Joshua M. Spin

All Publications

• Plasma proteomics reveals the potential causal impact of extracellular matrix proteins on abdominal aortic aneurysm. medRxiv : the preprint server for health sciences Khodursky, S., Yuan, S., Spin, J. M., Tsao, P. S., Levin, M. G., Damrauer, S. M. 2024

  Abstract

  Background: Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. Genetic studies have identified numerous associated loci, many potentially encoding plasma proteins. However, the causal effects of plasma proteins on AAA have not been thoroughly studied. We used genetic causal inference approaches to identify plasma proteins that have a potential causal impact on AAA.Methods: Causal inference was performed using two-sample Mendelian randomization (MR). For AAA, we utilized recently published summary statistics from a multi-population genome-wide association (GWAS) meta-analysis including 39,221 individuals with, and 1,086,107 individuals without AAA from 14 cohorts. We used protein quantitative trait loci (pQTLs) identified in two large-scale plasma-proteomics studies (deCODE and UKB-PPP) to generate genetic instruments. We tested 2,783 plasma proteins for possible causal effects on AAA using two-sample MR with inverse variance weighting and common sensitivity analyses to evaluate the MR assumptions. Bayesian colocalization and gene ontology (GO) enrichment analyses provided additional insights.Results: MR identified 90 plasma proteins associated with AAA at FDR<0.05, with 25 supported by colocalization analysis. Among those supported by both MR and colocalization were previously experimentally validated proteins such as PCSK9 (OR 1.3; 95%CI 1.2-1.4; P<1e-10), LTBP4 (OR 3.4; 95%CI 2.6-4.6; P<1e-10) and COL6A3 (OR 0.6; 95%CI 0.5-0.7; P<1e-6). GO analysis revealed enrichment of proteins found in extracellular matrix (ECM, OR 7.8; P<1e-4), some with maximal mRNA levels in aortic tissue. Bi-directional MR suggested plasma level changes were not caused by liability to AAA itself. We then investigated whether variants responsible for expression changes in the aorta also influenced plasma levels and AAA risk. Colocalization analysis showed that an aortic expression quantitative trait locus (eQTL) for COL6A3, and a splicing quantitative trait locus (sQTL) for LTBP4 colocalized with their respective plasma pQTLs and AAA signals (posterior probabilities 0.84 and 0.89, respectively).Conclusions: Our results highlight proteins and pathways with potential causal effects on AAA, providing a foundation for future functional experiments. These findings suggest a possible causal pathway whereby genetic variation affecting ECM proteins expressed in the aortic wall cause their levels to change in blood plasma, influencing development of AAA.

  View details for DOI 10.1101/2024.09.20.24314065

  View details for PubMedID 39398994

• Room temperature is a key factor for modeling human lower extremity artery disease with surgical murine hind limb ischemia. Journal of molecular and cellular cardiology Abe, Y., Javkhlant, A., Spin, J. M., Toyama, K. 2024

  View details for DOI 10.1016/j.yjmcc.2024.06.004

  View details for PubMedID 38848809

• Lack of ATP2B1 in CD4+ T Cells Causes Colitis. Inflammatory bowel diseases Javkhlant, A., Toyama, K., Abe, Y., Spin, J. M., Mogi, M. 2024

  Abstract

  The ATP2B1 gene encodes for a calcium pump, which plays a role in removing Ca2+ from cells and maintaining intracellular Ca2+ homeostasis. Reduction of the intracellular Ca2+ concentration in CD4+ T cells is thought to reduce the severity of colitis, while elevation of Ca2+ in CD4+ T cells induces T cell hyperactivity. Our aim was to clarify the role of ATP2B1 in CD4+ T cells and in inflammatory bowel disease development.A murine CD4+ T cell-specific knockout (KO) of ATP2B1 was created using a Cre-loxP system. CD4+ T cells were isolated from thymus, spleen, and blood using fluorescence-activated cell sorting. To quantify messenger RNA levels, quantitative real-time polymerase chain reaction was performed.Although the percentages of CD4+ T cells in both KO mouse spleen and blood decreased compared with those of the control samples, both T-bet (a T helper 1 [Th1] activity marker) and GATA3 (a Th2 activity marker) expression levels were further increased in KO mouse blood CD4+ T cells (vs control blood). Diarrhea and colonic wall thickening (with mucosal changes, including crypt distortion) were seen in KO mice but not in control mice. Prior to diarrhea onset, the KO mouse colon length was already noted to be shorter, and the KO mouse stool water and lipid content were higher than that of the control mice. Tumor necrosis factor α and gp91 expressions were increased in KO mouse colon.Lack of ATP2B1 in CD4+ T cells leads to Th1 and Th2 activation, which contributes to colitis via elevation of tumor necrosis factor α and oxidative stress.

  View details for DOI 10.1093/ibd/izae045

  View details for PubMedID 38507609

• Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm CARDIOVASCULAR RESEARCH Wagenhaeuser, M. U., Mulorz, J., Krott, K. J., Bosbach, A., Feige, T., Rhee, Y. H., Chatterjee, M., Petzold, N., Boeddeker, C., Ibing, W., Krueger, I., Popovic, A. M., Roseman, A., Spin, J. M., Tsao, P. S., Schelzig, H., Elvers, M. 2023

  Abstract

  Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases but their role in AAA is poorly understood.The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the ECM. They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodeling but also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity results in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity was also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodeling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall.In conclusion, our data strongly supports the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

  View details for DOI 10.1093/cvr/cvad168

  View details for Web of Science ID 001171481200001

  View details for PubMedID 37976180

• Genome-wide association meta-analysis identifies risk loci for abdominal aortic aneurysm and highlights PCSK9 as a therapeutic target NATURE GENETICS Roychowdhury, T., Klarin, D., Levin, M. G., Spin, J. M., Rhee, Y., Deng, A., Headley, C. A., Tsao, N. L., Gellatly, C., Zuber, V., Shen, F., Hornsby, W. E., Laursen, I., Verma, S. S., Locke, A. E., Einarsson, G., Thorleifsson, G., Graham, S. E., Dikilitas, O., Pattee, J. W., Judy, R. L., Pauls-Verges, F., Nielsen, J. B., Wolford, B. N., Brumpton, B. M., Dilme, J., Peypoch, O., Juscafresa, L., Edwards, T. L., Li, D., Banasik, K., Brunak, S., Jacobsen, R. L., Garcia-Barrio, M. T., Zhang, J., Rasmussen, L. M., Lee, R., Handa, A., Wanhainen, A., Mani, K., Lindholt, J. S., Obel, L. M., Strauss, E., Oszkinis, G., Nelson, C. P., Saxby, K. L., van Herwaarden, J. A., van der Laan, S. W., van Setten, J., Camacho, M., Davis, F. M., Wasikowski, R., Tsoi, L. C., Gudjonsson, J. E., Eliason, J. L., Coleman, D. M., Henke, P. K., Ganesh, S. K., Chen, Y., Guan, W., Pankow, J. S., Pankratz, N., Pedersen, O. B., Erikstrup, C., Tang, W., Hveem, K., Gudbjartsson, D., Gretarsdottir, S., Thorsteinsdottir, U., Holm, H., Stefansson, K., Ferreira, M. A., Baras, A., Kullo, I. J., Ritchie, M. D., Christensen, A. H., Iversen, K. K., Eldrup, N., Sillesen, H., Ostrowski, S. R., Bundgaard, H., Ullum, H., Burgess, S., Gill, D., Gallagher, K., Sabater-Lleal, M., Surakka, I., Jones, G. T., Bown, M. J., Tsao, P. S., Willer, C. J., Damrauer, S. M., Dudbridge, F., Samani, N. J., DiscovEHR, Regeneron Genetics Ctr, UK Aneurysm Growth Study, DBDS Genomic Consortium, VA Million Vet Program 2023

  View details for DOI 10.1038/s41588-023-01510

  View details for Web of Science ID 001085082700001

• Genome-wide association meta-analysis identifies risk loci for abdominal aortic aneurysm and highlights PCSK9 as a therapeutic target. Nature genetics Roychowdhury, T., Klarin, D., Levin, M. G., Spin, J. M., Rhee, Y. H., Deng, A., Headley, C. A., Tsao, N. L., Gellatly, C., Zuber, V., Shen, F., Hornsby, W. E., Laursen, I. H., Verma, S. S., Locke, A. E., Einarsson, G., Thorleifsson, G., Graham, S. E., Dikilitas, O., Pattee, J. W., Judy, R. L., Pauls-Verges, F., Nielsen, J. B., Wolford, B. N., Brumpton, B. M., Dilmé, J., Peypoch, O., Juscafresa, L. C., Edwards, T. L., Li, D., Banasik, K., Brunak, S., Jacobsen, R. L., Garcia-Barrio, M. T., Zhang, J., Rasmussen, L. M., Lee, R., Handa, A., Wanhainen, A., Mani, K., Lindholt, J. S., Obel, L. M., Strauss, E., Oszkinis, G., Nelson, C. P., Saxby, K. L., van Herwaarden, J. A., van der Laan, S. W., van Setten, J., Camacho, M., Davis, F. M., Wasikowski, R., Tsoi, L. C., Gudjonsson, J. E., Eliason, J. L., Coleman, D. M., Henke, P. K., Ganesh, S. K., Chen, Y. E., Guan, W., Pankow, J. S., Pankratz, N., Pedersen, O. B., Erikstrup, C., Tang, W., Hveem, K., Gudbjartsson, D., Gretarsdottir, S., Thorsteinsdottir, U., Holm, H., Stefansson, K., Ferreira, M. A., Baras, A., Kullo, I. J., Ritchie, M. D., Christensen, A. H., Iversen, K. K., Eldrup, N., Sillesen, H., Ostrowski, S. R., Bundgaard, H., Ullum, H., Burgess, S., Gill, D., Gallagher, K., Sabater-Lleal, M., Surakka, I., Jones, G. T., Bown, M. J., Tsao, P. S., Willer, C. J., Damrauer, S. M. 2023

  Abstract

  Abdominal aortic aneurysm (AAA) is a common disease with substantial heritability. In this study, we performed a genome-wide association meta-analysis from 14 discovery cohorts and uncovered 141 independent associations, including 97 previously unreported loci. A polygenic risk score derived from meta-analysis explained AAA risk beyond clinical risk factors. Genes at AAA risk loci indicate involvement of lipid metabolism, vascular development and remodeling, extracellular matrix dysregulation and inflammation as key mechanisms in AAA pathogenesis. These genes also indicate overlap between the development of AAA and other monogenic aortopathies, particularly via transforming growth factor β signaling. Motivated by the strong evidence for the role of lipid metabolism in AAA, we used Mendelian randomization to establish the central role of nonhigh-density lipoprotein cholesterol in AAA and identified the opportunity for repurposing of proprotein convertase, subtilisin/kexin-type 9 (PCSK9) inhibitors. This was supported by a study demonstrating that PCSK9 loss of function prevented the development of AAA in a preclinical mouse model.

  View details for DOI 10.1038/s41588-023-01510-y

  View details for PubMedID 37845353

  View details for PubMedCentralID 5800308

• Serum microRNA-501-3p is a potential diagnostic tool for detecting mild cognitive impairment: Ehime genome study. Journal of neurochemistry Toyama, K., Spin, J. M., Tsao, P. S., Maruyama, K., Osawa, H., Mogi, M., Takata, Y. 2023

  Abstract

  Tight junction disruption and dysfunction are involved in the progression of blood-brain barrier (BBB) breakdown. Recent investigations have revealed BBB disruption in patients with vascular cognitive decline. Our previous studies showed that miR-501-3p negatively regulates cerebral endothelial tight junction protein-1, resulting in the disruption of the BBB, and playing an important role in the development of vascular cognitive impairment. BBB breakdown in white matter lesions is often seen in the patients with vascular mild cognitive impairment (MCI). We therefore hypothesize that most early-phase MCI patients may demonstrate elevated expression of miR-501-3p and sought to investigate whether serum exosome miR-501-3p levels could be a clinical indicator for detecting mild cognitive impairment. One hundred and seventy-eight subjects (aged 73 [68-75] years, 53% male) were recruited for this study. The Japanese version of the Montreal Cognitive Assessment (MoCA-J) was used for detecting MCI. Serum exosome miR-501-3p expression levels were measured by qPCR methods. Patients were divided into two groups depending on whether their miR-501-3p ∆Ct values were above ("High"; n = 74) or below ("Low"; n = 104) cutoff levels determined by ROC curve. MCI was detected significantly more often in the miR-501-3p-High group (vs. -Low group, 63.5% vs. 47.1%, respectively; p < 0.05). Multivariate logistic regression analysis showed a significant association between MCI status and High miR-501-3p (odds ratio 2.662; p < 0.01), improved vs. known risk factors. In non-diabetic patients, High miR-501-3p was positively associated with MCI status (odds ratio 3.633; p < 0.01) and also positively associated with MCI status in those with atherosclerosis (odds ratio 3.219; p < 0.01). The present study demonstrates that elevated expression of blood exosomal miR-501-3p can indicate the presence of MCI in human patients. Early detection of vascular injuries may allow a reduction in progressive dementia through the management of vascular risk factors.

