonary vascular abnormalities associated with PAH. Emerging treatments are aimed at inducing apoptosis of abnormal vascular cells that obstruct blood flow and at promoting regeneration of "lost" distal vasculature.

  View details for DOI 10.1172/JCI33452

  View details for Web of Science ID 000257657400003

  View details for PubMedID 18596905

  View details for PubMedCentralID PMC2439479

• An antiproliferative BMP-2/PPAR gamma/apoE axis in human and murine SMCs and its role in pulmonary hypertension JOURNAL OF CLINICAL INVESTIGATION Hansmann, G., de Jesus Perez, V. A., Alastalo, T., Alvira, C. M., Guignabert, C., Bekker, J. M., Schellong, S., Urashima, T., Wang, L., Morrell, N. W., Rabinovitch, M. 2008; 118 (5): 1846-1857

  Abstract

  Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARgamma and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB-induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARgamma that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARgamma agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient-derived BMP-RII mutant PASMCs, a PPARgamma antagonist, and PASMCs isolated from PPARgamma- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARgamma, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARgamma in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARgamma-mediated events could protect against PAH, and PPARgamma agonists may reverse PAH in patients with or without BMP-RII dysfunction.

  View details for DOI 10.1172/JCI32503

  View details for PubMedID 18382765

• Pulmonary arterial remodeling induced by a Th2 immune response JOURNAL OF EXPERIMENTAL MEDICINE Daley, E., Emson, C., Guignabert, C., Malefyt, R. d., Louten, J., Kurup, V. P., Hogaboam, C., Taraseviciene-Stewart, L., Voelkel, N. F., Rabinovitch, M., Grunig, E., Grunig, G. 2008; 205 (2): 361-372

  Abstract

  Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4(+) T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule alpha, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

  View details for DOI 10.1084/jem.20071008

  View details for Web of Science ID 000253250300014

  View details for PubMedID 18227220

• Smooth muscle protein 22 alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling CIRCULATION RESEARCH El-Bizri, N., Wang, L., Merklinger, S. L., Guignabert, C., Desai, T., Urashima, T., Sheikh, A. Y., Knutsen, R. H., Mecham, R. P., Mishina, Y., Rabinovitch, M. 2008; 102 (3): 380-388

  Abstract

  Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension, but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of pulmonary arterial hypertension and associated right ventricular (RV) dysfunction, we deleted Bmpr1a in vascular smooth muscle cells and in cardiac myocytes in mice using the SM22alpha;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by Western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in RV systolic pressure and RV hypertrophy following 3 weeks in chronic hypoxia. However, both SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutant mice had fewer muscularized distal pulmonary arteries and attenuated loss of peripheral pulmonary arteries compared with age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by short interference RNA in cultured pulmonary arterial smooth muscle cells, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by short interference RNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RV systolic pressure and RV hypertrophy, despite the attenuated remodeling with chronic hypoxia in the flox/flox mutants versus controls, was not a function of elevated left ventricular end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially influencing vascular stiffness.

  View details for DOI 10.1161/CIRCRESAHA.107.161059

  View details for Web of Science ID 000253194600018

  View details for PubMedID 18079409

  View details for PubMedCentralID PMC2652676

• Reactivation of gamma HV68 induces neointimal lesions in pulmonary arteries of S100A4/Mts1-overexpressing mice in association with degradation of elastin AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Spiekerkoetter, E., Alvira, C. M., Kim, Y., Bruneau, A., Pricola, K. L., Wang, L., Ambartsumian, N., Rabinovitch, M. 2008; 294 (2): L276-L289

  Abstract

  S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the S100A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected S100A4/Mts1 mice with gammaherpesvirus 68 (gammaHV68). We observed, 6 mo after gammaHV68 [4 x 10(3) plaque-forming units (PFU)], perivascular inflammation in 10/15 S100A4/Mts1 mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose gammaHV68 (4 x 10(6) PFU) in C57Bl/6 and S100A4/Mts1 mice. In S100A4/Mts1 mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after gammaHV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-gammaHV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57Bl/6 (5-9% of vessels) and in 5/5 S100A4/Mts1 mice (13-40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mts1 mice may promote increased viral entry in the vessel wall. Using S100A4/Mts1 PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation.

  View details for DOI 10.1152/ajplung.00414.2007

  View details for PubMedID 18083765

• Developmental expression of LC3 alpha and beta: Absence of fibronectin or autophagy phenotype in LC3 beta knockout mice DEVELOPMENTAL DYNAMICS Cann, G. M., Guignabert, C., Ying, L., Deshpande, N., Bekker, J. M., Wang, L., Zhou, B., Rabinovitch, M. 2008; 237 (1): 187-195

  Abstract

  Murine light chain 3 (LC3) exists as two isoforms, LC3alpha and beta: LC3beta is an RNA-binding protein that enhances fibronectin (FN) mRNA translation, and is also a marker of autophagy. We report embryonic expression patterns for LC3alpha and LC3beta, with some overlap but notable differences in the brain, and in tissues of non-neuronal origin. LC3beta knockout (-/-) mice develop normally without a compensatory increase in LC3alpha. LC3beta-/- embryonic fibroblasts (MEFs) exhibit reduced FN synthesis but maintain wild type (WT) levels of FN protein. No significant changes in proteins associated with FN turnover, i.e., caveolin-1, LRP-1, or matrix metalloproteinases were identified. Autophagosomes form in amino acid-starved LC3beta-/-MEFs, and Caesarean-delivered pups survive as long as WT pups without an increase in LC3-related proteins linked to autophagy. These results suggest novel compensatory mechanisms for loss of LC3beta, ensuring proper FN accumulation and autophagy during fetal and neonatal life.

  View details for DOI 10.1002/dvdy.21392

  View details for Web of Science ID 000252386300018

  View details for PubMedID 18069693

• Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice. Prelude to defective alveolar septation during lung development? AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Bland, R. D., Ertsey, R., Mokres, L. M., Xu, L., Jacobson, B. E., Jiang, S., Alvira, C. M., Rabinovitch, M., Shinwell, E. S., Dixit, A. 2008; 294 (1): L3-L14

  Abstract

  Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

  View details for DOI 10.1152/ajplung.00362.2007

  View details for Web of Science ID 000252398600002

  View details for PubMedID 17934062

• Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Bland, R. D., Mokres, L. M., Ertsey, R., Jacobson, B. E., Jiang, S., Rabinovitch, M., Xu, L., Shinwell, E. S., Zhang, F., Beasley, M. A. 2007; 293 (5): L1099-L1110

  Abstract

  Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice. Am J Physiol Lung Cell Mol Physiol 293: , 2007. First published August 17, 2007; - Mechanical ventilation (MV) with O(2)-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% O(2) for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% O(2) for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% O(2) for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% O(2). These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with O(2)-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.

  View details for DOI 10.1152/ajplung.00217.2007

  View details for Web of Science ID 000250870700003

  View details for PubMedID 17704187

• Point: Counterpoint: Chronic hypoxia-induced pulmonary hypertension does/does not lead to loss of pulmonary vasculature JOURNAL OF APPLIED PHYSIOLOGY Rabinovitch, M., Chesler, N., Molthen, R. C. 2007; 103 (4): 1449-1451

  View details for DOI 10.1152/japplphysiol.00274.2007

  View details for Web of Science ID 000250087800047

  View details for PubMedID 17363624

• Dysregulation of pulmonary elastin synthesis and assembly in preterm lambs with chronic lung disease AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Bland, R. D., Xu, L., Ertsey, R., Rabinovitch, M., Albertine, K. H., Wynn, K. A., Kumar, V. H., Ryan, R. M., Swartz, D. D., Csiszar, K., Fong, K. S. 2007; 292 (6): L1370-L1384

  Abstract

  Failed alveolar formation and excess, disordered elastin are key features of neonatal chronic lung disease (CLD). We previously found fewer alveoli and more elastin in lungs of preterm compared with term lambs that had mechanical ventilation (MV) with O(2)-rich gas for 3 wk (MV-3 wk). We hypothesized that, in preterm more than in term lambs, MV-3 wk would reduce lung expression of growth factors that regulate alveolarization (VEGF, PDGF-A) and increase lung expression of growth factors [transforming growth factor (TGF)-alpha, TGF-beta(1)] and matrix molecules (tropoelastin, fibrillin-1, fibulin-5, lysyl oxidases) that regulate elastin synthesis and assembly. We measured lung expression of these genes in preterm and term lambs after MV for 1 day, 3 days, or 3 wk, and in fetal controls. Lung mRNA for VEGF, PDGF-A, and their receptors (VEGF-R2, PDGF-Ralpha) decreased in preterm and term lambs after MV-3 wk, with reduced lung content of the relevant proteins in preterm lambs with CLD. TGF-alpha and TGF-beta(1) expression increased only in lungs of preterm lambs. Tropoelastin mRNA increased more with MV of preterm than term lambs, and expression levels remained high in lambs with CLD. In contrast, fibrillin-1 and lysyl oxidase-like-1 mRNA increased transiently, and lung abundance of other elastin-assembly genes/proteins was unchanged (fibulin-5) or reduced (lysyl oxidase) in preterm lambs with CLD. Thus MV-3 wk reduces lung expression of growth factors that regulate alveolarization and differentially alters expression of growth factors and matrix proteins that regulate elastin assembly. These changes, coupled with increased lung elastase activity measured in preterm lambs after MV for 1-3 days, likely contribute to CLD.

  View details for DOI 10.1152/ajplung.00367.2006

  View details for PubMedID 17293375

• Nuclear factor-kappa B activation in neonatal mouse lung protects against lipopolysaccharide-induced inflammation AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Alvira, C. M., Abate, A., Yang, G., Dennery, P. a., Rabinovitch, M. 2007; 175 (8): 805-815

  Abstract

  Injurious agents often cause less severe injury in neonates as compared with adults.We hypothesized that maturational differences in lung inflammation induced by lipopolysaccharide (LPS) may be related to the nature of the nuclear factor (NF)-kappaB complex activated, and the profile of target genes expressed.Neonatal and adult mice were injected with intraperitoneal LPS. Lung inflammation was assessed by histology, and apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase UTP nick-end labeling). The expression of candidate inflammatory and apoptotic mediators was evaluated by quantitative real-time polymerase chain reaction and Western immunoblot.Neonates demonstrated reduced inflammation and apoptosis, 24 hours after LPS exposure, as compared with adults. This difference was associated with persistent activation of NF-kappaB p65p50 heterodimers in the neonates in contrast to early, transient activation of p65p50 followed by sustained activation of p50p50 in the adults. Adults had increased expression of a panel of inflammatory and proapoptotic genes, and repression of antiapoptotic targets, whereas no significant changes in these mediators were observed in the neonates. Inhibition of NF-kappaB activity in the neonates decreased apoptosis, but heightened inflammation, with increased expression of the same inflammatory genes elevated in the adults. In contrast, inhibition of NF-kappaB in the adults resulted in partial suppression of the inflammatory response.NF-kappaB activation in the neonatal lung is antiinflammatory, protecting against LPS-mediated lung inflammation by repressing similar inflammatory genes induced in the adult.

  View details for DOI 10.1164/rccm.200608-11620C

  View details for PubMedID 17255561

• Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-gamma activation CIRCULATION Hansmann, G., Wagner, R. A., Schellong, S., Perez, V. A., Urashima, T., Wang, L., Sheikh, A. Y., Suen, R. S., Stewart, D. J., Rabinovitch, M. 2007; 115 (10): 1275-1284

  Abstract

  Patients with pulmonary arterial hypertension (PAH) have reduced expression of apolipoprotein E (apoE) and peroxisome proliferator-activated receptor-gamma in lung tissues, and deficiency of both has been linked to insulin resistance. ApoE deficiency leads to enhanced platelet-derived growth factor signaling, which is important in the pathobiology of PAH. We therefore hypothesized that insulin-resistant apoE-deficient (apoE-/-) mice would develop PAH that could be reversed by a peroxisome proliferator-activated receptor-gamma agonist (eg, rosiglitazone).We report that apoE-/- mice on a high-fat diet develop PAH as judged by elevated right ventricular systolic pressure. Compared with females, male apoE-/- were insulin resistant, had lower plasma adiponectin, and had higher right ventricular systolic pressure associated with right ventricular hypertrophy and increased peripheral pulmonary artery muscularization. Because male apoE-/- mice were insulin resistant and had more severe PAH than female apoE-/- mice, we treated them with rosiglitazone for 4 and 10 weeks. This treatment resulted in markedly higher plasma adiponectin, improved insulin sensitivity, and complete regression of PAH, right ventricular hypertrophy, and abnormal pulmonary artery muscularization in male apoE-/- mice. We further show that recombinant apoE and adiponectin suppress platelet-derived growth factor-BB-mediated proliferation of pulmonary artery smooth muscle cells harvested from apoE-/- or C57Bl/6 control mice.We have shown that insulin resistance, low plasma adiponectin levels, and deficiency of apoE may be risk factors for PAH and that peroxisome proliferator-activated receptor-gamma activation can reverse PAH in an animal model.

  View details for DOI 10.1161/CIRCULATIONAHA.106.663120

  View details for Web of Science ID 000244864100017

  View details for PubMedID 17339547

• Pathobiology of pulmonary hypertension ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE Rabinovitch, M. 2007; 2: 369-399

  Abstract

  A variety of conditions can lead to the development of pulmonary arterial hypertension (PAH). Current treatments can improve symptoms and reduce the severity of the hemodynamic abnormality, but most patients remain quite limited, and deterioration in their condition necessitates a lung transplant. This review discusses current experimental and clinical studies that investigate the pathobiology of PAH. An emerging theme is the consideration of ways in which one might reverse the advanced occlusive structural changes in the pulmonary circulation causing PAH. The current debate concerning the role of regeneration through stem cells is presented. This review also highlights investigations in a number of laboratories relating the pathobiology of PAH to mutations causing loss of function of bone morphogenetic protein receptor II in patients with familial PAH, as well as sporadic cases.

  View details for DOI 10.1146/annurev.pathol.2.010506.092033

  View details for Web of Science ID 000245663900015

  View details for PubMedID 18039104

• PPAR gamma protects against pulmonary arterial hypertension (PAH) and is central in a novel antiproliferative BMP-2-PPAR gamma-ApoE-Axis in smooth muscle cells (SMC) 79th Annual Scientific Session of the American-Heart-Association Hansmann, G., Perez, V. A., Powers, J., Schellong, S., Wang, L., Urashima, T., Rabinovitch, M. LIPPINCOTT WILLIAMS & WILKINS. 2006: 37–37

  View details for Web of Science ID 000241792800192

• Altered expression of key growth factors (TGF alpha, TGF beta 1, PDGF-A) and flawed formation of alveoli and elastin (Eln) in lungs of preterm (PT) lambs with chronic lung disease (CLD) Experimental Biology 2006 Annual Meeting Xu, L. W., RABINOVITCH, M., Bland, R. FEDERATION AMER SOC EXP BIOL. 2006: A1442–A1443

  View details for Web of Science ID 000236326205356

• Cellular and molecular pathobiology of pulmonary hypertension conference summary 47th Annual Thomas L Petty Aspen Lung Conference Rabinovitch, M. AMER COLL CHEST PHYSICIANS. 2005: 642S–646S

  View details for Web of Science ID 000234371400068

  View details for PubMedID 16373886

• Mts1/S100A4 stimulates human pulmonary artery smooth muscle cell migration through multiple signaling pathways 47th Annual Thomas L Petty Aspen Lung Conference Spiekerkoetter, E., Lawrie, A., Merklinger, S., Ambartsumian, N., Lukanidin, D., Schmidt, A. A., Rabbiovitch, M. AMER COLL CHEST PHYSICIANS. 2005: 577S–577S

  View details for Web of Science ID 000234371400021

  View details for PubMedID 16373840

• Caspases from apoptotic myocytes degrade extracellular matrix: a novel remodeling paradigm. FASEB journal Cowan, K. N., Leung, W. C., Mar, C., Bhattacharjee, R., Zhu, Y., Rabinovitch, M. 2005; 19 (13): 1848-1850

  Abstract

  Induction of smooth muscle cell apoptosis is critical to the reversal of severe structural remodeling in hypertensive pulmonary arteries during disease regression. This process involves coordinated resorption of pathologically deposited extracellular matrix, including elastin, and occurs in the presence of serine elastase and matrix metalloproteinase inhibitors. Here, we show that apoptotic smooth muscle cells exhibit extensive degradation of elastin coincident with cell surface immunolocalization and release of caspases. We further document that recombinant caspase-2, -3, and -7 are potently elastolytic. These enzymes are present in an active form on apoptotic cell surfaces and caspase inhibitors attenuate their elastolytic activity. Our results reveal a previously undescribed function for apoptotic cells and a novel paradigm whereby removal of cells is coordinated with degradation of excess extracellular matrix during remodeling in development and disease.

  View details for PubMedID 16123172

• Gene transfer of prostaglandin synthase maintains patency of the newborn lamb arterial duct PEDIATRIC RESEARCH Humpl, T., Zaidi, S. H., Coe, J. Y., Russell, J., Kaneda, Y., Massaeli, H., Benson, L. N., Rabinovitch, M. 2005; 58 (5): 976-980

  Abstract

  In congenital heart disease with left- or right-sided obstruction, prostaglandin E (PGE)1 or PGE2 is infused to maintain ductus arteriosus (DA) patency. We hypothesized that transfection of the DA with PGE synthase would lead to a greater production of PGE2 in situ and, hence, patency of the DA. The cDNA for human prostaglandin synthase was sequenced and ligated into a eukaryotic expression vector. The negative control was created by ligating the cDNA encoding the bacterial protein chloramphenicol acetyltransferase into the same plasmid. Transfection (600 microg DNA) was achieved in lambs within the first 24 h of life using the hemagglutinating virus of Japan (HVJ)-liposome transfection method with a custom-made, basket-weave-perforated catheter. Echocardiography was performed to assess DA patency until the time of sacrifice. To confirm expression of the transgene, PGE2 concentration was measured in organ culture of the DA by immunoassay and by Western immunoblotting of homogenized DA tissue. Patency of the DA was demonstrated by color Doppler in all the lambs (7/7) in which the PGE synthase was delivered, whereas functional closure was seen in the control group (6/6). The PGE2 concentration in the culture medium of the explanted DA in the treatment group was 3-fold higher than that of the control groups. Western immunoblotting confirmed the presence of PGE synthase in the treatment group. Gene transfer of PGE synthase to the DA is feasible and will maintain patency for at least 1 wk.

  View details for DOI 10.1203/01.PDR.0000182820.20333.2A

  View details for Web of Science ID 000232779000027

  View details for PubMedID 16183805

• Chloride intracellular channel 4 (CLIC4), a novel downstream target of bone morphogenetic protein receptor II (BMPR-II) in human pulmonary artery smooth muscle cell (PASMC) migration 78th Annual Scientific Session of the American-Heart-Association Spiekerkoetter, E., Wang, L. L., Zamanian, R., Ambartsumian, N., Schmidt, A. M., Lukanidin, E., RABINOVITCH, M. LIPPINCOTT WILLIAMS & WILKINS. 2005: U210–U210

  View details for Web of Science ID 000232956401059

• Increased fibulin-5 and elastin in S100A4/Mts1 mice with pulmonary hypertension CIRCULATION RESEARCH Merklinger, S. L., Wagner, R. A., Spiekerkoetter, E., Hinek, A., Knutsen, R. H., Kabir, M. G., Desai, K., Hacker, S., Wang, L. L., Cann, G. M., Ambartsumian, N. S., Lukanidin, E., Bernstein, D., Husain, M., Mecham, R. P., Starcher, B., Yanagisawa, H., Rabinovitch, M. 2005; 97 (6): 596-604

  Abstract

  Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months "recovery" in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months "recovery" in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

  View details for DOI 10.1161/01.RES.00000182425.49768.8a

  View details for Web of Science ID 000231896500013

  View details for PubMedID 16109920

• Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease CIRCULATION RESEARCH Lawrie, A., Spiekerkoetter, E., Martinez, E. C., Ambartsumian, N., Sheward, W. J., MacLean, M. R., Harmar, A. J., Schmidt, A. M., Lukanidin, E., Rabinovitch, M. 2005; 97 (3): 227-235

  Abstract

  Heightened expression of the S100 calcium-binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100beta, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT-induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal-regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4-mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

  View details for DOI 10.1161/01.RES.0000176025.57706.1e

  View details for Web of Science ID 000230995100006

  View details for PubMedID 16002749

• Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary hypertension CIRCULATION Merklinger, S. L., Jones, P. L., Martinez, E. C., Rabinovitch, M. 2005; 112 (3): 423-431

  Abstract

  We previously reported that administration of elastase inhibitors reverses fatal pulmonary arterial hypertension (PAH) in rats by inducing smooth muscle cell (SMC) apoptosis. We showed in pulmonary artery (PA) organ culture that the mechanism by which elastase inhibitors induce SMC apoptosis involves repression of matrix metalloproteinase (MMP) activity and subsequent signaling through alphavbeta3-integrins and epidermal growth factor receptors (EGFRs). This suggests that blockade of these downstream effectors may also induce regression of PAH.In this study, we first showed in PA organ culture that MMP inhibition or alphavbeta3-integrin blockade with agents in clinical and preclinical use (SC-080 and cilengitide, respectively) mediates SMC apoptosis and regression of medial hypertrophy. We also documented similar results with an EGFR tyrosine kinase inhibitor. We then induced PAH in rats by injection of monocrotaline and, at day 21, began a 2-week treatment with SC-080, cilengitide, or the EGFR inhibitor PKI166. No vehicle- or cilengitide-treated animal survived beyond 2 weeks. Administration of SC-080 resulted in 44% survival at 2 weeks, and PKI166 therapy resulted in 78% and 54% survival in daily or 3-times-weekly treated animals, respectively. Four weeks after cessation of PKI166, we documented survivals of 50% and 23% in the 2 treatment groups, associated with reductions in pulmonary pressure, right ventricular hypertrophy, and abnormally muscularized distal arteries.We propose that selective blockade of EGFR signaling may be a novel strategy to reverse progressive, fatal PAH.

  View details for DOI 10.1161/CIRCULATIONHA.105.540542

  View details for Web of Science ID 000230597600019

  View details for PubMedID 16027270

• Subversion of cellular autophagosomal machinery by RNA viruses PLOS BIOLOGY Jackson, W. T., Giddings, T. H., Taylor, M. P., Mulinyawe, S., RABINOVITCH, M., Kopito, R. R., Kirkegaard, K. 2005; 3 (5): 861-871

  Abstract

  Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

  View details for DOI 10.1371/journal.pbio.0030156

  View details for Web of Science ID 000229125400014

  View details for PubMedID 15884975

  View details for PubMedCentralID PMC1084330

• Increased expression of genes associated with elastin synthesis and assembly in lungs of mechanically ventilated preterm lambs compared to term lambs Experimental Biology 2005 Meeting/35th International Congress of Physiological Sciences Bland, R. D., Xu, L. W., RABINOVITCH, M., Kumar, V., Ryan, R. M., Wynn, K. FEDERATION AMER SOC EXP BIOL. 2005: A1603–A1603

  View details for Web of Science ID 000227610904264

• The arginine rich motif (ARM) in light chain 3 (LC3) of microtubule associated proteins 1A and 1B is required for protein-RNA interaction and mRNA translation 44th Annual Meeting of the American-Society-for-Cell-Biology Ying, L., Lau, A., Sarnow, P., RABINOVITCH, M. AMER SOC CELL BIOLOGY. 2004: 216A–216A

  View details for Web of Science ID 000224648801543

• Transgenic mice with arterial smooth muscle cell-specific conditional deletion of the bone morphogenetic protein type IA receptor (BMPRIA/ALK3) are hyporesponsive to hypoxia 77th Scientific Meeting of the American-Heart-Association El-Bizri, N., Wang, L. L., Merklinger, S. L., Desai, K., Rountree, R. B., Mishina, Y., RABINOVITCH, M. LIPPINCOTT WILLIAMS & WILKINS. 2004: 132–32

  View details for Web of Science ID 000224783500624

• Apolipoprotein D and platelet-derived growth factor-BB synergism mediates vascular smooth muscle cell migration CIRCULATION RESEARCH Leung, W. C., Lawrie, A., Demaries, S., Massaeli, H., Burry, A., Yablonsky, S., Sarjeant, J. M., Fera, E., Rassart, E., Pickering, J. G., Rabinovitch, M. 2004; 95 (2): 179-186

  Abstract

  We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

  View details for DOI 10.1161/01.RES.0000135482.74178.14

  View details for Web of Science ID 000222835600010

  View details for PubMedID 15192024

• Elafin-overexpressing mice have improved cardiac function after myocardial infarction AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Ohta, K., Nakajima, T., Cheah, A. Y., Zaidi, S. H., Kaviani, N., Dawood, F., You, X. M., Liu, P., Husain, M., Rabinovitch, M. 2004; 287 (1): H286-H292

  Abstract

  Elevated serine elastase activity after myocardial infarction can contribute to remodeling associated with left ventricular dilatation and dysfunction. We therefore assessed the effects of overexpressing the selective serine elastase inhibitor elafin in transgenic mice in which a myocardial infarction was caused by ligation of the left anterior descending coronary artery (LAD). Elevated serine elastase activity was observed in nontransgenic littermates as early as 6 h after LAD ligation and persisted at 4 and 7 days but not in sham-operated or elafin-overexpressing transgenic mice. Myeloperoxidase activity (index of inflammatory cells) and matrix metalloproteinase 2 were also increased but only at 4 and 7 days and only in nontransgenic mice (P < 0.05 for both comparisons), and this increase correlated with inflammatory cell infiltration. Echocardiographic study at 4 days revealed indexes of diastolic dysfunction in nontransgenic versus elafin-overexpressing mice (P < 0.05). Morphometric and biochemical analyses at 28 days indicated impairment in cardiac performance, with greater scar thinning and infarct expansion in nontransgenic versus elafin transgenic littermates (P < 0.05 for all comparisons). Thus serine elastase inhibition appears to suppress inflammation, cardiac dilatation, and dysfunction after myocardial infarct.

  View details for DOI 10.1152/ajpheart.00479.2002

  View details for Web of Science ID 000222142100036

  View details for PubMedID 14693682

• Pulmonary arterial hypertension - Future directions - Report of a National Heart, Lung and Blood Institute/Office of Rare Diseases workshop CIRCULATION Newman, J. H., Fanburg, B. L., Archer, S. L., Badesch, D. B., Barst, R. J., Garcia, J. G., Kao, P. N., Knowles, J. A., Loyd, J. E., McGoon, M. D., Morse, J. H., Nichols, W. C., RABINOVITCH, M., Rodman, D. M., Stevens, T., Tuder, R. M., Voelkel, N. F., Gail, D. B. 2004; 109 (24): 2947-2952

  View details for DOI 10.1161/01.CIR.0000132476.87231.6F

  View details for Web of Science ID 000222178800007

  View details for PubMedID 15210611

• Cellular and molecular pathobiology of pulmonary arterial hypertension Third World Symposium on Pulmonary Arterial Hypertension Humbert, M., Morrell, N. W., Archer, S. L., Stenmark, K. R., MacLean, M. R., Lang, I. M., Christman, B. W., Weir, E. K., Eickelberg, O., Voelkel, N. F., RABINOVITCH, M. ELSEVIER SCIENCE INC. 2004: 13S–24S

  Abstract

  Pulmonary arterial hypertension (PAH) has a multifactorial pathobiology. Vasoconstriction, remodeling of the pulmonary vessel wall, and thrombosis contribute to increased pulmonary vascular resistance in PAH. The process of pulmonary vascular remodeling involves all layers of the vessel wall and is complicated by cellular heterogeneity within each compartment of the pulmonary arterial wall. Indeed, each cell type (endothelial, smooth muscle, and fibroblast), as well as inflammatory cells and platelets, may play a significant role in PAH. Pulmonary vasoconstriction is believed to be an early component of the pulmonary hypertensive process. Excessive vasoconstriction has been related to abnormal function or expression of potassium channels and to endothelial dysfunction. Endothelial dysfunction leads to chronically impaired production of vasodilators such as nitric oxide and prostacyclin along with overexpression of vasoconstrictors such as endothelin (ET)-1. Many of these abnormalities not only elevate vascular tone and promote vascular remodeling but also represent logical pharmacological targets. Recent genetic and pathophysiologic studies have emphasized the relevance of several mediators in this condition, including prostacyclin, nitric oxide, ET-1, angiopoietin-1, serotonin, cytokines, chemokines, and members of the transforming-growth-factor-beta superfamily. Disordered proteolysis of the extracellular matrix is also evident in PAH. Future studies are required to find which if any of these abnormalities initiates PAH and which ones are best targeted to cure the disease.

  View details for DOI 10.1016/j.jaac.2004.02.029

  View details for Web of Science ID 000222209300004

  View details for PubMedID 15194174

• The mouse through the looking glass - A new door into the pathophysiology of pulmonary hypertension CIRCULATION RESEARCH RABINOVITCH, M. 2004; 94 (8): 1001-1004

  View details for DOI 10.1161/01.RES.0000128079.89263.68

  View details for Web of Science ID 000221113700001

  View details for PubMedID 15117827

• Neonatal chronic lung disease (CLD) in terms vs. preterm lambs: Differing paradigms of lung injury and repair Annual Meeting of the Pediatric-Academic-Societies Bland, R., Xu, L., Kolansky, S., RABINOVITCH, M., Ryan, R., Kumar, V., Wynn, K., Swart, D., Albertine, K., Duhl, M., Williams, M. NATURE PUBLISHING GROUP. 2004: 437A–438A

  View details for Web of Science ID 000220591102551

• S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy AMERICAN JOURNAL OF PATHOLOGY Greenway, S., van Suylen, R. J., Sarvaas, G. D., Kwan, E., Ambartsumian, N., Lukanidin, E., Rabinovitch, M. 2004; 164 (1): 253-262

  Abstract

  S100A4/Mts1 confers a metastatic phenotype in tumor cells and may also be related to resistance to apoptosis and angiogenesis. Approximately 5% of transgenic mice overexpressing S100A4/Mts1 develop pulmonary arterial changes resembling human plexogenic arteriopathy with intimal hyperplasia leading to occlusion of the arterial lumen. To assess the pathophysiological significance of this observation, immunohistochemistry was applied to quantitatively analyze S100A4/Mts1 expression in pulmonary arteries in surgical lung biopsies from children with pulmonary hypertension secondary to congenital heart disease. S100A4/Mts1 was not detected in pulmonary arteries with low-grade hypertensive lesions but was expressed in smooth muscle cells of lesions showing neointimal formation and with increased intensity in vessels with an occlusive neointima and plexiform lesions. Putative downstream targets of S100A4/Mts1 include Bax, which is pro-apoptotic, and the pro-angiogenic vascular endothelial growth factor (VEGF). The increase in S100A4/Mts1 expression precedes heightened expression of Bax in progressively severe neointimal lesions but in non-S100A4/Mts1-expressing cells. VEGF immunoreactivity did not correlate with severity of disease. The relationship of increased S100A4/Mts1 to pathologically similar lesions in the transgenic mice and patients occurs despite differences in localization (endothelial versus smooth muscle cells).

  View details for Web of Science ID 000187560600027

  View details for PubMedID 14695338

• Apolipoprotein D and PDGF-BB synergism mediates vascular smooth muscle cell migration. Circ Res Leung W, Demaries S, Burry A, Yablonsky S, Sarjeant J, Fera E, Rassart E, Pickering JG, Rabinovitch M. 2004; 95 (2): 179-86
• Apolipoprotein D inhibits platelet-derived growth factor-BB-induced vascular smooth muscle cell proliferated by preventing translocation of phosphorylated extracellular signal regulated kinase 1/2 to the nucleus ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Sarjeant, J. M., Lawrie, A., Kinnear, C., Yablonsky, S., Leung, W., Massaeli, H., Prichett, W., Veinot, J. P., Rassart, E., RABINOVITCH, M. 2003; 23 (12): 2172-2177

  Abstract

  Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease.Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation.Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.

  View details for DOI 10.1161/01.ATV.0000100404.05459.39

  View details for Web of Science ID 000187236600010

  View details for PubMedID 14551159

• Low molecular weight heparin and unfractionated heparin are both effective at accelerating pulmonary vascular maturation in neonatal rabbits CIRCULATION O'Blenes, S. B., Merklinger, S. L., Jegatheeswaran, A., Campbell, A., RABINOVITCH, M., Rebeyka, I., Van Arsdell, G. 2003; 108 (10): 161-166

  View details for DOI 10.1161/01.cir.0000087380.36688.4c

  View details for Web of Science ID 000185265000028

• Temporal response and localization of integrins beta 1 and beta 3 in the heart after myocardial infarction - Regulation by cytokines CIRCULATION Sun, M., Opavsky, M. A., Stewart, D. J., RABINOVITCH, M., Dawood, F., Wen, W. H., Liu, P. P. 2003; 107 (7): 1046-1052

  Abstract

  Integrins are involved in structural remodeling and tissue repair. This study aimed to elucidate the role of the beta-integrins in cardiac remodeling after myocardial infarction (MI).The MI model was created by ligation of the left anterior descending coronary artery in rats. We detected cardiac integrins beta1 and beta3 gene expression (quantitative in situ hybridization) and protein production (Western blot and immunohistochemistry) and potential regulation by tumor necrosis factor (TNF) using neonatal ventricular myocytes and TNF-/- knockout mice. Integrins beta1 and beta3 gene expression and protein production were low in sham-operated hearts. After MI, the beta1 and beta3 mRNA and proteins were significantly increased at the site of MI at day 3, reached a peak at day 7, and gradually declined thereafter. Integrin beta1A localized primarily in fibroblasts and inflammatory cells, beta1D localized in myocytes, and integrin beta3 was associated primarily with endothelial and smooth muscle cells in peri-infarct vessels. In cultured myocytes, there was isoform transition from the adult beta1D to the fetal beta1A on exposure to TNF-alpha. This was confirmed in vivo in the peri-infarct myocytes, but the transition was voided in TNF-/--knockout mice.Integrins beta1 and beta3 are significantly activated in the infarcted myocardium. Integrin beta1 is active particularly at sites of inflammation and fibrosis, whereas integrin beta3 localizes to vessels in the peri-infarct zone in a temporally coordinated manner. Integrin beta1D to beta1A isoform transition in myocytes is regulated by TNF-alpha.