  View details for DOI 10.1111/jnc.15911

  View details for PubMedID 37439367

• Time-dependent effects of cellulose and gelatin-based hemostats on cellular processes of wound healing. Archives of medical science : AMS Wagenhäuser, M. U., Garabet, W., van Bonn, M., Ibing, W., Mulorz, J., Rhee, Y. H., Spin, J. M., Dimopoulos, C., Oberhuber, A., Schelzig, H., Simon, F. 2023; 19 (1): 194-202

  Abstract

  Oxidized regenerated cellulose-based (ORC - TABOTAMP), oxidized non-regenerated cellulose-based (ONRC - RESORBA CELL), and gelatin-based (GELA - GELITA TUFT-IT) hemostats are commonly used in surgery. However, their impact on the wound healing process remains largely unexplored. We here assess time-dependent effects of exposure to these hemostats on fibroblast-related wound healing processes.Hemostats were applied to fibroblast cell cultures for 5-10 (short-), 30 and 60 min (intermediate-) and 24 h (long-term). Representative images of the hemostat degradation process were obtained, and the pH value was measured. Cell viability, apoptosis and migration were analyzed after the above exposure times at 3, 6 and 24 h follow-up. Protein levels for tumor necrosis factor α (TNF-α) and transforming-growth factor β (TGF-β) were assessed.ORC and ONRC reduced pH values during degradation, while GELA proved to be pH-neutral. Hemostat structural integrity was prolonged for GELA (vs. ORC and ONRC). TGF-β and TNF-α levels were reduced for ORC and ONRC (vs. GELA and control) (p < 0.05). Further, exposure of ORC and ONRC for longer than 5-10 min reduced cell viability vs. GELA and control at 3 h post-exposure (p < 0.05). Similarly, cell migration was impaired with ORC and ONRC exposure longer than 60 min at 24 h follow-up (p < 0.05).Short-term exposure to ORC and ONRC impairs relevant wound healing-related processes in fibroblasts, and alters protein levels of key mediating cytokines. GELA does not show similar effects. We conclude that GELA may be preferred over ORC and ONRC over short-, intermediate- and long-term exposures. Future validation of the clinical relevance is warranted.

  View details for DOI 10.5114/aoms.2020.92830

  View details for PubMedID 36817681

  View details for PubMedCentralID PMC9897096

• Time-dependent effects of cellulose and gelatin-based hemostats on cellular processes of wound healing ARCHIVES OF MEDICAL SCIENCE Wagenhaeuser, M. U., Garabet, W., van Bonn, M., Ibing, W., Mulorz, J., Rhee, Y., Spin, J., Dimopoulos, C., Oberhuber, A., Schelzig, H., Simon, F. 2023; 19 (1): 194-202

  View details for DOI 10.5114/aoms.2020.92830

  View details for Web of Science ID 000921618400014

• IS IT POSSIBLE TO ACCELERATE SENESCENCE IN THE VASCULAR ENDOTHELIAL CELL BY MODULATING SEVERAL MICRORNAS? Toyama, K., Spin, J. M., Tsao, P. S., Mogi, M. LIPPINCOTT WILLIAMS & WILKINS. 2023: E171

  View details for Web of Science ID 000921768300399

• Message to researchers: the characteristic absence of a posterior communicating artery is easily lost in the gerbil. Anatomical science international Abe, Y., Toyama, K., Shinohara, A., Nagura-Kato, G. A., Ikai, Y., Koshimoto, C., Spin, J. M., Hato, N. 2022

  Abstract

  The Mongolian gerbil has historically been useful for brain ischemia experiments, owing to the gerbil's uniquely underdeveloped circle of Willis (CoW). This led to a gerbil model of cochlear ischemia being generated in our unit. However, we have found that the usual severe hearing loss seen in this model was not being induced consistently in recent experiments using the MON/Jms/GbsSlc gerbil (the sole commercially available gerbil in Japan). We set out to evaluate the posterior communicating artery (PcomA) in MON/Jms/GbsSlc, to re-establish whether this strain is appropriate for ischemia models. Having found that this unique feature is often lost, we then attempted to breed for the characteristic absent PcomA. India-ink perfusion revealed that the percentage of intact bilateral PcomA ("communicating type") in the MON/Jms/GbsSlc gerbil was 57%; unilateral only ("unilateral communicating type") was 39%; and completely absent PcomA ("non-communicating type") was 4%. We were able to obtain few examples of the indigenous old aged Japanese UNG/Mz gerbil strain (at University of Miyazaki). Unfortunately, the pure UNG/Mz female was too elderly for mating. Therefore, selective breeding crosses between MON/Jms/GbsSlc and male UNG/Mz were carried out. After five generations of selective breeding, the percentage of non-communicating type gerbils was significantly higher in the newly generated strain, MON/Jms/SlcMz (F6 generation; 63%) than in the MON/Jms/GbsSlc gerbil. Bilateral common carotid artery occlusion surgery demonstrated that the cerebral blood flow was significantly reduced in MON/Jms/SlcMz compared with MON/Jms/GbsSlc (p<0.0001) and induced more hippocampal injuries in MON/Jms/SlcMz than in MON/Jms/GbsSlc (p<0.01). In conclusion, the commercially available MON/Jms/GbsSlc gerbil can easily regain PcomA, and we established a new gerbil strain (MON/Jms/SlcMz) displaying non-PcomA.

  View details for DOI 10.1007/s12565-022-00698-z

  View details for PubMedID 36472757

• E-cigarette exposure augments murine abdominal aortic aneurysm development: role of Chil1. Cardiovascular research Mulorz, J., Spin, J. M., Mulorz, P., Wagenhauser, M., Deng, A., Mattern, K., Rhee, Y. H., Toyama, K., Adam, M., Schelzig, H., Maegdefessel, L., Tsao, P. S. 2022

  Abstract

  AIMS: Abdominal aortic aneurysm (AAA) is a common cardiovascular disease with a strong correlation to smoking, although underlying mechanisms have been minimally explored. Electronic cigarettes (e-cigs) have gained recent broad popularity and can deliver nicotine at comparable levels to tobacco cigarettes, but effects on AAA development are unknown.METHODS AND RESULTS: We evaluated the impact of daily e-cig vaping with nicotine on AAA using two complementary murine models and found that exposure enhanced aneurysm development in both models and genders. E-cigs induced changes in key mediators of AAA development including cytokine chitinase-3-like protein 1 (CHI3L1/Chil1) and its targeting microRNA-24 (miR-24). We show that nicotine triggers inflammatory signaling and reactive oxygen species while modulating miR-24 and CHI3L1/Chil1 in vitro, and that Chil1 is crucial to e-cig-augmented aneurysm formation using a knockout model.CONCLUSIONS: In conclusion our work shows increased aneurysm formation along with augmented vascular inflammation in response to e-cig exposure with nicotine. Further we identify Chil1 as a key mediator in this context. Our data raise concerns regarding the potentially harmful long-term effects of e-cig nicotine vaping.TRANSLATIONAL PERSPECTIVE: Smoking is one of the most hazardous modifiable risk factors, with clear links to abdominal aortic aneurysm. E-cig vaping has displayed explosive growth in popularity. Intended for smoking cessation, it has been taken up by millions with no such clinical need, delivering nicotine addiction to new generations. The presumption that vaping is safer than tobacco overlooks the potential cardiovascular risks of nicotine. This study shows for the first time that inhaled e-cig nicotine vapor augments experimental AAA and aortic inflammation, suggests a mechanistic role for the cytokine Chil1/CHI3L1 and its regulator microRNA-24, and raises red flags regarding longitudinal e-cig safety.

  View details for DOI 10.1093/cvr/cvac173

  View details for PubMedID 36413508

• Role of MicroRNAs in acceleration of vascular endothelial senescence. Biochemistry and biophysics reports Toyama, K., Spin, J. M., Deng, A. C., Abe, Y., Tsao, P. S., Mogi, M. 2022; 30: 101281

  Abstract

  Backgrounds: Many factors are involved in cellular aging, and senescence induction requires complex regulation of various signaling networks and processes. Specifically, in the area of aging-related vascular cognitive impairment, laboratory-based findings have not yet yielded agents of practical use for clinical settings. One possible reason is that the physiologic elements of aging have been insufficiently considered. We sought to establish techniques to better model cellular aging using modulation of microRNAs, aiming to identify key microRNAs capable of fine-tuning aging-associated genes, and thereby regulating the senescence of vascular endothelial cells.Methods: We utilized expression microRNA arrays to evaluate control and senescent vascular endothelial cells in order to identify testable candidates. Bioinformatic analysis was used to select key microRNAs. These candidates were then modulated in vitro using microRNA mimics and inhibitors in endothelial cells, and senescence-associated gene expression patterns were evaluated by qPCR.Results: Seventeen microRNAs were found to be significantly increased more than 2-fold in senescent cells. Of those, bioinformatic analysis concluded that miR-181a-5p, miR-30a-5p, miR-30a-3p, miR-100-5p, miR-21-5p, and miR-382-5p were likely associated with regulation of cellular senescence. We evaluated the potential targets of these six microRNAs by comparing them with cell-cycling and apoptosis-related genes from published mRNA transcriptional array data from aged tissues, and found that miR-181a-5p, miR-30a-5p and miR-30a-3p were enriched in overlapping targets compared with the other candidates. Modulation of these microRNAs in vascular endothelial cells revealed that over-expression of miR-30a-5p, and inhibition of both miR-30a-3p and miR-181a-5p, induced senescence.Conclusion: miR-181a-5p, miR-30a-5p and miR-30a-3p likely contribute to aging-associated vascular endothelial cell senescence.

  View details for DOI 10.1016/j.bbrep.2022.101281

  View details for PubMedID 35651952

• peri-Adventitial delivery of smooth muscle cells in porous collagen scaffolds for treatment of experimental abdominal aortic aneurysm. Biomaterials science Mulorz, J., Shayan, M., Hu, C., Alcazar, C., Chan, A. H., Briggs, M., Wen, Y., Walvekar, A. P., Ramasubramanian, A. K., Spin, J. M., Chen, B., Tsao, P. S., Huang, N. F. 2021

  Abstract

  Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

  View details for DOI 10.1039/d1bm00685a

  View details for PubMedID 34522940

• MicroRNA miR-29b regulates diabetic aortic remodeling and stiffening. Molecular therapy. Nucleic acids Schellinger, I. N., Wagenhauser, M., Chodisetti, G., Mattern, K., Dannert, A., Petzold, A., Jakubizka-Smorag, J., Emrich, F., Haunschild, J., Schuster, A., Schwob, E., Schulz, K., Maegdefessel, L., Spin, J. M., Stumvoll, M., HasenfuSS, G., Tsao, P. S., Raaz, U. 2021; 24: 188–99

  Abstract

  Patients with type 2 diabetes (T2D) are threatened by excessive cardiovascular morbidity and mortality. While accelerated arterial stiffening may represent a critical mechanistic factor driving cardiovascular risk in T2D, specific therapies to contain the underlying diabetic arterial remodeling have been elusive. The present translational study investigates the role of microRNA-29b (miR-29b) as a driver and therapeutic target of diabetic aortic remodeling and stiffening. Using a murine model (db/db mice), as well as human aortic tissue samples, we find that diabetic aortic remodeling and stiffening is associated with medial fibrosis, as well as fragmentation of aortic elastic layers. miR-29b is significantly downregulated in T2D and miR-29b repression is sufficient to induce both aortic medial fibrosis and elastin breakdown through upregulation of its direct target genes COL1A1 and MMP2 thereby increasing aortic stiffness. Moreover, antioxidant treatment restores aortic miR-29b levels and counteracts diabetic aortic remodeling. Concluding, we identify miR-29b as a comprehensive-and therefore powerful-regulator of aortic remodeling and stiffening in T2D that moreover qualifies as a (redox-sensitive) target for therapeutic intervention.

  View details for DOI 10.1016/j.omtn.2021.02.021

  View details for PubMedID 33767915

• Immunomodulation therapy using tolerogenic macrophages in a rodent model of pulmonary hypertension. Stem cells and development Guihaire, J., Deuse, T., Wang, D., Spin, J. M., Blankenberg, F. G., Fadel, E., Reichenspurner, H., Schrepfer, S. 2021

  Abstract

  Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group, n=6). Tolerogenic macrophages (toM) were generated from monocyte isolation and then injected either the day before semaxinib injection (Prevention group, n=6) or 3 weeks after (Reversion group, n=6). Six athymic nude rats were used as controls. In vivo trafficking of toM was investigated with bioluminescence imaging showing that toM were mainly located into the lungs until 48 hours after injection. Right ventricular end-systolic pressure (RVESP) and right ventricular systolic function were assessed at 4 weeks using echocardiography. Morphometric analysis and RNA sequencing of the lungs were realized at 4 weeks. Rats treated with toM (Prevention and Reversion groups) had a significantly lower RVESP at 4 weeks (respectively 25±8 and 30±6 vs. 67±9 mmHg, P<0.001), while right ventricular systolic dysfunction was observed in Su and Reversion groups... Mean medial wall thickness of small arterioles was lower in Prevention and Reversion groups compared to the Su group (respectively 10.9±0.8% and 16.4±1.3% vs. 28.2±2.1%, P<0.001). Similarly, cardiomyocyte area was decreased in rats treated with toM (150±18 and 160±86 vs. 279±50 m, P<0.001). A trend towards upregulation of genes involved in PAH pathobiology was found in Su rats, while KCNK3 was significantly downregulated (Fold-change=9.8, P<0.001). Injection of toM was associated with a less severe phenotype of PH in rats exposed to angioproliferative stress. Preserved expression of KCNK3 may explain the protective effect of toM.