  View details for DOI 10.1161/01.CIR.0000051363.86009.3C

  View details for Web of Science ID 000181427800035

  View details for PubMedID 12600920

• Elafin overexpressing mice have improved cardiac function after myocardial infarction. Am J Physiol Heart Circ Physiol Kunio O, Nakajima T, Cheah, AYL, Zaidi S, Kaviani N, Dawood, F, You, X, Liu, P, Husain, M, Rabinovitch M. 2003; 287: H286-H292
• Apolipoprotein D inhibits PDGF induced vascular smooth muscle cell proliferation by preventing translocation of P-ERK 1/2 to the nucleus. Arterioscler Thromb Vasc Biol Sarjeant JM, Kinnear C, Yablonsky S, Leung W, Maesseli, Rassart E, Rabinovitch M. 2003; 23: 2172-2177
• Dexfenfluramine protects against pulmonary hypertension in rats JOURNAL OF APPLIED PHYSIOLOGY Mitani, Y., Mutlu, A., Russell, J. C., Brindley, D. N., DeAlmeida, J., Rabinovitch, M. 2002; 93 (5): 1770-1778

  Abstract

  Dexfenfluramine (Dex), an appetite suppressant and serotonin reuptake inhibitor, is associated with pulmonary vascular disease (PVD) in some patients. The variability might be related to undetermined genetic abnormalities interacting with factors such as gender, weight loss, and vascular injury. We, therefore, assessed the effect of Dex (5 mg. kg(-1). day(-1)) in female obese rats, designated JCR:LA-cp or cp/cp; in lean rats, designated (+/?); and in normal Sprague-Dawley (S-D) rats under control conditions or after endothelial injury induced by monocrotaline (60 mg/kg). Pulmonary arterial pressure, right ventricular hypertrophy, percent medial wall thickness of muscular arteries, and muscularization of peripheral arteries were assessed as indexes of PVD. Although Dex reduced weight gain in cp/cp and S-D rats (P < 0.05 for both), it did not cause PVD. Moreover, PVD in S-D rats after monocrotaline injection was paradoxically ameliorated by Dex (P < 0.05) despite induction of pulmonary artery elastase (P < 0.05), which we showed is critical in inducing experimental PVD. Thus it is possible that Dex is concomitantly offsetting the sequelae of elastase activity.

  View details for DOI 10.1152/japplphysiol.00500.2002

  View details for Web of Science ID 000178744400026

  View details for PubMedID 12381765

• Understanding and treating vein graft atherosclerosis CARDIOVASCULAR PATHOLOGY Sarjeant, J. M., Rabinovitch, M. 2002; 11 (5): 263-271

  Abstract

  Vein grafts have been used as bypass conduits for coronary artery disease since the 1960s. This widely used treatment, however, is complicated by the development of changes in the vein graft, which resemble atherosclerosis and are often termed as such. They occur at about 10 years, which leads to the need for reoperation in some patients. The purpose of this review is to summarize the knowledge regarding the pathophysiology of vein graft "atherosclerosis," as well as promising new treatments for this disease.The relevant literature relating to the epidemiology, histology, cell and molecular pathophysiology and treatment of vein graft atherosclerosis is reviewed.The development of vein graft atherosclerosis differs from arterial atherosclerosis. Studies have examined the role of trauma, lipids, vasoactive mediators, smooth muscle cell mitogens, smooth muscle cells apoptosis, adhesion molecules and proteases. Therapies have been developed to prevent vein graft atherosclerosis based on these studies and have been tested using animal models and in patients.Promising new therapies have been developed based on current knowledge and further applications of genomics will allow for the further identification of risk factors and mechanistic insights. The use of arterial grafts such as the internal mammary artery, which have higher patency rates at 10 years compared with vein grafts as well as approaches to revascularize infarcted myocardium may one day replace the use of vascular conduits.

  View details for Web of Science ID 000178557800002

  View details for PubMedID 12361836

• Pulmonary arterial hypertension in congenital heart disease. Cardiology clinics Granton, J. T., Rabinovitch, M. 2002; 20 (3): 441-?

  Abstract

  Pulmonary arterial hypertension (PAH) is a recognized complication of congenital systemic to pulmonary arterial cardiac shunts. The prognosis of PAH in this situation is better than primary or other secondary forms of PAH. Our knowledge of the pathophysiology of PAH complicating congenital heart disease has evolved over the past decade. Despite differences in etiology and pathobiology, therapies that have proven successful for primary PAH may benefit this group of patients.

  View details for PubMedID 12371012

• Novel notions on newborn lung disease NATURE MEDICINE Rabinovitch, M., Bland, R. 2002; 8 (7): 664-666

  View details for DOI 10.1038/nm0702-664

  View details for Web of Science ID 000176495200017

  View details for PubMedID 12091898

• Enhanced ERK-1/2 activation in mice susceptible to coxsackievirus-induced myocarditis JOURNAL OF CLINICAL INVESTIGATION Opavsky, M. A., Martino, T., RABINOVITCH, M., Penninger, J., Richardson, C., Petric, M., Trinidad, C., Butcher, L., Chan, J., Liu, P. P. 2002; 109 (12): 1561-1569

  Abstract

  Group B coxsackieviral (CVB) infection commonly causes viral myocarditis. Mice are protected from CVB3 myocarditis by gene-targeted knockout of p56(Lck)(Lck), the Src family kinase (Src) essential for T cell activation. Extracellular signal-regulated kinase 1 and 2 (ERK-1/2) can influence cell function downstream of Lck. Using T cell lines and neonatal cardiac myocytes we investigated the role of ERK-1/2 in CVB3 infection. In Jurkat T cells ERK-1/2 is rapidly activated by CVB3; but, this response is absent in Lck-negative JCaM T cells. Inhibition of ERK-1/2 with UO126 reduced CVB3 titers in Jurkat cells, but not in JCaM cells. In cardiac myocytes CVB3 activation of ERK-1/2 is blocked by the Src inhibitor PP2. In addition, viral production in myocytes is decreased by Src or ERK-1/2 inhibition. In vitro, in both immune and myocardial cells, ERK-1/2 is activated by CVB3 downstream of Lck and other Src's and is necessary for efficient CVB3 replication. In vivo, following CVB3 infection, ERK-1/2 activation is evident in the myocardium. ERK-1/2 activation is intense in the hearts of myocarditis-susceptible A/J mice. In contrast, significantly less ERK-1/2 activation is found in the hearts of myocarditis-resistant C57BL/6 mice. Therefore, the ERK-1/2 response to CVB3 infection may contribute to differential host susceptibility to viral myocarditis.

  View details for DOI 10.1172/JCI200213971

  View details for Web of Science ID 000176318600009

  View details for PubMedID 12070303

• Fetal pulmonary artery diameters and their association with lung hypoplasia and postnatal outcome in congenital diaphragmatic hernia AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY Sokol, J., Bohn, D., Lacro, R. V., Ryan, G., Stephens, D., RABINOVITCH, M., Smallhorn, J., Hornberger, L. K. 2002; 186 (5): 1085-1090

  Abstract

  We hypothesized that fetal branch pulmonary artery (PA) diameters indirectly reflect lung mass and are associated with postnatal outcome in cases of isolated congenital diaphragmatic hernia (CDH).We retrospectively reviewed echocardiograms of fetuses with CDH, measuring branch PA diameters and other echocardiographic parameters. Antenatal parameters were correlated with postmortem lung weights in 5 fetuses after pregnancy termination. Fetal echocardiographic measures were correlated with outcome variables in 29 live-born infants with CDH to identify antenatal indices associated with postnatal death and respiratory morbidity.Antenatal branch PA size correlated with postmortem lung weights from 5 terminated fetuses (r = 0.87). In 26 cases of left CDH in which the fetus continued to term, the ipsilateral branch PA diameter was significantly smaller than the contralateral branch PA diameter at presentation (P <.001). In these fetuses, a larger contralateral PA diameter was associated with worse postnatal survival (P =.049). Among survivors with left CDH, the main PA z score and the discrepancy between right and left PA diameters correlated positively with duration of supplemental oxygen requirement (P =.019 and P =.022, respectively) and ventilation (P =.036 and P =.012, respectively). Serial antenatal studies in 8 of 10 cases revealed progressive ipsilateral PA hypoplasia.Antenatal branch PA size correlates with postmortem lung weight. A larger contralateral PA, and significant branch PA discrepancy and larger main PA diameter, best correlate with postnatal death and respiratory morbidity, respectively. Progressive ipsilateral PA hypoplasia suggests progressive in utero lung hypoplasia in cases of CDH.

  View details for DOI 10.1067/mob.2002.122413

  View details for Web of Science ID 000175761000048

  View details for PubMedID 12015541

• Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension CIRCULATION Zaidi, S. H., You, X. M., Ciura, S., Husain, M., Rabinovitch, M. 2002; 105 (4): 516-521

  Abstract

  Increased serine elastase activity has been implicated in the vascular remodeling associated with chronic hypoxia-related pulmonary hypertension in rats.In this study we determined the time course of hypoxia-induced serine elastase activity in the murine lung and related this to initiation of a proteolytic cascade characterized by an increase in matrix metalloproteinases (MMPs). We then used transgenic mice in which overexpression of the selective serine elastase inhibitor elafin was targeted to the cardiovascular system to determine whether upregulation of a naturally occurring serine elastase inhibitor suppresses MMPs and the hemodynamic and structural response to chronic hypoxia (air at 380 mm Hg). In nontransgenic but not in elafin-transgenic mice, we documented a transient increase in serine elastase activity after 12 hours of hypoxic exposure attributed to a 30-kDa protein as determined by elastin zymography and fluorophosphonate/fluorophosphate-biotin labeling. Two days after hypoxia, the pro-forms of MMP-2 and MMP-9 were induced in the nontransgenic mice, but MMP-9 was suppressed in elafin-transgenic mice. Acute hypoxic vasoconstriction was similar in nontransgenic and elafin-transgenic littermates. Chronic hypoxia for 26 days resulted in >1-fold increase in right ventricular pressure (P<0.004) in nontransgenic compared with control or elafin-transgenic littermates. In the latter mice, normalization of the right ventricular pressure was associated with reduced muscularization and preservation of the number of distal vessels (P<0.04 for both comparisons).Modulation of the severity of chronic hypoxia-induced pulmonary vascular disease could be a function of endogenously expressed serine elastase inhibitors.

  View details for Web of Science ID 000173600500032

  View details for PubMedID 11815437

• Linking a serotonin transporter polymorphism to vascular smooth muscle proliferation in patients with primary pulmonary hypertension JOURNAL OF CLINICAL INVESTIGATION RABINOVITCH, M. 2001; 108 (8): 1109-1111

  View details for Web of Science ID 000171614500004

  View details for PubMedID 11602617

• Pathobiology of pulmonary hypertension - Extracellular matrix CLINICS IN CHEST MEDICINE Rabinovitch, M. 2001; 22 (3): 433-?

  Abstract

  Changes in the extracellular matrix underlie the structural and functional abnormalities in the vessel wall that lead to progressive pulmonary vascular disease. Studies are reviewed aimed at addressing the cellular and molecular programs that regulate the production of the extracellular matrix describing new ways to arrest proliferation and migration of smooth muscle cells and to induce apoptosis. The latter can lead to the reversal of pathology at least in experimental animal models.

  View details for Web of Science ID 000171324900006

  View details for PubMedID 11590839

• Arterial elastase activity after balloon angioplasty and effects of elafin, an elastase inhibitor ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Barolet, A. W., Nili, N., Cheema, A., Robinson, R., Natarajan, M. K., O'Blenes, S., Li, J., Eskandarian, M. R., Sparkes, J., RABINOVITCH, M., Strauss, B. H. 2001; 21 (8): 1269-1274

  Abstract

  Increased proteolytic activity may be a factor in intimal hyperplasia after balloon angioplasty (BA). The objectives of this study were to assess elastase activity after BA in a rabbit arterial double-injury model and the effects of elastase inhibition. Elastase activity increased immediately after BA, reached an 8-fold peak at 1 week, and declined to baseline levels by 4 weeks. Elastin zymography showed that the elastase activity was associated predominantly with a molecular mass of 25 kDa. Elastase activity was significantly inhibited in vitro by elafin and phenylmethylsulfonyl fluoride, selective inhibitors of serine elastases. A second group of animals was transfected after BA with a plasmid containing the cDNA for either elafin or a control (chloramphenicol acetyltransferase, CAT) construct by using a hemagglutinating virus of Japan-liposome transfection technique. Arterial segments were obtained at 48 hours, 1 week, and 4 weeks to assess transgene expression, arterial wall elastase activity, and intimal cross-sectional area, respectively. Elafin transgene expression was evident at 48 hours and resulted in a significant (80%) inhibition of elastase activity compared with chloramphenicol acetyltransferase-transfected arteries. There was a 43% reduction in intimal cross-sectional area in elafin-transfected arteries (0.28+/-0.22 versus 0.16+/-0.07 mm(2) for CAT-transfected versus elafin-transfected arteries, respectively; P<0.05). These data suggest that an early increase in serine elastase activity after BA contributes to intimal hyperplasia. Serine elastase inhibition may be a potential therapeutic approach to inhibit intimal hyperplasia.

  View details for Web of Science ID 000170506000007

  View details for PubMedID 11498452

• Tumor necrosis factor-alpha induces fibronectin synthesis in coronary artery smooth muscle cells by a nitric oxide-dependent posttranscriptional mechanism CIRCULATION RESEARCH O'Blenes, C. A., Kinnear, C., Rabinovitch, M. 2001; 89 (1): 26-32

  Abstract

  Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (P<0.05). This correlated with increased NO production (P<0.05) and fibronectin synthesis (P<0.05). TNF-alpha induction of fibronectin synthesis was abrogated by the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (P<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (P<0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P<0.05). Northern blotting showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. TNF-alpha increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.

  View details for Web of Science ID 000169899500006

  View details for PubMedID 11440974

• Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ju, H. S., Gros, R., You, X. M., Tsang, S., Husain, M., RABINOVITCH, M. 2001; 98 (13): 7469-7474

  Abstract

  We cloned a rat vascular chymase (RVCH) from smooth muscle cells (SMCs) that converts angiotensin I to II and is up-regulated in SMC from spontaneously hypertensive vs. normotensive rats. To determine whether increased activity of RVCH is sufficient to cause hypertension, transgenic mice were generated with targeted conditional expression of RVCH to SMC, with the use of the tetracycline-controlled transactivator (tTA). We confirmed conditional expression of RVCH by mRNA, protein, and chymase activity in the absence, but not in the presence, of dietary doxycycline. The systolic blood pressure (mmHg), measured by carotid artery cannulation at 10-12 weeks of age, was higher in tTA+/RVCH+ mice than in nonbinary transgenic littermates (136 +/- 4 vs. 109 +/- 3) (P < 0.05), as were the diastolic and mean pressures. Hypertension was completely reversed by doxycycline, suggesting a causal link with chymase expression. Medial thickening of mesenteric arteries from tTA+/RVCH+ mice vs. littermates (0.82 +/- 0.1 vs. 0.42 +/- 0.02) (P < 0.05) was associated with increased SMC proliferation, as judged by positive immunoreactivity, with the use of an antibody to the proliferating cell nuclear antigen. These structural changes were prevented by doxycycline. Perfusion myography of mesenteric arteries from tTA+/RVCH+ mice also revealed increased vasoconstriction in response to phenylephrine and impaired metacholine-induced vasodilatation when compared with littermate controls or with the doxycyline-treated group. Our studies suggest that up-regulation of this vascular chymase is sufficient to cause a hypertensive arteriopathy, and that RVCH may be a candidate gene and a therapeutic target in patients with high blood pressure.

  View details for Web of Science ID 000169456600086

  View details for PubMedID 11416217

• A novel vascular smooth muscle chymase is upregulated in hypertensive rats JOURNAL OF CLINICAL INVESTIGATION Guo, C. Y., Ju, H. S., Leung, D., Massaeli, H., Shi, M. D., RABINOVITCH, M. 2001; 107 (6): 703-715

  Abstract

  While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell-expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.

  View details for Web of Science ID 000167604400010

  View details for PubMedID 11254670

• Hemodynamic unloading leads to regression of pulmonary vascular disease in rats 80th Annual Meeting of the American-Association-for-Thoracic-Surgery O'Blenes, S. B., Fischer, S., McIntyre, B., Keshavjee, S., RABINOVITCH, M. MOSBY-ELSEVIER. 2001: 279–89

  Abstract

  Treatment options for patients with advanced pulmonary vascular disease caused by a congenital heart defect are still mainly limited to heart-lung transplantation or lung transplantation with repair of the cardiac lesion. Because we have previously shown that the structural changes associated with pulmonary hypertension can be reversed by stress unloading in an organ culture model, we now investigate whether hemodynamic unloading will lead to regression of pulmonary vascular disease in the intact animal.Right middle and lower lobectomy and monocrotaline injection were performed in Lewis rats (n = 22) to cause pulmonary vascular disease from a combined hemodynamic and toxic injury. Twenty-eight days later the left lungs were examined (n = 10) or exposed to normal pulmonary artery pressure for an additional 14 (n = 5) or 28 (n = 7) days by transplantation into healthy recipients. Pulmonary artery pressure, ventricular weight, and pulmonary artery morphology were evaluated in each group.Pulmonary hypertension (50 vs 16 mm Hg; P <.001) and right ventricular hypertrophy (right ventricular/left ventricular weight 0.69 vs 0.32; P <.001) associated with pulmonary artery medial hypertrophy (28.2% vs 7.2% wall thickness; P <.001) and muscularization of small pulmonary arteries (92.3% vs 19.4%; P <.001) developed by day 28 (compared with untreated controls). However, transplantation into healthy recipients effectively unloaded the lungs (mean pulmonary artery pressure 17 and 24 mm Hg at 14 and 28 days after transplantation) and resulted in progressive normalization of medial hypertrophy (15.6% and 12.1% at 14 and 28 days) and muscularization (65.1% and 42.2% at 14 and 28 days) relative to nontransplanted controls (P <.005 in each case).Hemodynamic unloading of lungs with pulmonary vascular disease results in progressive normalization of pulmonary artery structure. These results are the first to provide a rationale for attempting to induce regression of pulmonary vascular disease by pressure unloading of the pulmonary circulation. Methods to mechanically unload the pulmonary circulation should be critically evaluated as a strategy for staged surgical repair of congenital heart defects despite presumed irreversible pulmonary hypertension.

  View details for Web of Science ID 000167014300013

  View details for PubMedID 11174733

• Gene transfer of the serine elastase inhibitor elafin protects against vein graft degeneration CIRCULATION O'Blenes, S. B., Zaidi, S. H., Cheah, A. Y., McIntyre, B., Kaneda, Y., Rabinovitch, M. 2000; 102 (19): 289-295

  View details for Web of Science ID 000165177300051

• Circulation research Editors' yearly report: 1999-2000 Circulation research Marban, E., Bolli, R., Breitwieser, G., Busse, R., Dietz, H., Endoh, M., Finkel, T., Kass, D., Lowenstein, C., Rabinovitch, M., Tomaselli, G. 2000; 87 (4): 261-3

  View details for PubMedID 10948056

• Pathobiology of pulmonary hypertension: Impact on clinical management. Seminars in thoracic and cardiovascular surgery. Pediatric cardiac surgery annual Rabinovitch, M. 2000; 3: 63-81

  Abstract

  Our previous studies showed how analysis of pulmonary vascular changes on lung biopsy tissue and on angiography added to the hemodynamic assessment of pulmonary vascular resistance in predicting the success of a surgical repair. Both the potential for heightened vasoreactivity in the early postoperative period and for reversibility of pulmonary vascular disease at later follow-up were correlated with qualitative and quantitative evaluation of arterial changes. The ability of continuous intravenous prostacylin to arrest progression and even induce regression of structurally advanced pulmonary vascular disease in some cases has led to rethinking how pathological material can be useful in clinical decision making. The presence of occlusive changes and particularly plexiform lesions was thought to represent irreversible disease, but the observation that ongoing cellular proliferation and connective tissue synthesis occurs even in advanced lesions thought to represent end stage 'burnt-out' lesions, led to re-evaluation of the potential of biologically reversing the disease process. Our laboratory has used clinical material, cultured cells, and studies in experimental animals to gain new insights into some of the mechanisms which lead to the progression of vascular changes, and has used this information in strategies aimed at arresting progression and, more recently, inducing regression of pulmonary hypertension and associated vascular lesions. Specifically, we have focused on the increased activity of an endogenous vascular elastase (EVE) and expression of the glycoproteins tenascin and fibronectin in the pathobiology of pulmonary hypertension. This report will first review our studies in children with congenital heart defects, assessment of reversibility of pulmonary hypertension, and then discuss more recent work addressing cellular and molecular mechanisms aimed at developing newer therapeutic strategies. Copyright 2000 by W.B. Saunders Company

  View details for PubMedID 11486187

• Isolated ductus arteriosus aneurysm in the fetus and infant: A multi-institutional experience JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Dyamenahalli, U., Smallhorn, J. F., Geva, T., Fouron, J. C., Cairns, P., Jutras, L., Hughes, V., RABINOVITCH, M., Mason, C. A., Hornberger, L. K. 2000; 36 (1): 262-269

  Abstract

  The purpose of this study was to describe the clinical characteristics and outcome and to elucidate the pathogenesis of ductus arteriosus aneurysm (DAA).Ductus arteriosus aneurysm is a rare lesion that can be associated with severe complications including thromboembolism, rupture and death.We reviewed the clinical records, diagnostic imaging studies and available histology of 24 cases of DAA, diagnosed postnatally (PD) in 15 and antenatally (AD) in 9 encountered in five institutions.Of PD cases, 13 presented at <2 months, and all AD cases were detected incidentally after 33 weeks of gestation during a late trimester fetal ultrasound study. Of the 24, only 4 had DAA-related symptoms and 6 had associated syndromes: Marfan, Smith-Lemli-Opitz, trisomies 21 and 13 and one possible Ehlers-Danlos. Three had complications related to the DAA: thrombus extension into the pulmonary artery, spontaneous rupture, and asymptomatic cerebral infarction. Six underwent uncomplicated DAA resection for ductal patency, DAA size or extension of thrombus. In the four examined, there was histologic evidence of reduced intimal cushions in two and abnormal elastin expression in two. Five of the 24 died, with only one death due to DAA. Of 19 survivors, all but one remain clinically asymptomatic at a median follow-up of 35 months; however, two have developed other cardiac lesions that suggest Marfan syndrome. A review of 200 consecutive third trimester fetal ultrasounds suggests an incidence of DAA of 1.5%.Ductus arteriosus aneurysm likely develops in the third trimester perhaps due to abnormal intimal cushion formation or elastin expression. Although it can be associated with syndromes and severe complications, many affected infants have a benign course. Given the potential for development of other cardiac lesions associated with connective tissue disease, follow-up is warranted.

  View details for Web of Science ID 000088105100040

  View details for PubMedID 10898444

• Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor NATURE MEDICINE Cowan, K. N., Heilbut, A., Humpl, T., Lam, C., Ito, S., Rabinovitch, M. 2000; 6 (6): 698-702

  Abstract

  Progression of pulmonary hypertension is associated with increased serine elastase activity and the proteinase-dependent deposition of the extracellular matrix smooth muscle cell survival factor tenascin-C (refs. 1,2). Tenascin-C amplifies the response of smooth muscle cells to growth factors, which are also liberated through matrix proteolysis. Recent organ culture studies using hypertrophied rat pulmonary arteries have shown that elastase inhibitors suppress tenascin-C and induce smooth muscle cell apoptosis. This initiates complete regression of the hypertrophied vessel wall by a coordinated loss of cellularity and extracellular matrix. We now report that elastase inhibitors can reverse advanced pulmonary vascular disease produced in rats by injecting monocrotaline, an endothelial toxin. We began oral administration of the peptidyl trifluoromethylketone serine elastase inhibitors M249314 or ZD0892 21 days after injection of monocrotaline. A 1-week treatment resulted in 92% survival, compared with 39% survival in untreated or vehicle-treated rats. Pulmonary artery pressure and muscularization were reduced by myocyte apoptosis and loss of extracellular matrix, specifically elastin and tenascin-C. After 2 weeks, pulmonary artery pressure and structure normalized, and survival was 86%, compared with 0% in untreated or vehicle-treated rats. Although concomitant treatment with various agents can reduce pulmonary hypertension, we have documented complete regression after establishment of malignant monocrotaline-induced disease.

  View details for Web of Science ID 000087438300043

  View details for PubMedID 10835689

• Suppressed smooth muscle proliferation and inflammatory cell invasion after arterial injury in elafin-overexpressing mice JOURNAL OF CLINICAL INVESTIGATION Zaidi, S. H., You, X. M., Ciura, S., O'Blenes, S., Husain, M., Rabinovitch, M. 2000; 105 (12): 1687-1695

  Abstract

  Elastases degrade the extracellular matrix, releasing growth factors and chemotactic peptides, inducing glycoproteins such as tenascin, and thereby promoting vascular cell proliferation and migration. Administration of serine elastase inhibitors reduces experimentally induced vascular disease. The ability to mount an intrinsic anti-elastase response may, therefore, protect against intimal/medial thickening after vascular injury. To investigate this, we showed that wire-induced endothelial denudation of the carotid artery is associated with transient elevation in elastase activity and confirmed that this is abolished in transgenic mice overexpressing the serine elastase inhibitor, elafin, targeted to the cardiovascular system. Ten days after injury, nontransgenic littermates show vessel enlargement, intimal thickening, increased medial area and cellularity, and 2-fold increase in tenascin. Injured vessels in transgenic mice become enlarged but are otherwise similar to sham-operated controls. Injury-induced vessel wall thickening, which is observed only in nontransgenic mice, is related to foci of neutrophils and macrophages, in addition to smooth muscle cells that fail to stain for alpha-actin and are likely dedifferentiated. Our study therefore suggests that a major determinant of the vascular response to injury is the early transient induction of serine elastase activity, which leads to cellular proliferation and inflammatory cell migration.

  View details for Web of Science ID 000087757400006

  View details for PubMedID 10862784

• Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMP-mediated suppression of ERK phosphorylation and AML1B nuclear partitioning FASEB JOURNAL Mitani, Y., Zaidi, S. H., Dufourcq, P., Thompson, K., Rabinovitch, H. 2000; 14 (5): 805-814

  Abstract

  Nitric oxide (NO) reduces the severity of pulmonary vascular disease in rats as do elastase inhibitors. We therefore hypothesized that NO inhibits elastase by suppressing mitogen-activated protein kinases that trans-activate AML1B, a transcription factor for elastase. We used cultured pulmonary artery smooth muscle cells in which serum-treated elastin (STE) induces a > threefold increase in elastase activity as evaluated by solubilization of [(3)H]-elastin. NO donors (SNAP and DETA NONOate) inhibited elastase in a dose-dependent manner as did a cGMP mimetic (8-pCPT-cGMP). SNAP inhibition of elastase was reversed by coadministration of a cGMP-PKG inhibitor (Rp-8-pCPT-cGMP). The STE-induced increase in phospho-ERK was suppressed by NO donors and the cGMP mimetic, and reversed by cGMP-PKG inhibitor, as was expression of AML1B and DNA binding in nuclear extracts. A concomitant increase in p38 phosphorylation was also inhibited by SNAP, but whereas MEK inhibitor (PD98059) suppressed elastase and AML1B-DNA binding, a p38 inhibitor (SB202190) did not. Our study uniquely links NO with inhibition of elastase-dependent matrix remodeling in vascular disease by suggesting a cGMP-PKG-related mechanism suppressing ERK-mediated partitioning of AML1B in nuclear extracts.

  View details for Web of Science ID 000086292700018

  View details for PubMedID 10744637

• Under new management - A six-month progress report on Circulation Research CIRCULATION RESEARCH Marban, E., Bolli, R., Breitwieser, G., Busse, R., Dietz, H., Endoh, M., Finkel, T., Kass, D., Lowenstein, C., RABINOVITCH, M., Tomaselli, G. 2000; 86 (2): 111-113

  View details for Web of Science ID 000085342000001

  View details for PubMedID 10666401

• Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease JOURNAL OF CLINICAL INVESTIGATION Cowan, K. N., Jones, P. L., Rabinovitch, M. 2000; 105 (1): 21-34

  Abstract

  Increased expression of the glycoprotein tenascin-C (TN) is associated with progression of clinical and experimental pulmonary hypertension. In cultured smooth muscle cells (SMCs) TN is induced by matrix metalloproteinases (MMPs) and amplifies the proliferative response to growth factors. Conversely, suppression of TN leads to SMC apoptosis. We now report that hypertrophied rat pulmonary arteries in organ culture, which progressively thicken in association with cell proliferation and matrix accumulation, can be made to regress by inhibiting either serine elastases or MMPs. This effect is associated with reduced TN, suppression of SMC proliferation, and induction of apoptosis. Selective repression of TN by transfecting pulmonary arteries with antisense/ribozyme constructs also induces SMC apoptosis and arrests progressive vascular thickening but fails to induce regression. This failure is related to concomitant expansion of a SMC population, which produces an alternative cell survival alpha(v)beta(3) ligand, osteopontin (OPN), in response to pro-proliferative cues provided by a proteolytic environment. OPN rescues MMP inhibitor-induced SMC apoptosis, and alpha(v)beta(3) blockade induces apoptosis in hypertrophied arteries. Our data suggest that proteinase inhibition is a novel strategy to induce regression of vascular disease because this overcomes the pluripotentiality of SMC-matrix survival interactions and induces coordinated apoptosis and resorption of matrix.

  View details for Web of Science ID 000084579300005

  View details for PubMedID 10619858

• Adventitial fibroblasts - Defining a role in vessel wall remodeling AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Strauss, B. H., RABINOVITCH, M. 2000; 22 (1): 1-3

  View details for Web of Science ID 000084783700001

  View details for PubMedID 10615057

• Pulmonary hypertension: Pathophysiology as a basis for clinical decision making JOURNAL OF HEART AND LUNG TRANSPLANTATION Rabinovitch, M. 1999; 18 (11): 1041-1053

  View details for Web of Science ID 000084008900002

  View details for PubMedID 10598727

• Functional and antigenic concentrations of alpha-1-proteinase inhibitor after administration for the prevention of chronic lung disease of prematurity 1996 Annual Meeting of the Pediatric-Academic-Societies Stiskal, J. A., Ito, S., Cox, D. W., Shennan, A. T., O'Brien, K. K., Kelly, E. N., Longley, T. B., RABINOVITCH, M., Dunn, M. S. KARGER. 1999: 134–43

  Abstract

  OBJECTIVE andAlpha-1-proteinase inhibitor (A1PI) supplementation has been used in adults with inherited alpha-1-antitrypsin (A1AT) deficiency to impede the development of emphysema. A1PI supplementation may also be useful for protecting premature neonates who receive mechanical ventilation from the development of chronic lung disease (CLD). However, the pharmacokinetics of exogenous A1PI in this population are unknown. We attempted to determine the disposition of A1PI in premature infants with birth weight 600-1,250 g who received 60 mg/kg on days 0, 4, 7 and 14 in a randomized, placebo-controlled, double-blind trial. Functional and antigenic plasma concentrations of A1PI were measured at specified time points.On both functional and antigenic assays, concentrations began in the normal adult range and rose from day 0 to 10 then fell slightly, but remained above initial values. The concentrations were not significantly different between the treatment and placebo groups.The results of this study indicate that neonatal pharmacokinetics of A1PI differ markedly from those of the adult. Total plasma clearance of exogenous A1PI seems high in the ventilated premature neonate. Higher or more frequent doses may be necessary to maintain A1PI plasma concentrations above baseline.

  View details for Web of Science ID 000082208400002

  View details for PubMedID 10460951

• Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation FASEB JOURNAL Mason, C. A., Chang, P., Fallery, C., RABINOVITCH, M. 1999; 13 (11): 1423-1434

  Abstract

  Ductus arteriosus intimal cushion formation is characterized by fibronectin-dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthesis in ductus SMC is regulated by the interaction of LC-3, a microtubule-associated protein, with an AU-rich element (ARE) in the 3'-untranslated region of fibronectin mRNA, facilitating its recruitment to polyribosomes for translation. Since nitric oxide (NO) is implicated in posttranscriptional gene regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis. NO production was sevenfold higher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronectin synthesis by approximately 45-50% (P<0.05), whereas the NO donor, SNAP, increased ductus fibronectin synthesis approximately onefold (P<0.05); neither agent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP increased and L-NMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to the fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production regulate the posttranscriptional increase in fibronectin synthesis required for SMC motility.

  View details for Web of Science ID 000082019100014

  View details for PubMedID 10428766

• EVE and beyond, retro and prospective insights Experimental Biology 96 Annual Meeting Rabinovitch, M. AMER PHYSIOLOGICAL SOC. 1999: L5–L12

  Abstract

  Our work has focused on the discovery that an endogenous vascular elastase (EVE) plays a pivotal role in the vascular changes associated with the development and progression of pulmonary hypertension. Recent studies have identified serum factors that stimulate transcription of this enzyme and have elucidated a signal transduction process involving activation of the mitogen-activated protein kinase pathway and nuclear expression of the transcription factor AML1. Proteases release and activate growth factors that are bound to the extracellular matrix and also induce, in a beta(3)-integrin-dependent manner, the transcription of the gene for the matrix glycoprotein tenascin. Tenascin alters smooth muscle cell shape and facilitates the proliferative response to growth factors by clustering and activating growth factor receptors. In addition, breakdown products of elastin, elastin peptides, can upregulate the production of fibronectin, a glycoprotein that is critical to smooth muscle cell migration. The mechanisms regulating enhanced fibronectin production have recently been successfully targeted to prevent the development of intimal lesions.

  View details for Web of Science ID 000081261000002

  View details for PubMedID 10409225

• Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages INFECTION AND IMMUNITY Gomes, M. S., Paul, S., Moreira, A. L., APPELBERG, R., RABINOVITCH, M., Kaplan, G. 1999; 67 (7): 3199-3206

  Abstract

  Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome.

  View details for Web of Science ID 000081063800004

  View details for PubMedID 10377091

• Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis CIRCULATION RESEARCH Cowan, K. N., Jones, P. L., Rabinovitch, M. 1999; 84 (10): 1223-1233

  Abstract

  Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.

  View details for Web of Science ID 000080575900012

  View details for PubMedID 10347097

• Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis JOURNAL OF CLINICAL INVESTIGATION Zaidi, S. H., Hui, C. C., Cheah, A. Y., You, X. M., Husain, M., Rabinovitch, M. 1999; 103 (8): 1211-1219

  Abstract

  Serine elastases degrade elastin, stimulate vascular smooth muscle cell migration and proliferation, and are associated with myocardial damage. To evaluate the impact of elastase inhibition on cardiovascular development and disease, transgenic mice were created in which the mouse preproendothelin-1 promoter was used to target elafin overexpression to the cardiovascular system. To distinguish the transgene from endogenous elafin, constructs were made incorporating a FLAG sequence; the COOH-terminus FLAG-tagged elafin construct produced a stable, functionally active gene product and was used to create transgenic mice. Consistent with endothelin expression, abundant elafin mRNA was observed in transgenic F1 embryos (embryonic day 13.5) and in adult transgenic mice heart, trachea, aorta, kidney, lung, and skin, but not in liver, spleen, and intestine. Functional activity of the transgene was confirmed by heightened myocardial elastase inhibitory activity. No tissue abnormalities were detected by light microscopy or elastin content. However, injection of 10 plaque-forming units (PFU) of encephalomyocarditis virus resulted in death within 11 days in 10 out of 12 nontransgenic mice compared with one out of nine transgenic littermates. This reduced mortality was associated with better cardiac function and less myocardial inflammatory damage. Thus, elafin expression may confer a protective advantage in myocarditis and other inflammatory diseases.

  View details for Web of Science ID 000079890400013

  View details for PubMedID 10207173

• Induction of vascular smooth muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a beta 3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element JOURNAL OF CELL SCIENCE Jones, P. L., Jones, F. S., Zhou, B., Rabinovitch, M. 1999; 112 (4): 435-445

  Abstract

  Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. In previous studies we showed that the presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Moreover, we demonstrated that denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. In the present study, we examine the pathway by which beta3 integrins stimulate expression of tenascin-C, and define a promoter sequence that is critical for its induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, we transfected A10 cells with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct and showed that, relative to native collagen, its activity is increased on denatured collagen. Next, to identify regions of the promoter involved, we examined a series of tenascin-C promoter constructs with 5' deletions and showed that denatured collagen-dependent promoter activity was retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element was suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. Our data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression.

  View details for Web of Science ID 000079021800002

  View details for PubMedID 9914156

• Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation NATURE MEDICINE Mason, C. A., Bigras, J. L., O'Blenes, S. B., Zhou, B., McIntyre, B., Nakamura, N., Kaneda, Y., RABINOVITCH, M. 1999; 5 (2): 176-182

  Abstract

  Closure of the ductus arteriosus requires prenatal formation of intimal cushions, which occlude the vessel lumen at birth. Survival of newborns with severe congenital heart defects, however, depends on ductal patency. We used a gene transfer approach to create a patent ductus arteriosus by targeting the fibronectin-dependent smooth muscle cell migration required for intimal cushion formation. Fetal lamb ductus arteriosus was transfected in utero with hemagglutinating virus of Japan liposomes containing plasmid encoding 'decoy' RNA to sequester the fibronectin mRNA binding protein. Fibronectin translation was inhibited and intimal cushion formation was prevented. We thus established the essential role of fibronectin-dependent smooth muscle cell migration in intimal cushion formation in the intact animal and the feasibility of incorporating biological engineering in the management of congenital heart disease.

  View details for Web of Science ID 000078274400026

  View details for PubMedID 9930865

• A serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally-induced murine myocarditis NATURE MEDICINE Lee, J. K., Zaidi, S. H., Liu, P., Dawood, F., Cheah, A. Y., Wen, W. H., Saiki, Y., RABINOVITCH, M. 1998; 4 (12): 1383-1391

  Abstract

  In viral myocarditis, inflammation and destruction of cardiac myocytes leads to fibrosis, causing progressive impairment in cardiac function. Here we show the etiologic importance of serine elastase activity in the pathophysiology of acute viral myocarditis and the therapeutic efficacy of an elastase inhibitor. In DBA/2 mice inoculated with the encephalomyocarditis virus, a more than 150% increase in myocardial serine elastase activity is observed. This is suppressed by a selective serine elastase inhibitor, ZD0892, which is biologically effective after oral administration. Mice treated with this compound had little evidence of microvascular constriction and obstruction associated with myocarditis-induced ischemia reperfusion injury, much less inflammation and necrosis, only mild fibrosis and myocardial collagen deposition, and normal ventricular function, compared with the infected nontreated group.

  View details for Web of Science ID 000077336900033

  View details for PubMedID 9846575

• Tissue-specific and developmental regulation of transforming growth factor-beta(1) expression in fetal lamb ductus arteriosus endothelial cells PEDIATRIC RESEARCH Zhou, B., Coulber, C., RABINOVITCH, M. 1998; 44 (6): 865-872

  Abstract

  We previously established that increased hyaluronan synthesis in ductus arteriosus (DA) compared with aorta (Ao) endothelial cells (EC) from early gestation fetal lambs (100-d, term = 145 d) is transforming growth factor-beta (TGF-beta)-dependent. We now address whether this is associated with tissue-specific and developmentally up-regulated expression of TGF-beta1 in the 100-d DA EC. Immunoprecipitation revealed TGF-beta synthesis doubled in DA versus Ao EC from 100-d gestation lambs (p < 0.05). In 138-d DA EC, TGF-beta protein levels were reduced (p < 0.05) and comparable to those in Ao cells. Western immunoblotting with a beta1 isoform-specific antibody confirmed these differences as being related to TGF-beta1. Northern blot analysis demonstrated that TGF-beta1 mRNA levels were slightly but not significantly increased in 100-d DA compared with Ao EC, despite its short half-life in DA (9.5 h) versus Ao EC (20 h). TGF-beta1 mRNA levels were reduced in 138-d DA and Ao EC (p < 0.05), and the mRNA half-life was comparable in DA (9 h) versus Ao (13 h). Nuclear run-on analysis confirmed increased TGF-beta1 mRNA transcription in 100-d DA versus Ao and 138-d DA EC. Thus, up-regulated TGF-beta1 expression in 100-d DA compared with Ao cells is due to increased transcription and translation of a relatively unstable mRNA, and its down-regulation in 138-d DA and Ao EC is related to reduced mRNA transcription and stability, respectively.

  View details for Web of Science ID 000077237800007

  View details for PubMedID 9853919

• Microtubule involvement in translational regulation of fibronectin expression by light chain 3 of microtubule-associated protein 1 in vascular smooth muscle cells CIRCULATION RESEARCH Zhou, B., RABINOVITCH, M. 1998; 83 (5): 481-489

  Abstract

  Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

  View details for Web of Science ID 000075690900002

  View details for PubMedID 9734470

• Elastase and the pathobiology of unexplained pulmonary hypertension Brenot Memorial Symposium on the Pathogenesis of Primary Pulmonary Hypertension Rabinovitch, M. AMER COLL CHEST PHYSICIANS. 1998: 213S–224S

  Abstract

  Our laboratory has focused on the increased activity of an endogenous vascular elastase in the pathobiology of pulmonary hypertension and on the mechanisms by which it is upregulated and by which it orchestrates abnormal remodeling of the vessel wall, specifically the induction of growth factors, the induction of the glycoprotein tenascin, which amplifies the proliferative response, and fibronectin, which is critical to the process of smooth muscle migration in the context of neointimal formation. We explore strategies by which targetting these processes might arrest progression or induce regression of pulmonary vascular disease associated with unexplained pulmonary hypertension.

  View details for Web of Science ID 000075942200007

  View details for PubMedID 9741572

• AML1-like transcription factor induces serine elastase activity in ovine pulmonary artery smooth muscle cells 36th Annual Meeting of the American-Society-for-Cell-Biology Wigle, D. A., Thompson, K. E., Yablonsky, S., Zaidi, S. H., Coulber, C., Jones, P. L., RABINOVITCH, M. LIPPINCOTT WILLIAMS & WILKINS. 1998: 252–63

  Abstract

  In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of approximately 2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells.

  View details for Web of Science ID 000075287300003

  View details for PubMedID 9710117

• Mechanisms of proliferative and obliterative vascular diseases - Insights from the pulmonary and systemic circulations AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Fishman, A. P., Fishman, M. C., Freeman, B. A., Gimbrone, M. A., RABINOVITCH, M., Robinson, D., Gail, D. B. 1998; 158 (2): 670-674

  View details for Web of Science ID 000075387700045

  View details for PubMedID 9700149

• Endothelial and serum factors which include apolipoprotein A1 tether elastin to smooth muscle cells inducing serine elastase activity via tyrosine kinase-mediated transcription and translation JOURNAL OF CELLULAR PHYSIOLOGY Thompson, K., Kobayashi, J., Childs, T., Wigle, D., RABINOVITCH, M. 1998; 174 (1): 78-89

  Abstract

  We previously reported that serine elastase activity is induced in cultured porcine pulmonary artery (PA) smooth muscle cells (SMC) following serum stimulation by a mechanism involving adhesion of elastin to an elastin binding protein and tyrosine kinase activity. The present study demonstrates that a PA endothelial cell factor also promotes a fourfold increase in elastin adhesion to PA SMC and a twofold increase in serine elastase activity. The mechanism involves tethering of the factor to SMC, since [3H]-elastin pre-incubated with serum or endothelial cell (EC)-conditioned medium or SMC pre-treated with serum accelerates binding of elastin and tyrosine-kinase related elastase activity. The serum factor appears to interact with integrins as elastase induction is partially inhibited by RGD peptides. The elastase-inducing properties of serum could not, however, be attributed to several RGD-containing proteins. While a 120 kD fibronectin fragment partially reproduced the effect, it was not found in the serum fraction containing elastase-inducing activity. Instead, a 27 kD serum protein was enriched by elastin affinity chromatography, identified as apolipoprotein (Apo) A1 by microsequence analysis, and found to have about 50% of the elastase-inducing activity of serum. Elastase induction is inhibited by actinomycin and cycloheximide, suggesting a requirement for mRNA transcription and protein synthesis. Our results suggest a novel cell-extracellular matrix interaction whereby a soluble factor, in this case a lipoprotein, binds and tethers a matrix component to the cell surface and induces tyrosine kinase-dependent transcription of mRNA culminating in substrate proteolysis.

  View details for Web of Science ID A1998YH65500009

  View details for PubMedID 9397158

• Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells JOURNAL OF CLINICAL INVESTIGATION Zhou, B., Boudreau, N., Coulber, C., Hammarback, J., RABINOVITCH, M. 1997; 100 (12): 3070-3082

  Abstract

  Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

  View details for Web of Science ID 000071149100020

  View details for PubMedID 9399954

• Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha(v)beta(3) integrin to promote epidermal growth factor receptor phosphorylation and growth JOURNAL OF CELL BIOLOGY Jones, P. L., Crack, J., RABINOVITCH, M. 1997; 139 (1): 279-293

  Abstract

  Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

  View details for Web of Science ID A1997YB44600023

  View details for PubMedID 9314546

• New insights and therapeutic strategies for postcardiac transplantation coronary artery disease VI Alexis Carrel Conference on Chronic Rejection and Graft Atherosclerosis RABINOVITCH, M. ELSEVIER SCIENCE INC. 1997: 2585–86

  View details for Web of Science ID A1997XV03200029

  View details for PubMedID 9290752

• Pulmonary hypertension: Updating a mysterious disease CARDIOVASCULAR RESEARCH RABINOVITCH, M. 1997; 34 (2): 268-272

  View details for Web of Science ID A1997XF20000003

  View details for PubMedID 9205539

• Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease AMERICAN JOURNAL OF PATHOLOGY Jones, P. L., Cowan, K. N., RABINOVITCH, M. 1997; 150 (4): 1349-1360

  Abstract

  Progressive pulmonary hypertension is characterized by smooth muscle cell proliferation and migration leading to occlusive arterial lesions. Previously, using cultured smooth muscle cells, we demonstrated that epidermal growth factor (EGF)-dependent proliferation and migration are dependent on tenascin-C (Tn) and cellular fibronectin (Fn), respectively. In this study we applied immunohistochemistry to lung biopsy tissue from patients with congenital heart defects and pulmonary hypertension to determine how the distribution and intensity of Tn, EGF, proliferating cell nuclear antigen (PCNA), and Fn expression related to arterial abnormalities. With mildly increased wall thickness, minimal Tn, PCNA, and EGF was evident. With progressive hypertrophy, moderately intense foci of Tn were apparent in the adventitia, periendothelium, and occasionally the media but not consistently co-distributing with EGF and PCNA. With obstructive lesions, intense neointimal Tn expression co-localized with EGF and PCNA. Fn accumulation in the periendothelium increased with medial hypertrophy and became more widespread in a diffuse pattern with neointimal formation. The neointima was predominantly composed of alpha-smooth-muscle-actin-positive cells, occasional inflammatory cells with no evidence of apoptosis. These studies are consistent with Tn modulating EGF-dependent neointimal smooth muscle cell proliferation and Fn providing a gradient for smooth muscle cell migration from media to neointima.

  View details for Web of Science ID A1997WR01200022

  View details for PubMedID 9094991

• The committed vascular smooth muscle cell: A question of ''timing'' or ''response to pressure'' or both AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY RABINOVITCH, M. 1997; 16 (4): 364-365

  View details for Web of Science ID A1997WR89200002

  View details for PubMedID 9115745

• Carbon monoxide formation in the ductus arteriosus in the lamb: Implications for the regulation of muscle tone BRITISH JOURNAL OF PHARMACOLOGY Coceani, F., Kelsey, L., Seidlitz, E., Marks, G. S., McLaughlin, B. E., Vreman, H. J., Stevenson, D. K., RABINOVITCH, M., Ackerley, C. 1997; 120 (4): 599-608

  Abstract

  1. We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2. The CO-forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3. CO was formed enzymatically from added substrate (hemin, 50 microM) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 microM). 4. ZnPP (10 microM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin-treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5. ZnPP (10 microM) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 microM)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6. We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

  View details for Web of Science ID A1997WJ00500011

  View details for PubMedID 9051297

  View details for PubMedCentralID PMC1564507

• Histological and immunohistochemical characteristics of eccentric coronary artery lesions retrieved by atherectomy from cardiac transplant recipients CARDIOVASCULAR PATHOLOGY Clausell, N., Daly, P. A., Molossi, S., Lima, V. C., Adelman, A. G., Gotlieb, A. I., RABINOVITCH, M. 1997; 6 (1): 23-29

  View details for Web of Science ID A1997WD88100003

• Cell-extracellular matrix interactions in the ductus arteriosus and perinatal pulmonary circulation SEMINARS IN PERINATOLOGY RABINOVITCH, M. 1996; 20 (6): 531-541

  Abstract

  Our studies and those of others have shown that changes in the extracellular matrix have profound effects on vascular remodeling. In the ductus, increased production of endothelial hyaluronan and smooth muscle cell chondroitin sulfate and fibronectin and impaired elastin fiber assembly are features critical to smooth muscle cell migration into the subendothelium and intimal cushion formation. There is a developmentally orchestrated process that involves post-transcriptional mechanisms of gene regulation. Closure of the ductus arteriosus is associated with further changes in matrix expression and programmed cell death. The changes in the extracellular matrix that induce neointimal formation are also observed in pathological conditions in pulmonary and coronary arteries. Plasticity of the pulmonary circulation in the perinatal period also involves matrix regulation, and processes that prevent the normal decrease in pulmonary vascular resistance will result in impaired matrix regulation, and in the development of structural changes in the pulmonary arteries, including abnormal smooth muscle cell differentiation, hypertrophy and proliferation, which sustain the elevation in pulmonary artery pressure.

  View details for Web of Science ID A1996WQ38300007

  View details for PubMedID 9090779

• Tenascin-C is induced with progressive pulmonary vascular disease in rats and is functionally related to increased smooth muscle cell proliferation CIRCULATION RESEARCH Jones, P. L., RABINOVITCH, M. 1996; 79 (6): 1131-1142

  Abstract

  Tenascin-C, an extracellular matrix glycoprotein prominent during tissue remodeling, has been linked to cell migration, proliferation, and apoptosis. To determine its potential role in the pathobiology of pulmonary hypertension, we compared tenascin expression in adult and infant rat pulmonary arteries (PAs) after injection of the toxin monocrotaline. Immunohistochemistry, in situ hybridization, and Northern blot analysis demonstrated induction of tenascin in adult rat central and peripheral PA. Tenascin was not, however, detected in infant vessels, which show spontaneous regression of vascular lesions. To determine a function for tenascin, we correlated its expression with evidence of apoptosis and cell proliferation using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and 5-bromo-2'-deoxyuridine labeling, respectively. Apoptosis was observed only in the adult rat PA endothelial cell layer, preceding the induction of tenascin, which colocalized both temporally and spatially with proliferating smooth muscle cells (SMCs). A cause-and-effect relationship was documented in cultured rat PA SMCs, where tenascin promoted growth in response to basic fibroblast growth factor and was a prerequisite for epidermal growth factor-induced proliferation. These data provide novel functional information suggesting that endothelial cell apoptosis precedes progressive pulmonary hypertension and that induction of tenascin may be critical to growth factor-dependent SMC proliferation.

  View details for Web of Science ID A1996VV61200008

  View details for PubMedID 8943951

• Gerbode's defect associated with acute sinus node dysfunction as a complication of infective endocarditis HEART Michel, C., Rabinovitch, M. A., Huynh, T. 1996; 76 (4): 379-379

  View details for Web of Science ID A1996VL97100023

  View details for PubMedID 8983692

• It all begins with eve (endogenous vascular elastase) 9th Annual Meeting of the Mediterranean-Association-of-Cardiology-and-Cardiac-Surgery RABINOVITCH, M. ISRAEL JOURNAL MED SCIENCES. 1996: 803–8

  View details for Web of Science ID A1996VU63200005

  View details for PubMedID 8950241

• Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: Cinemicrographic and ultrastructural observations BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH Veras, P. S., Topilko, A., Gouhier, N., Moreau, M. F., RABINOVITCH, M., Pouchelet, M. 1996; 29 (8): 1009-1018

  Abstract

  Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

  View details for Web of Science ID A1996VD70600012

  View details for PubMedID 9181083

• Elafin, a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits after heterotopic cardiac transplantation JOURNAL OF CLINICAL INVESTIGATION Cowan, B., Baron, O., Crack, J., Coulber, C., Wilson, G. J., RABINOVITCH, M. 1996; 97 (11): 2452-2468

  Abstract

  We have related experimentally induced post-cardiac transplant coronary arteriopathy to increased elastolytic activity, IL-1beta, fibronectin-mediated inflammatory and smooth muscle cell (SMC) migration, and SMC proliferation. Since our in vitro studies show that a serine elastase releases SMC mitogens and facilitates IL-lbeta induction of fibronectin, we hypothesized that administration in vivo of the specific serine elastase inhibitor, elafin, would decrease the post-cardiac transplant coronary arteriopathy. Cholesterol-fed rabbits underwent a heterotopic cardiac transplant without immunosuppression and received elafin (1.79 mg/kg per d continuous infusion after a 9 mg bolus, n = 6) or vehicle (n = 6). 1 wk later, hearts were harvested for morphometric, immunohistochemical, and biochemical analyses. A > 70% decrease in the total number of coronary arteries with intimal thickening in elafin-treated compared to control donor hearts (P < 0.002) was associated with reduced vascular elastolytic activity judged by fewer breaks in the internal elastic lamina (P < 0.03), less accumulation of immunoreactive fibronectin (P < 0.02), and reduced cell proliferation quantified by proliferating cell nuclear antigen (P < 0.0001). Despite myocardial lymphocytic infiltration, wet weight of elafin-treated donor hearts was reduced by 50% compared to untreated controls (P < 0.002) and associated with relative preservation of myocyte integrity, instead of extensive myocardial necrosis (P < 0.004). This protective effect correlated with decreased myocardial elastolytic activity (P < 0.0001) and inflammatory cell proliferation (P < 0.0001) and with an elafin-inhibitable elastase in lymphocytes. Serine elastase activity thus appears an important therapeutic target for post-cardiac transplant coronary arteriopathy and myocardial necrosis induced by rejection.

  View details for Web of Science ID A1996UQ23800009

  View details for PubMedID 8647937

• Functional interplay between interleukin-1 receptor and elastin binding protein regulates fibronectin production in coronary artery smooth muscle cells EXPERIMENTAL CELL RESEARCH Hinek, A., Molossi, S., RABINOVITCH, M. 1996; 225 (1): 122-131

  Abstract

  We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL)-1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL-1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production.

  View details for Web of Science ID A1996UM49100013

  View details for PubMedID 8635505

• Cohabitation of Leishmania amazonensis and Coxiella burnetii TRENDS IN MICROBIOLOGY RABINOVITCH, M., Veras, P. S. 1996; 4 (4): 158-161

  Abstract

  Intracellular pathogens customize the composition and function of the vacuoles they occupy, and can arrest or distort vacuolar maturation. In doubly infected cells, vacuoles that contain two different parasites can be used to test for exclusionary mechanisms, for expression of vacuolar phenotypes that permit or restrict fusion, and for the survival of pathogens targeted to an unusual cellular compartment.

  View details for Web of Science ID A1996UF17000010

  View details for PubMedID 8728610

• Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular matrix-bound basic fibroblast growth factor JOURNAL OF CELLULAR PHYSIOLOGY Thompson, K., RABINOVITCH, M. 1996; 166 (3): 495-505

  Abstract

  There is increasing evidence that extracellular matrix (ECM)-degrading proteinases contribute to the process of medial hypertrophy and neointimal proliferation in pulmonary vascular diseases. However, little is known about how proteinases, specifically elastases, induce vascular smooth muscle cell (SMC) hyperplasia. Our objective was to determine whether exogenous human leukocyte elastase (HLE), as well as endogenous vascular elastase, could release basic fibroblast growth factor (bFGF), a potent mitogen stored in the ECM surrounding SMCs. Cultured ovine and porcine pulmonary artery SMC were pre-incubated with [125I]-bFGF. After removal of unbound [125I]-bFGF, administration of HLE (0-1.0 microgram /ml, 1 h) resulted in a concentration-dependent accumulation of [125I]-bFGF in the conditioned medium, mirrored by depletion from the ECM. The serine elastase inhibitor elafin blocked this HLE-mediated action. Assessment by Western immunoblotting further demonstrated that HLE evoked the release of ECM-bound endogenous bFGF. When incubated with serum-starved SMC, conditioned medium from HLE-treated cells stimulated [3H]-thymidine incorporation, a feature neutralized by bFGF antibodies. In addition, SMC exposed to serum treated elastin (STE), previously shown to stimulate endogenous vascular elastase, liberated bioavailable bFGF from ECM stores, as determined by autoradiography, Western immunoblotting, and stimulation of DNA synthesis and SMC proliferation. Chondroitin sulfate, an inhibitor of STE-induced elastase activity, attenuated the release of bFGF. Our studies demonstrate that HLE, secreted by inflammatory cells, and endogenous vascular elastase release matrix-bound bFGF, suggesting a mechanism whereby elastases, through degradation of ECM, induce SMC proliferation associated with progressive vascular disease.

  View details for Web of Science ID A1996TX60300004

  View details for PubMedID 8600153

• Elastase and cell matrix interactions in the pathobiology of vascular disease. Acta paediatrica Japonica; Overseas edition RABINOVITCH, M. 1995; 37 (6): 657-666

  Abstract

  Ultrastructural observations in lung tissue implicated an endogenous vascular elastase (EVE), in the pathobiology of pulmonary vascular disease. In experimental rats, increased activity of a 20 kDa serine proteinase related to adipsin precedes the development of sustained pulmonary hypertension and vascular abnormalities. A further increase in activity is related to malignant progression of the disease. A cause and effect relationship was suggested by studies in which elastase inhibitors successfully prevented or retarded progression of pulmonary hypertension. In vitro studies have shown that both serum and endothelial factors induce EVE via tyrosine kinase intracellular signalling. Induction of EVE can release basic fibroblast growth factor from the extracellular matrix in an active form stimulating smooth muscle cell proliferation. Elastase activity was also observed in the process of smooth muscle cell migration and neointimal formation in coronary arteries following experimental cardiac transplantation. An immune/inflammatory response is observed with increased production of cytokines, tumor necrosis factor-alpha and interleukin (IL)-1 beta, reciprocally up-regulating production of fibronectin, a glycoprotein which mediated smooth muscle cell migration. The action of IL-1 beta in inducing fibronectin is facilitated by the production of elastin peptides generated by increased activity of an elastase in the coronary arteries. Our studies suggest that ligation of the elastin binding protein by elastin peptides unmasks IL-1 receptors. Fibronectin also stimulates transendothelial migration of lymphocytes which perpetuates the inflammatory response leading to neointimal formation in this model. Masking integrins on T cells with a decoy synthetic CS-1 (fibronectin) peptide largely prevented transendothelial migration and coronary neointimal formation following cardiac transplant.

  View details for PubMedID 8775547

• INCREASED ELASTIN-DEGRADING ACTIVITY AND NEOINTIMAL FORMATION IN PORCINE AORTIC ORGAN-CULTURE - REDUCTION OF BOTH FEATURES WITH A SERINE PROTEINASE-INHIBITOR ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Oho, S., DALEY, S. J., Koo, E. W., Childs, T., Gotlieb, A. I., RABINOVITCH, M. 1995; 15 (12): 2200-2206

  Abstract

  We investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [3H]elastin increased sixfold at day 3 after initiation of the culture (P < .01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P < .01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P < .05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P < .01). Addition of alpha 1-proteinase inhibitor for 14 days caused a 60% decrease in elastolytic activity in nondenuded organ cultures and a 27% reduction in ISMCs compared with untreated controls (P < .05 for both). The elastolytic activity, resolved as lytic bands on an elastin substrate gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at 43 and 50 kD. Our study suggests that endothelial cells release a soluble agent that enhances elastin-degrading activity in the aorta and may at least partially account for the initiation of neointimal formation.

  View details for Web of Science ID A1995TJ36800016

  View details for PubMedID 7489243

• Cytokine-mediated fibronectin production and transendothelial migration of lymphocytes in the mechanism of cardiac allograft vascular disease: Efficacy of novel therapeutic approaches 2nd International Symposium on Allograft Coronary Disease - A Basic Science and Clinical Review RABINOVITCH, M., Molossi, S., Clausell, N. ELSEVIER SCIENCE INC. 1995: S116–S123

  View details for Web of Science ID A1995TN11900003

  View details for PubMedID 8719473

• EXPERIENCE WITH REPAIR OF CONGENITAL HEART-DEFECTS USING ADJUNCTIVE ENDOVASCULAR DEVICES 75th Annual Meeting of the American-Association-for-Thoracic-Surgery Coles, J. G., Yemets, I., Najm, H. K., Lukanich, J. M., Perron, J., Wilson, G. J., RABINOVITCH, M., Nykanen, D. G., Benson, L. N., Rebeyka, I. M., TRUSLER, G. A., Freedom, R. M., Williams, W. G. MOSBY-YEAR BOOK INC. 1995: 1513–20

  Abstract

  The use of endovascular devices as an adjunct to repair of congenital heart anomalies represents a novel but unproven therapeutic approach. Intraoperative implantation of pulmonary arterial stents (5 to 15 mm diameter) was done in 11 patients with pulmonary atresia with ventricular septal defect (n = 4), classic tetralogy of Fallot (n = 2), truncus arteriosus (n = 1), hypoplastic left heart syndrome (stage II [n = 1] and stage III [n = 1] Norwood procedure), and miscellaneous pulmonary arterial stenoses (n = 3), as well as in patients with congenital (n = 1) and postoperative (n = 3) pulmonary venous obstruction and in 1 patient with combined pulmonary arterial and venous obstruction. The stents were effective at achieving immediate patency in all patients. There were two early deaths, one related to acute thrombosis of a small-diameter left pulmonary artery stent. Reintervention because of stent-related pulmonary arterial stenosis was frequently necessary. In five of seven patients who survived more than 1 month after implantation of stent size 8 mm or smaller severe stent-related pulmonary arterial obstruction developed. In four of the five patients with pulmonary vein stent implantation intractable obstruction developed, resulting in death in all three patients who had bilateral pulmonary vein stent implantation. Intraoperative occlusion of apical muscular ventricular septal defect with use of a clamshell device inserted from the right atrial approach was accomplished in four patients. One patient who underwent associated aortic arch reconstruction died as a result of left ventricular hypoplasia. The results in the remaining three patients were favorable on the basis of absence of significant late residual intraventricular shunting, left ventricular dysfunction, or arrhythmia. We conclude that recurrent intraluminal obstruction as a result of neointimal hyperplasia appears to be an eventual certainty in currently designed small-diameter endovascular stents. For this reason, we would recommend standard surgical techniques for repair of obstructive lesions of the pulmonary arterial confluence to maximize growth potential. Device occlusion of muscular ventricular septal defects is feasible but probably only indicated for complex cases of ventricular septal deficiency that otherwise necessitate a left ventriculotomy.

  View details for Web of Science ID A1995TG02000025

  View details for PubMedID 7475204

• INCREASED RESPIRATORY SYSTEM RESISTANCE AND BRONCHIAL SMOOTH-MUSCLE HYPERTROPHY IN CHILDREN WITH ACUTE POSTOPERATIVE PULMONARY-HYPERTENSION AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Schindler, M. B., Bohn, D. J., Bryan, A. C., Cutz, E., RABINOVITCH, M. 1995; 152 (4): 1347-1352

  Abstract

  Following surgery for congenital heart disease, there is often an increased reactivity of the pulmonary vasculature to stimuli, resulting in rapid increases in pulmonary artery pressure and a clinical impression of stiff lungs. Lung mechanics were measured in 30 children, mean age 6.7 +/- 4.1 mo, who were ventilated and had pulmonary artery pressure monitoring following surgery for congenital heart disease. A group of 15 patients developed postoperative pulmonary hypertension. In these patients, respiratory system resistance was 43% higher (p = 0.001) and compliance 11% lower (p = 0.004) during acute pulmonary hypertension compared with baseline pulmonary artery pressure. No changes in resistance or compliance were seen in the 15 patients who did not develop pulmonary hypertension. The changes in lung mechanics interfered with mechanical ventilation, resulting in a 9.4% rise in PaCO2 during pulmonary hypertension. The bronchial smooth muscle was found to be increased by 68%, and the vascular smooth muscle was more than twice normal in lung biopsies from 9 pulmonary hypertension patients compared with 6 age-matched postmortem controls patients who had no cardiac or pulmonary disease. The bombesin-immunoreactive pulmonary neuroendocrine cells (PNEC) were also increased in the pulmonary hypertension patients. These findings suggest a coconstriction and cohypertrophy of bronchial and vascular smooth muscle during pulmonary hypertension. Mediators, such as bombesin, endothelin-1, and serotonin, are known to be produced by PNEC and may be involved in the observed vasoconstriction, increased respiratory system resistance, and smooth muscle hypertrophy.

  View details for Web of Science ID A1995RX73100032

  View details for PubMedID 7551393

• LYMPHOCYTE TRANSENDOTHELIAL MIGRATION TOWARD SMOOTH-MUSCLE CELLS IN INTERLEUKIN-1-BETA-STIMULATED COCULTURES IS RELATED TO FIBRONECTIN INTERACTIONS WITH ALPHA(4)BETA(1) AND ALPHA(5)BETA(1) INTEGRINS JOURNAL OF CELLULAR PHYSIOLOGY Molossi, S., Elices, M., Arrhenius, T., RABINOVITCH, M. 1995; 164 (3): 620-633

  Abstract

  We previously reported infiltration of immune-inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)-1 beta. We now investigate, using a porcine endothelial-smooth muscle cell co-culture system, whether IL-1 beta-stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL-2, were characterized by fluorescence-activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL-1 beta. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking alpha 4 beta 1 and alpha 5 beta 1 integrin receptors on lymphocyte surfaces, respectively. A 55-70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium, we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine-mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface alpha 4 beta 1 and alpha 5 beta 1 integrins.

  View details for Web of Science ID A1995RR00200020

  View details for PubMedID 7650068

• ABNORMALITIES IN INTRAMYOCARDIAL ARTERIES DETECTED IN CARDIAC TRANSPLANT BIOPSY SPECIMENS AND LACK OF CORRELATION WITH ABNORMAL INTRACORONARY ULTRASOUND OR ENDOTHELIAL DYSFUNCTION IN LARGE EPICARDIAL CORONARY-ARTERIES JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Clausell, N., Butany, J., Molossi, S., Lonn, E., Gladstone, P., RABINOVITCH, M., Daly, P. A. 1995; 26 (1): 110-119

  Abstract

  We sought to determine whether abnormalities in small intramyocardial vessels could be detected on routine cardiac transplant biopsy specimens and whether these features correlate with intimal thickening by intracoronary ultrasound and endothelial dysfunction in large epicardial vessels.Variability in clinical presentation of allograft vasculopathy suggests differential involvement of large and small vessels. Intracoronary ultrasound and endothelial function studies detect large-vessel abnormalities but may not reflect changes in small intramyocardial arteries. The latter could be detected in routine cardiac biopsy specimens by histologic and immunohistochemical studies.Thirty-nine cardiac transplant recipients underwent intracoronary ultrasound and acetylcholine studies 5 to 7 days after endomyocardial biopsy. Biopsy tissue was evaluated for coronary artery endothelial plumping and intimal thickening and increased immunostaining for fibronectin, tumor necrosis factor-alpha and receptor for hyaluronan-mediated motility. Large-vessel disease was assessed by calculating an average intimal index from intracoronary ultrasound of the left anterior descending coronary artery. Endothelial function was determined by quantitative coronary analysis after acetylcholine challenge.Coronary arteries were found in the biopsy tissue of 30 (76%) of the 39 patients who formed the study group. Fourteen of 30 patients had abnormal histologic findings. Immunohistochemical analysis for fibronectin, possible in 20 of 30 patients, was positive in 14 (70%) of 20 and correlated with abnormal histologic findings (p = 0.01). Immunostaining was positive for tumor necrosis factor-alpha and receptor for hyaluronan-mediated motility in 12 (40%) and 13 (43%) of 30 patients, respectively. All patients had intimal thickening by intracoronary ultrasound, but intimal index did not correlate significantly with small-artery disease by histologic or immunohistochemical analysis. Large-vessel endothelial dysfunction in 13 patients (43%) did not correlate with either abnormal ultrasound findings or small-vessel disease.Intramyocardial arteries are readily observed in biopsy specimens from cardiac transplant recipients and provide useful information about allograft vasculopathy. Lack of correlation between intramyocardial and epicardial vessel disease suggests discordant progression of allograft vasculopathy.