  View details for DOI 10.1089/scd.2021.0007

  View details for PubMedID 33726521

• Exosome miR-501-3p Elevation Contributes to Progression of Vascular Stiffness. Circulation reports Toyama, K., Igase, M., Spin, J. M., Abe, Y., Javkhlant, A., Okada, Y., Wagenhauser, M. U., Schelzig, H., Tsao, P. S., Mogi, M. 2021; 3 (3): 170–77

  Abstract

  Background: Tight junction (TJ) disruption and dysfunction are involved in the progression of arteriosclerosis. miR-501-3p regulates endothelial TJ protein-1, resulting in TJ disruption. Because exosomal microRNAs can travel to distant tissues and influence cell behavior, patients with elevated miR-501-3p may experience accelerated vascular disease progression secondary to miR-501-3p-induced reductions in TJ. This study investigated whether plasma exosome miR-501-3p levels are associated with vascular stiffness, an indicator for arteriosclerotic changes. Methods and Results: Fifty-one subjects (mean [±SD] age 70±8 years, 37% male) enrolled in a medical checkup program were recruited to the study. Brachial-ankle arterial pulse wave velocity (baPWV) and plasma exosome miR-501-3p expression were measured. Patients were divided into 2 groups depending on whether their miR-501-3p ∆Ct values were above ("High"; n=24) or below ("Low"; n=27) the cut-off levels determined by receiver operating characteristic (ROC) curve analysis. Median (interquartile range) baPWV levels were significantly higher in the miR-501-3p High than Low group (1,664 [1,496-1,859] vs. 1,450 [1,353-1,686] cm/s, respectively; P<0.05). Multivariate logistic regression analysis showed a significant association between increased baPWV and High miR-501-3p expression (odds ratio 4.66). At follow-up visits (mean 62 months later), baPWV remained significantly higher in the miR-501-3p High than Low group (1,830 [1,624-2,056] vs. 1,620 [1,377-1,816] cm/s, respectively; P<0.05). Conclusions: High expression levels of exosome miR-501-3p contribute to arteriosclerotic changes.

  View details for DOI 10.1253/circrep.CR-20-0135

  View details for PubMedID 33738350

• Genetic Architecture of Abdominal Aortic Aneurysm in the Million Veteran Program. Circulation Klarin, D. n., Verma, S. S., Judy, R. n., Dikilitas, O. n., Wolford, B. N., Paranjpe, I. n., Levin, M. G., Pan, C. n., Tcheandjieu, C. n., Spin, J. M., Lynch, J. n., Assimes, T. L., Nyrønning, L. Å., Mattsson, E. n., Edwards, T. L., Denny, J. n., Larson, E. n., Lee, M. T., Carrell, D. n., Zhang, Y. n., Jarvik, G. P., Gharavi, A. G., Harley, J. n., Mentch, F. n., Pacheco, J. A., Hakonarson, H. n., Skogholt, A. H., Thomas, L. n., Gabrielsen, M. E., Hveem, K. n., Nielsen, J. B., Zhou, W. n., Fritsche, L. n., Huang, J. n., Natarajan, P. n., Sun, Y. V., DuVall, S. L., Rader, D. J., Cho, K. n., Chang, K. M., Wilson, P. W., O'Donnell, C. J., Kathiresan, S. n., Scali, S. T., Berceli, S. A., Willer, C. n., Jones, G. T., Bown, M. J., Nadkarni, G. n., Kullo, I. J., Ritchie, M. n., Damrauer, S. M., Tsao, P. S. 2020

  View details for DOI 10.1161/CIRCULATIONAHA.120.047544

  View details for PubMedID 32981348

• Hyperlipidemia does not affect development of elastase-induced abdominal aortic aneurysm in mice. Atherosclerosis Mulorz, J. n., Spin, J. M., Beck, H. C., Tha Thi, M. L., Wagenhäuser, M. U., Rasmussen, L. M., Lindholt, J. S., Tsao, P. S., Steffensen, L. B. 2020; 311: 73–83

  Abstract

  Hyperlipidemia is a suggested risk factor for abdominal aortic aneurysm (AAA). However, whether hyperlipidemia is causally involved in AAA progression remains elusive. Here, we tested the hypothesis that hyperlipidemia aggravates AAA formation in the widely used porcine pancreatic elastase (PPE) model of AAA in mice with varying levels of plasma lipids.Prior to PPE-surgery, 8-week-old male C57BL/6J mice (n = 32) received 1·1011 viral genomes of rAAV8-D377Y-mPcsk9 or control rAAV8 via the tail vein. Mice were fed either western type diet or regular chow. At baseline and during the 28 days following PPE-surgery, mice underwent weekly ultrasonic assessment of AAA progression. Experiments were repeated using Apolipoprotein E knockout (ApoE-/-) mice (n = 7) and wildtype C57BL/6J mice (n = 5).At sacrifice, maximal intergroup plasma cholesterol and non-HDL/HDL ratio differences were >5-fold and >20-fold, respectively. AAA diameters expanded to 150% of baseline, but no intergroup differences were detected. This was verified in an independent experiment comparing 8-week-old male ApoE-/- mice with wildtype mice. Histological evaluation of experimental AAA lesions revealed accumulated lipid in neointimal and medial layers, and analysis of human AAA lesions (n = 5) obtained from open repair showed medial lipid deposition.In summary, we find that lipid deposition in the aortic wall is a feature of PPE-induced AAA in mice as well as human AAA lesions. Despite, our data do not support the hypothesis that hyperlipidemia contributes to AAA progression.

  View details for DOI 10.1016/j.atherosclerosis.2020.08.012

  View details for PubMedID 32949946

• Low-Normal Platelets and Decreasing Platelets Are Risk Factors for Hearing Impairment Development. The Laryngoscope Abe, Y. n., Toyama, K. n., Kazurayama, M. n., Tanaka, S. n., Yamaizumi, M. n., Ueno, M. n., Spin, J. M., Hato, N. n., Mogi, M. n. 2020

  Abstract

  Identification of undefined risk factors will be crucial for the development of therapeutic strategies in hearing impairment. Platelets are likely to affect the development of sudden sensorineural hearing loss, which is a primary risk factor for permanent hearing impairment. This implies that abnormal platelets might contribute to long-term hearing loss. This study investigated the role of platelets in the development of hearing impairment over a 5-year period.This study was a retrospective cohort study and consisted of a population-based survey, which was performed for 1,897 participants in 2014 to 2019. To evaluate the effect of platelet level on hearing ability, the subjects were divided into two groups: a high-normal platelet group (25 ∼ 40 × 104 cells/μL) and a low-normal platelet group (15 ∼ 25 × 104 cells/μL). Subjects were defined as having hearing impairment when pure tone audiometry was over 25 dB HL in either ear (tested in 2017 and 2019). Incidence of hearing impairment was analyzed.Incidence of hearing impairment at low frequencies was significantly higher in the low-normal platelet group than in the high-normal group year over year. Low-normal platelet count associated with low-frequency hearing impairment (LFHI) incidence (odds ratio [OR], 2.34; 95% confidence interval [CI], 1.15-4.76). In the low-normal platelet group, subjects whose counts declined from baseline developed more LFHI than those whose counts increased over time. Further, decreasing platelets appeared to be an independent risk factor contributing to the incidence of LFHI (OR, 2.10; 95%CI, 1.09-4.06) in the low-normal platelet group.Both a low-normal platelet and a declining platelet count were independently associated with the incidence of LFHI.3 Laryngoscope, 2020.

  View details for DOI 10.1002/lary.28970

  View details for PubMedID 32835430

• Involvement of Myeloid Cells and Non-Coding RNA in Abdominal Aortic Aneurysm Disease. Antioxidants & redox signaling Knappich, C. n., Spin, J. M., Eckstein, H. H., Tsao, P. S., Maegdefessel, L. n. 2020

  Abstract

  Abdominal aortic aneurysm (AAA) is a potentially fatal condition, featuring the possibility of high-mortality rupture. To date, prophylactic surgery by means of open surgical repair (OSR) or endovascular aortic repair (EVAR) at specific thresholds are considered standard therapy. Both surgical options hold different risk profiles of short- and long-term morbidity and mortality. Targeting early stages of AAA development in order to decelerate disease progression is desirable. Recent Advances: Understanding the pathomechanisms that initiate formation, maintain growth, and promote rupture of AAA is crucial to developing new medical therapeutic options. Inflammatory cells, in particular macrophages, have been investigated for their contribution to AAA disease for decades, while evidence on lymphocytes, mast cells, and neutrophils is sparse. Recently, there has been increasing interest in non-coding RNAs (ncRNA) and their involvement in disease development, including AAA.The current evidence on myeloid cells and ncRNAs in AAA largely originates from small animal models, making clinical extrapolation difficult. Although it is feasible to collect surgical human AAA samples, these tissues reflect end-stage disease, preventing examination of critical mechanisms behind early AAA formation.Gaining more insight into how myeloid cells and ncRNAs contribute to AAA disease, particularly in early stages, might suggest non-surgical AAA treatment options. The utilization of large animal models might be helpful in this context to help bridge translational results to humans.

  View details for DOI 10.1089/ars.2020.8035

  View details for PubMedID 31989839

• Controlled isoflurane anesthesia exposure is required for reliable behavioral testing in murine surgical models. Journal of pharmacological sciences Toyama, K., Spin, J. M., Abe, Y., Suzuki, Y., Deng, A. C., Wagenhauser, M. U., Yoshino, T., Mulorz, J., Liu, S., Tsao, P. S., Mogi, M. 2019

  Abstract

  We investigated the effects of variations in anesthesia exposure time prior to conducting anxiety response behavioral testing in sham controls from an experimental murine model. The staying time in the center area of the Open Field test in the "long exposure" group was significantly decreased compared to that of the "short exposure" group. Significant correlation was found between anesthesia time and the duration of staying time in the center area. We conclude that anesthesia time may have a significant impact on behavioral anxiety testing in this context, and advise careful control of this parameter in protocol optimization in related surgical animal models.

  View details for DOI 10.1016/j.jphs.2019.03.007

  View details for PubMedID 31133404

• Clinical outcomes after direct and indirect surgical venous thrombectomy for inferior vena cava thrombosis. Journal of vascular surgery. Venous and lymphatic disorders Wagenhauser, M. U., Dimopoulos, C., Antakyali, K., Meyer-Janiszewski, Y. K., Mulorz, J., Ibing, W., Ertas, N., Spin, J. M., Schelzig, H., Duran, M. 2019

  Abstract

  OBJECTIVE: Inferior vena cava thrombosis is rare, but patients are at high risk for development of a post-thrombotic syndrome (PTS) in the long term. Surgical approaches include indirect transfemoral venous thrombectomy (iTFVT) and direct open venous thrombectomy (dOVT). This study reports patient outcomes after iTFVT and dOVT for inferior vena cava thrombosis covering a 25-year follow-up period.METHODS: The study period was from January 1, 1982, to December 31, 2013. Data were retrieved from archived medical records, and patients were invited for a detailed phlebologic follow-up examination (DPFE). Health-related quality of life was assessed with the 36-Item Short Form Health Survey questionnaire. Patient survival, patency rates, and freedom from PTS were calculated using Kaplan-Meier estimation with log-rank testing. The chi2 test with Yates continuity correction and logistic regression analysis were applied to identify associations between risk factors or coagulation disorders, mortality, and PTS.RESULTS: Complete medical records were available for 152 patients. Patients' 5-year survival was 91%± 3%, and 5-year primary and secondary patency rates were 80%± 3% and 94%± 2%. Freedom from PTS after 25years was 84%± 6%. No differences for patient survival, patency rates, or freedom from PTS were identified between iTFVT, dOVT, and a combination of both procedures. Antithrombin III deficiency was the most common coagulation disorder, and patients' physical function and social function were impaired compared with those found in German normative data (P< .05). Norisk factor or coagulation disorder was associated with survival or PTS.CONCLUSIONS: Open surgical venous thrombectomy is safe and delivers satisfying short- and long-term outcomes compared with endovascular approaches. It remains valuable for patients who are not eligible for other interventional therapies.

  View details for PubMedID 30853561

• Non-coding RNAs in aneurysmal aortopathy VASCULAR PHARMACOLOGY Spin, J. M., Li, D. Y., Maegdefessel, L., Tsao, P. S. 2019; 114: 110–21

  View details for DOI 10.1016/j.vph.2018.06.008

  View details for Web of Science ID 000463464100010

• Long noncoding RNAs in key cellular processes involved in aortic aneurysms. Atherosclerosis Wu, Z. Y., Trenner, M. n., Boon, R. A., Spin, J. M., Maegdefessel, L. n. 2019; 292: 112–18

  Abstract

  Aortic aneurysm (AA) is a complex and dangerous vascular disease, featuring progressive and irreversible vessel dilatation. AA is typically detected either by screening, or identified incidentally through imaging studies. To date, no effective pharmacological therapies have been identified for clinical AA management, and either endovascular repair or open surgery remains the only option capable of preventing aneurysm rupture. In recent years, multiple research groups have endeavored to both identify noncoding RNAs and to clarify their function in vascular diseases, including aneurysmal pathologies. Notably, the molecular roles of noncoding RNAs in AA development appear to vary significantly between thoracic aortic aneurysms (TAAs) and abdominal aortic aneurysms (AAAs). Some microRNAs (miRNA - a non-coding RNA subspecies) appear to contribute to AA pathophysiology, with some showing major potential for use as biomarkers or as therapeutic targets. Studies of long noncoding RNAs (lncRNAs) are more limited, and their specific contributions to disease development and progression largely remain unexplored. This review aims to summarize and discuss the most current data on lncRNAs and their mediation of AA pathophysiology.