  View details for Web of Science ID A1995RG11000015

  View details for PubMedID 7541058

• EXPRESSION OF TUMOR-NECROSIS-FACTOR-ALPHA AND ACCUMULATION OF FIBRONECTIN IN CORONARY-ARTERY RESTENOTIC LESIONS RETRIEVED BY ATHERECTOMY BRITISH HEART JOURNAL Clausell, N., DELIMA, V. C., Molossi, S., Liu, P., Turley, E., Gotlieb, A. I., Adelman, A. G., RABINOVITCH, M. 1995; 73 (6): 534-539

  Abstract

  The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury.Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility.The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta.Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury.

  View details for Web of Science ID A1995RD75900011

  View details for PubMedID 7626352

• BLOCKADE OF VERY LATE ANTIGEN-4 INTEGRIN BINDING TO FIBRONECTIN WITH CONNECTING SEGMENT-1 PEPTIDE REDUCES ACCELERATED CORONARY ARTERIOPATHY IN RABBIT CARDIAC ALLOGRAFTS JOURNAL OF CLINICAL INVESTIGATION Molossi, S., Elices, M., Arrhenius, T., Diaz, R., Coulber, C., RABINOVITCH, M. 1995; 95 (6): 2601-2610

  Abstract

  Graft arteriopathy, a leading cause of cardiac allograft failure, is associated with increased intimal smooth muscle cells, inflammatory cells, and accumulation of extracellular matrix. We hypothesized that cellular fibronectin plays a pivotal role in the progression of the allograft arteriopathy by directing the transendothelial trafficking of inflammatory cells through interaction of the connecting segment-1 (CS1) motif with the very late antigen-4 (VLA-4) integrin, and tested this in vivo using a blocking peptide. Cholesterol-fed rabbits underwent heterotopic cardiac transplantation without immunosuppression. The treatment group (n = 7) received a synthetic CS1 peptide (1 mg/kg per d, subcutaneously), and the controls (n = 7) received an inactive peptide (1 mg/kg per d, subcutaneously). At 7-8 d after transplantation, hearts were harvested and sectioned for morphometric analysis and immunohistochemical studies. We observed a > 50% decrease in the incidence (P < 0.001) and severity (P < 0.001) of donor coronary artery intimal thickening in the CS1-treated compared with the control group. These findings correlated with reduced infiltration of T cells (P < 0.05), a trend toward decreased expression of adhesion molecules (P < 0.06), and less accumulation of fibronectin (P < 0.03). Our data suggest that the VLA-4-fibronectin interaction is critical to the progression of the allograft arteriopathy by perpetuating the immune-inflammatory response in the vessel wall.

  View details for Web of Science ID A1995RB21200025

  View details for PubMedID 7539456

• ICAM-1 AND VCAM-1 EXPRESSION IN ACCELERATED CARDIAC ALLOGRAFT ARTERIOPATHY AND MYOCARDIAL REJECTION ARE INFLUENCED DIFFERENTLY BY CYCLOSPORINE-A AND TUMOR-NECROSIS-FACTOR-ALPHA BLOCKADE JOURNAL OF PATHOLOGY Molossi, S., Clausell, N., Sett, S., RABINOVITCH, M. 1995; 176 (2): 175-182

  Abstract

  Expression of the vascular cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) occurs in allograft myocardium and in coronary arteries, promoting adhesion and transendothelial migration of inflammatory cells. We therefore investigated, in cholesterol-fed rabbits 9-10 days following heterotopic cardiac transplantation, whether the reduction of both myocardial rejection and graft arteriopathy with cyclosporine A (CsA) or graft arteriopathy alone with tumour necrosis factor-alpha soluble receptor (TNFsr) was associated with suppression of ICAM-1 and VCAM-1 expression. Host hearts showed negative immunostaining for these adhesion molecules, whereas donor specimens from untreated (control) rabbits showed moderate immunostaining for ICAM-1 and weaker immunostaining for VCAM-1 in the coronary arteries, myocardium (cardiac myocytes), and perivenular regions. The selective reduction of the coronary arteriopathy with TNFsr was associated with somewhat reduced expression of these adhesion molecules in the arteries, whereas CsA also suppressed myocardial rejection and markedly decreased both vascular and myocyte expression of ICAM-1 and VCAM-1.

  View details for Web of Science ID A1995RF91000010

  View details for PubMedID 7543564

• RECIPROCAL INDUCTION OF TUMOR-NECROSIS-FACTOR-ALPHA AND INTERLEUKIN-1-BETA ACTIVITY MEDIATES FIBRONECTIN SYNTHESIS IN CORONARY-ARTERY SMOOTH-MUSCLE CELLS JOURNAL OF CELLULAR PHYSIOLOGY Molossi, S., Clausell, N., RABINOVITCH, M. 1995; 163 (1): 19-29

  Abstract

  We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-1 beta and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-1 beta was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-1 beta and tumor necrosis factor (TNF)-alpha has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-alpha expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-1 beta, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-alpha antibodies, and exogenous TNF-alpha upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-alpha-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-1 beta antibodies. Likewise, IL-1 beta-stimulated fibronectin synthesis was downregulated to control levels when TNF-alpha neutralizing antibodies were added. Combining TNF-alpha and IL-1 beta enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-1 beta and TNF-alpha activity and provide the first demonstration of a 'cytokine loop' modulating matrix production.

  View details for Web of Science ID A1995QN68600003

  View details for PubMedID 7896895

• VLA-4 AND LYMPHOCYTE TRAFFICKING IN IMMUNE-INFLAMMATORY STATES - NOVEL THERAPEUTIC APPROACHES IN ALLOGRAFT ARTERIOPATHY SPRINGER SEMINARS IN IMMUNOPATHOLOGY Molossi, S., RABINOVITCH, M. 1995; 16 (4): 443-465

  View details for Web of Science ID A1995QT02400009

  View details for PubMedID 7570295

• ALPHA(1)-ANTITRYPSIN PROTECTS NEONATAL RATS FROM PULMONARY VASCULAR AND PARENCHYMAL EFFECTS OF OXYGEN-TOXICITY PEDIATRIC RESEARCH KOPPEL, R., Han, R. N., Cox, D., Tanswell, A. K., RABINOVITCH, M. 1994; 36 (6): 763-770

  Abstract

  We investigated whether alpha 1-antitrypsin (alpha 1-AT) might protect neonatal rats from the pulmonary parenchymal and vascular effects resulting from hyperoxic exposure. Neonatal rats born into and maintained in hyperoxia (60% fraction of inspired oxygen) or room air were injected with a loading dose of alpha 1-AT (72 mg/kg) followed by 36 mg/kg every 72 h or with vehicle during the first 14 d of life. At the end of the experimental period, we measured body weight, lung compliance, lung volume, alveoli per mm2, and total number of alveoli and assessed right ventricular hypertrophy and vascular changes consisting of medial hypertrophy, muscular extension into peripheral, normally nonmuscular arteries, and number of peripheral arteries relative to alveoli. Our data show that alpha 1-AT treatment prevented the reduced lung compliance observed in the untreated hyperoxia-exposed neonatal rats, as well as the right ventricular hypertrophy and the associated vascular changes of medial hypertrophy of muscular arteries and muscularization of distal arteries. Reduced lung compliance in the hyperoxic but alpha 1-AT-untreated rats was associated with a reduction in lung elastin compared with room-air or alpha 1-AT-treated rats. In room-air rats, alpha 1-AT treatment increased lung compliance but also reduced the number of arteries relative to the number of alveoli, a feature that was not, however, associated with right ventricular hypertrophy. Our data suggest that supplemental alpha 1-AT might restore the imbalance in elastolytic activity induced by hyperoxia and thereby alleviate the toxic effects on lung parenchymal and vascular development.

  View details for Web of Science ID A1994PV11100013

  View details for PubMedID 7898985

• THE ENDOGENOUS VASCULAR ELASTASE THAT GOVERNS DEVELOPMENT AND PROGRESSION OF MONOCROTALINE-INDUCED PULMONARY-HYPERTENSION IN RATS IS A NOVEL ENZYME RELATED TO THE SERINE PROTEINASE ADIPSIN JOURNAL OF CLINICAL INVESTIGATION Zhu, L., Wigle, D., Hinek, A., Kobayashi, J., Ye, C. L., Zuker, M., Dodo, H., Keeley, F. W., RABINOVITCH, M. 1994; 94 (3): 1163-1171

  Abstract

  We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

  View details for Web of Science ID A1994PF35400032

  View details for PubMedID 8083356

• 67-KD ELASTIN-BINDING PROTEIN IS A PROTECTIVE COMPANION OF EXTRACELLULAR INSOLUBLE ELASTIN AND INTRACELLULAR TROPOELASTIN JOURNAL OF CELL BIOLOGY Hinek, A., RABINOVITCH, M. 1994; 126 (2): 563-574

  Abstract

  The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S-GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S-GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.

  View details for Web of Science ID A1994NX54700026

  View details for PubMedID 8034752

• POST-CARDIAC TRANSPLANT ARTERIOPATHY IN PIGLETS IS ASSOCIATED WITH FRAGMENTATION OF ELASTIN AND INCREASED ACTIVITY OF A SERINE ELASTASE AMERICAN JOURNAL OF PATHOLOGY Oho, S., RABINOVITCH, M. 1994; 145 (1): 202-210

  Abstract

  In experimental piglets after heterotopic heart transplant, we observed an immune/inflammatory response in the coronary arteries with increased expression of interleukin-1 beta and accumulation of fibronectin and smooth muscle cells in the subendothelium (N. Clausell, S. Molossi, M. Rabinovitch, Am J Pathol 1993, 142, 1772-1786). Proteolytic enzymes including elastases regulate cytokine activity and are associated with the development of neointimal proliferation. We now report ultrastructural evidence of elastolytic activity in the donor compared to host coronary arteries judged by a fivefold increase in the breaks in the internal elastic lamina, (P < 0.01) correlating with a 10-fold increase in elastase activity per mg tissue (P < 0.01). The enzyme activity is serine elastase, i.e., inhibited by phenylmethyl sulfonyl fluoride, and elafin but not EDTA. Using a novel strategy that greatly increases the activity extractable from the tissue, we resolved the enzyme on an elastin substrate gel as a protein of approximately 23 kd. Ours is the first report and characterization of increased elastase activity associated with the development of the post-cardiac transplant coronary arteriopathy. The source may be inflammatory or smooth muscle cells, and elastase may play a pathophysiological role in neointimal proliferation by activating cytokines and growth factors and by release of chemotactic peptides.

  View details for Web of Science ID A1994NV86100025

  View details for PubMedID 8030749

• SERUM-INDUCED VASCULAR SMOOTH-MUSCLE CELL ELASTOLYTIC ACTIVITY THROUGH TYROSINE KINASE INTRACELLULAR SIGNALING JOURNAL OF CELLULAR PHYSIOLOGY Kobayashi, J., Wigle, D., Childs, T., Zhu, L., Keeley, F. W., RABINOVITCH, M. 1994; 160 (1): 121-131

  Abstract

  In previous studies, we related increased elastolytic activity in pulmonary arteries (PA) with endothelial injury to the later development of PA hypertension in rats. As the mechanism causing the increased PA elastase was unknown, we hypothesized that serum factors which are accessible to vascular smooth muscle cells (SMC) following endothelial injury stimulate their elastolytic activity. To test this, we developed an in vitro assay in which we added [3H]-elastin to cultured vascular SMC after 24 h serum starvation and monitored elastolysis following a further 24 h incubation with fetal bovine serum (FBS). We observed that serum induced increased elastolytic activity in both PA and aorta-derived SMC but not in endothelial cells or SMC with low basal levels of elastolytic activity. Maximum stimulation of SMC elastolytic activity occurred with a concentration as low as 1% FBS and despite elastase inhibitors in serum, suggesting that the activity is confined to the immediate pericellular region where enzyme concentration is high. Serum-stimulated elastolytic activity was not reproduced by growth factors or cytokines known to be associated with vascular disease or to induce release of elastases in other cells. The serum inducing elastolytic activity was heat and acid labile. It was associated with increased elastin adhesion to the 67 kD elastin binding protein on SMC surfaces and was prevented by tyrosine kinase inhibitors but not protein kinase C or A inhibitors. Our studies therefore suggest a mechanism whereby serum induction of SMC elastase requires signalling through the elastin binding protein and activation of tyrosine kinase.

  View details for Web of Science ID A1994NU58600014

  View details for PubMedID 8021292

• IN-VIVO BLOCKADE OF TUMOR NECROSIS FACTOR-A IN CHOLESTEROL-FED RABBITS AFTER CARDIAC TRANSPLANT INHIBITS ACUTE CORONARY-ARTERY NEOINTIMAL FORMATION CIRCULATION Clausell, N., Molossi, S., Sett, S., RABINOVITCH, M. 1994; 89 (6): 2768-2779

  Abstract

  We previously identified in piglet cardiac allografts an immunoinflammatory response in coronary arteries in which increased fibronectin regulated by interleukin-1 beta was associated with early evidence of intimal thickening. In the present study, we used rabbits to assess whether acute neointimal formation after cardiac transplantation was reduced by blockade of tumor necrosis factor (TNF)-alpha, which modulates interleukin-1 beta, or by cyclosporine A.Sixteen rabbits underwent heterotopic cardiac transplantation and were given saline, TNF-soluble receptor (sr), or cyclosporine A. In host hearts from saline- or TNFsr-treated groups, few coronary arteries (approximately 13% to 16%) had intimal thickening, whereas values were higher in the cyclosporine A-treated group (approximately 30%). In donor hearts from the saline-treated group, however, approximately 68% of vessels had intimal thickening versus approximately 32% in TNFsr- and approximately 30% in cyclosporine A-treated groups (P < .01 for both). Severity of intimal thickening assessed quantitatively as percent vessel area was approximately 38% in the saline-treated group but reduced in TNFsr- and cyclosporine A-treated groups to approximately 22% and 18%, respectively (P < .01 for each). Immunohistochemistry revealed increased staining for major histocompatibility complex II, T cells, interleukin-1 beta, TNF-alpha, and fibronectin in donor coronary arteries from saline-treated animals when compared with TNFsr- and cyclosporine A-treated animals. Grade 3 myocardial rejection was observed in both saline- and TNFsr-treated groups, but only grade 1 was apparent in the cyclosporine A-treated group.In vivo blockade of TNF-alpha suppresses the acute development of neointimal formation by selectively reducing the vascular immunoinflammatory reaction and accumulation of fibronectin, whereas cyclosporine A suppresses both the myocardial and the vascular immune reaction.

  View details for Web of Science ID A1994NQ83000043

  View details for PubMedID 8205691

• PULMONARY-HYPERTENSION AND VASCULAR REMODELING IN FETAL SHEEP AMERICAN JOURNAL OF PHYSIOLOGY Belik, J., Keeley, F. W., Baldwin, F., RABINOVITCH, M. 1994; 266 (6): H2303-H2309

  Abstract

  Vascular remodeling is commonly associated with pulmonary hypertension (PH) postnatally, but little is known about its presence in fetuses. In sixteen fetal sheep (126 wk gestation), the ductus arteriosus (DA) was ligated, and the animals were studied at 4, 8, and 14 days after surgery. The uninstrumented twins served as controls. Four days post-DA ligation PH resulted in an increase in the right and left ventricular free wall weight ratio (1.2 +/- 0.1 vs. 1.0 +/- 0.1 in the controls; P < 0.01), with a further progressive increase at 8 (1.4 +/- 0.1 vs. 1.0 +/- 0.1; P < 0.01) and 14 days (1.5 +/- 0.2 vs. 1.0 +/- 0.1; P < 0.01). An increase in vascular percent medial thickness was observed after 4 days of DA ligation and was restricted to small vessels. The large arteries collagen and elastin contents were 28.9 +/- 3.4 and 27.1 +/- 3.4 micrograms/micrograms of DNA, respectively, and were not significantly different from control values even after 14 days DA ligation. We further compared elastin synthesis in fetal and neonatal arteries in vitro. Synthesis in the fetus was greater than the newborn (10.6 +/- 1.4 vs. 4.6 +/- 0.7 cpm.mg wet wt-1.h-1; P < 0.01). Vessel endothelium denudation reduced synthesis to 60 +/- 8% of controls in the fetus, whereas no change was seen in the newborn. After an increase in wall stress, synthesis increased in the fetus (194 +/- 28% of control P < 0.01) and newborn (173 +/- 25%; P < 0.01). Removal of the endothelium abolished the response.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1994NV81000019

  View details for PubMedID 8023991

• CYTOCHROME-P450 DURING ONTOGENIC DEVELOPMENT - OCCURRENCE IN THE DUCTUS-ARTERIOSUS AND OTHER TISSUES CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY Coceani, F., Kelsey, L., Ackerley, C., RABINOVITCH, M., Gelboin, H. 1994; 72 (3): 217-226

  Abstract

  Our previous investigations have implicated a cytochrome P450 mechanism in the oxygen contraction of the ductus arteriosus and, by extension, in the closure of the vessel at birth. This study was undertaken in fetal and newborn sheep to characterize the ductal cytochrome and gather insight into its operation. Other tissues, vascular and extravascular, were used as a reference. Benzo[a]pyrene hydroxylation (a marker for the 3-methylcholanthrene-inducible isozyme) and aminopyrine N-demethylation (a marker for the glucocorticoid-inducible isozyme) had insignificant activity in the ductus and aorta from fetal sheep, and no increase was noted after exposing the animal in utero to beta-naphthoflavone or dexamethasone, both alone and in combination with phenobarbital. However, dexamethasone and, particularly, dexamethasone plus phenobarbital produced a variable constriction of the fetal ductus. No monooxygenase activity was found in the naturally closing ductus of the newborn. Conversely, both enzyme reactions were measurable in the fetal liver, and they became more active after treatment with the inducers or at birth. Scanning immunoelectron microscopy of cultured endothelial and muscle cells from both ductus and aorta showed specific gold labelling for the glucocorticoid-inducible cytochrome P450 only in ductal muscle. By transmission electron microscopy, this immunoreactivity was located along the sarcolemma and in the sarcoplasmic reticulum. These findings indicate the presence in the ductus arteriosus of a cytochrome P450 belonging to the 3A subfamily. However, considering the uneven action of the inducers on enzyme activity in ductal tissue and muscle tone, the role of this cytochrome in closure of the vessel at birth remains to be ascertained.

  View details for Web of Science ID A1994NM23900005

  View details for PubMedID 8069768

• GAS BUBBLE EMBOLI DETECTED BY TRANSCRANIAL DOPPLER SONOGRAPHY IN PATIENTS WITH PROSTHETIC HEART-VALVES - A PRELIMINARY-REPORT JOURNAL OF ULTRASOUND IN MEDICINE Dauzat, M., Deklunder, G., Aldis, A., RABINOVITCH, M., BURTE, F., BRET, P. M. 1994; 13 (2): 129-135

  Abstract

  After observing spikes in the Doppler signal of cerebral arteries of patients with neurologic symptoms and prosthetic heart valves, we then studied two groups of patients with prosthetic heart valves: seven patients with neurologic symptoms and 65 asymptomatic patients. Using transcranial Doppler sonography of the middle cerebral artery, we found Doppler spikes in six symptomatic and 24 asymptomatic patients with mechanical heart valves. No spikes were found in one symptomatic and 21 asymptomatic patients with biological valves or in 20 asymptomatic patients with mechanical valves. We concluded that gas cavitation during the opening or closure of the valve, producing bubble emboli, is the most probable explanation for these Doppler spikes in patients with mechanical prosthetic heart valves.

  View details for Web of Science ID A1994MX02700012

  View details for PubMedID 7932957

• Coronary artery endothelial interleukin-1 beta mediates enhanced fibronectin production related to post-cardiac transplant arteriopathy in piglets. Circulation Molossi, S., Clausell, N., RABINOVITCH, M. 1993; 88 (5): II248-56

  Abstract

  Graft coronary arteriopathy has become the major complication observed in the late follow-up of cardiac transplant patients. We investigated this process experimentally in piglets after a heterotopic cardiac transplant and observed early changes in donor coronary arteries compatible with an immune-inflammatory process, ie, there is increased expression of interleukin-1 beta (IL-1 beta), fibronectin, and activated lymphocytes associated with intimal thickening.In this study, we cultured porcine coronary artery endothelial cells from host and donor hearts and found similarities in morphology and uptake of acetylated low-density lipoprotein (LDL). As well, host and donor cells showed similar patterns of growth, protein, and glycosaminoglycan synthesis. Endothelial cell fibronectin synthesis was determined after radiolabeling with [35S]-methionine in serum-free medium, gelatin-sepharose extraction of the culture medium and resolution on 5% SDS-PAGE. Donor coronary artery endothelial cell fibronectin synthesis was up to fivefold higher than that of host but was not associated with comparable increased levels of fibronectin mRNA. IL-1 beta appeared to mediate this enhanced fibronectin production, since the IL-1 receptor antagonist caused a 50% decrease in this feature, a change not observed in host cells. Furthermore, donor endothelial cells produced twice the amount of IL-1 beta compared with host cells as judged by immunoprecipitation.Increased donor coronary artery endothelial cell fibronectin appears to be regulated at least partly by an autocrine mechanism involving IL-1 beta, and fibronectin may mediate lymphocyte trafficking and smooth muscle cell migration related to graft arteriopathy.

  View details for PubMedID 8222162

• CORONARY-ARTERY ENDOTHELIAL INTERLEUKIN-1-BETA MEDIATES ENHANCED FIBRONECTIN PRODUCTION RELATED TO POST-CARDIAC TRANSPLANT ARTERIOPATHY IN PIGLETS CIRCULATION Molossi, S., Clausell, N., RABINOVITCH, M. 1993; 88 (5): 248-256

  View details for Web of Science ID A1993ME83400035

• Pulmonary vascular obstructive disease. Cardiology clinics Collins-Nakai, R. L., RABINOVITCH, M. 1993; 11 (4): 675-687

  Abstract

  The history, clinical course, pathophysiology, pathology, diagnosis, and therapy of pulmonary vascular obstructive disease are presented. An explanation is provided for the terms Eisenmenger syndrome, Eisenmenger complex, and pulmonary vascular disease. The complications frequently seen in the natural history of the condition are presented, as are the therapeutic options. Despite intensive investigation of new therapeutic products, the most promising treatment for established pulmonary vascular obstructive disease remains heart-lung operation or single-lung transplant with repair of cardiac defects. Even with this option, the long-term outlook for this progressive condition remains dismal.

  View details for PubMedID 8252567

• HYPERPLASIA OF PULMONARY ARTERIAL MEDIA IN INFANTILE FAMILIAL PULMONARY-HYPERTENSION ASSOCIATED WITH SEVERE METABOLIC-ACIDOSIS MODERN PATHOLOGY Cullinane, C., Clarke, J. T., RABINOVITCH, M., Bohn, D., Silver, M. M. 1993; 6 (6): 699-706

  Abstract

  Two female siblings, offspring of consanguinous parents, died at 10 and 12 wk of age following short illnesses characterized clinically by hypoxia and severe metabolic acidosis. Cardiac catheterization confirmed severe pulmonary hypertension in the second infant, who survived 6 wk after onset of symptoms and 4 wk after admission to hospital; extensive investigations failed to identify any inborn metabolic error. At autopsy, small pulmonary arteries/arterioles in both cases showed marked medial thickening due to smooth muscle hyperplasia; concentric intimal fibrosis was present focally in the older infant. Compared morphometrically with small pulmonary arteries in 20 infantile controls who died of Reye's syndrome (n = 8), a Reye's-like illness (n = 5) or an identified metabolic error associated with metabolic acidosis (n = 7), intraacinar pulmonary arteries in both cases, were significantly more numerous and had a significantly greater relative medial thickness. We suggest that an autosomal recessive gene caused or potentiated the pulmonary medial hyperplasia. The latter finding, compared with medial hypertrophy, is rarely described as the morphologic basis of pulmonary hypertension, and its occurrence may be restricted to early infancy. In this family, the relationship between hypertensive pulmonary arteriopathy and severe metabolic acidosis remains speculative.

  View details for Web of Science ID A1993MK43100012

  View details for PubMedID 8302812

• PROFILE OF PEDIATRIC-PATIENTS WITH PULMONARY-HYPERTENSION JUDGED BY RESPONSIVENESS TO VASODILATORS BRITISH HEART JOURNAL Houde, C., Bohn, D. J., Freedom, R. M., RABINOVITCH, M. 1993; 70 (5): 461-468

  Abstract

  To describe the demographic and haemodynamic variables of children presenting with primary pulmonary hypertension or pulmonary hypertension appearing or persisting after surgical correction of congenital heart defects and to assess the acute effect of vasodilator drugs on their pulmonary vascular bed.Retrospective review.Paediatric cardiology department and intensive care unit of a large tertiary centre.Fourteen consecutive patients presenting with primary pulmonary hypertension (group 1) or pulmonary hypertension persisting or appearing late after complete surgical repair (group 2).Baseline haemodynamic measurements were taken in room air at rest and repeated in 100% oxygen. With constant monitoring of heart rate and pulmonary and systemic arterial pressures, patients were given serial intravenous, sublingual, or oral incremental doses of vasodilators (mean 4.1 trials per patient). The maximum effect of the drug was charted.A positive response to acute vasodilator tests was defined as a decrease in mean pulmonary or mean systemic arterial pressure > 15% with the mean pulmonary artery pressure not reaching the systemic level and either no change or an increase in mean systemic arterial pressure. Haemodynamic variables between groups (1 v 2, responders v non-responders, patients experiencing or not experiencing adverse effects to vasodilators) were compared by a two tailed unpaired Student's t test, and their survival curves were compared by the log rank statistic.Groups are small and definitive conclusions are difficult to draw, but the baseline haemodynamic assessments were not significantly different between group 1 and 2 or between responders and non-responders to vasodilators. Patients experiencing adverse effects had a higher pulmonary vascular resistance (p = 0.04) and wedge pressure (p = 0.02) than those without adverse effects. Of the vasodilators used, tolazoline, hydralazine, salbutamol, phentolamine, and phenoxybenzamine were ineffective. A positive response was seen in five of 13 patients given oxygen, in one of eight given prostacyclin, four of 12 given nifedipine, four of eight given diltiazem, one of six given captopril, and two of seven given glyceryl trinitrate. Estimates of survival of the population with primary pulmonary hypertension were 37% at one year and 12% at 2.5 years. Survival was significantly shorter in the non-responders than in the responders (p = 0.005).Children with primary pulmonary hypertension present to the cardiologist at a young age (five of eight were younger than 7 years) but with advanced pulmonary vascular disease and have a poor prognosis. 64% of group 1 and group 2 patients had a positive response to acute treatment with at least one vasodilator. Calcium channel blockers were the most effective agents. There was a positive response to drugs despite a negative response to acute treatment with oxygen. The survival of non-responders was shorter than that of the responders.

  View details for Web of Science ID A1993MF26800014

  View details for PubMedID 8260279

• UP-REGULATION OF FIBRONECTIN SYNTHESIS BY INTERLEUKIN-1-BETA IN CORONARY-ARTERY SMOOTH-MUSCLE CELLS IS ASSOCIATED WITH THE DEVELOPMENT OF THE POST-CARDIAC TRANSPLANT ARTERIOPATHY IN PIGLETS JOURNAL OF CLINICAL INVESTIGATION Clausell, N., RABINOVITCH, M. 1993; 92 (4): 1850-1858

  Abstract

  We previously described in piglets after heterotopic cardiac transplantation the early development of a coronary arteriopathy characterized by increased immunostaining for fibronectin and interleukin-1 beta (IL-1 beta) in the vessel wall. The objective of this study was to culture smooth muscle cells from donor and host coronary arteries in these piglets to determine whether donor cells produce more fibronectin than host cells as judged by increased protein and mRNA levels, and whether IL-1 beta may be regulating this increase by an autocrine mechanism involving increased production of the cytokine. We documented increased donor coronary artery smooth muscle cell fibronectin protein synthesis and mRNA compared to host. By using neutralizing antibodies to IL-1 beta, fibronectin protein synthesis and mRNA levels were reduced in donor cells to the levels observed in the host cells and a similar reduction in synthesis was observed with the IL-1 receptor antagonist. Immunoprecipitation of newly synthesized IL-1 beta revealed increased endogenous levels in donor compared to host cells. We therefore suggest in the coronary arteriopathy a pathophysiologic mechanism whereby IL-1 beta-mediated increased fibronectin synthesis may promote lymphocyte trapping and migration of medial smooth muscle cells leading to progressive intimal thickening associated with the post-cardiac transplant coronary arteriopathy.

  View details for Web of Science ID A1993MB16600036

  View details for PubMedID 8408637

• SCIMITAR SYNDROME IN INFANCY JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Gao, Y. A., Burrows, P. E., Benson, L. N., RABINOVITCH, M., Freedom, R. M. 1993; 22 (3): 873-882

  Abstract

  The objectives of this study were to determine the anatomic and physiological factors most responsible for the severe symptoms and poor prognosis of infants with scimitar syndrome.Whereas the diagnosis of scimitar syndrome is often made incidentally in older children and adults who undergo chest radiography for diverse reasons, infants in whom the diagnosis is made typically present with severe symptoms and have a poor prognosis.The clinical, catheterization and imaging data of 13 consecutive infants with scimitar syndrome who underwent cardiac catheterization in the 1st 6 months of life were reviewed, with emphasis on the pulmonary artery pressure, pulmonary and cardiovascular anatomy, therapeutic interventions and outcome.Twelve of the 13 infants had pulmonary hypertension at the time of diagnosis. Six patients died despite specific treatment. Eleven of 13 infants had associated cardiac malformations and 9 had large systemic arterial collateral channels to the right lung. Seven patients had anomalies involving the left side of the heart, especially varying degrees of hypoplasia of the left heart or aorta, and six of these patients died. Ten patients underwent surgical or transcatheter therapy in the 1st year of life. Systemic arteries to the right lung were ligated in three patients and occluded by transcatheter embolization in four. Balloon angioplasty was carried out in two patients, one with stenosis of the left-sided pulmonary veins and one with stenosis of the anomalous right pulmonary vein. The latter had placement of a balloon-expandable stent. In both patients, pulmonary vein stenosis progressed. Six patients had surgical repair of associated cardiovascular anomalies, and two required repair of extracardiac congenital anomalies. Occlusion of the anomalous systemic arteries was generally associated with clinical improvement, but congestive heart failure and pulmonary hypertension recurred in those patients with associated cardiovascular anomalies, whose condition subsequently responded after correction of the shunt lesions.The severe symptoms and pulmonary hypertension found in infants with scimitar syndrome have many causes. Anomalous systemic arterial supply, pulmonary vein stenosis and associated cardiovascular anomalies play a significant role, and the ultimate outcome of individual infants depends on the feasibility of treating these anomalies in early infancy.

  View details for Web of Science ID A1993MQ36100038

  View details for PubMedID 8354827

• INCREASED INTERLEUKIN-1-BETA AND FIBRONECTIN EXPRESSION ARE EARLY FEATURES OF THE DEVELOPMENT OF THE POSTCARDIAC TRANSPLANT CORONARY ARTERIOPATHY IN PIGLETS AMERICAN JOURNAL OF PATHOLOGY Clausell, N., Molossi, S., RABINOVITCH, M. 1993; 142 (6): 1772-1786

  Abstract

  The mechanism causing intimal thickening in the postcardiac transplant coronary arteriopathy (PCTCA) is associated with interactions between inflammatory cells and vascular cells. Our previous studies related intimal thickening to fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, and others have shown that cytokines, eg, interleukin (IL)-1 beta, up-regulate SMC fibronectin synthesis. In this study, we identified, in piglets, features compatible with early development of the PCTCA. Ultrastructure revealed increased SMC and inflammatory cells in the subendothelium. Immunohistochemistry showed major histocompatibility complex II presentation in the endothelium and adventitia, associated with infiltration of different subsets of inflammatory cells; increased IL-1 beta, particularly in the endothelium; and fibronectin, in the subendothelium and inner media, the latter confirmed by quantitative immunoelectron microscopy. In the PCTCA, increases in IL-1 beta and fibronectin could mediate adherence, transendothelial migration and trapping of inflammatory cells, and SMC migration into the subendothelium.

  View details for Web of Science ID A1993LF97800012

  View details for PubMedID 8506947

• REGULATION OF FETAL LAMB DUCTUS-ARTERIOSUS SMOOTH-MUSCLE CELL-MIGRATION BY INDOMETHACIN AND DEXAMETHASONE PEDIATRIC RESEARCH KOPPEL, R., RABINOVITCH, M. 1993; 33 (4): 352-358

  Abstract

  Postnatal closure of the ductus arteriosus (DA) requires the development, during late gestation, of "intimal cushions." These structures, which partially occlude the vessel lumen, are characterized by smooth muscle cell (SMC) migration into an expanded subendothelium. DA SMC migration is dependent upon increased fibronectin (FN) production. We hypothesized that indomethacin (INDO) or dexamethasone, which could influence FN production, may affect SMC migration and DA intimal cushion formation. SMC harvested from the DA and aorta of 100-d fetal lambs were seeded onto three-dimensional collagen gels. Migration into the gels was quantitatively assessed by phase-contrast microscopy. INDO retarded DA SMC migration (p < 0.05), but it had no effect on aorta SMC migration. Synthesis of FN was measured after [35S]-methionine radiolabeling of cells, gelatin sepharose extraction of conditioned media, and resolution by SDS-PAGE. Despite the decrease in migration, INDO did not affect FN synthesis in DA SMC, whereas dexamethasone, a stabilizer of FN mRNA that increased DA FN synthesis by 44%, had no further effect on SMC migration. We then investigated whether INDO might be influencing SMC migration by decreasing collagenase production or altering cell shape through changes in F-actin polymerization. Collagenase activity, assessed by zymography using collagen types I and IV, was similar in control and treated DA and aorta cells. Image analysis of the actin cytoskeleton after rhodamine-phalloidin staining of DA SMC, however, revealed significant shortening of INDO-treated DA SMC relative to control, consistent with the observed reduction in migration.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1993KV79100009

  View details for PubMedID 8479815

• THE 67-KD ELASTIN LAMININ-BINDING PROTEIN IS RELATED TO AN ENZYMATICALLY INACTIVE, ALTERNATIVELY SPLICED FORM OF BETA-GALACTOSIDASE JOURNAL OF CLINICAL INVESTIGATION Hinek, A., RABINOVITCH, M., Keeley, F., OKAMURAOHO, Y., Callahan, J. 1993; 91 (3): 1198-1205

  Abstract

  We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.

  View details for Web of Science ID A1993KR46100056

  View details for PubMedID 8383699

• A DEVELOPMENTALLY REGULATED PROGRAM RESTRICTING INSOLUBILIZATION OF ELASTIN AND FORMATION OF LAMINAE IN THE FETAL LAMB DUCTUS-ARTERIOSUS LABORATORY INVESTIGATION Zhu, L., Dagher, E., Johnson, D. J., BEDELLHOGAN, D., Keeley, F. W., Kagan, H. M., RABINOVITCH, M. 1993; 68 (3): 321-331

  Abstract

  The ductus arteriosus (DA) is a fetal vessel in which the elastic laminae fail to assemble normally in late gestation. This feature is associated with the development of intimal cushions, structures that partially occlude the DA lumen and assure that the vessel will close completely when it constricts postnatally.We studied the fetal lamb DA at two different gestational time-points, 100 days before, and 138 days coincident with intimal cushion formation (term = 145 days) to establish the ultrastructural basis for the 'disassembly' of elastic laminae apparent on light microscopy and to determine further whether the mechanism was due to increased elastolytic activity, decreased synthesis of tropoelastin, or impaired insolubilization of tropoelastin.Morphometric ultrastructural analyses of tissue from the 138-day gestation fetal lambs revealed that the volume density of elastin in the DA vessel wall was only 40% of that in the aorta (Ao) and 50% of that in the pulmonary artery (PA). Moreover, only 16% of the elastin present contributed to the formation of laminae when compared to 80% in the Ao and 50% in the PA. Despite the morphologic appearance of 'fragmented' elastin, there was no evidence of increased elastolytic activity in the DA at either gestational time-point as judged by solubilization of a [3H] elastin substrate. The reduced elastin apparent was morphologically accompanied by an increase in soluble (tropo) elastin in DA compared with Ao and PA, as measured by enzyme linked immunosorbent assay, in tissue from both 100- and 138-day gestation lambs. Lack of differences in tropoelastin mRNA levels when comparing the 3 vessels suggested that the enzyme linked immunosorbent assay measurements reflected increased DA tropoelastin accumulation owing to lack of insolubilization rather than an increase in synthesis. Reduced insolubilization of newly synthesized elastin was evident in the DA compared with the Ao at 100 days gestation and in the DA compared with both Ao and PA at 138 days gestation in association with reduced desmosine levels.The mechanism of the decrease in tropoelastin insolubilization was unrelated to lysyl oxidase activity in the tissue and represents a unique developmental program.

  View details for Web of Science ID A1993KT43800009

  View details for PubMedID 8095564

• CORONARY ARTERIOPATHY POSTCARDIAC TRANSPLANT IN A HETEROTOPIC PIGLET MODEL - THE ROLE OF MATRIX-CYTOKINE INTERACTION JEAN HAMBURGER MEMORIAL CONGRESS / 14TH INTERNATIONAL CONGRESS OF THE TRANSPLANTATION SOC Clausell, N., Coles, J., RABINOVITCH, M. ELSEVIER SCIENCE INC. 1993: 859–60

  View details for Web of Science ID A1993KN62200004

  View details for PubMedID 8442248

• THE DUCTUS-ARTERIOSUS MIGRATORY SMOOTH-MUSCLE CELL PHENOTYPE PROCESSES TROPOELASTIN TO A 52-KDA PRODUCT ASSOCIATED WITH IMPAIRED ASSEMBLY OF ELASTIC LAMINAE JOURNAL OF BIOLOGICAL CHEMISTRY Hinek, A., RABINOVITCH, M. 1993; 268 (2): 1405-1413

  Abstract

  We established the identity of a 52-kDa protein secreted by fetal lamb ductus arteriosus (DA) smooth muscle cells (SMC) and suggest how it might be related to structural changes unique to DA development, i.e. reduced assembly of elastic laminae and associated formation of intimal cushions. We produced a monoclonal antibody (HI-20) to the 52-kDa protein and observed, by electron microscopy, immunogold labeling of elastin in both DA and aorta vessel walls. Western immunoblotting showed that HI-20, as well as antibodies to tropoelastin, reacted with the 52-kDa protein secreted by DA SMC, as well as with 68-kDa tropoelastin. The highly specific antibody to the carboxyl-terminal sequence of tropoelastin failed, however, to recognize the 52-kDa protein, although it reacted well with the 68-kDa tropoelastin. Amino acid analysis and sequencing data confirmed the identity of the affinity-purified 52-kDa protein as truncated tropoelastin with an intact amino terminus. Cell-free translation of mRNA extracted from DA and aorta SMC produced a 68-kDa, but not a 52-kDa, immunoprecipitated tropoelastin. When DA and aorta SMC were pulsed with [14C]valine, we immunoprecipitated, after only a 15-min chase, both 68-kDa and 52-kDa tropoelastin from cell extracts of DA SMC, but only the 68-kDa tropoelastin was present in aorta SMC. There was no evidence of proteolytic degradation of radiolabeled aorta 68-kDa tropoelastin to a 52-kDa species when mixed with DA SMC conditioned medium. This suggests that the 52-kDa tropoelastin is the result of cell-associated processing or degradation of an original 68-kDa product of translation. Furthermore, pulse-chase experiments showed initial secretion of equivalent amounts of 68-kDa and 52-kDa tropoelastins by cultured DA SMC with increasing accumulation of the 52-kDa species, suggesting its impaired insolubilization. The production, in high concentration, of a 52-kDa tropoelastin product that lacks the carboxyl terminus, may prevent its alignment on the microfibrillar scaffold, resulting in abnormal assembly of elastic laminae in the DA. The accumulation of this soluble tropoelastin may be associated with the previously described property of chemotaxis resulting in the increased SMC migration into the subendothelium associated with DA intimal thickening.

  View details for Web of Science ID A1993KG07700096

  View details for PubMedID 8419341

• RELATIVE EFFICIENCY OF INCORPORATION OF NEWLY SYNTHESIZED ELASTIN AND COLLAGEN INTO AORTA, PULMONARY-ARTERY AND PULMONARY VEIN OF GROWING PIGS CONNECTIVE TISSUE RESEARCH Johnson, D. J., LABOURENE, J., RABINOVITCH, M., Keeley, F. W. 1993; 29 (3): 213-221

  Abstract

  It is generally accepted that, once laid down in the extracellular matrix, elastin turns over only very slowly if at all. However, much of the evidence for minimal turnover of elastin comes from aortic tissue. In this study we have compared the relative synthesis rates of elastin and collagen with their relative accumulation rates in segments of aorta (AO), pulmonary artery (PA), and pulmonary vein (PV) of young, growing pigs. While rates of elastin synthesis are comparable in the AO and PA, the PA accumulates only 33% of the elastin deposited in the AO. Similarly, while the rate of elastin synthesis in the PV is 60% of that in the AO, accumulation of elastin is only 20% of that in the AO. Similar discrepancies between collagen synthesis and accumulation were seen among these three vessels. These data suggest that, at least in growing animals, the efficiency of permanent incorporation of elastin and collagen into the extracellular matrix may be an important factor in determining the final contents of these connective tissue proteins in vascular tissues.

  View details for Web of Science ID A1993LQ41100005

  View details for PubMedID 8222648

• VASCULAR SMOOTH-MUSCLE CELL DETACHMENT FROM ELASTIN AND MIGRATION THROUGH ELASTIC LAMINAE IS PROMOTED BY CHONDROITIN SULFATE-INDUCED SHEDDING OF THE 67-KDA CELL-SURFACE ELASTIN BINDING-PROTEIN EXPERIMENTAL CELL RESEARCH Hinek, A., BOYLE, J., RABINOVITCH, M. 1992; 203 (2): 344-353

  Abstract

  Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in N-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a three-dimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.

  View details for Web of Science ID A1992KD84200008

  View details for PubMedID 1333980

• TRANSFORMING GROWTH-FACTOR-BETA REGULATES INCREASED DUCTUS-ARTERIOSUS ENDOTHELIAL GLYCOSAMINOGLYCAN SYNTHESIS AND A POSTTRANSCRIPTIONAL MECHANISM CONTROLS INCREASED SMOOTH-MUSCLE FIBRONECTIN, FEATURES ASSOCIATED WITH INTIMAL PROLIFERATION LABORATORY INVESTIGATION Boudreau, N., Clausell, N., BOYLE, J., RABINOVITCH, M. 1992; 67 (3): 350-359

  Abstract

  In previous studies we established that there are developmentally regulated increases in endothelial hyaluronan (HA) and heparan sulfate (HS), and smooth muscle cell fibronectin (FN) related to the formation of intimal cushions, structures essential to the postnatal closure of the ductus arteriosus (DA). In this report, we investigated the mechanisms underlying these features to ascertain whether they were independently or coordinately regulated.We determined by assessing HA polymer size and by pulse labeling with [3H]glucosamine whether the increased glycosaminoglycans (GAGs) incorporated in DA compared with aorta (Ao) endothelial cell matrices reflected increased synthesis of HA and HS rather than decreased degradation. We assessed whether transforming growth factor-beta (TGF-beta) may be responsible for the increased DA endothelial GAGs and smooth muscle FN production by confirming the presence of TGF-beta in DA tissue using immunohistochemistry and by assessing the effect of adding neutralizing antibodies to the cell cultures. We next determined whether the level of regulation of the increase in FN in DA smooth muscle cells was transcriptional or post-transcriptional by relating protein synthesis to steady state mRNA levels and to mRNA levels after serum stimulation. Using northern blot analyses with specific probes, we also explored the possibility that the FN produced by the DA and Ao was qualitatively different in the proportion of isoforms containing the V95+ region associated with secretion or the EIIIB+ region that has been related to migration.We observed that HA polymer size produced by DA and Ao endothelial cells was similar and we further verified using pulse labeling that the increase in DA compared with Ao endothelial GAGs reflected increased synthesis of HA and HS rather than decreased degradation. There was increased immunostaining for TGF-beta in DA compared with Ao tissue and we showed that TGF-beta neutralizing antibodies reduced synthesis of GAGs by the DA endothelial cells to the level of that seen in the Ao cells, but did not reduce DA smooth muscle cell FN synthesis. The increase in FN synthesis by DA compared to Ao smooth muscle cells was not associated with increased levels of steady-state mRNA for FN. Furthermore, following serum-stimulated increases in FN mRNA, the DA yielded greater amounts of FN protein compared to Ao smooth muscle cells. The increased FN production by the DA smooth muscle cells could not be attributed to a relative lack of degradation of FN protein or mRNA, or to qualitative differences which might influence secretion, as both cell types contained similar proportions of EIIIB+ and V95+ isoforms of FN.These results would suggest that, in contrast to the TGF-beta dependent increase in DA endothelial GAG synthesis, the increase in DA smooth muscle FN synthesis arises through differences in post-transcriptional regulation that are likely independent of TGF-beta.

  View details for Web of Science ID A1992JP57200008

  View details for PubMedID 1405493

• INCREASED PULMONARY-ARTERY ELASTOLYTIC ACTIVITY IN ADULT-RATS WITH MONOCROTALINE-INDUCED PROGRESSIVE HYPERTENSIVE PULMONARY VASCULAR-DISEASE COMPARED WITH INFANT RATS WITH NONPROGRESSIVE DISEASE AMERICAN REVIEW OF RESPIRATORY DISEASE TODOROVICHHUNTER, L., Dodo, H., Ye, C. L., McCready, L., Keeley, F. W., RABINOVITCH, M. 1992; 146 (1): 213-223

  Abstract

  In a rat model of pulmonary hypertension induced by monocrotaline, medial hypertrophy of the pulmonary arteries is associated with enhanced production (synthesis) of insoluble elastin relative to accumulation and an increased number of elastin fragments, features suggestive of an elastolytic process. In the present study, we measured and characterized pulmonary artery (PA) elastolytic activity at time points before as well as coincident with the progression of medial hypertrophy in monocrotaline-injected adult male Sprague-Dawley rats. We also determined whether medial hypertrophy is preceded by ultrastructural changes in elastin. Since medial hypertrophy develops but fails to progress in rats injected with monocrotaline at 8 days of age, we assessed whether, compared with adult rats, there were also structural and biochemical differences in elastin and elastolytic activity. A twofold increase in elastolytic activity per milligram tissue was observed 2 days after monocrotaline injection in adult rats (p less than 0.01), and there was an increased number of breaks in the internal elastic lamina (IEL) at 4 days (p less than 0.05) (i.e., before the development of medial hypertrophy). Associated with the progression of medial hypertrophy between 16 and 28 days after monocrotaline injection, there was a further threefold increase in elastolytic activity per milligram tissue by 28 days (p less than 0.01). Susceptibility of the elastolytic activity to specific inhibitors suggested that one or more serine elastases is involved. In infant rats in which medial and right ventricular hypertrophy fail to progress in severity between 16 and 28 days after monocrotaline injection, we did not measure an increase in elastolytic activity, nor was there evidence of an increase in the number of breaks in the IEL at 4 days, suggesting a lack of increased elastolytic activity at an earlier time point. The total content of PA elastin in infant rats, although increased compared with control rats (p less than 0.01), was not associated with heightened production and appeared ultrastructurally as thicker laminae (p less than 0.05) rather than as fragments previously reported in adult rats.

  View details for Web of Science ID A1992JC56900039

  View details for PubMedID 1626806

• FACTORS INFLUENCING THE IDIOPATHIC DEVELOPMENT OF PULMONARY-HYPERTENSION IN THE FAWN HOODED RAT AMERICAN REVIEW OF RESPIRATORY DISEASE Sato, K., Webb, S., TUCKER, A., RABINOVITCH, M., OBRIEN, R. F., McMurtry, I. F., Stelzner, T. J. 1992; 145 (4): 793-797

  Abstract

  Fawn hooded rats (FHR), a strain of rat with a hereditary bleeding tendency due to a genetic defect in platelet aggregation, have recently been found to develop pulmonary hypertension. However, whether the pulmonary hypertension in FHR has a genetic basis or simply reflects the influence of extrinsic factors known to increase pulmonary artery pressure in other rat strains has not been fully evaluated. To further examine the structural and hemodynamic changes of pulmonary hypertension in FHR, and to investigate the extent to which alveolar hypoxia may have promoted these abnormalities, hemodynamic and morphometric measurements were made in FHR (4 to 24 wk) and compared with age-matched Sprague-Dawley (SDR) control rats. Increases in mean pulmonary artery pressure, total pulmonary resistance, and right ventricular enlargement were present in both male and female FHR and were evident at an early age (4 wk). Morphometric analysis of barium gelatin-infused lungs revealed marked pulmonary vascular remodelling in FHR characterized as extension of muscle into more peripheral pulmonary vessels, medial hypertrophy of proximal vessels, and reduced number of barium-filled arteries. The increases in pulmonary artery pressure in FHR were not due to the influence of more severe hypoxia, hypoventilation, or polycythemia, as blood gas tension and hematocrit were similar in FHR and SDR. Moreover, we found that pulmonary hypertension could be transmitted to backcross and second filial generation offspring arising from selective matings between FHR and control Wistar Kyoto rats, confirming the heritable basis for pulmonary hypertension in the FHR.

  View details for Web of Science ID A1992HM12000013

  View details for PubMedID 1554204

• NEW DEVELOPMENTS IN THE PULMONARY CIRCULATION IN CHILDREN CURRENT OPINION IN CARDIOLOGY Ye, C. L., RABINOVITCH, M. 1992; 7 (1): 124-133

  Abstract

  This article reviews new advances in the diagnosis, treatment, and pathophysiology of pulmonary hypertension. In diagnosis, Doppler echocardiography has been refined and its accuracy increased, and magnetic resonance imaging is being developed as a new tool. Prostacyclin and calcium channel blockers remain mainstays of short-term therapy, and single-lung transplantation has become a new therapeutic option. Newer pharmacotherapeutics are largely directed at reversing acute vasoconstriction and include approaches related to ATP and endothelial-derived relaxing factor. In pathophysiology, recent studies have shown the role of elastase in mediating the initiation and progression of pulmonary hypertension, the regulation of gene expression of collagen, elastin, endothelin, and growth factors including transforming growth factor-B and insulinlike growth factor-I, and mechanisms of signal transduction of smooth muscle cell proliferation. The migratory smooth muscle cell phenotype has been studied in terms of altered expression of endothelial and smooth muscle extracellular matrix components, including hyaluronan and fibronectin, respectively.

  View details for Web of Science ID A1992HG23900018

  View details for PubMedID 10149847

• ASSOCIATION OF PULMONARY-HYPERTENSION WITH CONGENITAL PORTAL-HYPERTENSION IN A CHILD JOURNAL OF PEDIATRICS Silver, M. M., Bohn, D., Shawn, D. H., Shuckett, B., Eich, G., RABINOVITCH, M. 1992; 120 (2): 321-329

  View details for Web of Science ID A1992HC90100031

  View details for PubMedID 1735836

• ABNORMALITIES IN VONWILLEBRAND-FACTOR AND ANTITHROMBIN-III AFTER CARDIOPULMONARY BYPASS OPERATIONS FOR CONGENITAL HEART-DISEASE JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY TURNERGOMES, S. O., Andrew, M., Coles, J., TRUSLER, G. A., Williams, W. G., RABINOVITCH, M. 1992; 103 (1): 87-97

  Abstract

  In patients with congenital heart disease two poorly understood postoperative complications are pulmonary hypertensive crises after repair of large atrioventricular or ventricular septal defects and right atrial and pulmonary thrombi after the Fontan operation. In this study we assessed whether cardiopulmonary bypass in these patients is associated with the release of agents that might induce platelet aggregation and vasoconstriction, such as biologically active von Willebrand factor and platelet-activating factor. In addition, we measured levels of anticoagulants such as antithrombin III and proteins C and S. Three groups of patients with congenital heart disease undergoing cardiopulmonary bypass were monitored through the perioperative period for secundum atrial septal defects, large atrioventricular or ventricular septal defects, and tricuspid atresia or univentricular heart (Fontan candidates). Control values were obtained from age-matched patients; patients requiring major noncardiac operations and those with cardiac disease not requiring cardiopulmonary bypass were also studied. After cardiopulmonary bypass in all three groups biologic activity of von Willebrand factor increased markedly in the immediate and early postoperative periods compared with preoperative values, whereas antithrombin III values were decreased. Platelet-activating factor was detected in only two patients with congenital heart disease, both in the early postoperative period. In contrast, patients who did not have cardiopulmonary bypass did not show these abnormalities. All measured parameters normalized at late follow-up (6 to 18 months after operation). Although cardiopulmonary bypass in these patients resulted in increased von Willebrand factor activity and decreased antithrombin III, changes that may predispose the patient to platelet aggregation and thrombus formation, absolute values in individual patients alone were not predictive of pulmonary hypertensive crises or detectable thrombi. This suggests that these hematologic abnormalities may contribute to but are not by themselves a cause of morbidity in the early postoperative period. Moreover, the increased von Willebrand factor biologic activity seen postoperatively in patients with congenital heart disease suggests that use of synthetic vasopressin may be ineffective and potentially detrimental.

  View details for Web of Science ID A1992GY98200015

  View details for PubMedID 1728719

• AUTOIMMUNE-DISEASE AND UNEXPLAINED PULMONARY-HYPERTENSION CIRCULATION RABINOVITCH, M. 1992; 85 (1): 380-381

  View details for Web of Science ID A1992GY58200050

  View details for PubMedID 1728476

• IMPAIRED ELASTIN FIBER ASSEMBLY RELATED TO REDUCED 67-KD ELASTIN-BINDING PROTEIN IN FETAL LAMB DUCTUS-ARTERIOSUS AND IN CULTURED AORTIC SMOOTH-MUSCLE CELLS TREATED WITH CHONDROITIN SULFATE JOURNAL OF CLINICAL INVESTIGATION Hinek, A., Mecham, R. P., Keeley, F., RABINOVITCH, M. 1991; 88 (6): 2083-2094

  Abstract

  In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.

  View details for Web of Science ID A1991GU96700040

  View details for PubMedID 1661296

• CHRONIC HYPOXIC PULMONARY-HYPERTENSION IN RATS AND INCREASED ELASTOLYTIC ACTIVITY AMERICAN JOURNAL OF PHYSIOLOGY Maruyama, K., Ye, C. L., Woo, M., VENKATACHARYA, H., Lines, L. D., Silver, M. M., RABINOVITCH, M. 1991; 261 (6): H1716-H1726

  Abstract

  Previously in rats injected with the toxin monocrotaline and administered SC-39026, a serine elastase inhibitor, pulmonary hypertension was decreased in association with reduced muscularization of peripheral pulmonary arteries. To determine whether inhibition of elastolytic activity might prevent this vascular change in other conditions producing pulmonary hypertension, we administered SC-39026 to rats during a 10-day exposure to chronic hypobaric hypoxia. We also measured elastolytic activity in the central pulmonary arteries of rats using [3H]elastin substrate and determined whether there was an increase in activity either as early as 2 days or at completion of the hypoxic exposure, which could be inhibited by SC-39026. to further determine whether the mechanism of muscularization of peripheral arteries is modulated by degradation of elastin or other elastase-susceptible extracellular matrix proteins, we assessed desmosine excretion and ultrastructural alterations in elastin as well as in type IV collagen, fibronectin, and laminin. SC-39026 reduced the number of muscularized arteries and the level of pulmonary arterial pressure during exposure to chronic hypoxia. Elastolytic activity was fourfold higher in central pulmonary arteries 2 days after hypoxia when compared with values in control vessels, and the activity was inhibited by SC-39026. In small peripheral pulmonary arteries there were no significant changes with hypoxia reflected in desmosines or in the immunocytochemistry of elastase-susceptible glycoproteins, with the exception of decreased laminin. This feature was not inhibited by SC-39026. To further assess whether the protective effect of SC-39026 was related to its inhibition of elastase, an extended study was carried out using a different elastase inhibitor, alpha 1-proteinase inhibitor. An even greater reduction in hypoxia-induced pulmonary hypertension and vascular changes was observed with this elastase inhibitor and the latter included medial hypertrophy.

  View details for Web of Science ID A1991GX68700006

  View details for PubMedID 1836309

• LEISHMANIA-AMAZONENSIS - SPECIFIC LABELING OF AMASTIGOTE CYSTEINE PROTEINASES BY RADIOIODINATED N-BENZYLOXYCARBONYL-TYROSYL-ALANYL DIAZOMETHANE EXPERIMENTAL PARASITOLOGY Alfieri, S. C., Pral, E. M., Shaw, E., Ramazeilles, C., RABINOVITCH, M. 1991; 73 (4): 424-432

  Abstract

  Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.

  View details for Web of Science ID A1991GP83400005

  View details for PubMedID 1959570

• INHIBITION OF ELASTOLYSIS BY SC-37698 REDUCES DEVELOPMENT AND PROGRESSION OF MONOCROTALINE PULMONARY-HYPERTENSION AMERICAN JOURNAL OF PHYSIOLOGY Ye, C. L., RABINOVITCH, M. 1991; 261 (4): H1255-H1267

  Abstract

  Our previous studies showed that increased pulmonary artery elastolytic activity is associated with monocrotaline-induced pulmonary hypertension in rats, and the latter is reduced by the elastase inhibitor SC-39026. This agent, given orally, decreases monocrotaline-induced muscularization of normally nonmuscular peripheral arteries but not medial hypertrophy of muscular arteries. To establish whether constant infusion of an elastase inhibitor would reduce both vascular lesions induced by monocrotaline injection, SC-37698 (an analogue of SC-39026) was given intravenously by osmopump. To separately assess whether SC-37698 would inhibit development of the vascular changes as well as their progression, SC-37698 or vehicle was infused for the first 2 wk (2-wk study) or was delayed until 1 wk after monocrotaline injection (3-wk study). Hemodynamic data were recorded from indwelling catheters, and the lungs were evaluated morphologically. Saline-injected control rats given SC-37698 or vehicle were similar at both time points. Monocrotaline-injected rats given SC-37698 compared with those given vehicle alone had lower pulmonary artery pressures, 17.9 +/- 0.5 vs. 23.7 +/- 0.8 mmHg (P less than 0.01) in the 2-wk study and 24.0 +/- 1.8 vs. 33.5 +/- 3.1 mmHg (P less than 0.05) in the 3-wk study. This was associated with significant decreases in muscularization of peripheral arteries and reductions in medial hypertrophy of muscular arteries. In the hilar pulmonary arteries assessed at 3 wk only, SC-37698 significantly decreased monocrotaline-induced endothelial injury, subendothelial edema, migration of smooth muscle cells into subendothelium, medial hypertrophy, collagen accumulation, and abnormal distribution of elastin as interlamellar islands. Pulmonary artery elastolytic activity was reduced in SC-37698-treated compared with untreated monocrotaline-injected rats (P less than 0.05). Thus infusion of SC-37698 reduces monocrotaline-induced pulmonary hypertension when administered before or even after development of early vascular changes.

  View details for Web of Science ID A1991GK86900038

  View details for PubMedID 1928407

• EFFECT OF AMBIENT OXYGEN CHANGES ON PLATELET-ACTIVATING-FACTOR PRODUCTION BY FETAL OVINE ENDOTHELIAL-CELLS PROSTAGLANDINS TURNERGOMES, S. O., Boudreau, N., RABINOVITCH, M. 1991; 41 (5): 463-472

  Abstract

  To determine whether platelet activating factor (PAF) plays a role in the responses seen in the fetal and transitional circulations, we assessed endogenous release of PAF in cultured fetal ovine endothelial cells from the pulmonary artery (PA), ductus arteriosus (DA) and aorta (Ao) under basal conditions and following exposure to hypoxia or hyperoxia. The cells were prelabeled with [3H] acetate and subsequently exposed to different ambient oxygen concentrations, i.e., 95% O2 or 95% N2, balance CO2, using calcium ionophore as a positive control. The effect of indomethacin on DA endothelial PAF production following stimulation with ionophore was also established. Synthesis of [3H] PAF was measured by counts comigrating on TLC with unlabeled PAF. We found that PAF production by fetal ovine PA, Ao and DA cells was similar and unaffected by hypoxia or hyperoxia. Exposure of ionophore stimulated DA cells to indomethacin was, however, associated with a decrease in PAF production (p less than 0.05). We speculate that in vitro alterations in ambient O2 concentration do not influence fetal ovine endothelial PAF production but indomethacin may decrease PAF production in the DA.

  View details for Web of Science ID A1991FK93900005

  View details for PubMedID 1862226

• FIBRONECTIN, HYALURONAN, AND A HYALURONAN BINDING-PROTEIN CONTRIBUTE TO INCREASED DUCTUS-ARTERIOSUS SMOOTH-MUSCLE CELL-MIGRATION DEVELOPMENTAL BIOLOGY Boudreau, N., Turley, E., RABINOVITCH, M. 1991; 143 (2): 235-247

  Abstract

  "Intimal cushions" which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previous in vitro studies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded Da or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 +/- 3.1% vs 16.2 +/- 2.3%, P less than 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5-1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 +/- 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 micrograms/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observed in vivo.

  View details for Web of Science ID A1991EV51200004

  View details for PubMedID 1703972

• DEVELOPMENTALLY REGULATED CHANGES IN EXTRACELLULAR-MATRIX IN ENDOTHELIAL AND SMOOTH-MUSCLE CELLS IN THE DUCTUS-ARTERIOSUS MAY BE RELATED TO INTIMAL PROLIFERATION LABORATORY INVESTIGATION Boudreau, N., RABINOVITCH, M. 1991; 64 (2): 187-199

  Abstract

  In the late gestation fetal lamb ductus arteriosus (DA), intimal proliferation is observed, characterized by smooth muscle migration and proliferation in the subendothelium. The nature of changes in the endothelial and smooth muscle extracellular matrix associated with the development of this feature are not known. We assessed the production of glycoproteins (fibronectin, laminin, and type IV collagen) and glycosaminoglycans (GAGs) (hyaluronic acid, heparan sulfate, and chondroitin sulfate) in endothelial and smooth muscle cells harvested from the DA, aorta (Ao), and pulmonary artery of fetal lambs at 100 days gestation, before the appearance of DA intimal proliferation, and at 138 days, when well-developed intimal 'cushions' are seen. In passage 3 cells, glycoprotein synthesis was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after 48 hours incubation with [35S]methionine, and GAGs were assessed by labeling with [3H] glucosamine and separation on DEAE ion-exchange high performance liquid chromatography. Analyses were carried out on culture medium, cell layer, and solubilized matrix. Fibronectin secretion by DA smooth muscle cells from 100-day lambs was found to be twice that of Ao or pulmonary artery cells. No significant differences were seen in smooth muscle cells from 138-day lambs or when comparing endothelial cells from each of the vascular sites at both gestational ages. As well, there were no DA-specific differences in laminin or type IV collagen. No significant differences in endothelial GAG secretion were observed comparing each vascular site at both gestational ages. Analysis of endothelial-derived matrices, however, revealed increased incorporation of hyaluronic acid in the DA from 100-day lambs, 10-fold that of the pulmonary artery and Ao, and increased heparan sulfate. These differences were still present in cell matrices from late gestation animals, but were less marked. No differences in GAGs were seen when comparing smooth muscle cells. Incubation of 100-day DA and Ao smooth muscle cells with endothelial conditioned medium however, resulted in a 2-fold increase in chondroitin sulfate in DA, compared with Ao. These results indicate that distinct, developmentally regulated patterns of extracellular matrix production are related to vascular site and specific features appear to precede intimal proliferation in the DA.

  View details for Web of Science ID A1991EY30800006

  View details for PubMedID 1997732

• INVESTIGATIONAL APPROACHES TO PULMONARY-HYPERTENSION 10TH INTERNATIONAL SYMP OF THE SOC OF TOXICOLOGIC PATHOLOGISTS : PULMONARY TOXICOLOGIC PATHOLOGY RABINOVITCH, M. SOC TOXICOLOGIC PATHOLOGISTS. 1991: 458–69

  Abstract

  Pulmonary vascular disease (PVD) revolves around a series of switches in the smooth muscle cell (SMC) phenotype. Differentiation of SMC from precursor cells causes muscularization of normally non-muscular peripheral arteries; hypertrophy and hyperplasia of existing SMC and increased connective tissue protein synthesis cause thickening of the wall, and migration of SMC into the subendothelial space is the basis of intimal proliferation. To uncover the pathophysiologic mechanisms of these changes, we have used a variety of animal models and cell culture systems. From rats in which hypertensive PVD was induced by exposure to chronic hypoxia or following injection of the pyrrolizidine alkaloid, monocrotaline, we have identified increased pulmonary artery (PA) elastolytic activity which occurs early and which accompanies progressive rather than reversible PVD. Inhibition of elastolytic activity prevents or reduces PVD. We are cloning the gene for this new enzyme to study its regulation in PVD. To address the mechanism of SMC proliferation under conditions of high PA pressure and flow, we cultured endothelial cells on polyvinylchloride membranes and pulsated them at high pressure. This caused reduced synthesis of heparan sulfate. The resulting decrease binding of fibroblast growth factor would lessen its mitogenic effect and modulate SMC proliferation in response to other growth factors from platelets or serum. To study SMC migration, we cultured endothelial and SMC from the ductus arteriosus (a fetal vessel which spontaneously develops intimal proliferation in late gestation). The migratory SMC phenotype is a function of increased production of fibronectin governed by a translational control mechanism, and increased endothelial hyaluronan regulated by transforming growth factor beta. SMC migration is also related to impaired assembly of elastin, the result of a chondroitin sulfate-induced decrease in elastin binding proteins and the production of a novel 'defunct' 52 kD tropoelastin.