  View details for DOI 10.1016/j.atherosclerosis.2019.11.013

  View details for PubMedID 31785492

• Therapeutic perspective on vascular cognitive impairment. Pharmacological research Toyama, K. n., Spin, J. M., Mogi, M. n., Tsao, P. S. 2019: 104266

  Abstract

  Dementia is one of the greatest public health concerns for the modern aging world. Over the last decade, most researchers developing new therapeutic strategies for dementia have focused on amyloid-β. In contrast, numerous recent studies have indicated that vascular risk factors are associated with various forms of dementia, and that in fact most forms of dementia can be considered an extension of vascular disease. Accordingly, it is sensible to pursue treatment approaches that focus on the blood vessels. Blood-brain barrier (BBB) disruptions in the white matter of patients with vascular cognitive impairment (VCI) have been observed using imaging analysis, and might be potential targets for novel VCI treatment. Tight junctions between cerebral endothelial cells play an important role in the function of the BBB, and recent studies have demonstrated the essential role of microRNAs in regulating tight junctions. Further elucidation of the mechanisms of tight junction-disruption in dementia are likely to lead to promising novel treatments. In this article, we summarize current knowledge regarding microRNAs and vascular cognitive impairment and the possibility of utilizing microRNAs as biomarkers for BBB dysfunction, and seek to envision future therapeutic strategies.

  View details for DOI 10.1016/j.phrs.2019.104266

  View details for PubMedID 31108183

• Chronic Nicotine Exposure Induces Murine Aortic Remodeling and Stiffness Segmentation-Implications for Abdominal Aortic Aneurysm Susceptibility. Frontiers in physiology Wagenhäuser, M. U., Schellinger, I. N., Yoshino, T., Toyama, K., Kayama, Y., Deng, A., Guenther, S. P., Petzold, A., Mulorz, J., Mulorz, P., Hasenfuß, G., Ibing, W., Elvers, M., Schuster, A., Ramasubramanian, A. K., Adam, M., Schelzig, H., Spin, J. M., Raaz, U., Tsao, P. S. 2018; 9: 1459

  Abstract

  Aim: Arterial stiffness is a significant risk factor for many cardiovascular diseases, including abdominal aortic aneurysms (AAA). Nicotine, the major active ingredient of e-cigarettes and tobacco smoke, induces acute vasomotor effects that may temporarily increase arterial stiffness. Here, we investigated the effects of long-term nicotine exposure on structural aortic stiffness. Methods: Mice (C57BL/6) were infused with nicotine for 40 days (20 mg/kg/day). Arterial stiffness of the thoracic (TS) and abdominal (AS) aortic segments was analyzed using ultrasound (PWV, pulse wave velocity) and ex vivo pressure myograph measurements. For mechanistic studies, aortic matrix-metalloproteinase (MMP) expression and activity as well as medial elastin architecture were analyzed. Results: Global aortic stiffness increased with nicotine. In particular, local stiffening of the abdominal segment occurred after 10 days, while thoracic aortic stiffness was only increased after 40 days, resulting in aortic stiffness segmentation. Mechanistically, nicotine exposure enhanced expression of MMP-2/-9 and elastolytic activity in both aortic segments. Elastin degradation occurred in both segments; however, basal elastin levels were higher in the thoracic aorta. Finally, MMP-inhibition significantly reduced nicotine-induced MMP activity, elastin destruction, and aortic stiffening. Conclusion: Chronic nicotine exposure induces aortic MMP expression and structural aortic damage (elastin fragmentation), irreversibly increasing aortic stiffness. This process predominantly affects the abdominal aortic segment, presumably due in part to a lower basal elastin content. This novel phenomenon may help to explain the role of nicotine as a major risk factor for AAA formation and has health implications for ECIGs and other modes of nicotine delivery.

  View details for DOI 10.3389/fphys.2018.01459

  View details for PubMedID 30429794

  View details for PubMedCentralID PMC6220086

• Decoding the Genomics of Abdominal Aortic Aneurysm. Cell Li, J., Pan, C., Zhang, S., Spin, J. M., Deng, A., Leung, L. L., Dalman, R. L., Tsao, P. S., Snyder, M. 2018; 174 (6): 1361

  Abstract

  A key aspect of genomic medicine is to make individualized clinical decisions from personal genomes. We developed a machine-learning framework to integrate personal genomes and electronic health record (EHR) data and used this framework to study abdominal aortic aneurysm (AAA), a prevalent irreversible cardiovascular disease with unclear etiology. Performing whole-genome sequencing on AAA patients and controls, we demonstrated its predictive precision solely from personal genomes. By modeling personal genomes with EHRs, this framework quantitatively assessed the effectiveness of adjusting personal lifestyles given personal genome baselines, demonstrating its utility as a personal health management tool. We showed that this new framework agnostically identified genetic components involved in AAA, which were subsequently validated in human aortic tissues and in murine models. Our study presents a new framework for disease genome analysis, which can be used for both health management and understanding the biological architecture of complex diseases. VIDEO ABSTRACT.

  View details for PubMedID 30193110

• Non-coding RNAs in aneurysmal aortopathy. Vascular pharmacology Spin, J. M., Li, D. Y., Maegdefessel, L., Tsao, P. S. 2018

  Abstract

  Aortic aneurysms represent a major public health burden, and currently have no medical treatment options. The pathophysiology behind these aneurysms is complex and variable, depending on location and underlying cause, and generally involves progressive dysfunction of all elements of the aortic wall. Changes in smooth muscle behavior, endothelial signaling, extracellular matrix remodeling, and to a variable extent inflammatory signaling and cells, all contribute to the dilation of the aorta, ultimately resulting in high mortality and morbidity events including dissection and rupture. A large number of researchers have identified non-coding RNAs as crucial regulators of aortic aneurysm development, both in humans and in animal models. While most work to-date has focused on microRNAs, intriguing information has also begun to emerge regarding the role of long-non-coding RNAs. This review summarizes the currently available data regarding the involvement of non-coding RNAs in aneurysmal aortopathies. Going forward, these represent key potential therapeutic targets that might be leveraged in the future to slow or prevent aortic aneurysm formation, progression and rupture.

  View details for PubMedID 29909014

• MicroRNA-Mediated Therapy Modulating Blood-Brain Barrier Disruption Improves Vascular Cognitive Impairment. Arteriosclerosis, thrombosis, and vascular biology Toyama, K., Spin, J. M., Deng, A. C., Huang, T., Wei, K., Wagenhauser, M. U., Yoshino, T., Nguyen, H., Mulorz, J., Kundu, S., Raaz, U., Adam, M., Schellinger, I. N., Jagger, A., Tsao, P. S. 2018

  Abstract

  OBJECTIVE: There are currently no effective treatments for the prevention of dementia associated with vascular cognitive impairment. MicroRNAs regulate gene expression at the post-transcriptional level and play key roles in vascular disorders. TNFalpha (tumor necrosis factor-alpha) regulates blood-brain barrier breakdown through modification of cerebral tight junctions. Here, we sought key TNFalpha-responsive microRNAs that might influence blood-brain barrier breakdown via cerebral tight junction disruption in vascular cognitive impairment.APPROACH AND RESULTS: Using a mouse model of vascular cognitive impairment, chronic cerebral hypoperfusion within the white matter was induced with bilateral common carotid artery stenosis (BCAS) surgery. TNFalpha gene expression was increased in white matter post-BCAS surgery, and TNFalpha stimulation decreased claudin-5, ZO-1 (tight-junction protein 1), and occludin gene expression in murine brain endothelial cells. In silico analysis predicted 8 candidate microRNAs as regulators of claudin-5, ZO-1, and occludin gene expression. Of these, only miR-501-3p was upregulated by TNFalpha in vitro and was upregulated in the white matter after BCAS surgery. Further, miR-501-3p directly bound to the 3'-untranslated region of human ZO-1 and downregulated transendothelial electric resistance. In vivo administration of a locked nucleic acid -modified antisense oligonucleotide versus miR-501-3p suppressed BCAS-induced reduction of ZO-1 gene expression and blood-brain barrier disruption within the white matter and significantly ameliorated working memory deficits after BCAS surgery.CONCLUSIONS: We here provide the first evidence that the TNFalpha-miR-501-3p-ZO-1 axis plays an important role in the pathogenesis of cerebral hypoperfusion-induced working memory deficits and white matter lesions, as a result of blood-brain barrier breakdown via tight junction disruption. Therapeutic manipulation of miR-501-3p holds promise for limiting vascular cognitive impairment progression.

  View details for PubMedID 29650692

• Systemic Upregulation of IL-10 (Interleukin-10) Using a Nonimmunogenic Vector Reduces Growth and Rate of Dissecting Abdominal Aortic Aneurysm. Arteriosclerosis, thrombosis, and vascular biology Adam, M. n., Kooreman, N. n., Jagger, A. n., Wagenhaeuser, M. U., Mehrkens, D. n., Wang, Y. n., Kayama, Y. n., Toyama, K. n., Raaz, U. n., Schellinger, I. N., Maegdefessel, L. n., Spin, J. M., Hamming, J. F., Quax, P. H., Baldus, S. n., Wu, J. C., Tsao, P. S. 2018

  Abstract

  Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression.Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE-/- infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4+/CD25+/Foxp3 (forkhead box P3)+ regulatory T cells, with fewer CD8+/Granzyme B+ cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-α (tumor necrosis factor-α), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)-positive alternatively activated macrophages. These results concurred with gene expression analysis of LPS-stimulated and Ang II-primed human peripheral blood mononuclear cells.Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.

  View details for PubMedID 29880489

• H19 Induces Abdominal Aortic Aneurysm Development and Progression. Circulation Li, D. Y., Busch, A. n., Jin, H. n., Chernogubova, E. n., Pelisek, J. n., Karlsson, J. n., Sennblad, B. n., Liu, S. n., Lao, S. n., Hofmann, P. n., Bäcklund, A. n., Eken, S. M., Roy, J. n., Eriksson, P. n., Dacken, B. n., Ramanujam, D. n., Dueck, A. n., Engelhardt, S. n., Boon, R. A., Eckstein, H. H., Spin, J. M., Tsao, P. S., Maegdefessel, L. n. 2018

  Abstract

  Background -Long noncoding RNAs (lncRNAs) have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize lncRNAs as potential mediators in abdominal aortic aneurysm (AAA) development. Methods -We profiled RNA transcript expression in two murine AAA models, Angiotensin II (ANGII) infusion in ApoE-/- mice (n=8) and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice (n=12). The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and in situ hybridization. Results -Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical LDLR-/- Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1-alpha (HIF1α) as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and HIF1α and sequential p53 stabilization. Additionally, H19 induced transcription of HIF1α via recruiting the transcription factor specificity protein 1 (Sp1) to the promoter region. Conclusions -The lncRNA H19 is a novel regulator of SMC survival in AAA development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.

  View details for PubMedID 29669788

• Chronic Mesenteric Ischemia: Patient Outcomes Using Open Surgical Revascularization. Digestive surgery Wagenhäuser, M. U., Meyer-Janiszewski, Y. K., Dueppers, P., Spin, J. M., Floros, N., Schelzig, H., Duran, M. 2017

  Abstract

  Chronic mesenteric ischemia (CMI) is a rare disease. Open treatment (OT) remains a valuable treatment option. We analyzed patient outcomes after OT and investigated health-related quality of life (HRQoL).Data were analyzed retrospectively. The investigation period was from January 1, 2001, to December 31, 2014. We investigated mortality and patency rates using Kaplan-Meier analysis. HRQoL was measured using a 36-item health survey. Various statistical methods were employed.A total of 100 patients (celiac trunk [TC: n = 23], superior mesenteric artery [SMA: n = 26], or both [n = 51]) were included. Median follow-up was 5 ± 35 months. One-year survival rate for TC was 75 ± 11%, for SMA: 79 ± 10%, and for both: 96 ± 3%. TC 5-year survival was 75 ± 11% (SMA: 57 ± 16%: both: 80 ± 8%). Obesity and the length of hospital stay were independently associated with patient survival (p < 0.05). Primary 1-year patency rate was 60 ± 13% for TC (SMA: 86 ± 10%; both: 71 ± 8%) and secondary 1-year patency rate was 84 ± 9% for TC (SMA: 100%; both: 79 ± 7%). HRQoL was inferior compared to the German normative data (p < 0.05).CMI overlaps between gastrointestinal and vascular surgery. OT is safe, and simultaneous revascularization of the TC and the SMA does not affect mortality. Patients would not necessarily benefit from OT in terms of HRQoL.

  View details for DOI 10.1159/000464413

  View details for PubMedID 28301853

• Open surgery for iliofemoral deep vein thrombosis with temporary arteriovenous fistula remains valuable. Phlebology Wagenhäuser, M. U., Sadat, H. n., Dueppers, P. n., Meyer-Janiszewski, Y. K., Spin, J. M., Schelzig, H. n., Duran, M. n. 2017: 268355517736437

  Abstract

  Objective We assessed outcomes of open surgical venous thrombectomy with temporary arteriovenous fistula, and the procedure's effect on health-related quality of life. Method We retrospectively analyzed 48 (26 at long-term) patient medical records. Mortality rates, patency, and risk of post-thrombotic syndrome were analyzed using Kaplan-Meier estimation. The association between risk factors/coagulation disorders and patency/post-thrombotic syndrome along with patient health-related quality of life at long-term was analyzed employing various statistical methods. Results Patient one-year survival rate was 93 ± 4% and primary one-year patency rate was 89 ± 5% (secondary one-year patency rate 97 ± 3%). Freedom from post-thrombotic syndrome after eight years was 80 ± 12% (post-thrombotic syndrome rate 20 ± 12%). Health-related quality of life was impaired vs. normative data in the physical and social subscales, and in the mental component score ( p < .05). Conclusions Open surgical venous thrombectomy appears safe compared with literature-reported outcomes in similar patients using alternative approaches. Iliofemoral deep vein thrombosis impairs physical, social, and mental health-related quality of life.