  View details for Web of Science ID A1991HQ74900014

  View details for PubMedID 1813989

• Pathophysiology of dynamic cardiomyoplasty: a clinico-pathological case study. Journal of cardiac surgery Odim, J. N., BURGESS, J. H., Williams, B. H., Blundell, P. E., Rabinovitch, M. A., Stewart, J. A., LOUGH, J. O., Chiu, R. C. 1990; 5 (4): 336-346

  Abstract

  A 58-year-old man with end-stage ischemic cardiomyopathy underwent dynamic cardiomyoplasty. "On" and "off" studies with the cardiac assist device failed to show any significant hemodynamic changes despite improvement in functional status. The patient's late postoperative course was complicated by two episodes of acute pulmonary edema followed by cardiac arrest. These events were precipitated by ventricular tachycardia. The last episode led to myocardial infarction requiring diastolic counterpulsation and inotropic support. He died 4 1/2 months following the cardiomyoplasty. Postmortem findings revealed an anterior left ventricular infarct with aneurysm. There was fusion of skeletal muscle to the epicardium with minimal fibrosis and atrophy. The latissimus dorsi (LD) flap was viable, but myofibrillar ATPase stain revealed incomplete transformation. Several clinical observations have emerged from the early experience with dynamic cardiomyoplasty: (1) Important arrhythmias and cardiac arrest compromise the vascular supply and thus power of the muscular flap; (2) Resting ejection fraction does not correlate with exercise tolerance, therefore, other parameters must be sought to explain improved functional status; (3) Uniform muscle transformation in humans may be unpredictable with current clinical stimulation protocols. The conformation of LD to the epicardium underscores a potential remodeling phenomenon which may ultimately spare the diseased myocardium by altering its oxygen supply/demand ratio and thus the natural history.

  View details for PubMedID 2133867

• William Glenn lecture. The cavopulmonary shunt. Evolution of a concept. Circulation TRUSLER, G. A., Williams, W. G., Cohen, A. J., RABINOVITCH, M., MOES, C. A., Smallhorn, J. F., Coles, J. G., Lightfoot, N. E., Freedom, R. M. 1990; 82 (5): IV131-8

  Abstract

  A bold and imaginative development, the cavopulmonary anastomosis, appeared to originate in several centers almost simultaneously. After extensive research on right heart bypass, Glenn was the first in North America to perform a successful experimental cavopulmonary shunt, and it became known by his name. In properly selected patients, palliation success was excellent, and mortality rates were low. From 1961 through 1988, we used a cavopulmonary anastomosis for palliation in 139 infants and children. There were eight hospital deaths, and most occurred early in the series. Palliation generally lasted 6-8 years-until the child outgrew the blood supply to the contralateral lung. Palliation could be restored by increased flow to that lung with another shunt. Six otherwise inoperable patients received benefit from the addition of an axillary arteriovenous fistula. Late pulmonary arteriovenous fistulas were identified in 11% of our patients by angiography, but with more sensitive testing, the incidence rate may be as high as 21%. The occurrence of pulmonary arteriovenous fistulas caused general concern and less frequent use of the shunt. Recent application of an end-to-side anastomosis, creating a bidirectional shunt, has restored interest. A major legacy of the cavopulmonary anastomosis was demonstration of the feasibility of partial right heart bypass, which paved the way for the Fontan operation, and it is frequently constructed as part of that operation. Currently, the Glenn shunt is most often used as a temporary or permanent alternative to a Fontan repair if there appears to be significant risk. The risk factors usually encountered include small pulmonary arteries, young age, poor ventricular function, atrioventricular valve incompetence, and myocardial hypertrophy-sometimes alone but often in combination.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for PubMedID 1699683

• THE CAVOPULMONARY SHUNT - EVOLUTION OF A CONCEPT CIRCULATION TRUSLER, G. A., Williams, W. G., Cohen, A. J., RABINOVITCH, M., MOES, C. A., Smallhorn, J. F., Coles, J. G., Lightfoot, N. E., Freedom, R. M. 1990; 82 (5): 131-138

  View details for Web of Science ID A1990EJ51000018

• Effective endothelialization of polyurethane surfaces. Response to shear stress and platelet adhesion. ASAIO transactions / American Society for Artificial Internal Organs Zhu, L., Williams, W. G., BELLHOUSE, B., Pugh, S., RABINOVITCH, M. 1990; 36 (4): 811-816

  Abstract

  Biomer and Mitrathane are thromboresistant polyurethane ureas that are suitable for coating artificial valves. Degeneration of the surface coatings, however, does occur over time, and in experimental animal studies adherent thrombi have been observed as a complication. In this study, morphometric techniques applied to scanning electron microscopy were used to assess whether vascular endothelial cells harvested from jugular veins of fetal lambs could be grown to confluence on these polyurethane surfaces, whether the monolayer would remain intact under conditions of shear stress (104 dynes/cm2), and whether this would result in decreased platelet adherence of sheep platelets relative to nonendothelialized surfaces. The results have shown that both Biomer and Mitrathane could be endothelialized: 88.8 +/- 5.1% of the Biomer surface consisted of intact confluent endothelial cells, as did 95.45 +/- 1.7% of the Mitrathane surface. After 6 hr of shear stress, a significant reduction in this feature was observed (p = 0.02), but both materials still maintained a high percentage of confluent endothelial cells (78.65% for Biomer and 85.58% for Mitrathane). After 48 hr of shear stress, however, the percent confluence was similar to control values, which suggested new cell growth. Endothelialized Biomer compared with Mitrathane had fewer adherent single platelets, or small or large aggregates (p = 0.001, p = 0.01, and p = 0.05, respectively); this feature was not affected by shear stress. Whereas endothelialization of the surface clearly decreased platelet adherence on Mitrathane (p = 0.01), only a trend was seen with Biomer. These studies therefore show that endothelialization of these polyurethane surfaces is feasible.

  View details for PubMedID 2268484

• ALTERATIONS IN ELASTIN AND COLLAGEN RELATED TO THE MECHANISM OF PROGRESSIVE PULMONARY VENOUS OBSTRUCTION IN A PIGLET MODEL - A HEMODYNAMIC, ULTRASTRUCTURAL, AND BIOCHEMICAL-STUDY CIRCULATION RESEARCH LaBourene, J. I., Coles, J. G., Johnson, D. J., Mehra, A., Keeley, F. W., RABINOVITCH, M. 1990; 66 (2): 438-456

  Abstract

  We created an animal model to understand better the pathogenesis and underlying mechanism of progressive central pulmonary venous (PV) obstruction, a condition not amenable to current therapy. Twenty piglets underwent banding of their PVs, 18 had a sham operation, and 12 were nonoperated controls. After 1, 3, and 6 weeks hemodynamic data were obtained and correlated with ventricular weights, PV and pulmonary artery (PA) distensibilities (at 1 week), morphometric structural and ultrastructural analyses, and biochemical assessment of elastin determined gravimetrically (and by desmosine level at 1 week), collagen, and elastase activity. At 1 week, PV banding was associated with increased PV compliance (p less than 0.05). At 3 weeks, an increased PA pressure (Ppa) (p less than 0.05) was observed, unaccompanied by a rise in PV pressure (Pcw). In the PV, however, there was breakdown of the internal elastic lamina with apparent migration of smooth muscle cells from media to subendothelium. At 6 weeks, a rise in Pcw (p less than 0.01), a further rise in Ppa (p less than 0.01), and right ventricular hypertrophy (p less than 0.005) were observed. We also observed mild PV intimal thickening (p less than 0.01), complete degradation of elastic laminae (p less than 0.05), and an increase in collagen assessed morphometrically (p less than 0.01). The banding procedure resulted in an overall increase in PV elastin synthesis and in the proportion of elastin determined gravimetrically (p less than 0.05 for both) but not by desmosine level, suggesting the possibility of poor cross-linking of elastin, which might account for the early increased distensibility of the PV. However, our assay could not detect an increase in elastase activity associated with either the increased distensibility or the ultrastructural changes of elastin degradation. The increased Ppa was not associated with significant PA biochemical or structural changes. We speculate that in response to distal venous obstruction, early remodeling of the PVs increases distensibility, protecting the lung from venous congestion and blunting a rise in Pcw. PA hypertension precedes the rise in Pcw, likely because of reflex vasoconstriction. The subsequent modest rise in Pcw is already associated with extensive fibrosis of the PV, suggesting a reason for unsuccessful current therapy and a need for consideration of earlier assessment and intervention.

  View details for Web of Science ID A1990CM15800018

  View details for PubMedID 2297812

• New developments in the pathogenesis of pulmonary hypertension in the newborn and child. Acta paediatrica Japonica; Overseas edition RABINOVITCH, M. 1989; 31 (6): 631-640

  Abstract

  We have identified several features which appear to play a key role in the pathogenesis of pulmonary vascular disease. Future work will involve exploring the regulation of elastase and elastin synthesis, control of smooth muscle proliferation and production of extracellular matrix. Application of cellular and molecular techniques in vascular research should ultimately lead to new and improved therapeutic approaches.

  View details for PubMedID 2516392

• Structure and function of the pulmonary vascular bed: an update. Cardiology clinics RABINOVITCH, M. 1989; 7 (4): 895-914

  Abstract

  This article discusses the recent information regarding normal and abnormal structural maturation of the pulmonary vascular bed, features that govern the functional behavior of the pulmonary circulation. Special emphasis is given to the cell biology of the vessel wall.

  View details for PubMedID 2688888

• PREDICTION OF THE LEFT-VENTRICULAR EJECTION FRACTION RESPONSE TO DOXORUBICIN USING A MULTIPLE LINEAR-REGRESSION MODEL CANADIAN JOURNAL OF CARDIOLOGY RABINOVITCH, M., Suissa, S., Stern, J., Adam, J., Mettauer, B., Boileau, G., ELSTEIN, J., Rouleau, J. 1989; 5 (8): 382-388

  Abstract

  The aim of this study was to develop a model for predicting clinically significant deterioration in the left ventricular ejection fraction due to chronic doxorubicin administration. Twenty-six patients were monitored during their courses of doxorubicin chemotherapy with serial gated equilibrium radionuclide angiography. Multiple linear regression analysis was used to derived the best combination of clinical and radionuclide angiographic predictors of resting left ventricular ejection fraction at any point during the course of chemotherapy. The final model consisted of five variables: left ventricular ejection fraction at the previous monitoring point; cumulative dose of doxorubicin achieved at the previous monitoring point; increment in dose from the previous monitoring point; age of the patient; and time to peak left ventricular emptying at the previous monitoring point. The cumulative dose, the ejection fraction at the previous monitoring point and the final model, respectively, explained 11%, 33% and 53% of the variability in ejection fraction determinations during the 26 patient courses. The final model also forecast a potentially very low resting left ventricular ejection fraction (less than 35%) at the cumulative doses of doxorubicin which provoked serious clinical cardiotoxicity in two patients. A multivariate model is a useful aid in timing discontinuation of doxorubicin prior to the development of a clinically significant deterioration in left ventricular ejection fraction.

  View details for Web of Science ID A1989CG90200005

  View details for PubMedID 2605548

• OXYGEN-RELATED PROSTAGLANDIN SYNTHESIS IN DUCTUS-ARTERIOSUS AND OTHER VASCULAR CELLS PEDIATRIC RESEARCH RABINOVITCH, M., Boudreau, N., Vella, G., Coceani, F., Olley, P. M. 1989; 26 (4): 330-335

  Abstract

  We compared oxygen-related prostaglandin synthesis in fetal lamb ductus arteriosus (DA) pulmonary artery (PA) and aorta endothelial and smooth muscle cells. We measured basal synthesis of 6-keto-PGF1 alpha and PGE2, the response to calcium ionophore (A23187), a nonspecific stimulus of prostaglandin production, as well as the response to oxygen, a perinatal stimulus, monitoring both the effects of hyperoxia (95% O2) and hypoxia (2% O2). In addition, we established whether differences observed in fetal lamb PA cells related to oxygen tension were also observed in newborn central and microvessel PA cells. Our results indicate that DA endothelial cells increase 6-keto-PGF1 alpha in response to ionophore (p less than 0.05). With hyperoxia, DA endothelial cells increase PGE2 synthesis and DA smooth muscle cells increase 6-keto-PGF1 alpha (p less than 0.05 and 0.02, respectively). Aorta smooth muscle cells increase 6-keto-PGF1 alpha in response to ionophore and hyperoxia (p less than 0.003 and 0.05, respectively). PA endothelial and smooth muscle cells have higher levels of basal prostaglandin synthesis when compared with DA and aorta. In response to ionophore, increased 6-keto-PGF1 alpha is observed in both PA endothelial and smooth muscle cells (p less than 0.02 and 0.0004, respectively), and PGE2 is increased in PA smooth muscle cells (p less than 0.003). Hypoxia, however, decreases PA smooth muscle production of both 6-keto-PGF1 alpha and PGE2 (p less than 0.02 and 0.01, respectively). Similar observations were made in newborn lamb central and microvessel PA cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1989AR78400009

  View details for PubMedID 2508051

• Structure and function of the pulmonary vascular bed: an update. Cardiology clinics RABINOVITCH, M. 1989; 7 (2): 227-238

  Abstract

  This article discusses recent information regarding the normal and abnormal structural maturation of the pulmonary vascular bed, features that govern the functional behavior of the pulmonary circulation. Special emphasis is given to the cell biology of the vessel wall.

  View details for PubMedID 2659172

• SC-39026, A SERINE ELASTASE INHIBITOR, PREVENTS MUSCULARIZATION OF PERIPHERAL ARTERIES, SUGGESTING A MECHANISM OF MONOCROTALINE-INDUCED PULMONARY-HYPERTENSION IN RATS CIRCULATION RESEARCH ILKIW, R., TODOROVICHHUNTER, L., Maruyama, K., Shin, J., RABINOVITCH, M. 1989; 64 (4): 814-825

  Abstract

  In rats injected with the toxin monocrotaline, altered synthesis and distribution of pulmonary artery elastin suggest that increased elastase activity may be important in the development of vascular changes and progressive pulmonary hypertension. To test this hypothesis, male Sprague-Dawley rats (250-300 g) were given 40 mg/kg of the elastase inhibitor SC-39026 in a carboxymethylcellulose vehicle or vehicle only by gavage, 12 hours before and twice daily for 8 days after a single subcutaneous injection of either monocrotaline (60 mg/kg) or saline. Thirteen days after injection, indwelling cardiovascular catheters were inserted under pentobarbital anesthesia, and at 15 days after injection, pulmonary and systemic hemodynamic measurements were recorded with the animals awake. At post-mortem examination, the lungs were perfused and morphometric techniques applied for light and electron microscopic evaluation. Saline-injected rats given either SC-39026 or vehicle were similar in all features assessed. In contrast, monocrotaline-injected rats given SC-39026 had significantly lower mean pulmonary artery pressure than those given vehicle (21.0 +/- 1.6 vs. 27.5 +/- 0.8 mm Hg, p less than 0.05), and this correlated with a significant reduction in the number of abnormally muscularized arteries at alveolar wall level (r2 = 0.89, p less than 0.001). SC-39026 did not significantly reduce monocrotaline-induced medial hypertrophy of muscular arteries, endothelial injury, and associated subendothelial edema; nor was there a significant increase in the proportion of the medial elastin, although a trend was apparent. Additional groups of monocrotaline injected rats were followed 3 weeks after injection, but both SC-39026 and vehicle-treated rats were similar at this point. Our data suggest that increased serine elastase activity associated with endothelial injury may mediate early abnormal pulmonary vascular smooth muscle differentiation resulting in muscularization of normally nonmuscular peripheral arteries and pulmonary hypertension induced in rats by injection of the toxin monocrotaline. Lack of persistence of this protective effect suggests that there may be continued elastase activity in this model. Failure to inhibit medial hypertrophy with SC-39026 suggests that a different mechanism or a different elastase may be involved in this structural change.

  View details for Web of Science ID A1989U129000018

  View details for PubMedID 2495195

• Platelet-endothelial factors. Advances in pediatrics RABINOVITCH, M., Turner-Gomes, S. O. 1989; 36: 91-115

  View details for PubMedID 2675578

• ENDOTHELIAL INJURY AND VASCULAR REACTIVITY IN MONOCROTALINE PULMONARY-HYPERTENSION AMERICAN JOURNAL OF PHYSIOLOGY Rosenberg, H. C., RABINOVITCH, M. 1988; 255 (6): H1484-H1491

  Abstract

  We used awake, unsedated rats with indwelling cardiovascular catheters to study the role of endothelial injury and increased pulmonary vascular reactivity in the pathogenesis of monocrotaline (MC)-induced pulmonary hypertension. Hemodynamic findings were correlated with morphometric analyses of alterations in the vascular endothelium assessed by electron microscopy and of muscularization of pulmonary arteries assessed by light microscopy. Male Sprague-Dawley rats (200-250 g) were injected with MC (60 mg/kg) or with saline vehicle. The hemodynamic response to acute hypoxia (10% O2 for 10 min) was studied at 4, 8, and 12 days postinjection. Pulmonary artery pressures and resistances (Ppa, Rp) were similar in saline- and MC-injected rats at 4 and 8 days postinjection. In response to acute hypoxia, the rise in Ppa was also similar, but there was a slight but significant rise in Rp, (P less than 0.05) in the 8-day group, due largely to a decrease in cardiac output. At 12 days after injection, base-line Ppa was increased in MC-injected rats (P less than 0.01) and there was a heightened response to hypoxia assessed both as a significant increase in Ppa and Rp (P less than 0.05 each). Endothelial injury was observed as early as 4 days postinjection with pallor and swelling evident qualitatively and by a decreased proportion of microfilaments (P less than 0.05) assessed quantitatively. By light microscopy, extension of smooth muscle into normally nonmuscularized pulmonary arteries was evident by 8 days postinjection. By 12 days postinjection there was marked extension of smooth muscle present (P less than 0.01) with medial hypertrophy of muscular arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1988R495800030

  View details for PubMedID 3144186

• INTRALIPID EFFECT ON NORMAL AND HYPOXIC REMODELED RAT PULMONARY VASCULATURE AMERICAN JOURNAL OF PHYSIOLOGY ILKIW, R., Maruyama, K., RABINOVITCH, M. 1988; 255 (6): H1499-H1508

  Abstract

  Intralipid (IL) infusion has been associated with pulmonary vasoconstriction and decreased oxygenation and may worsen preexisting pulmonary vascular changes. To investigate this, we infused IL or 0.9% saline for 1 wk in normal Sprague-Dawley rats and in rats with vascular changes induced by a previous 2-wk exposure to chronic hypobaric hypoxia (air at 380 mmHg). At postmortem we quantitatively evaluated arterial changes in the left lung by light microscopy and alterations in endothelial cells in the right lung by electron microscopy. In rats maintained in room air, 1-wk IL infusion resulted in extension of muscle into alveolar wall and duct arteries (P less than 0.001 for both), medial hypertrophy of arteries 50-99 microns external diameter (P less than 0.01), reduced arterial density (P less than 0.05), and an increase in volume density of endothelial smooth endoplasmic reticulum (P less than 0.05). In post-hypoxia rats, however, IL infusion did not induce further progression of the more severe arterial and endothelial changes observed. To determine whether IL-associated vascular abnormalities may be related to vasoconstriction, different rats were instrumented under pentobarbital sodium anesthesia with indwelling cardiovascular catheters and, 2 days later, with the animals fully conscious, the hemodynamic response assessed. An acute 15-min IL infusion caused a significant increase in pulmonary artery pressure and mild hypoxemia (P less than 0.05 for both) in room air rats but not in the posthypoxia group. This response was not, however, sustained over a 2-day IL infusion. Thus IL induces pulmonary vascular abnormalities that do not appear to be related to vasoconstriction or hypoxemia. We speculate that differences in endothelial metabolism of IL in room air and posthypoxia rats may explain the lack of IL-related abnormalities in the latter group.

  View details for Web of Science ID A1988R495800032

  View details for PubMedID 2974251

• QUALITATIVE AND QUANTITATIVE DIFFERENCES IN PROTEIN-SYNTHESIS COMPARING FETAL LAMB DUCTUS-ARTERIOSUS ENDOTHELIUM AND SMOOTH-MUSCLE WITH CELLS FROM ADJACENT VASCULAR SITES DEVELOPMENTAL BIOLOGY RABINOVITCH, M., Beharry, S., Bothwell, T., Jackowski, G. 1988; 130 (1): 250-258

  Abstract

  During late gestation, intimal cushions form in the ductus arteriosus (DA) and these cause the vessel to close when it constricts in the postnatal period. The formation of intimal cushions suggests highly specialized functions of DA endothelial and smooth muscle cells. To investigate these properties, we established, from fetal lambs on Day 138 of a 148-day term gestation, primary cell cultures of DA endothelium and smooth muscle and compared them to cells derived from the adjacent pulmonary artery and aorta. Purity of the endothelial cell cultures from each vascular site was assessed by the contact inhibited "cobblestone" monolayer phenotype, by positive immunofluorescence for factor VIII and by angiotensin converting enzyme activity. Purity of smooth muscle cell cultures at each vascular site was assessed by the "hills and valleys" phenotype and by positive immunofluorescence with a smooth muscle actin specific monoclonal antibody. Endothelial and smooth muscle cells had different growth curves, ultrastructural features, and protein profiles on single and two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE), but vascular sites were similar. To further determine whether differences related to DA origin were indeed present, endothelial and smooth muscle cells from all three vascular sites were incubated with the radiolabeled amino acids [14C]leucine, [14C]proline, and [14C]valine and the proteins in both the cells and the conditioned medium were analyzed by autoradiography after SDS-PAGE. A dense band corresponding to a 42-kDa protein was observed in valine-labeled DA endothelial cells and conditioned medium and a 52-kDa protein was observed in the conditioned medium of leucine-labeled DA smooth muscle cells only. Further isolation and characterization of these endothelial and smooth muscle proteins will be necessary to determine whether they are related to the mechanism of intimal cushion formation in the late gestation DA or are present abnormally in association with the intimal proliferation observed in pulmonary and systemic vascular disease.

  View details for Web of Science ID A1988Q930200024

  View details for PubMedID 2846387

• PROTEINASE-INHIBITORS PROTECT LEISHMANIA-AMAZONENSIS AMASTIGOTES FROM DESTRUCTION BY AMINO-ACID ESTERS MOLECULAR AND BIOCHEMICAL PARASITOLOGY Alfieri, S. C., Ramazeilles, C., Zilberfarb, V., Galpin, I., Norman, S. E., RABINOVITCH, M. 1988; 29 (2-3): 191-201

  Abstract

  Lysosomotropic amino acid esters and amides kill Leishmania amazonensis amastigotes by a mechanism which probably involves enzymatic hydrolysis of the compounds and rapid accumulation of less permeant amino acid within the parasites. We show here that, in agreement with this model, the proteinase inhibitors antipain and chymostatin prevented the killing of intracellular and isolated parasites by L-leucine methyl ester (Leu-OMe). Survival of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by tetrazolium (MTT) reduction. Near maximal protection of intracellular parasites was obtained after 24 h incubation of macrophage cultures with 50 micrograms ml-1 antipain or chymostatin. Incubation for greater than 1 h with chymostatin or greater than 4 h with antipain alone resulted in loss of viability of the parasites. Protective activity was only slightly diminished by 20 h chase of isolated parasites in inhibitor-free medium. Two synthetic chymostatin analogues, Z-Val-Phe-Sc and Z-Ile-Phe-Sc, protected isolated amastigotes at 4 or 10 micrograms ml-1. With the exception of Trp-NH2, the toxicity of which was only minimally inhibited, antipain and chymostatin also prevented parasite destruction by other amino acid derivatives. Finally, in concentration-dependent fashion, the inhibitors reduced the accumulation of [3H]leucine in isolated amastigotes incubated with [3H]Leu-OMe. Since uptake of labelled ester was unaffected, we postulate that protection involves inhibition of the parasite enzymes which hydrolyse the amino acid derivatives.

  View details for Web of Science ID A1988N920500010

  View details for PubMedID 3412375

• REPAIR OF TRICUSPID ATRESIA - UTILITY OF RIGHT VENTRICULAR INCORPORATION ANNALS OF THORACIC SURGERY Coles, J. G., LEUNG, M., KIELMANOWICZ, S., Freedom, R. M., Benson, L. N., RABINOVITCH, M., SHERRET, H., DASMAHAPATRA, H., TRUSLER, G. A., McLaughlin, P. R., Williams, W. G. 1988; 45 (4): 384-389

  Abstract

  During a 10-year period, 62 patients underwent the following modifications of the Fontan operation for repair of tricuspid atresia: direct atriopulmonary connection (N = 15), atriopulmonary connection using a conduit (N = 5), direct atrioventricular (AV) connection (N = 22), and AV connections with a valved conduit (N = 20), including 2 with combined Fontan-arterial switch procedures. The overall hospital mortality was 16.1% (10/62) (70% confidence limits, 11.2 to 22.4%). By multivariate analysis, the risk factors for early and late death included increasing right atrial pressure after repair, use of an atriopulmonary connection, and previous pulmonary artery banding (all variables, p less than 0.05). Postoperative catheterization was performed in 22 patients including 15 with AV valved-conduit connections. Right ventricular (RV) work based on pulmonary artery pressure minus right atrial pressure was correlated with the preoperative RV to left ventricular volume ratio computed from the four-chamber angiographic projection (p = 0.025), and was appreciable only with ratios exceeding about 30%. In 6 of 19 eligible patients, severe conduit obstruction has developed. Considering the survival data, the risk of reoperation, and postoperative hemodynamic findings, analysis of our experience supports the preferential use of nonvalved AV connections in most patients with tricuspid atresia and ventriculoarterial concordance.

  View details for Web of Science ID A1988M824300008

  View details for PubMedID 3355280

• Problems of pulmonary hypertension in children with congenital cardiac defects. Chest RABINOVITCH, M. 1988; 93 (3): 119S-126S

  View details for PubMedID 3277814

• ANGIOTENSIN-II PREVENTS HYPOXIC PULMONARY-HYPERTENSION AND VASCULAR CHANGES IN RAT AMERICAN JOURNAL OF PHYSIOLOGY RABINOVITCH, M., Mullen, M., Rosenberg, H. C., Maruyama, K., OBRODOVICH, H., Olley, P. M. 1988; 254 (3): H500-H508

  Abstract

  Angiotensin II, a vasoconstrictor, has been previously demonstrated to produce a secondary vasodilatation due to release of prostaglandins. Because of this effect, we investigated whether infusion of exogenous angiotensin II via miniosmopumps in rats during a 1-wk exposure to chronic hypobaric hypoxia might prevent pulmonary hypertension, right ventricular hypertrophy, and vascular changes. We instrumented the rats with indwelling cardiovascular catheters and compared the hemo-dynamic and structural response in animals given angiotensin II, indomethacin in addition to angiotensin II (to block prostaglandin production), or saline with or without indomethacin. We then determined whether angiotensin II infusion also prevents acute hypoxic pulmonary vasoconstriction. We observed that exogenous angiotensin II infusion abolished the rise in pulmonary artery pressure, the right ventricular hypertrophy, and the vascular changes induced during chronic hypoxia in control saline-infused rats with or without indomethacin. The protective effect of angiotensin II was lost when indomethacin was given to block prostaglandin synthesis. During acute hypoxia, both angiotensin II and prostacyclin infusions similarly prevented the rise in pulmonary artery pressure observed in saline-infused rats and in rats given indomethacin or saralasin in addition to angiotensin II. Thus exogenous angiotensin II infusion prevents chronic hypoxic pulmonary hypertension, associated right ventricular hypertrophy, and vascular changes and blocks acute hypoxic pulmonary hypertension, and this is likely related to its ability to release vasodilator prostaglandins.

  View details for Web of Science ID A1988M591900014

  View details for PubMedID 3348429

• HIGH-PRESSURE PULSATION OF CENTRAL AND MICROVESSEL PULMONARY-ARTERY ENDOTHELIAL-CELLS AMERICAN JOURNAL OF PHYSIOLOGY RABINOVITCH, M., Bothwell, T., Mullen, M., HAYAKAWA, B. N. 1988; 254 (2): C338-C343

  Abstract

  We developed an in vitro method of pulsating central and microvessel pulmonary artery endothelial cells that would allow us to study the effects of increased distending pressures over a prolonged period of time. Preservation of the contact-inhibited monolayer was assessed on phase contrast microscopy and, in addition, scanning and transmission electron microscopy (SEM, TEM) were used to determine whether there were alterations in the surface characteristics or intracytoplasmic organelles that suggested cellular damage. The cells used were obtained from Rambouillet lambs, age 3-5 days, anesthetized with halothane and ventilated. The endothelium was harvested from the central pulmonary artery (CPA) by scraping the luminal surface and from the microvessels (MPA) by infusing microcarrier beads 40-140 microns external diameter. After the second passage in culture, the cells were seeded onto the translucent, flexible polyvinylchloride membrane of a transducer dome and grown to confluence. The cell dome was then connected to a blank dome with an attached quartz transducer, to a reservoir, and to stainless steel bellows tubing, all filled with culture medium and affixed to a pulsation generator. By varying the height of the reservoir, the amplitude of excursion of the bellows tubing, and the rate, the cells could be pulsated at a given distending pressure and frequency. Confluent CPA endothelial cells from three lambs and MPA cells from two others were studied after pulsation at both 100/60 and 20/10 mmHg, 60 times/min for 48 h and after nonpulsation. On phase contrast light microscopy and on SEM, the cells remained confluent.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1988M371700016

  View details for PubMedID 3348366

• ALTERED ELASTIN AND COLLAGEN-SYNTHESIS ASSOCIATED WITH PROGRESSIVE PULMONARY-HYPERTENSION INDUCED BY MONOCROTALINE - A BIOCHEMICAL AND ULTRASTRUCTURAL-STUDY LABORATORY INVESTIGATION TODOROVICHHUNTER, L., Johnson, D. J., Ranger, P., Keeley, F. W., RABINOVITCH, M. 1988; 58 (2): 184-195

  Abstract

  Changes in elastin and collagen synthesis in the pulmonary artery wall, assessed both biochemically and ultrastructurally, were related to the development of progressive pulmonary hypertension induced by the toxin monocrotaline. Male Sprague-Dawley rats (200 to 225 gm) were injected subcutaneously in the hind flank with either monocrotaline (60 mg/kg) or an equivalent volume of saline and studied 8, 16 and 28 days later. At each time point, the right ventricle and left ventricle with septum were separated and weighed to follow the development of right ventricular hypertrophy. The hilar pulmonary artery was assessed by light microscopy for medial hypertrophy and by electron microscopy for changes in the endothelium, subendothelium and media. The mainstem pulmonary artery was used to determine synthesis of elastin and collagen by in vitro incorporation of [14C]proline into nonelastin, [14C]hydroxyproline into collagen, and [3H]valine into cyanogen bromide-insoluble elastin. In addition, total content of insoluble elastin was determined by weight of the residue after cyanogen bromide digestion and of collagen by total hydroxyproline content in sodium dodecyl sulfate and cyanogen bromide extracts. Eight days after monocrotaline injection, there was pulmonary artery endothelial swelling and a significant decrease in the number of myoendothelial junctions (p less than 0.05) associated with a decreased proportion of amorphous elastin in the media (p less than 0.01). Sixteen days after monocrotaline injection, a decrease in the proportion of elastin in the media was still evident (p less than 0.01) despite an apparent increase in insoluble elastin synthesis. Moreover, the amorphous elastin was distributed preferentially in small islands rather than in laminae (p less than 0.05). Twenty-eight days after monocrotaline injection, medial and right ventricular hypertrophy had developed (p less than 0.05 and p less than 0.01, respectively). At the same time, there was a striking increase in both insoluble elastin synthesis and total insoluble elastin content (p less than 0.01 for both) and an increase in collagen synthesis and total collagen content (p less than 0.05 for both). In addition, the ratio of insoluble elastin synthesis to collagen synthesis was greater than in controls (p less than 0.01), whereas the ratio of total insoluble elastin to total collagen did not change. On ultrastructural analysis, the proportion of amorphous elastin in the vessel wall relative to other elements remained low (p less than 0.01) and was distributed throughout the media as increased numbers of small islands (p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1988M298300009

  View details for PubMedID 3123799

• METAIODOBENZYLGUANIDINE [I-131] SCINTIGRAPHY DETECTS IMPAIRED MYOCARDIAL SYMPATHETIC NEURONAL TRANSPORT FUNCTION OF CANINE MECHANICAL-OVERLOAD HEART-FAILURE CIRCULATION RESEARCH Rabinovitch, M. A., Rose, C. P., Rouleau, J. L., Chartrand, C., Wieland, D. M., Lepanto, L., Legault, F., Suissa, S., Rosenthall, L., BURGESS, J. H. 1987; 61 (6): 797-804

  Abstract

  In heart failure secondary to chronic mechanical overload, cardiac sympathetic neurons demonstrate depressed catecholamine synthetic and transport function. To assess the potential of sympathetic neuronal imaging for detection of depressed transport function, serial scintigrams were acquired after the intravenous administration of metaiodobenzylguanidine [131I] to 13 normal dogs, 3 autotransplanted (denervated) dogs, 5 dogs with left ventricular failure, and 5 dogs with compensated left ventricular hypertrophy due to a surgical arteriovenous shunt. Nine dogs were killed at 14 hours postinjection for determination of metaiodobenzylguanidine [131I] and endogenous norepinephrine content in left atrium, left ventricle, liver, and spleen. By 4 hours postinjection, autotransplanted dogs had a 39% reduction in mean left ventricular tracer accumulation, reflecting an absent intraneuronal tracer pool. Failure dogs demonstrated an accelerated early mean left ventricular tracer efflux rate (26.0%/hour versus 13.7%/hour in normals), reflecting a disproportionately increased extraneuronal tracer pool. They also showed reduced late left ventricular and left atrial concentrations of tracer, consistent with a reduced intraneuronal tracer pool. By contrast, compensated hypertrophy dogs demonstrated a normal early mean left ventricular tracer efflux rate (16.4%/hour) and essentially normal late left ventricular and left atrial concentrations of tracer. Metaiodobenzylguanidine [131I] scintigraphic findings reflect the integrity of the cardiac sympathetic neuronal transport system in canine mechanical-overload heart failure. Metaiodobenzylguanidine [123I] scintigraphy should be explored as a means of early detection of mechanical-overload heart failure in patients.