  View details for PubMedID 29065779

• A Pilot Study: The Beneficial Effects of Combined Statinexercise Therapy on Cognitive Function in Patients with Coronary Artery Disease and Mild Cognitive Decline INTERNAL MEDICINE Toyama, K., Sugiyama, S., Oka, H., Hamada, M., Iwasaki, Y., Horio, E., Rokutanda, T., Nakamura, S., Spin, J. M., Tsao, P. S., Ogawa, H. 2017; 56 (6): 641-649

  Abstract

  Objective Hypercholesterolemia, a risk factor in cognitive impairment, can be treated with statins. However, cognitive decline associated with "statins" (HMG-CoA reductase inhibitors) is a clinical concern. This pilot study investigated the effects of combining statins and regular exercise on cognitive function in coronary artery disease (CAD) patients with prior mild cognitive decline. Methods We recruited 43 consecutive CAD patients with mild cognitive decline. These patients were treated with a statin and weekly in-hospital aerobic exercise for 5 months. We measured serum lipids, exercise capacity, and cognitive function using the mini mental state examination (MMSE). Results Low-density lipoprotein cholesterol levels were significantly decreased, and maximum exercise capacity (workload) was significantly increased in patients with CAD and mild cognitive decline after treatment compared with before. Combined statin-exercise therapy significantly increased the median (range) MMSE score from 24 (22-25) to 25 (23-27) across the cohort (p<0.01). Changes in body mass index (BMI) were significantly and negatively correlated with changes in the MMSE. After treatment, MMSE scores in the subgroup of patients that showed a decrease in BMI were significantly improved, but not in the BMI-increased subgroup. Furthermore, the patients already on a statin at the beginning of the trial displayed a more significant improvement in MMSE score than statin-naïve patients, implying that exercise might be the beneficial aspect of this intervention as regards cognition. In a multivariate logistic regression analysis adjusted for age >65 years, sex, and presence of diabetes mellitus, a decrease in BMI during statin-exercise therapy was significantly correlated with an increase in the MMSE score (odds ratio: 4.57, 95% confidence interval: 1.05-20.0; p<0.05). Conclusion Statin-exercise therapy may help improve cognitive dysfunction in patients with CAD and pre-existing mild cognitive decline.

  View details for DOI 10.2169/internalmedicine.56.7703

  View details for Web of Science ID 000398893200010

  View details for PubMedID 28321063

• Role of microRNAs on Blood Brain Barrier Dysfunction in Vascular Cognitive Impairment. Current drug delivery Toyama, K., Spin, J. M., Tsao, P. S. 2016: -?

  Abstract

  Dementia cases are increasing as the population ages, leading to increased financial costs. Several neuronal diseases including ischemic and hemorrhagic stroke involve cerebrovascular injury or pathophysiology. Cerebrovascular injury is closely tied to blood brain barrier (BBB) disruption. Many studies have shown a significant association between BBB dysfunction and neurological diseases. Therefore, an understanding of the molecular mechanisms which regulate BBB permeability and disruption is essential for establishing future therapeutic strategies to alter dementia disease progression related to cerebrovascular injury,so-called vascular cognitive impairment (VCI). microRNAs (miRs) are small noncodingRNAs that regulate gene expression through targeting of mRNA transcripts.miRs have been implicated in the development and progression of various illnesses, including vascular disease. However, the role of miRs in BBB breakdown or permeability and VCI development has not yet been well clarified.Research content related to the origins of VCI and the role of the BBB in pathologic development and therapeutic targeting are reviewed, including current relevant animal models. We draw from the published literature regarding microRNA candidates that are associated with modulation of BBB structure and function.In this review, we summarize the current knowledge about VCI, explore the potential role of miRs in BBB breakdown and VCI progression, and identify potential candidate miRs for development of new treatment strategies.miRs constitute a promising novel avenue as future therapeutic options for alteration of both BBB permeability and development of VCI.

  View details for PubMedID 27572324

• Oxidized (non)-regenerated cellulose affects fundamental cellular processes of wound healing SCIENTIFIC REPORTS Wagenhaeuser, M. U., Mulorz, J., Ibing, W., Simon, F., Spin, J. M., Schelzig, H., Oberhuber, A. 2016; 6

  Abstract

  In this study we investigated how hemostats such as oxidized regenerated cellulose (ORC, TABOTAMP) and oxidized non-regenerated cellulose (ONRC, RESORBA CELL) influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated in vitro with ORC and ONRC. Cell proliferation was assayed over time, and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC, and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p < 0.05). When control media was pH-lowered to match the lower pH values typically seen with ORC and ONRC, significant differences in cell proliferation and migration were still observed between ONRC and ORC (p < 0.05). However, in these pH conditions, inhibition of matrix contraction was only significant for ONRC (p < 0.05). We find that ORC and ONRC inhibit fibroblast proliferation, migration and matrix contraction, and stronger inhibition of these essential cellular processes of wound healing were observed for ONRC when compared with ORC. These results will require further validation in future in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing.

  View details for DOI 10.1038/srep32238

  View details for Web of Science ID 000381867000001

  View details for PubMedID 27557881

  View details for PubMedCentralID PMC4997603

• The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation TRANSPLANTATION Deuse, T., Hua, X., Stubbendorff, M., Spin, J. M., Neofytou, E., Taylor, V., Chen, Y., Park, G., Fink, J. B., Renne, T., Kiefmann, M., Kiefmann, R., Reichenspurner, H., Robbins, R. C., Schrepfer, S. 2016; 100 (5): 1022-1031

  Abstract

  The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models.Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells.In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1β.The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.

  View details for DOI 10.1097/TP.0000000000001110

  View details for Web of Science ID 000377126000016

  View details for PubMedID 26910327

• CDKN2B Regulates TGFß Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels. Circulation research Nanda, V., Downing, K. P., Ye, J., Xiao, S., Kojima, Y., Spin, J. M., DiRenzo, D., Nead, K. T., Connolly, A. J., Dandona, S., Perisic, L., Hedin, U., Maegdefessel, L., Dalman, J., Guo, L., Zhao, X., Kolodgie, F. D., Virmani, R., Davis, H. R., Leeper, N. J. 2016; 118 (2): 230-240

  Abstract

  Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown.To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism.Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β (TGFβ) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro.These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFβ signaling and hypoxic neovessel maturation.

  View details for DOI 10.1161/CIRCRESAHA.115.307906

  View details for PubMedID 26596284

  View details for PubMedCentralID PMC4740238

• Response to Letters Regarding Article, "Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm Development" CIRCULATION Raaz, U., Zoellner, A. M., Schellinger, I. N., Toh, R., Nakagami, F., Brandt, M., Emrich, F. C., Kayama, Y., Eken, S., Adam, M., Maegdefessel, L., Hertel, T., Deng, A., Jagger, A., Buerke, M., Dalman, R. L., Spin, J. M., Kuhl, E., Tsao, P. S. 2016; 133 (1): E11–E12

  View details for PubMedID 26719393

  View details for PubMedCentralID PMC4704124

• Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression. PloS one Azuma, J., Wong, R. J., Morisawa, T., Hsu, M., Maegdefessel, L., Zhao, H., Kalish, F., Kayama, Y., Wallenstein, M. B., Deng, A. C., Spin, J. M., Stevenson, D. K., Dalman, R. L., Tsao, P. S. 2016; 11 (2)

  Abstract

  Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

  View details for DOI 10.1371/journal.pone.0149288

  View details for PubMedID 26894432

• Diabetic Cardiovascular Disease Induced by Oxidative Stress INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Kayama, Y., Raaz, U., Jagger, A., Adam, M., Schellinger, I. N., Sakamoto, M., Suzuki, H., Toyama, K., Spin, J. M., Tsao, P. S. 2015; 16 (10): 25234-25263

  Abstract

  Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). DM can lead to multiple cardiovascular complications, including coronary artery disease (CAD), cardiac hypertrophy, and heart failure (HF). HF represents one of the most common causes of death in patients with DM and results from DM-induced CAD and diabetic cardiomyopathy. Oxidative stress is closely associated with the pathogenesis of DM and results from overproduction of reactive oxygen species (ROS). ROS overproduction is associated with hyperglycemia and metabolic disorders, such as impaired antioxidant function in conjunction with impaired antioxidant activity. Long-term exposure to oxidative stress in DM induces chronic inflammation and fibrosis in a range of tissues, leading to formation and progression of disease states in these tissues. Indeed, markers for oxidative stress are overexpressed in patients with DM, suggesting that increased ROS may be primarily responsible for the development of diabetic complications. Therefore, an understanding of the pathophysiological mechanisms mediated by oxidative stress is crucial to the prevention and treatment of diabetes-induced CVD. The current review focuses on the relationship between diabetes-induced CVD and oxidative stress, while highlighting the latest insights into this relationship from findings on diabetic heart and vascular disease.

  View details for DOI 10.3390/ijms161025234

  View details for PubMedID 26512646

• Local MicroRNA Modulation Using a Novel Anti-miR21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Wang, D., Deuse, T., Stubbendorff, M., Chernogubova, E., Erben, R. G., Eken, S. M., Jin, H., Li, Y., Busch, A., Heeger, C., Behnisch, B., Reichenspurner, H., Robbins, R. C., Spin, J. M., Tsao, P. S., Schrepfer, S., Maegdefessel, L. 2015; 35 (9): 1945-1953

  Abstract

  Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication.We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed.This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

  View details for DOI 10.1161/ATVBAHA.115.305597

  View details for Web of Science ID 000360497600008

  View details for PubMedCentralID PMC4552606

• Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus CIRCULATION RESEARCH Raaz, U., Schellinger, I. N., Chernogubova, E., Warnecke, C., Kayama, Y., Penov, K., Hennigs, J. K., Salomons, F., Eken, S., Emrich, F. C., Zheng, W. H., Adam, M., Jagger, A., Nakagami, F., Toh, R., Toyama, K., Deng, A., Buerke, M., Maegdefessel, L., Hasenfuss, G., Spin, J. M., Tsao, P. S. 2015; 117 (6): 513-524

  Abstract

  Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date.The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus.Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter.In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

  View details for DOI 10.1161/CIRCRESAHA.115.306341

  View details for Web of Science ID 000360142000007

  View details for PubMedCentralID PMC4553105

• Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm Development CIRCULATION Raaz, U., Zoellner, A. M., Schellinger, I. N., Toh, R., Nakagami, F., Brandt, M., Emrich, F. C., Kayama, Y., Eken, S., Adam, M., Maegdefessel, L., Hertel, T., Deng, A., Jagger, A., Buerke, M., Dalman, R. L., Spin, J. M., Kuhl, E., Tsao, P. S. 2015; 131 (20): 1783-1795

  Abstract

  Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined.Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening.The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.

  View details for DOI 10.1161/CIRCULATIONAHA.114.012377

  View details for PubMedID 25904646

• MicroRNAs in Abdominal Aortic Aneurysm. Current vascular pharmacology Adam, M., Raaz, U., Spin, J. M., Tsao, P. S. 2015; 13 (3): 280-290

  Abstract

  Abdominal aortic aneurysms (AAA) are an important source of morbidity and mortality in the U.S. and worldwide. Treatment options are limited, with open surgery or endovascular repair remaining the only curative treatments. Classical cardiovascular medications have generally failed to prevent or significantly alter AAA formation or progression. Therefore, there is a tremendous need for better therapeutic approaches. With increasing knowledge of microRNA (miR) regulation in the context of cardiovascular disease, and with improving technical options permitting alteration of miR-expression levels in vitro and in vivo, we are offered a glimpse into the diagnostic and therapeutic possibilities of using miRs to treat vascular pathobiology. This review focuses on the role of miRs in aneurysmal disease of the abdominal aorta, summarizing recent publications regarding this topic, and outlining known effects of relevant miRs in AAA formation, including miR-21 and miR-29b. Despite there being only limited studies available, several other miRs also display clear potential for alteration of the disease process including miR-26a, the miR-17-92-cluster, miRs-221/222, miR-133 and miR-146a. While studies have shown that miRs can regulate the activity and interplay of vascular inflammatory cells, endothelial cells, smooth muscle cells and fibroblasts, all key elements leading to AAA formation, much work remains to be done.

  View details for PubMedID 23713862

• Levosimendan displays anti-inflammatory effects and decreases MPO bioavailability in patients with severe heart failure. Scientific reports Adam, M., Meyer, S., Knors, H., Klinke, A., Radunski, U. K., Rudolph, T. K., Rudolph, V., Spin, J. M., Tsao, P. S., Costard-Jäckle, A., Baldus, S. 2015; 5: 9704-?

  Abstract

  Treatment of decompensated heart failure often includes administration of levosimendan. Myeloperoxidase (MPO) is released during polymorphonuclear neutrophil (PMN) degranulation, and mediates dysregulation of vascular tone in heart failure. We evaluated the effects of levosimendan-treatment on MPO in patients with acute decompensation of chronic heart failure over a one week course. Plasma MPO levels were significantly decreased after levosimendan treatment (from 252.1 ± 31.1 pmol/l at baseline to 215.02 ± 27.96 pmol/l at 6 h, p < 0.05). Ex vivo incubation of whole blood with levosimendan decreased MPO release after PMN-stimulation (8.2 ± 1.4-fold increase at baseline vs. 6.0 ± 1.1-fold increase with levosimendan). MPO levels also significantly correlated with diastolic blood pressure over the time course. In a multivariate linear model, the main contributor to systolic, diastolic and mean blood pressure was level of PMN elastase. MPO contributed only in heparin-treated patients, suggesting a more significant role for endothelial-bound MPO than for circulating MPO or elastase with respect to blood pressure regulation. We here provide the first evidence that levosimendan treatment inhibits MPO release by PMNs in decompensated heart failure patients. This mechanism may regulate endothelial function and vascular tone in heart failure patients.

  View details for DOI 10.1038/srep09704

  View details for PubMedID 25867530

• Battle of the bulge: miR-195 versus miR-29b in aortic aneurysm. Circulation research Spin, J. M., Tsao, P. S. 2014; 115 (10): 812-813

  View details for DOI 10.1161/CIRCRESAHA.114.305233

  View details for PubMedID 25342766

  View details for PubMedCentralID PMC4323148

• miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development NATURE COMMUNICATIONS Maegdefessel, L., Spin, J. M., Raaz, U., Eken, S. M., Toh, R., Azuma, J., Adam, M., Nagakami, F., Heymann, H. M., Chernugobova, E., Jin, H., Roy, J., Hultgren, R., Caidahl, K., Schrepfer, S., Hamsten, A., Eriksson, P., McConnell, M. V., Dalman, R. L., Tsao, P. S. 2014; 5

  Abstract

  Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

  View details for DOI 10.1038/ncomms6214

  View details for Web of Science ID 000343982800003

  View details for PubMedCentralID PMC4217126

• New ways to dismantle a ticking time bomb: microRNA 712/205 and abdominal aortic aneurysm development. Arteriosclerosis, thrombosis, and vascular biology Maegdefessel, L., Spin, J. M., Tsao, P. S. 2014; 34 (7): 1339-1340

  View details for DOI 10.1161/ATVBAHA.114.303952

  View details for PubMedID 24951652

• Dichloroacetate prevents restenosis in preclinical animal models of vessel injury. Nature Deuse, T., Hua, X., Wang, D., Maegdefessel, L., Heeren, J., Scheja, L., Bolaños, J. P., Rakovic, A., Spin, J. M., Stubbendorff, M., Ikeno, F., Länger, F., Zeller, T., Schulte-Uentrop, L., Stoehr, A., Itagaki, R., Haddad, F., Eschenhagen, T., Blankenberg, S., Kiefmann, R., Reichenspurner, H., Velden, J., Klein, C., Yeung, A., Robbins, R. C., Tsao, P. S., Schrepfer, S. 2014; 509 (7502): 641-644

  Abstract

  Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.

  View details for DOI 10.1038/nature13232

  View details for PubMedID 24747400

• Hemodynamic regulation of reactive oxygen species: implications for vascular diseases. Antioxidants & redox signaling Raaz, U., Toh, R., Maegdefessel, L., Adam, M., Nakagami, F., Emrich, F. C., Spin, J. M., Tsao, P. S. 2014; 20 (6): 914-928

  Abstract

  Significance: Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Whereas physiological mechanical stress helps to maintain vascular structure and function, pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes which drive disease. Recent advances: Reactive oxygen species (ROS) may represent an intriguing class of mechanically- regulated second messengers. Chronically enhanced ROS-generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. Critical issues and Future directions: This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress or cyclic stretch) as modulators of ROS- driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano)signaling may represent a key for successful translation of experimental findings into clinical practice.

  View details for DOI 10.1089/ars.2013.5507

  View details for PubMedID 23879326

• miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development. Nature communications Maegdefessel, L., Spin, J. M., Raaz, U., Eken, S. M., Toh, R., Azuma, J., Adam, M., Nakagami, F., Heymann, H. M., Chernogubova, E., Jin, H., Roy, J., Hultgren, R., Caidahl, K., Schrepfer, S., Hamsten, A., Eriksson, P., McConnell, M. V., Dalman, R. L., Tsao, P. S. 2014; 5: 5214-?

  Abstract

  Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

  View details for DOI 10.1038/ncomms6214

  View details for PubMedID 25358394

• Loss of somatostatin receptor subtype 2 in prostate cancer is linked to an aggressive cancer phenotype, high tumor cell proliferation and predicts early metastatic and biochemical relapse. PloS one Hennigs, J. K., Müller, J., Adam, M., Spin, J. M., Riedel, E., Graefen, M., Bokemeyer, C., Sauter, G., Huland, H., Schlomm, T., Minner, S. 2014; 9 (7)

  Abstract

  Somatostatin receptor subtype 2 (SSTR2) is the most frequently expressed SSTR subtype in normal human tissues. SSTR2 expression is differentially regulated in various tumor types and therapeutic somatostatin analogs binding to SSTR2 are in clinical use. In prostate cancers highly contradictory results in terms of SSTR2 expression and its consequences have been published over the past years. The aim of this study was to clarify prevalence and clinical significance of SSTR2 expression in prostate cancer. Therefore, quantitative immunohistochemistry (IHC) using a tissue microarray containing samples from 3,261 prostate cancer patients with extensive clinical and molecular cancer characteristics and oncological follow-up data was performed. IHC data was compared to publicly available Gene Expression Omnibus datasets of human prostate cancer gene expression arrays. While membranous SSTR2 staining was always seen in normal prostate epithelium, SSTR2 staining was absent in more than half (56.1%) of 2,195 interpretable prostate cancer samples. About 13% of all analyzed prostate cancers showed moderate to strong cytoplasmic and membranous SSTR2 staining. Staining intensities were inversely correlated with high Gleason grade, advanced pT category, high tumor cell proliferation (p<0.0001 each), high pre-operative PSA levels, (p = 0.0011) and positive surgical margins (p = 0.006). In silico analysis confirmed lower SSTR2 gene expression in prostate cancers vs. normal adjacent tissue (p = 0.0424), prostate cancer metastases vs. primary cancers (p = 0.0011) and recurrent vs. non-recurrent prostate cancers (p = 0.0438). PSA-free survival gradually declined with SSTR2 staining intensity (p<0.0001). SSTR2-negative cancers were more likely to develop metastases over time (p<0.05). In conclusion, most prostate cancers are indeed SSTR2-negative and loss of SSTR2 strongly predicts an unfavorable tumor phenotype and poor prognosis. Therefore, SSTR2 expression seems an important factor in the pathogenesis of prostate cancer and re-introduction of the receptor in SSTR2-negative prostate cancers may feature a promising target for novel gene therapy approaches.

  View details for DOI 10.1371/journal.pone.0100469

  View details for PubMedID 25010045

• Micromanaging Abdominal Aortic Aneurysms INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Maegdefessel, L., Spin, J. M., Adam, M., Raaz, U., Toh, R., Nakagami, F., Tsao, P. S. 2013; 14 (7): 14374-14394

  Abstract

  The contribution of abdominal aortic aneurysm (AAA) disease to human morbidity and mortality has increased in the aging, industrialized world. In response, extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of the diseased aorta. This work aims to develop novel diagnostic and therapeutic strategies to limit AAA expansion and, ultimately, rupture. Contributions from multiple research groups have uncovered a complex transcriptional and post-transcriptional regulatory milieu, which is believed to be essential for maintaining aortic vascular homeostasis. Recently, novel small noncoding RNAs, called microRNAs, have been identified as important transcriptional and post-transcriptional inhibitors of gene expression. MicroRNAs are thought to "fine tune" the translational output of their target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. With the discovery that microRNAs act as powerful regulators in the context of a wide variety of diseases, it is only logical that microRNAs be thoroughly explored as potential therapeutic entities. This current review summarizes interesting findings regarding the intriguing roles and benefits of microRNA expression modulation during AAA initiation and propagation. These studies utilize disease-relevant murine models, as well as human tissue from patients undergoing surgical aortic aneurysm repair. Furthermore, we critically examine future therapeutic strategies with regard to their clinical and translational feasibility.

  View details for DOI 10.3390/ijms140714374

  View details for Web of Science ID 000322171700085

  View details for PubMedID 23852016

• Physiologic and molecular characterization of a murine model of right ventricular volume overload. American journal of physiology. Heart and circulatory physiology Reddy, S., Zhao, M., Hu, D., Fajardo, G., Katznelson, E., Punn, R., Spin, J. M., Chan, F. P., Bernstein, D. 2013; 304 (10): H1314-27

  Abstract

  Pulmonary insufficiency (PI) is a common long-term sequel after repair of tetralogy of Fallot, causing progressive right ventricular (RV) dilation and failure. We describe the physiologic and molecular characteristics of the first murine model of RV volume overload. PI was created by entrapping the pulmonary valve leaflets with sutures. Imaging, catheterization, and exercise testing were performed at 1, 3, and 6 mo and compared with sham controls. RNA from the RV free wall was hybridized to Agilent whole genome oligonucleotide microarrays. Volume overload resulted in RV enlargement, decreased RV outflow tract shortening fraction at 1 mo followed by normalization at 3 and 6 mo (39 ± 2, 44 ± 2, and 41 ± 2 vs. 46 ± 3% in sham), early reversal of early and late diastolic filling velocities (E/A ratio) followed by pseudonormalization (0.87 ± 0.08, 0.82 ± 0.08, and 0.96 ± 0.08 vs. 1.04 ± 0.03; P < 0.05), elevated end-diastolic pressure (7.6 ± 0.7, 6.9 ± 0.8, and 7 ± 0.5 vs. 2.7 ± 0.2 mmHg; P < 0.05), and decreased exercise duration (26 ± 0.4, 26 ± 1, and 22 ± 1.3 vs. 30 ± 1.1 min; P < 0.05). Subendocardial RV fibrosis was evident by 1 mo. At 1 mo, 372 genes were significantly downregulated. Mitochondrial pathways and G protein-coupled receptor signaling were the most represented categories. At 3 mo, 434 genes were upregulated and 307 downregulated. While many of the same pathways continued to be downregulated, TNF-α, transforming growth factor-β(1) (TGF-β(1)), p53-signaling, and extracellular matrix (ECM) remodeling transitioned from down- to upregulated. We describe a novel murine model of chronic RV volume overload recapitulating aspects of the clinical disease with gene expression changes suggesting early mitochondrial bioenergetic dysfunction, enhanced TGF-β signaling, ECM remodeling, and apoptosis.

  View details for DOI 10.1152/ajpheart.00776.2012

  View details for PubMedID 23504182

• MicroRNA-24 controls abdominal aortic aneurysm development through regulation of YKL-40 Maegdefessel, L., Raaz, U., Adam, M., Spin, J., Eriksson, P., Hamsten, A., Tsao, P. SPRINGER. 2013: 10–10

  View details for Web of Science ID 000317299700013

• Loss of CDKN2B promotes p53-dependent smooth muscle cell apoptosis and aneurysm formation. Arteriosclerosis, thrombosis, and vascular biology Leeper, N. J., Raiesdana, A., Kojima, Y., Kundu, R. K., Cheng, H., Maegdefessel, L., Toh, R., Ahn, G., Ali, Z. A., Anderson, D. R., Miller, C. L., Roberts, S. C., Spin, J. M., de Almeida, P. E., Wu, J. C., Xu, B., Cheng, K., Quertermous, M., Kundu, S., Kortekaas, K. E., Berzin, E., Downing, K. P., Dalman, R. L., Tsao, P. S., Schadt, E. E., Owens, G. K., Quertermous, T. 2013; 33 (1): e1-e10

  Abstract

  Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear.Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model.These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

  View details for DOI 10.1161/ATVBAHA.112.300399

  View details for PubMedID 23162013

• Vascular smooth muscle cell phenotypic plasticity: focus on chromatin remodelling CARDIOVASCULAR RESEARCH Spin, J. M., Maegdefessel, L., Tsao, P. S. 2012; 95 (2): 147-155

  Abstract

  Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.

  View details for DOI 10.1093/cvr/cvs098

  View details for Web of Science ID 000306141100004

  View details for PubMedID 22362814

  View details for PubMedCentralID PMC3388815

• MicroRNA-21 Blocks Abdominal Aortic Aneurysm Development and Nicotine-Augmented Expansion SCIENCE TRANSLATIONAL MEDICINE Maegdefessel, L., Azuma, J., Toh, R., Deng, A., Merk, D. R., Raiesdana, A., Leeper, N. J., Raaz, U., Schoelmerich, A. M., McConnell, M. V., Dalman, R. L., Spin, J. M., Tsao, P. S. 2012; 4 (122)

  Abstract

  Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

  View details for DOI 10.1126/scitranslmed.3003441

  View details for PubMedID 22357537

• Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development JOURNAL OF CLINICAL INVESTIGATION Maegdefessel, L., Azuma, J., Toh, R., Merk, D. R., Deng, A., Chin, J. T., Raaz, U., Schoelmerich, A. M., Raiesdana, A., Leeper, N. J., McConnell, M. V., Dalman, R. L., Spin, J. M., Tsao, P. S. 2012; 122 (2): 497-506

  Abstract

  MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

  View details for DOI 10.1172/JCI61598

  View details for PubMedID 22269326

• miR-29b Participates in Early Aneurysm Development in Marfan Syndrome CIRCULATION RESEARCH Merk, D. R., Chin, J. T., Dake, B. A., Maegdefessel, L., Miller, M. O., Kimura, N., Tsao, P. S., Iosef, C., Berry, G. J., Mohr, F. W., Spin, J. M., Alvira, C. M., Robbins, R. C., Fischbein, M. P. 2012; 110 (2): 312-?

  Abstract

  Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-β (TGF-β) signaling. Although TGF-β blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-β causes aneurysms remain ill-defined.We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS.Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-β, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-β blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies.We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.