  View details for Web of Science ID A1987L149600005

  View details for PubMedID 2960468

• ABNORMAL ENDOTHELIAL FACTOR-VIII ASSOCIATED WITH PULMONARY-HYPERTENSION AND CONGENITAL HEART-DEFECTS CIRCULATION RABINOVITCH, M., Andrew, M., Thom, H., TRUSLER, G. A., Williams, W. G., Rowe, R. D., Olley, P. M. 1987; 76 (5): 1043-1052

  Abstract

  In patients with pulmonary hypertension associated with congenital heart defects, ultrastructural abnormalities are observed in endothelial cells, which suggest heightened metabolic function. If endothelial production of the von Willebrand factor (vWF) is increased, this may be associated with abnormal interactions with platelets leading to worsening of the pulmonary hypertension. We therefore evaluated vWF in 30 patients with pulmonary hypertension (25 with congenital heart defects) and in 30 individuals with normal pulmonary arterial pressure (12 with congenital heart defects). We measured the antigenic (vWF: Ag) and biologic (VWF: rist) activity of vWF in plasma and assessed endothelial vWF: Ag directly by an immunoperoxidase stain applied to lung biopsy tissue. Because of considerable variance and small size, the group of five patients with pulmonary hypertension and without congenital heart defects were excluded from statistical analyses. Patients with pulmonary hypertension and congenital heart defects had significant higher vWF: Ag levels than individuals with normal pulmonary arterial pressure without congenital heart defects (p less than .05), whereas values in those with normal pressure and congenital heart defects were intermediate. In lung biopsy tissue available from 29 patients in this study and from 11 others we previously reported, immunostain of pulmonary arterial endothelium for vWF was intense (suggesting increased production) in 29 of 32 with pulmonary hypertension and congenital heart defects and in only one of eight with normal pulmonary arterial pressure and congenital heart defects (p less than .01). Only three patients with congenital heart defects and pulmonary hypertension and increased vWF: Ag, however, had increased vWF: rist. Compatible with this discrepancy was a loss of vWF high-molecular weight forms as determined by both crossed immunoelectrophoresis and multimeric analysis. Our results suggest that increased vWF in most patients with congenital heart defects and pulmonary hypertension is associated with increased production of a biologically deficient molecule lacking high-molecular weight forms.

  View details for Web of Science ID A1987K711700011

  View details for PubMedID 3117409

• STRUCTURAL COMPOSITION OF CENTRAL PULMONARY-ARTERIES - GROWTH-POTENTIAL AFTER SURGICAL SHUNTS JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY ROSENBERG, H. G., Williams, W. G., TRUSLER, G. A., Higa, T., RABINOVITCH, M. 1987; 94 (4): 498-503

  Abstract

  Failure to achieve adequate pulmonary artery growth in patients with cyanotic congenital heart disease is a major obstacle to surgical correction. To assess whether differences in structural composition of central pulmonary arteries influence their growth potential after surgically created shunts, we obtained full-thickness biopsy specimens from the hilar pulmonary arteries of eight patients with pulmonary atresia or tetralogy of Fallot undergoing modified Blalock-Taussig shunts under 1 year of age. Tissue was processed for electron microscopic studies and a morphometric assessment was made of the volume proportions of smooth muscle, collagen, ground substance, and elastin. Initial pulmonary artery size was determined angiographically during the diagnostic cardiac catheterization. Pulmonary artery size was determined by cross-sectional echocardiograms 7 to 27 months later (mean 19 months). Pulmonary artery growth did not correlate with the interval between examinations but did correlate with the volume proportion of elastin (r = 0.73, standard error of the estimate = 1.45, p less than 0.05). Thus the structural composition of pulmonary arteries may influence their potential for growth after surgical shunts. In particular, an inadequate proportion of elastin may be a hindrance to growth.

  View details for Web of Science ID A1987K347200003

  View details for PubMedID 3657252

• VENTILATORY PREDICTORS OF PULMONARY HYPOPLASIA IN CONGENITAL DIAPHRAGMATIC-HERNIA, CONFIRMED BY MORPHOLOGICAL ASSESSMENT JOURNAL OF PEDIATRICS Bohn, D., Tamura, M., Perrin, D., Barker, G., RABINOVITCH, M. 1987; 111 (3): 423-431

  Abstract

  We carried out a prospective study in 66 infants with congenital diaphragmatic hernia within the first 6 hours of life to determine whether outcome is related to the degree of underlying pulmonary hypoplasia, as predicted by preoperative PaCO2, when correlated with an index of ventilation (VI = mean airway pressure X respiratory rate) and confirmed by postmortem analysis of the lung. Those infants with PaCO2 greater than 40 mm Hg before surgery had a 77% mortality; when PaCO2 reduction could be achieved only with VI greater than 1000, the mortality was still greater than 50%. After repair, however, the ability to hyperventilate to PaCO2 less than 40 mm Hg proved to be an important determinant of survival; only one of 31 infants in this group died, whereas only two of 27 infants with PaCO2 greater than 40 mm Hg survived. In 16 infants with PaCO2 greater than 40 mm Hg despite hyperventilation, high-frequency oscillatory ventilation was started. This resulted in a rapid fall in PaCO2, but 14 of the 16 infants had only temporary improvement in oxygenation, and died. In five of the infants who died, alveolar number was assessed by postmortem morphometric analysis; there was a severe reduction to less than 10% of published normal neonatal values. Pulmonary vascular changes of increased muscularization were less remarkable than those observed in infants with persistent pulmonary hypertension. Our findings suggest that the degree of pulmonary hypoplasia (which would not be influenced by surgical repair), rather than the pulmonary vascular abnormality, mainly determines survival. Consideration could therefore be given to an initial nonsurgical approach to congenital diaphragmatic hernia, with the expectation that pulmonary function might improve and pulmonary vascular resistance decrease.

  View details for Web of Science ID A1987J892000027

  View details for PubMedID 3625414

• SURGICAL EXPERIENCE WITH THE MODIFIED FONTAN PROCEDURE CIRCULATION Coles, J. G., KIELMANOWICZ, S., Freedom, R. M., Benson, L. N., MOES, F., Olley, P. M., RABINOVITCH, M., ROSENBERG, H., SHERRET, H., Rowe, R. D., McLaughlin, P. R., TRUSLER, G. A., Williams, W. G. 1987; 76 (3): 61-66

  View details for Web of Science ID A1987J906500011

• PROSTAGLANDINS AND STRUCTURAL-CHANGES IN PULMONARY-ARTERIES AMERICAN REVIEW OF RESPIRATORY DISEASE RABINOVITCH, M. 1987; 136 (3): 777-779

  View details for Web of Science ID A1987J974300048

  View details for PubMedID 3631748

• Surgical experience with the modified Fontan procedure. Circulation Coles, J. G., KIELMANOWICZ, S., Freedom, R. M., Benson, L. N., MOES, F., Olley, P. M., RABINOVITCH, M., ROSENBERG, H., SHERRET, H., Rowe, R. D. 1987; 76 (3): III61-6

  Abstract

  During a 10 year period 109 patients (3 months to 47 years old) underwent modifications of the Fontan procedure for repair of classic tricuspid atresia (TA) (n = 58), univentricular atrioventricular connection (UVH) (n = 38), or other complex malformations (CM) (n = 13). Among patients with TA, an atriopulmonary connection was used in 19 (33%) and incorporation of the right ventricle with the Björk modification and with a right atrial-to-right ventricular valved conduit was used in 20 (34%) and in 19 (33%), respectively. Three of the latter 19 also underwent a combined Fontan-switch procedure. The hospital mortality rate was 13.8% (70% confidence limits, 9.3% to 18.3%) for patients with TA, 28.9% (70% confidence limits, 21.3% to 37.0%) for patients with UVH, and 7.7% (70% confidence limits, 0% to 15.4%) for patients with CM. Multivariate analysis identified with the following variables as risk factors for both early and late deaths: diagnosis of UVH, previous pulmonary artery banding (PAB), and postrepair right atrial pressure, and, in patients with TA, the use of a direct atriopulmonary connection (all variables, p less than .05). Morphometric lung biopsy scores were not different in patients with PAB, implicating the role of ventricular hypertrophy rather than pulmonary vascular disease as the mechanism for the adverse effect of PAB. Right atrial pressure was a predictor of serious late cardiac symptoms, which were present in 10% of eligible patients (p = .032). This review demonstrates a survival advantage with modifications of the Fontan procedure that incorporate the hypoplastic right ventricle in patients with TA.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for PubMedID 2441898

• A PULMONARY-ARTERY ENDOTHELIAL FACTOR CAUSES UNIDIRECTIONAL ALIGNMENT OF SMOOTH-MUSCLE CELLS TISSUE & CELL Wilson, W., Bothwell, T., RABINOVITCH, M. 1987; 19 (2): 177-182

  Abstract

  Pulmonary artery and aorta endothelial and smooth muscle cells harvested from near term Rambouillet lambs were cultured. To determine whether cultured endothelial cells produce a factor which alters the shape and alignment of smooth muscle cells, rhodamine phalloidin staining of F-actin filaments was carried out, as well as time-lapse cinematography. We were able to demonstrate, with pulmonary artery but not with aortic cells in culture, an endothelial-derived factor which causes the smooth muscle cells to migrate and align in uniform direction. The effect could not be enhanced by making the endothelial cells hypoxic and was similar to that produced by exposing smooth muscle cells to KCl or directly to hypoxia.

  View details for Web of Science ID A1987G952600003

  View details for PubMedID 3590148

• Sex-specific criteria for interpretation of thallium-201 myocardial uptake and washout studies. Journal of nuclear medicine RABINOVITCH, M., Suissa, S., ELSTEIN, J., STANILOFF, H., Tang, A., Rush, C., Aldis, A., Tannous, R., Turek, M., ADDAS, A. 1986; 27 (12): 1837-1841

  Abstract

  A study was undertaken to determine the effect of gender on criteria for the quantitative analysis of exercise-redistribution 201Tl myocardial scintigraphy. The studies of 26 normal females and 23 normal males were subjected to bilinear interpolative background subtraction and horizontal profile analysis. Significant sexual differences were found in both regional uptake ratios and washout rates. These differences primarily reflected a proportionately decreased anterior and upper septal uptake in females, and faster washout in females. Faster myocardial 201Tl washout rates in females could not be clearly ascribed to either a physiological or artifactual explanation. It is concluded that since important differences exist between males and females in the detected pattern of 201Tl myocardial uptake and washout, sex-specific criteria may enhance the predictive accuracy of exercise-redistribution 201Tl myocardial scintigraphy.

  View details for PubMedID 3783272

• SEX-SPECIFIC CRITERIA FOR INTERPRETATION OF TL-201 MYOCARDIAL UPTAKE AND WASHOUT STUDIES JOURNAL OF NUCLEAR MEDICINE RABINOVITCH, M., Suissa, S., ELSTEIN, J., STANILOFF, H., Tang, A., Rush, C., Aldis, A., Tannous, R., Turek, M., ADDAS, A., Obrien, C., BURGESS, J. H., Rosenthall, L. 1986; 27 (12): 1837-1841

  View details for Web of Science ID A1986F152900009

• PULMONARY-ARTERY ENDOTHELIAL ABNORMALITIES IN PATIENTS WITH CONGENITAL HEART-DEFECTS AND PULMONARY-HYPERTENSION - A CORRELATION OF LIGHT WITH SCANNING ELECTRON-MICROSCOPY AND TRANSMISSION ELECTRON-MICROSCOPY LABORATORY INVESTIGATION RABINOVITCH, M., Bothwell, T., HAYAKAWA, B. N., Williams, W. G., TRUSLER, G. A., Rowe, R. D., Olley, P. M., Cutz, E. 1986; 55 (6): 632-653

  Abstract

  Scanning electron microscopy and transmission electron microscopy were applied to lung biopsy specimens from patients with congenital heart defects, and pulmonary artery endothelium was analyzed for alterations in surface characteristics and intracytoplasmic composition which might reflect abnormal function. The patients were divided into four groups distinguished by increasing severity of pulmonary vascular changes on light microscopy graded both morphometrically and by the Heath-Edwards classification; group 1, normal vasculature or only abnormal extension of muscle into peripheral arteries; group 2, medial hypertrophy; group 3, medial hypertrophy +/- decreased artery number + intimal hyperplasia; group 4, decreased artery number + occlusive intimal hyperplasia. On scanning electron microscopy, the pulmonary artery endothelial surface in group 1 patients was "crinkled" or "corduroy-like", i.e., composed of narrow, even ridges; in groups 2 and 3, it was "cable-like", i.e., comprised of deep intertwined ridges; in group 4 it was "chenille" in texture, i.e., high ridges alternated with low, uneven, and twisted ones. There was significant increased density of surface microvilli in groups 2 and 3 patients when compared to groups 1 and 4 (p less than 0.05 for each comparison). On transmission electron microscopy pulmonary artery endothelial cells in groups 2 and 3 patients were also characterized by a significant increase in the volume density of rough endoplasmic reticulum (p less than 0.01) and microfilament bundles (p less than 0.05). The coarse endothelial surface characteristics associated with pulmonary vascular changes may result in abnormal interaction with blood elements and release of vasoactive substances. The increased microvilli, rough endoplasmic reticulum, and microfilament bundles in patients with moderate but not advanced arterial changes suggest a phase where increased endothelial metabolic function and alterations in the cytoskeleton may also contribute to heightened pulmonary vascular reactivity.

  View details for Web of Science ID A1986F245300006

  View details for PubMedID 3784535

• PULMONARY-HYPERTENSION IN CHILDREN - PERIOPERATIVE MANAGEMENT CANADIAN ANAESTHETISTS SOCIETY JOURNAL Burrows, F. A., Klinck, J. R., RABINOVITCH, M., Bohn, D. J. 1986; 33 (5): 606-628

  View details for Web of Science ID A1986E230500012

  View details for PubMedID 3533237

• PULMONARY ARTERIAL CHANGES IN PATIENTS WITH VENTRICULAR SEPTAL-DEFECTS AND SEVERE PULMONARY-HYPERTENSION PEDIATRIC CARDIOLOGY Fried, R., FALKOVSKY, G., Newburger, J., Gorchakova, A. I., RABINOVITCH, M., GORDONOVA, M. I., FYLER, D., Reid, L., BURAKOVSKY, V. 1986; 7 (3): 147-154

  Abstract

  In 25 patients, aged eight months to 31 years, with ventricular septal defect (VSD; isolated in 15, the others with atrial septal defect, PDA, coarctation or patent ductus arteriosus + coarctation), each with severe pulmonary artery hypertension (pulmonary artery systolic pressure [Ppa] at least 75% of systemic and an elevated pulmonary vascular resistance), we related morphologic and morphometric data from open-lung biopsy to hemodynamic measurements obtained at cardiac catheterization during the same hospital admission. Of the hemodynamic features measured, only the ratios of pulmonary-to-systemic flow and pulmonary-to-systemic resistance correlated significantly with structure. Neither pulmonary artery pressure (Ppa) nor pulmonary vascular resistance correlated significantly with any structural feature studied. The increased external diameter of respiratory bronchiolar arteries in those with the more advanced Heath-Edwards grades reflects dilatation and suggests that it is in the small arteries of the distal arterial bed that the changes of pulmonary hypertension are most significant. Neither age nor body weight correlated significantly with the degree of structural or hemodynamic abnormality. In the ten patients who underwent VSD closure, Ppa was measured postoperatively. The Heath-Edwards grade (no more than one grade-III lesion) and arterial density (at least one-half that normal for age) were the best correlates of the difference between preoperative Ppa and Ppa immediately after corrective surgery. The presurgical catheterization data, including pulmonary resistance and the resistance ratio, did not correlate significantly with change in Ppa following VSD closure.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1986F425900004

  View details for PubMedID 3808993

• ABNORMAL DISTRIBUTION OF PULMONARY BLOOD-FLOW AFTER THE GLENN SHUNT OR FONTAN PROCEDURE - RISK OF DEVELOPMENT OF ARTERIOVENOUS-FISTULAS CIRCULATION Cloutier, A., Ash, J. M., Smallhorn, J. F., Williams, W. G., TRUSLER, G. A., Rowe, R. D., RABINOVITCH, M. 1985; 72 (3): 471-479

  Abstract

  Since the Fontan procedure results in low pulsatile pulmonary blood flow similar to that seen in patients with a Glenn shunt, it may also be associated with abnormal distribution of flow to the lower lung lobes and with the development of pulmonary arteriovenous fistulae (PAVF). In 12 patients 0.8 to 4.5 years after Fontan procedure and in 20 patients 0.2 to 18 years after receipt of Glenn shunts we assessed ventilation (with 133Xe) and perfusion (after a peripheral injection of 99mTc-macroaggregated albumin) to compare upper to lower lobe distribution of blood flow with that in a control group. The presence of PAVF was assessed by radionuclide activity in kidneys and the brain and by a two-dimensional echocardiographic contrast study. A decreased upper/lower lobe perfusion ratio was noted in 13 of 20 patients with Glenn shunts (65%) and correlated with the time after surgery (p less than .05). Despite the shorter follow-up period, two of 12 (16%) patients who had undergone the Fontan procedure also had a decreased upper/lower lobe perfusion ratio, and one of these developed right heart failure. Brain and kidney radionuclide counts above control values were observed in all patients with Glenn shunts and in 11 of 12 patients who had the Fontan operation. However, in only five of 20 (25%) patients with Glenn shunts were PAVF confirmed by the two-dimensional echocardiographic contrast study. Three of the five patients with PAVF had Glenn shunts of long duration.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1985APY7700005

  View details for PubMedID 4017202

• MORPHOLOGY OF THE DEVELOPING PULMONARY BED - PHARMACOLOGIC IMPLICATIONS PEDIATRIC PHARMACOLOGY RABINOVITCH, M. 1985; 5 (1): 31-48

  View details for Web of Science ID A1985AEL1900005

  View details for PubMedID 3887312

• PULMONARY TOXICITY OF MONOCROTALINE DIFFERS AT CRITICAL PERIODS OF LUNG DEVELOPMENT PEDIATRIC RESEARCH Todd, L., Mullen, M., Olley, P. M., RABINOVITCH, M. 1985; 19 (7): 731-737

  Abstract

  We injected 3-day-old (neonatal), 8-day-old (infant), and 8-wk-old (adult) Sprague-Dawley rats with monocrotaline to examine the effect of a toxic agent at various stages of lung development. Two and four weeks after injection the rats were killed and the heart and lungs removed: the right and left ventricles were separated and weighed, the pulmonary artery was injected with barium-gelatin, and the lung was fixed in the inflated state. Morphometric techniques were applied to assess lung volume, alveolar size and number, and arterial size, muscularity, and concentration relative to alveolar. Rats injected with monocrotaline in the neonatal period did not survive to 3 wk. After 2 wk, there was no significant right ventricular hypertrophy and pulmonary vascular changes were no worse than in the other rat groups injected with monocrotaline, but alveolar development was severely impaired; less than one-third the normal number was present. Rats injected with monocrotaline in infancy had normal alveolar development. After 2-wk, the arterial changes, i.e. extension of muscle into peripheral arteries, medial hypertrophy of muscular arteries, and decreased arterial concentration relative to alveolar were similar to those observed in adult rats. After 4 wk, there was a decrease in medial hypertrophy associated with growth in artery size and only a lack of regression of right ventricular weight. In adult rats, after 4 wk medial hypertrophy became progressively more severe, the arterial concentration relative to alveolar decreased further, and right ventricular hypertrophy developed.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1985AKT7700019

  View details for PubMedID 3161000

• HYPEROXIA-INDUCED PULMONARY VASCULAR AND LUNG ABNORMALITIES IN YOUNG-RATS AND POTENTIAL FOR RECOVERY PEDIATRIC RESEARCH Wilson, W. L., Mullen, M., Olley, P. M., RABINOVITCH, M. 1985; 19 (10): 1059-1067

  Abstract

  We carried out morphometric studies to assess the effects of increasing durations of hyperoxic exposure on the developing rat lung and to evaluate the potential for new growth and for regression of structural abnormalities on return to room air. From day 10 of life Sprague-Dawley rats were either exposed to hyperoxia (0.8FIO2) for 2-8 wk or were removed after 2 wk and allowed to "recover" in room air for 2-6 wk. Litter mates maintained in room air served as age matched controls. Every 2 wk experimental and control rats from each group were weighed and killed. The heart and lungs were removed, the pulmonary artery was injected with barium-gelatin, and the lung was fixed in formalin in the inflated state. Morphometric assessments were made of right and left ventricular weights, lung volume, axial artery lumen diameter, alveolar number and concentration, and arterial number, concentration and muscularity. Rats continuously exposed to hyperoxia and rats exposed for only 2 wk showed the same degree of impaired parenchymal lung growth, as judged by a decrease in the concentration and number of alveoli. A significant decrease in arterial concentration, increase in muscularization of peripheral arteries, and medial hypertrophy of muscular arteries occurred after 2 wk of hyperoxia. Despite an initial trend toward regression, these features became progressively severe with continued hyperoxic exposure and by 8 wk were associated with a decreased arterial lumen diameter, with right ventricular hypertrophy and with failure to thrive.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1985ARG4300020

  View details for PubMedID 2932674

• THE PULMONARY CIRCULATION IN CHILDREN WITH CONGENITAL HEART-DISEASE - MORPHOLOGIC AND MORPHOMETRIC CONSIDERATIONS CANADIAN ANAESTHETISTS SOCIETY JOURNAL Burrows, F. A., RABINOVITCH, M. 1985; 32 (4): 364-373

  View details for Web of Science ID A1985ANE7200009

  View details for PubMedID 3896433

• VARIATIONS ON CALCULATING LEFT-VENTRICULAR VOLUME WITH THE RADIONUCLIDE COUNT-BASED METHOD MEDICAL PHYSICS Koral, K. F., Rabinovitch, M. A., Kalff, V., Chan, W., Juni, J. E., Lerman, B., Lampman, R., Walton, J., Vogel, R., Pitt, B., Thrall, J. H. 1985; 12 (1): 93-98

  Abstract

  Various methods for the calculation of left-ventricular volume by the count-based method utilizing red-blood-cell labeling with 99mTc and a parallel-hole collimator are evaluated. Attenuation correction, linked to an additional left posterior oblique view, is utilized for all 26 patients. We examine (1) two methods of calculating depth, (2) the use of a pair of attenuation coefficients, (3) the optimization of attenuation coefficients, and (4) the employment of an automated program for expansion of the region of interest. The standard error of the estimate (SEE) from the correlation of the radionuclide volumes with the contrast-angiography volumes, and the root-mean-square difference between the two volume sets at the minimum SEE are computed. It is found that optimizing a single linear attenuation coefficient assumed for attenuation correction best reduces the value of the SEE. The average of the optimum value from the end-diastolic data and that from the end-systolic data is 0.11 cm-1. This value agrees with the mean minus one standard deviation value determined independently from computed tomography scans (0.13-0.02 cm-1). It is also found that expansion of the region of interest beyond the second-derivative edge with an automated program, in order to correctly include more counts, does not lower the SEE as hoped. This result is in contrast to the results of others with different data and a manual method. Possible causes for the difference are given.

  View details for Web of Science ID A1985ABK7000017

  View details for PubMedID 3838357

• THE EFFECT OF DOBUTAMINE ON EXERCISE PERFORMANCE IN PATIENTS WITH SYMPTOMATIC ISCHEMIC-HEART-DISEASE AMERICAN HEART JOURNAL Rabinovitch, M. A., Kalff, V., Chan, W., Schork, A., Gross, M. D., Vogel, R. A., Thrall, J. H., Pitt, B. 1984; 107 (1): 81-85

  Abstract

  The effect of dobutamine on exercise performance was assessed in 20 patients with ischemic heart disease (CAD) and a positive stress test. These patients had a wide range of resting left ventricular ejection fraction (range 22% to 69%, mean 42%). Each patient entered a double-blind crossover study in which two identical exercise radionuclide ventriculograms were performed in patients on dobutamine, 5 micrograms/kg/min intravenously, or placebo. Dobutamine increased resting left ventricular ejection fraction. Although ejection fraction fell with dobutamine during submaximal exercise, it remained higher than with placebo. At peak exercise, ejection fraction fell to the same level on dobutamine as with placebo. Dobutamine diminished exercise time and time to ischemia while peak pressure-rate product was unchanged. Four of 20 patients developed complex ventricular premature beats, all while on dobutamine. Although useful when administered to resting patients with acute left ventricular failure, dobutamine's effects may be deleterious in exercising patients with chronic ischemic heart disease.

  View details for Web of Science ID A1984RZ08700014

  View details for PubMedID 6691244

• CONGENITAL UNILATERAL PULMONARY VEIN STENOSIS COMPLICATING TRANSPOSITION OF THE GREAT-ARTERIES AMERICAN JOURNAL OF CARDIOLOGY Vogel, M., Ash, J., Rowe, R. D., TRUSLER, G. A., RABINOVITCH, M. 1984; 54 (1): 166-171

  Abstract

  Four patients with transposition of the great arteries and unilateral pulmonary vein (PV) stenosis, all left-sided, were studied. Two patients had an intact ventricular septum (1 with a patent ductus arteriosus), 1 patient had a ventricular septal defect and 1 had a ventricular septal defect with pulmonary stenosis. Clinical signs, such as recurrent pneumonia, unilateral pulmonary edema and pleural effusion, were present preoperatively in 2 patients. Diagnosis was made at cardiac catheterization by cineangiography in 2 patients and at Mustard operation when the PV orifices were inspected in the other 2. PV dilatation was attempted in 3 patients, 1 before correction (age 6 months), 1 during and after it (ages 1 and 3 years, respectively) and 1 during corrective surgery (age 15 months). In the fourth patient only the intracardiac baffle was enlarged near the left PV orifices. In the first patient, at Mustard operation (age 18 months), only a fibrotic PV without an orifice was found; this patient died after surgery. The mean follow-up in the 3 survivors was 3.2 years (range 2 months to 7.6 years). All have severe residual PV obstruction documented by technetium-99m lung perfusion scans that show decreased flow to the left lung (0 to 16% total counts; normal 45%); 2 have unilateral pulmonary edema and 1 has pulmonary artery pressure at systemic level. It is believed that in patients with transposition of the great arteries, left-sided unilateral PV stenosis is a congenital anomaly that becomes progressive as a result of postnatal preferential flow to the right lung.

  View details for Web of Science ID A1984SY86600029

  View details for PubMedID 6741809

• BILATERAL DUCTUS-ARTERIOSUS (OR REMNANT) - AN ANALYSIS OF 27 PATIENTS AMERICAN JOURNAL OF CARDIOLOGY Freedom, R. M., MOES, C. A., Pelech, A., Smallhorn, J., RABINOVITCH, M., Olley, P. M., Williams, W. G., TRUSLER, G. A., Rowe, R. D. 1984; 53 (7): 884-891

  Abstract

  Bilateral ductus arteriosus (DA) was clinically recognized in 27 patients studied angiographically from 1963 through May 1983. Distal bilateral DA origin of non-confluent pulmonary arteries was identified in 15 patients, ectopic or distal ductal origin of 1 pulmonary artery in 9 patients (5 without evidence of intracardiac disease) and isolation of the left subclavian artery in 3 (all 3 of whom had a right aortic arch). Other conditions reported to be associated with bilateral DA include interruption of the aortic arch with isolation of a subclavian artery, aortic atresia with interruption of the aortic arch in which bilateral DA supports the entire systemic circulation, bilateral DA complicating forms of congenitally malformed hearts other than those just stated, and, rarely, bilateral DA in isolation. Understanding the symmetric or paired nature of the primitive aortic arch system in the developing human heart facilitates recognition of the patterns of fourth and sixth arch anomalies seen with bilateral DA.

  View details for Web of Science ID A1984SJ08400002

  View details for PubMedID 6702642

• VASCULAR STRUCTURE IN LUNG-TISSUE OBTAINED AT BIOPSY CORRELATED WITH PULMONARY HEMODYNAMIC-FINDINGS AFTER REPAIR OF CONGENITAL HEART-DEFECTS CIRCULATION RABINOVITCH, M., Keane, J. F., Norwood, W. I., Castaneda, A. R., Reid, L. 1984; 69 (4): 655-667

  Abstract

  At the time of surgical repair, a lung biopsy was performed on patients with congenital heart defects who either had pulmonary hypertension or in whom it would be likely to develop if the lesion were not corrected. Pulmonary vascular changes, assessed morphometrically and also according to the classification of Heath and Edwards (Circulation 18: 533, 1958), were correlated with the postoperative pulmonary hemodynamic findings: mean pulmonary arterial pressure the day after correction and mean pulmonary arterial pressure and pulmonary vascular resistance measured 1 year later. On the first postoperative day, increased mean pulmonary arterial pressure was uncommon in patients with morphometric grade A or B (mild) biopsy findings and Heath-Edwards grade N (normal), and if it was present it was of a mild degree. Mean pulmonary arterial pressure was commonly elevated in those with grade B (severe) or C (mild or severe) and Heath-Edwards grade I biopsy results and was more frequently elevated in those with grade II findings. Moderate-to-severe elevation of mean pulmonary arterial pressure was invariable in patients with Heath-Edwards grade III changes regardless of the morphometric grade. One year after repair, mean pulmonary arterial pressure and/or pulmonary vascular resistance were normal in all patients whose conditions were corrected surgically before 9 months of age regardless of the severity of the pulmonary vascular changes. Values were normal in patients whose conditions were repaired surgically at 9 months of age or later who had grade A or B (mild) morphometric findings with any Heath-Edwards grade or grade B (severe) morphometric findings with Heath-Edwards grade I but were increased in half of the patients with grade B (severe) morphometric findings and Heath-Edwards grade II or with grade C (mild or severe) and Heath-Edwards grade I or II changes. Pulmonary arterial pressure and pulmonary vascular resistance were increased in all patients whose conditions were repaired after 2 years of age with grade C morphometric findings and to a severe degree if associated with Heath-Edwards grade III. Thus, although the Heath-Edwards grade can usually be used to identify patients at risk for pulmonary hypertension in the early postoperative period, both the morphometric and the Heath-Edwards grades as well as the age of the patient at the time of repair can be used to determine whether pulmonary arterial pressure and resistance eventually return to normal or remain elevated.

  View details for Web of Science ID A1984SJ86000002

  View details for PubMedID 6697454

• MEASUREMENT OF LEFT-VENTRICULAR EJECTION FRACTION IN PEDIATRIC-PATIENTS USING THE NUCLEAR STETHOSCOPE AMERICAN JOURNAL OF CARDIOLOGY Spicer, R. L., RABINOVITCH, M., Rosenthal, A., Pitt, B. 1984; 53 (1): 211-214

  Abstract

  Left ventricular (LV) ejection fraction (EF) was measured in 25 patients, aged 2 weeks to 20 years (mean 8.6 years), using a portable nonimaging scintillation stethoscope. Technically satisfactory studies were obtained in 23 patients. LVEF was validated by cineangiography in 19 patients and by standard gated blood pool scintigraphy in 4. EF measured by the nuclear stethoscope correlated well with values obtained by cineangiography or scintigraphy (r = 0.869, p less than 0.001) over a wide range of EF values (18 to 79%). In children younger than 5 years (n = 11), the correlation (r = 0.728, p less than 0.02) was less satisfactory than in those older than 5 years (r = 0.926; p less than 0.001). Although modifications in the instrument and further clinical trials with the stethoscope are needed before the device becomes clinically useful to pediatric cardiologists, our data indicate that the nuclear stethoscope can provide reliable assessment of LVEF in pediatric patients.

  View details for Web of Science ID A1984RX73900040

  View details for PubMedID 6318544

• CHANGES IN PULMONARY BLOOD-FLOW AFFECT VASCULAR-RESPONSE TO CHRONIC HYPOXIA IN RATS CIRCULATION RESEARCH RABINOVITCH, M., Konstam, M. A., Gamble, W. J., Papanicolaou, N., Aronovitz, M. J., Treves, S., Reid, L. 1983; 52 (4): 432-441

  Abstract

  We banded the left pulmonary artery in rats to investigate, in the same animal, the effect of both increased and decreased flow on the lung vasculature and to determine how these hemodynamic states modify the structural changes produced by a 2-week exposure to hypobaric hypoxia. In unanesthetized rats, pressures were recorded from the main pulmonary artery and aorta via indwelling catheters, cardiac output was calculated by the Fick principle, and pulmonary and systemic vascular resistance estimated. Technetium-99m macroaggregated albumin was injected and radionuclide activity counted separately over the right and left lungs as a measure of flow. At postmortem, right and left ventricles of the heart were weighed and the lungs injected to permit analysis of arteriograms and morphometric assessment of structural changes in the pulmonary vascular bed. Flow in the left lung was reduced to one-fifth normal in rats with left pulmonary artery bands. In "room air" rats, pressure proximal to the left pulmonary artery band and in the right lung was slightly higher than in nonbanded controls, but not as high as in nonbanded or banded hypoxic rats. Changes in flow and pressure in both lungs of "room air" rats with left pulmonary artery bands were associated with a mild degree of extension of muscle into peripheral pulmonary arteries normally nonmuscular, medial hypertrophy of normally muscular arteries, and reduced arterial density. These three structural changes were present in both lungs of "hypoxic" rats but were much more severe. High flow in the right lungs of "hypoxic rats" with left pulmonary artery bands worsened only the degree of extension. Decreased flow and pressure in the left lungs of these animals prevented both the extension and the medial hypertrophy of hypoxia, but not the severe reduction in arterial density. It seems that the latter may occur as a direct response to low oxygen tension, whereas extension and medial hypertrophy are influenced by altered flow and pressure, respectively.

  View details for Web of Science ID A1983QN28700007

  View details for PubMedID 6831659

• POLYCYTHEMIA AND THE ACUTE HYPOXIC RESPONSE IN AWAKE RATS FOLLOWING CHRONIC HYPOXIA JOURNAL OF APPLIED PHYSIOLOGY Fried, R., Meyrick, B., RABINOVITCH, M., Reid, L. 1983; 55 (4): 1167-1172

  Abstract

  The acute hypoxic pressor response was studied in 22 chronically catheterized awake rats, 13 in whom the pulmonary arterial circulation had been remodeled by 10 days of exposure to hypobaric hypoxia. Five of these had their hematocrit lowered to normocytic levels after the chronic hypoxic exposure. Nine were controls. After 24 h in room air the pulmonary arterial pressure (Ppa) and pulmonary vascular resistance (Rp) of hypoxic-polycythemic rats was at least twice the control value; in the hypoxic-normocytic rats Ppa and Rp were less than that of hypoxic-polycythemic animals and greater than that of controls. Cardiac index, heart rate, and O2 saturation were similar in all groups. In 10% O2 a rise in Ppa and Rp occurred in all groups; in absolute terms the rise was greater in hypoxic rats than in controls and greater in polycythemic than in normocytic animals. In the intact animal the acute hypoxic pressor response can still be elicited in a pulmonary vascular bed structurally altered by chronic hypoxia. When calculated as a percent increase over base line, its intensity was greater than in room air controls and for Ppa was independent of hematocrit.