  View details for DOI 10.1161/CIRCRESAHA.111.253740

  View details for PubMedID 22116819

• In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Sheikh, A. Y., Huber, B. C., Narsinh, K. H., Spin, J. M., van der Bogt, K., de Almeida, P. E., Ransohoff, K. J., Kraft, D. L., Fajardo, G., Ardigo, D., Ransohoff, J., Bernstein, D., Fischbein, M. P., Robbins, R. C., Wu, J. C. 2012; 32 (1): 92-102

  Abstract

  Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model.We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.

  View details for DOI 10.1161/ATVBAHA.111.238618

  View details for Web of Science ID 000298288700014

  View details for PubMedID 22034515

  View details for PubMedCentralID PMC3241895

• Microrna-21 Regulates Expansion of Abdominal Aortic Aneurysms Through the PTEN/PI3K/AKT Pathway Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Maegdefessel, L., Azuma, J., Deng, A., Toh, R. M., Merk, D. R., Raiesdana, A., Leeper, N. J., Spin, J. M., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738707282

• Nicotine-Augmented Abdominal Aortic Aneurysms are Regulated by MicroRNA-29b Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Maegdefessel, L., Azuma, J., Merk, D. R., Toh, R. M., Deng, A., Chin, J. P., Spin, J. M., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738707360

• Cardiac pressure overload hypertrophy is differentially regulated by beta-adrenergic receptor subtypes AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Zhao, M., Fajardo, G., Urashima, T., Spin, J. M., Poorfarahani, S., Rajagopalan, V., Diem Huynh, D., Connolly, A., Quertermous, T., Bernstein, D. 2011; 301 (4): H1461-H1470

  Abstract

  In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.

  View details for DOI 10.1152/ajpheart.00453.2010

  View details for Web of Science ID 000295360100028

  View details for PubMedID 21705675

  View details for PubMedCentralID PMC3197363

• Transcriptional profiling and network analysis of the murine angiotensin II-induced abdominal aortic aneurysm PHYSIOLOGICAL GENOMICS Spin, J. M., Hsu, M., Azuma, J., Tedesco, M. M., Deng, A., Dyer, J. S., Maegdefessel, L., Dalman, R. L., Tsao, P. S. 2011; 43 (17): 993-1003

  Abstract

  We sought to characterize temporal gene expression changes in the murine angiotensin II (ANG II)-ApoE-/- model of abdominal aortic aneurysm (AAA). Aortic ultrasound measurements were obtained over the 28-day time-course. Harvested suprarenal aortic segments were evaluated with whole genome expression profiling at 7, 14, and 28 days using the Agilent Whole Mouse Genome microarray platform and Statistical Analysis of Microarrays at a false discovery rate of <1%. A group of angiotensin-treated mice experienced contained rupture (CR) within 7 days and were analyzed separately. Progressive aortic dilatation occurred throughout the treatment period. However, the numerous early expression differences between ANG II-treated and control were not sustained over time. Ontologic analysis revealed widespread upregulation of inflammatory, immune, and matrix remodeling genes with ANG II treatment, among other pathways such as apoptosis, cell cycling, angiogenesis, and p53 signaling. CR aneurysms displayed significant decreases in TGF-β/BMP-pathway signaling, MAPK signaling, and ErbB signaling genes vs. non-CR/ANG II-treated samples. We also performed literature-based network analysis, extracting numerous highly interconnected genes associated with aneurysm development such as Spp1, Myd88, Adam17 and Lox. 1) ANG II treatment induces extensive early differential expression changes involving abundant signaling pathways in the suprarenal abdominal aorta, particularly wide-ranging increases in inflammatory genes with aneurysm development. 2) These gene expression changes appear to dissipate with time despite continued growth, suggesting that early changes in gene expression influence disease progression in this AAA model, and that the aortic tissue adapts to prolonged ANG II infusion. 3) Network analysis identified nexus genes that may constitute aneurysm biomarkers or therapeutic targets.

  View details for DOI 10.1152/physiolgenomics.00044.2011

  View details for Web of Science ID 000294730000002

  View details for PubMedID 21712436

  View details for PubMedCentralID PMC3180735

• MicroRNA-26a Is a Novel Regulator of Vascular Smooth Muscle Cell Function JOURNAL OF CELLULAR PHYSIOLOGY Leeper, N. J., Raiesdana, A., Kojima, Y., Chun, H. J., Azuma, J., Maegdefessel, L., Kundu, R. K., Quertermous, T., Tsao, P. S., Spin, J. M. 2011; 226 (4): 1035-1043

  Abstract

  Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.

  View details for DOI 10.1002/jcp.22422

  View details for Web of Science ID 000287258800019

  View details for PubMedID 20857419

  View details for PubMedCentralID PMC3108574

• Gene Coexpression Network Topology of Cardiac Development, Hypertrophy, and Failure CIRCULATION-CARDIOVASCULAR GENETICS Dewey, F. E., Perez, M. V., Wheeler, M. T., Watt, C., Spin, J., Langfelder, P., Horvath, S., Hannenhalli, S., Cappola, T. P., Ashley, E. A. 2011; 4 (1): 26-U129

  Abstract

  Network analysis techniques allow a more accurate reflection of underlying systems biology to be realized than traditional unidimensional molecular biology approaches. Using gene coexpression network analysis, we define the gene expression network topology of cardiac hypertrophy and failure and the extent of recapitulation of fetal gene expression programs in failing and hypertrophied adult myocardium.We assembled all myocardial transcript data in the Gene Expression Omnibus (n=1617). Because hierarchical analysis revealed species had primacy over disease clustering, we focused this analysis on the most complete (murine) dataset (n=478). Using gene coexpression network analysis, we derived functional modules, regulatory mediators, and higher-order topological relationships between genes and identified 50 gene coexpression modules in developing myocardium that were not present in normal adult tissue. We found that known gene expression markers of myocardial adaptation were members of upregulated modules but not hub genes. We identified ZIC2 as a novel transcription factor associated with coexpression modules common to developing and failing myocardium. Of 50 fetal gene coexpression modules, 3 (6%) were reproduced in hypertrophied myocardium and 7 (14%) were reproduced in failing myocardium. One fetal module was common to both failing and hypertrophied myocardium.Network modeling allows systems analysis of cardiovascular development and disease. Although we did not find evidence for a global coordinated program of fetal gene expression in adult myocardial adaptation, our analysis revealed specific gene expression modules active during both development and disease and specific candidates for their regulation.

  View details for DOI 10.1161/CIRCGENETICS.110.941757

  View details for PubMedID 21127201

• Gene Mutations and Familial Thoracic Aortic Aneurysms A Walk on the Mild Side CIRCULATION-CARDIOVASCULAR GENETICS Spin, J. M. 2011; 4 (1): 4-6

  View details for DOI 10.1161/CIRCGENETICS.110.959171

  View details for Web of Science ID 000287353200010

  View details for PubMedID 21325162

• Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300 PLOS ONE Spin, J. M., Quertermous, T., Tsao, P. S. 2010; 5 (12)

  Abstract

  Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.

  View details for DOI 10.1371/journal.pone.0014301

  View details for Web of Science ID 000285246900013

  View details for PubMedID 21179216

  View details for PubMedCentralID PMC3001469

• Nicotine Accelerates the Expansion of Abdominal Aortic Aneurysms in Mice; A Potential Role for miR-21 and miR-26a Maegdefessel, L., Azuma, J., Spin, J. M., Deng, A., McConnell, M. V., Dalman, R. L., Tsao, P. S. LIPPINCOTT WILLIAMS & WILKINS. 2010

  View details for Web of Science ID 000208231602032

• Microarray Analysis Identifies miRNA-26a as a Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology Leeper, N. J., Raiesdana, A., Kojima, Y., Chun, H. J., Azuma, J., Kundu, R. K., Quertermous, T., Tsao, P. S., Spin, J. M. LIPPINCOTT WILLIAMS & WILKINS. 2010: E244–E244

  View details for Web of Science ID 000283234800283

• New options with dabigatran etexilate in anticoagulant therapy. Vascular health and risk management Maegdefessel, L., Spin, J. M., Azuma, J., Tsao, P. S. 2010; 6: 339-349

  Abstract

  Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.

  View details for PubMedID 20531953

• Analysis of In Situ and Ex Vivo Vascular Endothelial Growth Factor Receptor Expression During Experimental Aortic Aneurysm Progression ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tedesco, M. M., Terashima, M., Blankenberg, F. G., Levashova, Z., Spin, J. M., Backer, M. V., Backer, J. M., Sho, M., Sho, E., McConnell, M. V., Dalman, R. L. 2009; 29 (10): 1452-?

  Abstract

  Mural inflammation and neovascularization are characteristic pathological features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E-deficient (Apo E(-/-)) murine AAA model.Apo E(-/-) mice maintained on a high-fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n=16 mice, 3 to 4 time points per mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n=9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy 5.5 fluorescent tracer (7 to 18 microg/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 microg/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression. Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31(+) cell density.Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.

  View details for DOI 10.1161/ATVBAHA.109.187757

  View details for PubMedID 19574559

• Frontiers in nephrology: Genomic approaches to understanding the molecular basis of atherosclerosis JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Ashley, E. A., Spin, J. M., Tabibiazar, R., Quertermous, T. 2007; 18 (11): 2853-2862

  Abstract

  Atherosclerosis is a complex multicellular disease that is responsible for pathology in various organ systems. The understanding of its initiation and progression has been enhanced in recent years by the application of high-throughput genomic tools such as the microarray. Increasing in genomic coverage, such tools allow a view of the disease unaffected by previous conjecture as to the primary signal of interest. New statistical tools and pathway modeling techniques have established definitively for the first time the central role of inflammation in this process. This article reviews the genomic literature relating to atherosclerosis from cell culture, animal models, and human tissues. In this comparison of these differing approaches, the available data are synthesized to reach a new understanding of the complex interplay between vascular wall and immune system components.

  View details for DOI 10.1681/ASN.2007040514

  View details for Web of Science ID 000250737600012

  View details for PubMedID 17942952

• Network analysis of human in-stent restenosis CIRCULATION Ashley, E. A., Ferrara, R., King, J. Y., Vailaya, A., Kuchinsky, A., He, X., Byers, B., Gerckens, U., Oblin, S., Tsalenko, A., Soito, A., Spin, J. M., Tabibiazar, R., Connolly, A. J., Simpson, J. B., Grube, E., Quertermous, T. 2006; 114 (24): 2644-2654

  Abstract

  Recent successes in the treatment of in-stent restenosis (ISR) by drug-eluting stents belie the challenges still faced in certain lesions and patient groups. We analyzed human coronary atheroma in de novo and restenotic disease to identify targets of therapy that might avoid these limitations.We recruited 89 patients who underwent coronary atherectomy for de novo atherosclerosis (n=55) or in-stent restenosis (ISR) of a bare metal stent (n=34). Samples were fixed for histology, and gene expression was assessed with a dual-dye 22,000 oligonucleotide microarray. Histological analysis revealed significantly greater cellularity and significantly fewer inflammatory infiltrates and lipid pools in the ISR group. Gene ontology analysis demonstrated the prominence of cell proliferation programs in ISR and inflammation/immune programs in de novo restenosis. Network analysis, which combines semantic mining of the published literature with the expression signature of ISR, revealed gene expression modules suggested as candidates for selective inhibition of restenotic disease. Two modules are presented in more detail, the procollagen type 1 alpha2 gene and the ADAM17/tumor necrosis factor-alpha converting enzyme gene. We tested our contention that this method is capable of identifying successful targets of therapy by comparing mean significance scores for networks generated from subsets of the published literature containing the terms "sirolimus" or "paclitaxel." In addition, we generated 2 large networks with sirolimus and paclitaxel at their centers. Both analyses revealed higher mean values for sirolimus, suggesting that this agent has a broader suppressive action against ISR than paclitaxel.Comprehensive histological and gene network analysis of human ISR reveals potential targets for directed abrogation of restenotic disease and recapitulates the results of clinical trials of existing agents.

  View details for DOI 10.1161/CIRCULATIONAHA.106.637025

  View details for Web of Science ID 000243477800015

  View details for PubMedID 17145989

• Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes - Distinct responses to stimuli ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Deng, D. X., Spin, J. M., Tsalenko, A., Vailaya, A., Ben-Dor, A., Yakhini, Z., Tsao, P., Bruhn, L., Quertermous, T. 2006; 26 (5): 1058-1065

  Abstract

  Phenotypic differences between vascular smooth muscle cell (VSMC) subtypes lead to diverse pathological processes including atherosclerosis, postangioplasty restenosis and vein graft disease. To better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes, we compared gene expression profiles and functional responses to oxidized low-density lipoprotein (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM).OxLDL and PDGF elicited markedly different functional responses and expression profiles between the 2 SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration and modified gene expression of chemokines (CXCL10, CXCL11 and CXCL12), proinflammatory cytokines (IL-1, IL-6, and IL-18), insulin-like growth factor binding proteins (IGFBPs), and both endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-kappaB and phosphatidylinositol signaling pathways, and upregulated prostaglandin (PG) receptors and synthases. In untreated cells, alpha-chemokines, proinflammatory cytokines, and genes associated with apoptosis, inflammation, and lipid biosynthesis were higher in CASM, whereas some beta-chemokines, metalloproteinase inhibitors, and IGFBPs were higher in SVSM. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. In summary, our results suggest dramatic differences in gene expression patterns and functional responses to OxLDL and PDGF between venous and arterial SMCs, with venous SMCs having stronger proliferative/migratory responses to stimuli but also higher expression of atheroprotective genes at baseline.These results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.

  View details for DOI 10.1161/01.ATV.0000208185.16371.97

  View details for Web of Science ID 000236942400017

  View details for PubMedID 16456091

• Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation PHYSIOLOGICAL GENOMICS Wu, J. C., Spin, J. M., Cao, F., Lin, S. A., Xie, X. Y., Gheysens, O., Chen, I. Y., Sheikh, A. Y., Robbins, R. C., Tsalenko, A., Gambhir, S. S., Quertermous, T. 2006; 25 (1): 29-38

  Abstract

  Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.

  View details for DOI 10.1152/physiolgenomics.00254.2005

  View details for Web of Science ID 000236722700004

  View details for PubMedID 16390873

• Pathway analysis of coronary atherosclerosis PHYSIOLOGICAL GENOMICS King, J. Y., Ferrara, R., Tabibiazar, R., Spin, J. M., Chen, M. M., Kuchinsky, A., Vailaya, A., Kincaid, R., Tsalenko, A., Deng, D. X., Connolly, A., Zhang, P., Yang, E., Watt, C., Yakhini, Z., Ben-Dor, A., Adler, A., Bruhn, L., Tsao, P., Quertermous, T., Ashley, E. A. 2005; 23 (1): 103-118

  Abstract

  Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease.

  View details for DOI 10.1152/physiolgenomics.00101.2005

  View details for Web of Science ID 000232065200012

  View details for PubMedID 15942018

• Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease PHYSIOLOGICAL GENOMICS Tabibiazar, R., Wagner, R. A., Ashley, E. A., King, J. Y., Ferrara, R., Spin, J. M., Sanan, D. A., Narasimhan, B., Tibshirani, R., Tsao, P. S., Efron, B., Quertermous, T. 2005; 22 (2): 213-226

  Abstract

  The propensity for developing atherosclerosis is dependent on underlying genetic risk and varies as a function of age and exposure to environmental risk factors. Employing three mouse models with different disease susceptibility, two diets, and a longitudinal experimental design, it was possible to manipulate each of these factors to focus analysis on genes most likely to have a specific disease-related function. To identify differences in longitudinal gene expression patterns of atherosclerosis, we have developed and employed a statistical algorithm that relies on generalized regression and permutation analysis. Comprehensive annotation of the array with ontology and pathway terms has allowed rigorous identification of molecular and biological processes that underlie disease pathophysiology. The repertoire of atherosclerosis-related immunomodulatory genes has been extended, and additional fundamental pathways have been identified. This highly disease-specific group of mouse genes was combined with an extensive human coronary artery data set to identify a shared group of genes differentially regulated among atherosclerotic tissues from different species and different vascular beds. A small core subset of these differentially regulated genes was sufficient to accurately classify various stages of the disease in mouse. The same gene subset was also found to accurately classify human coronary lesion severity. In addition, this classifier gene set was able to distinguish with high accuracy atherectomy specimens from native coronary artery disease vs. those collected from in-stent restenosis lesions, thus identifying molecular differences between these two processes. These studies significantly focus efforts aimed at identifying central gene regulatory pathways that mediate atherosclerotic disease, and the identification of classification gene sets offers unique insights into potential diagnostic and therapeutic strategies in atherosclerotic disease.

  View details for DOI 10.1152/physiolgenomics.00001.2005

  View details for Web of Science ID 000230987900011

  View details for PubMedID 15870398

• Mouse strain-specific differences in vascular wall gene expression and their relationship to vascular disease ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tabibiazar, R., Wagner, R. A., Spin, J. M., Ashley, E. A., Narasimhan, B., Rubin, E. M., Efron, B., Tsao, P. S., Tibshirani, R., Quertermous, T. 2005; 25 (2): 302-308

  Abstract

  Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice.We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis.Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.

  View details for DOI 10.1161/011.ATV.0000151372.86863.a5

  View details for Web of Science ID 000226594000009

  View details for PubMedID 15550693

• Transcriptional profiling of in vitro smooth muscle cell differentiation identifies specific patterns of gene and pathway activation PHYSIOLOGICAL GENOMICS Spin, J. M., Nallamshetty, S., Tabibiazar, R., Ashley, E. A., King, J. Y., Chen, M., Tsao, P. S., Quertermous, T. 2004; 19 (3): 292-302

  Abstract

  Mesodermal and epidermal precursor cells undergo phenotypic changes during differentiation to the smooth muscle cell (SMC) lineage that are relevant to pathophysiological processes in the adult. Molecular mechanisms that underlie lineage determination and terminal differentiation of this cell type have received much attention, but the genetic program that regulates these processes has not been fully defined. Study of SMC differentiation has been facilitated by development of the P19-derived A404 embryonal cell line, which differentiates toward this lineage in the presence of retinoic acid and allows selection for cells adopting a SMC fate through a differentiation-specific drug marker. We sought to define global alterations in gene expression by studying A404 cells during SMC differentiation with oligonucleotide microarray transcriptional profiling. Using an in situ 60-mer array platform with more than 20,000 mouse genes derived from the National Institute on Aging clone set, we identified 2,739 genes that were significantly upregulated after differentiation was completed (false-detection ratio <1). These genes encode numerous markers known to characterize differentiated SMC, as well as many unknown factors. We further characterized the sequential patterns of gene expression during the differentiation time course, particularly for known transcription factor families, providing new insights into the regulation of the differentiation process. Changes in genes associated with specific biological ontology-based pathways were evaluated, and temporal trends were identified for functional pathways. In addition to confirming the utility of the A404 model, our data provide a large-scale perspective of gene regulation during SMC differentiation.

  View details for DOI 10.1152/physiolgenomics.00148.2004

  View details for PubMedID 15340120

• Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice JOURNAL OF BIOLOGICAL CHEMISTRY Ishida, T., Choi, S. S., Kundu, R. K., Spin, J., Yamashita, T., Hirata, K., Kojima, Y., Yokoyama, M., Cooper, A. D., Quertermous, T. 2004; 279 (43): 45085-45092

  Abstract

  Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.

  View details for DOI 10.1074/jbc.M406360200

  View details for Web of Science ID 000224505600101

  View details for PubMedID 15304490

• Treadmill scores in elderly men JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Lai, S., Kaykha, A., Yamazaki, T., Goldstein, M., Spin, J. M., Myers, J., Froelicher, V. F. 2004; 43 (4): 606-615

  Abstract

  This study seeks to further characterize the role of exercise testing in the elderly for prognosis and diagnosis of coronary artery disease.Recent exercise testing guidelines have recognized that statements regarding the elderly do not have an adequate evidence-based quality because the studies they are based on have limitations in sample size and design. The Duke Treadmill Score has been recommended for risk stratification, but recent evidence has suggested that it does not function in the elderly.The study population consisted of male veterans (1872 patients >or=65 years; 3798 patients <65 years) who underwent routine clinical exercise testing with a mean follow-up of six years. A subset who underwent coronary angiography as clinically indicated (elderly, n = 405; younger, n= 809) were included. The primary outcome for all subjects was cardiovascular mortality with coronary angiographic findings as the outcome in those selected for angiography.In survival analysis, exercise-induced ST depression was prognostic in both age groups only when cardiovascular death was considered as the end point. Peak metabolic equivalents were the most significant predictor for both age groups only when all-cause death was considered as the end point. New age-specific prognostic scores were developed and found to be predictive for cardiovascular mortality in the elderly. Moreover, in the angiographic subset of the elderly, a specific diagnostic score provided significantly better discrimination than exercise ST measurements alone. For any new score, there is a need for validation in another elderly population.The mortality end point affected the choice of prognostic variables. This study demonstrates that exercise test scores can be helpful for the diagnosis and prognosis of coronary disease in the elderly.

  View details for DOI 10.1016/j.jacc.2003.07.051

  View details for Web of Science ID 000188944600017

  View details for PubMedID 14975471

• Retinal evaluation of patients on chronic amiodarone therapy RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES Shaikh, S., Shaikh, N., Chun, S. H., Spin, J. M., Blumenkranz, M. S., Marmor, M. F. 2003; 23 (3): 354-359

  Abstract

  To determine whether retinal electrophysiologic changes can be detected and correlated with funduscopic findings in patients with the long-term use of amiodarone.Eleven patients ranging in age from 52 to 67 years were recruited from the Stanford University Medical Center Department of Cardiology for ophthalmologic examination. Patients had received amiodarone at various dosages ranging from 100 to 800 mg daily for at least 15 months. Clinical indications for the use of amiodarone included atrial fibrillation, ventricular arrhythmias, and congestive heart failure. All patients underwent retinal electrophysiology studies (full-field and multifocal electroretinograms) in addition to a complete ophthalmologic examination and fluorescein angiography.No patients were found to have significant vision loss. Funduscopic examination and fluorescein angiography showed mild age-related changes in four patients, three of whom had nonspecific foveal pigmentary alterations. Multifocal and full-field electroretinograms were mostly unremarkable, and the mildly subnormal findings in a few patients showed no consistent pattern to suggest a toxic cause. Dosage, duration of amiodarone exposure, patient age, and underlying cardiac disease did not appear to correlate with these findings.No significant adverse retinal funduscopic changes or electrophysiologic effects could be correlated with amiodarone exposure in this small series of patients. Routine electrophysiologic and funduscopic screening of patients receiving amiodarone does not seem warranted, although future prospective controlled studies may be required to exclude the possibility of progressive abnormalities in patients with preexisting age-related macular degeneration.

  View details for PubMedID 12824836

• Is the Duke Treadmill Score appropriate in the elderly? Annual Meeting of the American-Geriatrics-Society Lai, S. R., Kaykha, A., Yamazaki, T., Spin, J., Goldstein, M., Froelicher, V. F., Myers, J. WILEY-BLACKWELL. 2003: S213–S213

  View details for Web of Science ID 000182255100551

• Early use of statins in acute coronary syndromes. Current cardiology reports Spin, J. M., Vagelos, R. H. 2002; 4 (4): 289-297

  Abstract

  This review examines the use of HMG-CoA reductase inhibitor (statin) medications early in the clinical course of acute coronary syndrome. Available data demonstrate that there are clear clinical benefits to this practice. Numerous previous studies have documented the primary and secondary benefits of statins in the prevention of coronary events. Recent trials show that when statins are used during hospital admissions for acute coronary syndrome (ACS), patients experience decreased recurrent myocardial infarction, lower death rates, and fewer repeat hospitalizations for ischemia or revascularization. Several studies suggest that the positive effects of statins on plaque stabilization, inflammation, thrombosis, and endothelial function may be independent of lipid levels. There is also an emerging view that beneficial lipid-lowering with statins in high-risk patients has no lower limit. This information suggests that all patients admitted for ACS should be treated with statins, regardless of cholesterol levels.

  View details for PubMedID 12052268

• The prognostic value of exercise testing in elderly men AMERICAN JOURNAL OF MEDICINE Spin, J. M., PRAKASH, M., Froelicher, V. F., Partington, S., Marcus, R., Do, D., Myers, J. 2002; 112 (6): 453-459

  Abstract

  Our purposes were to compare the responses to exercise testing in elderly (> or =65 years of age) and younger men, and to investigate whether exercise testing has similar prognostic value in the two age groups.We included all elderly (n = 1185) and younger (n = 2789) male veterans without established coronary heart disease who underwent routine clinical exercise testing between 1987 and 2000 at two academically affiliated Veteran's Affairs medical center laboratories. Measurements included a standardized medical history, exercise testing, and all-cause mortality.Compared with younger patients, elderly patients achieved a lower workload (a mean [+/- SD] of 7 +/- 3 vs. 10 +/- 4 metabolic equivalents [METs], P <0.001) and were more likely to have abnormal ST depression (27% [n = 324] vs. 16% [n = 436], P <0.001). During the mean follow-up of 6 years, annual mortality was twice as high among elderly patients as among younger patients (4% vs. 2%, P <0.001). The only exercise test variable that was associated significantly with time to death in both age groups was maximal METs achieved: each 1 MET increase in exercise capacity was associated with an 11% reduction in annual mortality. Exercise-induced ST depression was more common in those who subsequently died, but was not an independent predictor of mortality.In elderly men, exercise testing provided prognostic information incremental to clinical data. Achieved workload (in METs) was the major exercise testing variable associated with all-cause mortality. Its prognostic importance was the same in elderly as in younger men.

  View details for PubMedID 11959055

• Abnormal ECG after extensive maxillofacial surgery HOSPITAL PRACTICE Spin, J., Hancock, E. W. 1999; 34 (11): 27-28

  View details for Web of Science ID 000083062900003

  View details for PubMedID 10887427

• CRYOELECTRON MICROSCOPY OF LOW-DENSITY-LIPOPROTEIN IN VITREOUS ICE BIOPHYSICAL JOURNAL Spin, J. M., Atkinson, D. 1995; 68 (5): 2115-2123

  Abstract

  In this report, images of low density lipoprotein (LDL) in vitreous ice at approximately 30 A resolution are presented. These images show that LDL is a quasi-spherical particle, approximately 220-240 A in diameter, with a region of low density (lipid) surrounded by a ring (in projection) of high density believed to represent apolipoprotein B-100. This ring is seen to be composed of four or five (depending on view) large regions of high density material that may represent protein superdomains. Analysis of LDL images obtained at slightly higher magnification reveals that areas of somewhat lower density connect these regions, in some cases crossing the projectional interiors of the LDL particles. Preliminary image analysis of LDL covalently labeled at Cys3734 and Cys4190 with 1.4-nm Nanogold clusters demonstrates that this methodology will provide an important site-specific marker in studies designed to map the organization of apoB at the surface of LDL.

  View details for Web of Science ID A1995RH65800052

  View details for PubMedID 7612855