  View details for Web of Science ID A1983RL96500019

  View details for PubMedID 6629949

• COMPUTER-ANALYSIS OF CARDIAC RADIONUCLIDE DATA PROGRESS IN CARDIOVASCULAR DISEASES Froelich, J. W., Thrall, J. H., Kalff, V., Rogers, W. L., RABINOVITCH, M. 1983; 26 (1): 43-74

  View details for Web of Science ID A1983RA16700004

  View details for PubMedID 6346394

• IMPACT OF RADIONUCLIDE TECHNIQUES ON EVALUATION OF PATIENTS WITH ISCHEMIC-HEART-DISEASE JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Pitt, B., Kalff, V., Rabinovitch, M. A., Buda, A. J., COLFER, H. T., Vogel, R. A., Thrall, J. H. 1983; 1 (1): 63-72

  Abstract

  Radiotracer studies of the heart have become clinically important in the last decade, especially for evaluation of patients with known or suspected ischemic heart disease. Radionuclide ventriculography provides quantitative measures of biventricular function and regional wall motion. Recent technical advances include the development of computer programs for analyzing diastolic function, parametric imaging methods such as "phase" analysis and methods for calculating absolute ventricular volumes. Thallium-201 scans provide maps of regional myocardial perfusion. Recent advances include development of computer programs to quantitate regional thallium-201 uptake and to calculate thallium-201 turnover rates and the development of tomographic imaging systems. Technetium-99m pyrophosphate localizes in irreversibly damaged myocardium and provides a method for diagnosing, localizing and sizing acute myocardial infarcts. Recent applications include tomographic imaging to improve image contrast and development of criteria to identify high risk patients after infarction. Two important trends affecting the application of all the radionuclide studies in clinical cardiologic practice are the increasing use of decision analysis for incorporating results of multiple tests into single diagnostic probability statements, and the use of diagnostic algorithms that include the radionuclide studies to optimize the cost effectiveness of evaluation of patients with ischemic heart disease.

  View details for Web of Science ID A1983QD13700007

  View details for PubMedID 6826946

• SELECTIVE RIGHT VENTRICULAR DYSFUNCTION AFTER CORONARY-ARTERY BYPASS-GRAFTING JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Rabinovitch, M. A., ELSTEIN, J., Chiu, R. C., Rose, C. P., Arzoumanian, A., BURGESS, J. H. 1983; 86 (3): 444-446

  Abstract

  Preoperative and postoperative right (RVEF) and left ventricular ejection fractions (LVEF) were studied by means of radionuclide techniques in 15 patients undergoing coronary bypass operations. Three of them, all with right coronary artery lesions, had postoperative depression of RVEF without concomitant decrease in LVEF. In contrast to those with left ventricular dysfunction, the patients with selective RVEF depression did not have significant elevation of myocardial injury index calculated from creatine kinase isoenzyme (CK-MB) curves.

  View details for Web of Science ID A1983RG46200016

  View details for PubMedID 6604200

• ANEURYSM OF THE PULMONARY-ARTERY AS A CAUSE OF SEVERE CHEST PAIN ARCHIVES OF INTERNAL MEDICINE Jaffin, B. W., GUNDEL, W. D., CAPELESS, M. A., Castaneda, A. R., RABINOVITCH, M., Wackers, F. J. 1983; 143 (7): 1484-1485

  Abstract

  A 35-year-old man came to the emergency room with severe prolonged precordial chest pain. Serial chest roentgenograms obtained over a ten-year period revealed gradual development of marked aneurysmal dilatation of the pulmonary artery. Impending rupture of the artery was feared in spite of the presence of normal pulmonary artery pressure. At surgery, a markedly dilated pulmonary artery without evidence of rupture was found and the size was reduced by aneurysmorrhaphy. Prompt and continued relief of symptoms was achieved thereafter. Severe chest pain can be an initial symptom of pulmonary artery aneurysm in the absence of rupture, such pain arising either from pain receptors in the wall of the pulmonary artery or possibly from pressure on contiguous mediastinal structures.

  View details for Web of Science ID A1983QX50200033

  View details for PubMedID 6870423

• EVALUATION OF CARDIAC-FUNCTION IN THE PRESENCE OF FREQUENT VENTRICULAR PREMATURE BEATS RADIOLOGY Chan, W., Kalff, V., Rabinovitch, M. A., CLARE, J. M., Thrall, J. H., Pitt, B. 1983; 146 (2): 551-552

  Abstract

  Frequent ventricular ectopia in an otherwise healthy person is often benign. Nevertheless, for optimal patient management, serious underlying heart disease should be excluded. The authors illustrate the use of list-mode equilibrium blood-pool ventriculography for defining left ventricular function.

  View details for Web of Science ID A1983PZ13800053

  View details for PubMedID 6184745

• TOPOGRAPHY OF PREEMPTYING VENTRICULAR SEGMENTS IN PATIENTS WITH WOLFF-PARKINSON-WHITE SYNDROME USING SCINTIGRAPHIC PHASE MAPPING AND ESOPHAGEAL PACING CIRCULATION CHAN, W. W., Kalff, V., Dick, M., Rabinovitch, M. A., Jenkins, J., Thrall, J. H., Pitt, B. 1983; 67 (5): 1139-1146

  Abstract

  We analyzed the sequence of ventricular emptying using the phase image in 10 patients with accessory pathways and in 15 normal subjects. In normal subjects, the earliest emptying occurred in ventricular septal, apical and left basal segments. Eight patients had manifest preexcitation; the earliest emptying occurred ectopically in the right ventricle in one of these patients and in the left ventricle in five. The remaining two patients had normal phase maps. Two patients had concealed left-sided pathways. Their phase maps showed earliest emptying in left basal segments. Six of the 10 patients underwent electrophysiologic mapping. There was complete agreement between phase and electrophysiologic maps. Transesophageal atrial pacing increased preexcitation in one patient, normalized the ECG in another and precipitated narrow QRS tachycardia in four patients. Phase maps then showed enlargement, reduction and loss of the ectopic earliest emptying segments, respectively. We conclude that this technique in conjunction with pacing is successful in lateralizing accessory pathways.

  View details for Web of Science ID A1983QM76300029

  View details for PubMedID 6831675

• TRANSPOSITION OF THE GREAT-ARTERIES AND VENTRICULAR SEPTAL-DEFECT - RESULTS WITH THE SENNING OPERATION AND CLOSURE OF THE VENTRICULAR SEPTAL-DEFECT IN INFANTS ANNALS OF THORACIC SURGERY Penkoske, P. A., WESTERMAN, G. R., Marx, G. R., RABINOVITCH, M., Freed, M. D., Norwood, W. I., Castaneda, A. R. 1983; 36 (3): 281-288

  Abstract

  From May, 1978, to July, 1982, 46 infants ranging in age from 12 days to 12 months and in weight from 2.1 to 8.4 kg underwent repair of dextrotransposition of the great arteries (D-TGA) and ventricular septal defect (VSD) using a Senning repair and closure of the VSD. Ventricular septal defects were classified as membranous (47.8%), malaligned (28.3%), atrioventricular (AV) canal type (13.0%), subarterial (2.2%), muscular (2.2%), and multiple (6.5%). Hospital mortality was 15.2% and late mortality, 5.1%. Postoperative complications included tricuspid regurgitation (mild in 3 and severe, requiring tricuspid valve replacement, in 3), residual VSD (pulmonary/systemic flow ratio of greater than 2:1) in 3 patients (2, AV canal type and 1, multiple VSDs), pulmonary venous obstruction in 3 patients, and permanent complete heart block in 4 patients (2, AV canal type of VSD also requiring tricuspid valve replacement). Lung biopsy studies showed reversible Heath-Edwards and morphometric changes. No patient was seen with Heath-Edwards III or greater changes. In 10 patients, right ventricular end-diastolic pressures and pulmonary artery pressures at rest were within normal limits one year after operation. As the operative mortality of atrial inversion and arterial switch operations for D-TGA with VSD tends to become comparable, more extensive follow-up data, including cardiac catheterization and coronary arteriography in a large number of patients, will be necessary to establish the superiority of one approach over the other.

  View details for Web of Science ID A1983RG76400008

  View details for PubMedID 6615066

• WALK-THROUGH ANGINA PHENOMENON DEMONSTRATED BY GRADED-EXERCISE RADIONUCLIDE VENTRICULOGRAPHY - POSSIBLE CORONARY SPASM MECHANISMS AMERICAN HEART JOURNAL RIZI, H. R., Kline, R. C., BESOZZI, M. C., Kalf, V., RABINOVITCH, M., Chan, W., Thrall, J. H., Pitt, B. 1982; 103 (2): 292-294

  View details for Web of Science ID A1982NA67000016

  View details for PubMedID 7055060

• BASELINE LEFT-VENTRICULAR FUNCTION DURING FREQUENT VENTRICULAR OR ATRIAL ECTOPIC BEATS - CONCISE COMMUNICATION JOURNAL OF NUCLEAR MEDICINE Kalff, V., Rabinovitch, M. A., Chan, W., ONEILL, W., STEWART, J., Walton, J., Pitt, B., Thrall, J. H. 1982; 23 (12): 1076-1079

  View details for Web of Science ID A1982PT01700004

  View details for PubMedID 7143088

• COMPRESSION OF INTRA-PULMONARY BRONCHI BY ABNORMALLY BRANCHING PULMONARY-ARTERIES ASSOCIATED WITH ABSENT PULMONARY VALVES AMERICAN JOURNAL OF CARDIOLOGY RABINOVITCH, M., Grady, S., David, I., VANPRAAGH, R., Sauer, U., Buhlmeyer, K., Castaneda, A. R., Reid, L., Silva, D. K. 1982; 50 (4): 804-813

  Abstract

  In 3 patients with absent pulmonary valve syndrome and absent ductus arteriosus, the lungs were injected and analyzed postmortem using morphometric techniques. Two patients had tetralogy of Fallot and 1 had D-transposition of the great arteries, the latter being the first autopsy-proved case of absent pulmonary valve with transposition. In addition to the expected dilatation of the central pulmonary arteries and compression of the mainstem bronchi, postmortem pulmonary arteriography revealed a bizarre pattern of hilar branching. Instead of single segmental arteries, tufts of arteries arose which entwined and compressed the intrapulmonary bronchi. In all 3 patients the histologic structure of the pulmonary arteries was abnormal. The elastic lamina of the media of the right and left pulmonary arteries were increased in number outside the lung, but were decreased within the lung. At both sites, the elastic laminae were thickened and fragmented. In the 2 ventilator-dependent patients, there was slight medial hypertrophy and extension of muscle into normally nonmuscular arteries. In 1 of the 2 cases in which the number of bronchial generations was counted, they were decreased, and in the 1 case in which bronchial count was unknown, alveolar multiplication was severely impaired. Therefore, our data may explain why, in some patients with absent pulmonary valve syndrome, relief of compression of the mainstem bronchi alone does not appreciably alleviate or reverse severe respiratory disease.

  View details for Web of Science ID A1982PK60700023

  View details for PubMedID 7124639

• SCINTIGRAPHIC DEMONSTRATION OF VENTRICULO ATRIAL CONDUCTION IN THE VENTRICULAR PACEMAKER SYNDROME JOURNAL OF NUCLEAR MEDICINE Rabinovitch, M. A., STEWART, J., Chan, W., DUNLAP, T. E., Kalff, V., Clare, J., Thrall, J. H., Pitt, B. 1982; 23 (9): 795-798

  Abstract

  A patient with the sick-sinus syndrome was evaluated because of symptomatic deterioration after insertion of a ventricular demand pacemaker. Clinical features of the ventricular pacemaker syndrome were recognized and confirmed by electrophysiological and hemodynamic studies. Phase analysis--a new technique for detecting patterns of cardiac emptying from gated cardiac blood-pool scintigrams--demonstrated a pattern consistent with 1:1 ventriculo-atrial conduction. Phase analysis of the gated cardiac blood-pool scintigram may be useful in the assessment of patients with ventricular demand pacemakers who complain of fatigue and effort intolerance.

  View details for Web of Science ID A1982PF56300008

  View details for PubMedID 7108627

• RADIONUCLIDE EVALUATION OF POST-EXTRASYSTOLIC POTENTIATION OF LEFT-VENTRICULAR FUNCTION INDUCED BY ATRIAL AND VENTRICULAR STIMULATION AMERICAN JOURNAL OF CARDIOLOGY Kalff, V., Chan, W., RABINOVITCH, M., ONEILL, W., Walton, J., STEWART, J., Thrall, J. H., Pitt, B. 1982; 50 (1): 106-111

  Abstract

  Postextrasystolic potentiation of left ventricular function induced by ventricular and atrial stimulation was compared in 10 patients using radionuclide ventriculography. After insertion of pacing wires, a preliminary radionuclide ventriculogram was obtained and then ventricular and atrial trigeminy was induced in random order, each with identical R-R coupling intervals, each for 6 to 10 minutes. During the stimulation studies, radionuclide data were acquired in electrocardiographic gated list mode format. Left ventricular ejection fraction and relative end-diastolic and end-systolic volume changes were measured for each reformatted composite sinus, atrial and ventricular premature beat and potentiated beat. The volume changes were normalized to the count-based values obtained for the sinus beat of the appropriate study. Postextrasystolic potentiation induced by either ventricular or atrial stimulation was characterized by similar significant increases in left ventricular ejection fraction (mean +/- standard deviation 7 +/- 3 percent, p less than 0.01 versus 7 +/- 5 percent, p less than 0.01; difference not significant [NS]) and decreases in relative end-systolic volume (-12 +/- 12 percent, p less than 0.01 versus -12 +/- 8 percent, p less than 0.01; NS) but little change in relative end-diastolic volume (+5 +/- 10 percent, NS versus +4 +/- 7 percent, NS; NS). This was despite a longer compensatory pause (1,120 +/- 220 versus 1,050 +/- 190 ms, p less than 0.01) after the ventricular premature beat. It is concluded that there is no difference in the postextrasystolic potentiation induced by atrial or ventricular premature stimulation.

  View details for Web of Science ID A1982NW95900014

  View details for PubMedID 7090992

• RADIONUCLIDE ANGIOCARDIOGRAPHIC ASSESSMENT OF PULMONARY VASCULAR REACTIVITY IN PATIENTS WITH LEFT TO RIGHT SHUNT AND PULMONARY-HYPERTENSION AMERICAN JOURNAL OF CARDIOLOGY Fujii, A. M., RABINOVITCH, M., Keane, J. F., Fyler, D. C., Treves, S. 1982; 49 (2): 356-361

  Abstract

  Radionuclide angiocardiography was used to assess pulmonary vascular reactivity in eight patients (nine studies) with a large, relatively unrestrictive intracardiac defect and pulmonary arterial hypertension. Radionuclide angiocardiograms, using technetium-99m pertechnetate, were performed first with the patient breathing room air and then after 10 minutes of breathing a mixture containing 90 percent or more of oxygen. The pulmonary to systemic flow ratios obtained by gamma variate analysis of the radionuclide time-activity curves were compared with those calculated with the Fick principle at the time of cardiac catheterization. There was a good correlation between the two methods both in room air studies (r = 0.88) and in those obtained with 90 percent or more of oxygen (r = 0.94). All six studies (in five patients) with a reactive pulmonary vasculature (judged by a pulmonary vascular resistance at cardiac catheterization of less than 6 units/m2 with oxygen or after tolazoline) had a radionuclide pulmonary to systemic flow ratio of 3.0 or greater with oxygen. The three patients with a nonreactive pulmonary vasculature had a radionuclide pulmonary to systemic flow ratio of 2.3 or less with oxygen, a value that was unchanged from the room air value. These data suggest that radionuclide angiocardiography may be a useful, relatively noninvasive method of assessing pulmonary vascular reactivity in patients with a large, relatively unrestrictive intracardiac defect.

  View details for Web of Science ID A1982NA58200014

  View details for PubMedID 6277174

• Quantitative structural analysis of the pulmonary vascular bed in congenital heart defects. Cardiovascular clinics RABINOVITCH, M., Reid, L. M. 1981; 11 (2): 149-169

  View details for PubMedID 7011541

• THE STRUCTURAL BASIS OF PERSISTENT PULMONARY-HYPERTENSION OF THE NEWBORN-INFANT JOURNAL OF PEDIATRICS Murphy, J. D., RABINOVITCH, M., Goldstein, J. D., Reid, L. M. 1981; 98 (6): 962-967

  Abstract

  Six neonates of 35 to 42 weeks' gestation had findings of persistent pulmonary hypertension and died between one and 6 days of age despite intensive medical therapy. Each patient had pulmonary artery pressure near or above systemic level, with a right-to-left shunt via the foramen ovale and/or ductus arteriosus. At postmortem, morphometric analysis of the peripheral pulmonary vascular bed revealed extension of muscle into small arteries, which was severe in five of six patients; all alveolar duct and wall arteries (less than 30 micrometers external diameter), normally nonmuscular, were fully muscularized. In these five patients medial wall thickness of the normally muscular intra-acinar arteries was doubled; arterial size and number, however, were normal in all. This striking structural maldevelopment of the peripheral pulmonary arterial bed occurred or was initiated in utero and does not merely represent a failure of the fetal pattern to regress. We suggest that this particular group of patients remained refractory to all current modes of therapy because of these severe structural pulmonary vascular changes.

  View details for Web of Science ID A1981LU03800025

  View details for PubMedID 7229803

• AGE AND SEX INFLUENCE ON PULMONARY-HYPERTENSION OF CHRONIC HYPOXIA AND ON RECOVERY AMERICAN JOURNAL OF PHYSIOLOGY RABINOVITCH, M., Gamble, W. J., Miettinen, O. S., Reid, L. 1981; 240 (1): H62-H72

  Abstract

  To study the influence of age and sex on the hemodynamic and structural response of the pulmonary vascular bed to chronic hypobaric hypoxia, "infant" Sprague-Dawley rats from 8 days old and "adult" rats from 9 wk old, each group including both sexes, were exposed to half atmospheric pressure for 1 mo and then allowed to recover in room air for up to 3 mo. During hypoxic exposure, pulmonary artery hypertension (Ppa) developed in all groups. The level of Ppa was similar in both male and female infant and in male adult rats but was significantly lower (P < 0.01) in the female adult rats. After recovery in room air, only partial regression of Ppa had occurred in all groups (P < 0.001). In male and female adult rats, recovery values were similar but infant rats had more residual Ppa than adults (P < 0.001). The structural changes that developed during hypoxia, especially the abnormal presence of muscle in small and peripheral intra-acinar arteries, were more severe in male adult rats compared with female adults (P < 0.01) and in infants of both sexes compared with male adults (P < 0.01). After recovery, residual structural changes were present in all rat groups but were most severe in the infants (P < 0.01).

  View details for Web of Science ID A1981KZ88300045

  View details for PubMedID 6450541

• GROWTH AND DEVELOPMENT OF THE PULMONARY VASCULAR BED IN PATIENTS WITH TETRALOGY OF FALLOT WITH OR WITHOUT PULMONARY ATRESIA CIRCULATION RABINOVITCH, M., HERRERADELEON, V., Castaneda, A. R., Reid, L. 1981; 64 (6): 1234-1249

  View details for Web of Science ID A1981MT14700023

  View details for PubMedID 7296796

• QUANTITATIVE-ANALYSIS OF THE PULMONARY WEDGE ANGIOGRAM IN CONGENITAL HEART-DEFECTS - CORRELATION WITH HEMODYNAMIC DATA AND MORPHOMETRIC FINDINGS IN LUNG-BIOPSY TISSUE CIRCULATION RABINOVITCH, M., Keane, J. F., Fellows, K. E., Castaneda, A. R., Reid, L. 1981; 63 (1): 152-164

  View details for Web of Science ID A1981KV09600020

  View details for PubMedID 7470217

• QUANTITATIVE TL-201 MYOCARDIAL IMAGING IN ASSESSING RIGHT VENTRICULAR PRESSURE IN PATIENTS WITH CONGENITAL HEART-DEFECTS BRITISH HEART JOURNAL RABINOVITCH, M., Fischer, K. C., Treves, S. 1981; 45 (2): 198-205

  Abstract

  Thallium-201 myocardial scintigraphy was performed in patients with congenital heart defects to determine whether, by quantification of right ventricular isotope uptake, one could assess the degree of right ventricular hypertrophy and so predict the level of right ventricular pressure. A total of 24 patients ranging in age from 7 months to 30 years was studied; 18 were studied before corrective surgery and six after operation. All but three had congenital heart defects which had resulted in pressure and/or volume-overload of the right ventricle. At routine cardiac catheterisation, 20 microCi/kg thallium-201 as thallous chloride was injected through the venous catheter and myocardial images were recorded in anterior and left anterior oblique projections; these were subsequently analysed quantitatively and qualitatively. Insignificant right ventricular thallium-201 counts judged as being less than 1 per cent of the injected dose or less than 0.3 of the left ventricular counts were present in six patients all with right ventricular peak systolic pressure less than 30 mmHg. In the remaining 18 patients there was a good correlation between the right ventricular/left ventricular peak systolic pressure ratio and the right ventricular/left ventricular thallium-201 counts ratio. All patients with right ventricular/left ventricular peak systolic pressure less than 0.5 had right ventricular/left ventricular thallium-201 counts less than 0.4. Qualitative evaluation of right ventricular isotope intensity proved helpful mainly in distinguishing the patients with right ventricular pressures at or above systemic levels. Thus quantitative analysis of myocardial imaging with thallium-201 is of use clinically in patients with congenital heart defects, in assessing the severity of pulmonary stenosis or the presence of pulmonary artery hypertension.

  View details for Web of Science ID A1981LC72400014

  View details for PubMedID 7459178

• LUNG-BIOPSY WITH FROZEN SECTION AS A DIAGNOSTIC-AID IN PATIENTS WITH CONGENITAL HEART-DEFECTS AMERICAN JOURNAL OF CARDIOLOGY RABINOVITCH, M., Castaneda, A. R., Reid, L. 1981; 47 (1): 77-84

  Abstract

  In eight patients with congenital heart defects and equivocal preoperative hemodynamic data, lung biopsy with frozen section was performed and quantitative morphometric analysis carried out to help select a palliative or corrective surgical procedure. During catheterization it had been possible in only six of the eight patients to enter the pulmonary artery and obtain pressure measurements; four patients had moderate to severe elevation of pulmonary vascular resistance whereas the other two had only mild elevation of pulmonary arterial pressure, but they were being considered for a Fontan procedure. The lung biopsy tissue was taken inflated and fixed inflated in hot glutaraldehyde. From cryostat sections arterial concentration was assessed relative to alveolar concentration, and the degree of arterial muscularity was assessed by wall thickness and extension of muscle; the more advanced Health-Edwards arterial changes were also evaluated. On the basis of the morphologic assessment, six of the eight patients underwent corrective surgery and two underwent a palliative procedure. Postoperative hemodynamic data available in four of the patients who underwent corrective surgery revealed either a significant reduction in pulmonary vascular resistance or return to a normal level of pulmonary arterial pressure, whereas no change has occurred in the one patient studied who underwent a palliative procedure. Assessment of pulmonary arterial development and structure on lung biopsy with frozen section proved helpful in deciding between a palliative and corrective surgical procedure.

  View details for Web of Science ID A1981KX77800013

  View details for PubMedID 7457413

• A CASE OF SPONTANEOUS RESOLUTION OF IDIOPATHIC PULMONARY-HYPERTENSION BRITISH HEART JOURNAL Fujii, A., RABINOVITCH, M., MATTHEWS, E. C. 1981; 46 (5): 574-577

  View details for Web of Science ID A1981MU37700018

  View details for PubMedID 7317224

• EARLY PULMONARY VASCULAR CHANGES IN CONGENITAL HEART-DISEASE STUDIED IN BIOPSY-TISSUE HUMAN PATHOLOGY RABINOVITCH, M., Haworth, S. G., VANCE, Z., VAWTER, G., Castaneda, A. R., NADAS, A. S., Reid, L. M. 1980; 11 (5): 499-509

  Abstract

  Patients with congenital heart defects who had or were at risk for developing pulmonary artery hypertension underwent lung biopsy at the time of intracardiac repair. In 95 consecutive patients with either ventricular septal defect, d-transposition of the great arteries, or a defect of the atrioventricular canal, the pulmonary arteries were evaluated microscopically by quantitative morphologic techniques, and the findings were correlated with hemodynamic data obtained at a recent preoperative cardiac catheterization. Three grades of severity of early pulmonary vascular changes were identified, which correlated with hemodynamic evidence of progressive functional impairment. Grade A denotes cases with abnormal extension of muscle into peripheral arteries only; pulmonary blood flow is increased but pulmonary artery pressure is normal. In grade B an increased medial wall thickness of the normally muscular arteries is also present, and in these cases pulmonary artery pressure is also increased. Grade C denotes cases in which in addition to the findings in grade B disease there is a reduction in the number of small peripheral arteries; in these cases pulmonary vascular resistance is increased. Follow-up postoperative hemodynamic evaluation will reveal the significance of these changes in reflecting irreversible functional impairment of the pulmonary vascular bed.

  View details for Web of Science ID A1980KP08600002

  View details for PubMedID 7429501

• RAT PULMONARY CIRCULATION AFTER CHRONIC HYPOXIA - HEMODYNAMIC AND STRUCTURAL FEATURES AMERICAN JOURNAL OF PHYSIOLOGY RABINOVITCH, M., Gamble, W., NADAS, A. S., Miettinen, O. S., Reid, L. 1979; 236 (6): H818-H827

  Abstract

  In 55 Sprague-Dawley rats (mean wt, 277 +/- 6.2 g) exposed to hypobaric hypoxia (air at 380 mmHg), and 23 weight-matched controls kept in room air, pulmonary and systemic artery pressures were measured daily for 2 wk via indwelling catheters. After each day of exposure, 1 or 2 hypoxic rats, to a total of 20, and 5 control rats were killed during the experiment. In these rats, the pulmonary arterial tree was injected post mortem with barium-gelatin and inflated with formaldehyde solution, and three structural features were quantified microscopically: 1) abnormal extension of muscle into peripheral arteries where it is not normally present (EMPA); 2) increased wall thickness of the normally muscular arteries, expressed as a percentage of external diameter (%WT); and 3) reduction in artery number expressed as an increase in the ratio of alveoli to arteries (A/a). Mean pulmonary artery pressure (Ppa) rose significantly after day 3 of hypoxic exposure (P less than 0.05) and had doubled by day 14; the mean systemic artery pressure (Psa) of hypoxic rats and Ppa and Psa of control rats were unchanged. The level of Ppa correlated with the degree of structural changes; for EMPA, r = 0.84; for %WT, r = 0.64; and for A/a, r = 0.73 (P less than 0.001 in all.

  View details for Web of Science ID A1979GZ71600036

  View details for PubMedID 443445

• TL-201 SCINTIGRAPHY IN PATIENTS WITH ANOMALOUS ORIGIN OF THE LEFT CORONARY-ARTERY FROM THE MAIN PULMONARY-ARTERY JOURNAL OF PEDIATRICS RABINOVITCH, M., Rowland, T. W., Castaneda, A. R., Treves, S. 1979; 94 (2): 244-247

  View details for Web of Science ID A1979GG11300017

  View details for PubMedID 762617

• UNUSUAL VASCULAR ANOMALIES CAUSING PERSISTENT PULMONARY-HYPERTENSION IN A NEWBORN AMERICAN JOURNAL OF CARDIOLOGY Goldstein, J. D., RABINOVITCH, M., VANPRAAGH, R., Reid, L. 1979; 43 (5): 962-968

  Abstract

  A unique case of pulmonary vascular anomalies causing persistent pulmonary hypertension in a newborn is described. The child died 3 days after birth. Necropsy revealed marked hypoplasia of the right and left pulmonary arteries with a normal main pulmonary artery, patent ductus arteriosus, bilateral systemic arteries to the lungs from the abdominal aorta, and partial anomalous pulmonary venous connection. Quantitative morphometric techniques demonstrated slight abnormalities of alveolar development and severe arterial medial hypertrophy with abnormal extension of muscle into small peripheral arteries. Bronchopulmonary development appeared relatively normal in spite of the vascular abnormalities.

  View details for Web of Science ID A1979GT79500013

  View details for PubMedID 433778

• TL-201 - QUANTITATION OF RIGHT VENTRICULAR HYPERTROPHY IN CHRONICALLY HYPOXIC RATS RADIOLOGY RABINOVITCH, M., Fisher, K., Gamble, W., Reid, L., Treves, S. 1979; 130 (1): 223-225

  Abstract

  Sprague Dawley rats were divided into two groups. Ten were kept in room air and 10 in hypobaric hypoxia (air at 380 mm Hg). After two weeks all were injected intravenously with 50 muCi of 201Tl and sacrificed. The right and left ventricles were separated, weighed, and measured for radioactivity in a gamma well counter. Left and right ventricular mass ratios (MR) correlated with 201Tl radioactivity ratios (TAR) in both control and hypoxic rats: r = 0.962 where MR = 0.863 TAR + 0.27. Myocardial 201Tl uptake reflects and quantitates normal and abnormal ventricular mass, the abnormal mass in this model consisting of right ventricular hypertrophy associated with hypoxic pulmonary hypertension.

  View details for Web of Science ID A1979GB83700040

  View details for PubMedID 152938

• LUNG-BIOPSY IN CONGENITAL HEART-DISEASE - MORPHOMETRIC APPROACH TO PULMONARY VASCULAR-DISEASE CIRCULATION RABINOVITCH, M., Haworth, S. G., Castaneda, A. R., NADAS, A. S., Reid, L. M. 1978; 58 (6): 1107-1122

  Abstract

  Fifty patients with congenital heart disease, ages 2 days-30 years (median 12 months) at cardiac surgery, underwent lung biopsy to assess pulmonary vascular disease (PVD). Twenty-six had ventricular septal defects (VSD), 17 d-transposition of the great arteries (D-TGA), and seven, defects of the atrioventricular canal (AVC). Quantitative morphologic data was correlated with hemodynamic data. Three new grades of PVD were observed. Abnormal extension of muscle into peripheral arteries (grade A) was found in all patients; all had increased pulmonary blood flow. In addition, 38 of 50 patients had an increase in percentage arterial wall thickness (grade B); this correlated with elevation in pulmonary artery (PA) pressure (r = 0.59). Another 10 of 50 patients had, in addition to A and B, a reduction in the number of small arteries (grade C); nine of 10 were patients with elevated PA resistance greater than 3.5 mu/m2 (P less than 0.005). All three patients with Heath-Edwards changes of grade III or worse also had grade C. Reduction in peripheral arterial number probably precedes obliterative PVD and may identify those patients in whom, despite corrective surgery, PVD will progress.

  View details for Web of Science ID A1978GA34300017

  View details for PubMedID 709766

• REGIONAL LUNG-FUNCTION STUDIES AND RADIONUCLIDE ANGIOGRAPHY IN D-TRANSPOSITION OF GREAT ARTERIES PEDIATRIC RESEARCH RABINOVITCH, M., Rosenthal, A., Sade, R. M., Castaneda, A. R., Treves, S., NADAS, A. S. 1977; 11 (10): 1117-1121

  Abstract

  Thirty-nine ventilation and perfusion lung studies with xenon 313 were perfomed in 29 patients with D-transposition of the great arteries (DTGA). Fifteen patients had simple DTGA and 14 more complex anatomic features. Inequalitiies of ventilation (V) were present in 23% (9/39) and perfusion (Q) in 44% (17/39) of studies. Six patients with simple DTGA showed abnormal distribution of perfusion, all with increased flow to the right lung. Among the 14 patients with complex DTGA, eight (57%) had abnormal distribution of perfusion. In patients with pulmonary artery band, increased flow was directed toward the left lung, and in those with an aorticopulmonary anastomosis, increased flow was to the side of the shunt. Radionuclide angiograms were performed in 17 patients with DTGA, both before and after surgical repair. In all, an abnormal circulatory pattern was noted. Complete superior vena cava obstruction present in four patients and residual left to right shunt (Qp/qs greater than 1.7/liter) in three was confirmed at cardiac catheterization. Ventilation perfusion studies with xenon-313 and technetium-99m radionuclide angiogram were effective noninvasive techniques in assessing pulmonary function and hemodynamics in patients with DTGA.

  View details for Web of Science ID A1977DY24200010

  View details for PubMedID 904978

• INTRACARDIAC ELECTROGRAPHY IN ENDOCARDIAL CUSHION DEFECTS CIRCULATION Jacobsen, J. R., Gillette, P. C., CORBETT, B. N., RABINOVITCH, M., McNamara, D. G. 1976; 54 (4): 599-603

  Abstract

  Conduction of the sino-atrial impulse from the high right atrium to the ventricles was studied by intracardiac electrography in 21 unoperated patients, age 3 months to 11 years, with endocardial cushion defects (ECD). The high right atrium-to-low right atrium conduction time was prolonged in 15 of 18 subjects (mean 57 +/- 20 msec). The low right atrium-to-His bundle conduction time (LRA-H) was normal in 16 of 17 subjects (mean 82 +/- 30 msec), prolonged in one. The His-to-ventricle conduction time (H-V) was normal in 16 of 17 subjects (mean 37 +/- 8 msec), equivocally short in one. Nine patients with ECD, age 3 to 21 years, were studied postoperatively. One had an acquired complete atrioventricular block in the His bundle. Two had prolonged LRA-H and two prolonged LRA-H and two prolonged H-V. The surface ECG failed to identify accurately either prolonged atrioventricular conduction or the site of prolongation.

  View details for Web of Science ID A1976CE72200010

  View details for PubMedID 786499