Kristy Red-Horse

All Publications

• Fibrin drives thromboinflammation and neuropathology in COVID-19. Nature Ryu, J. K., Yan, Z., Montano, M., Sozmen, E. G., Dixit, K., Suryawanshi, R. K., Matsui, Y., Helmy, E., Kaushal, P., Makanani, S. K., Deerinck, T. J., Meyer-Franke, A., Rios Coronado, P. E., Trevino, T. N., Shin, M. G., Tognatta, R., Liu, Y., Schuck, R., Le, L., Miyajima, H., Mendiola, A. S., Arun, N., Guo, B., Taha, T. Y., Agrawal, A., MacDonald, E., Aries, O., Yan, A., Weaver, O., Petersen, M. A., Meza Acevedo, R., Alzamora, M. D., Thomas, R., Traglia, M., Kouznetsova, V. L., Tsigelny, I. F., Pico, A. R., Red-Horse, K., Ellisman, M. H., Krogan, N. J., Bouhaddou, M., Ott, M., Greene, W. C., Akassoglou, K. 2024

  Abstract

  Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

  View details for DOI 10.1038/s41586-024-07873-4

  View details for PubMedID 39198643

  View details for PubMedCentralID 9362465

• Eph-ephrin signaling couples endothelial cell sorting and arterial specification. Nature communications Stewen, J., Kruse, K., Godoi-Filip, A. T., Jeong, H. W., Adams, S., Berkenfeld, F., Stehling, M., Red-Horse, K., Adams, R. H., Pitulescu, M. E. 2024; 15 (1): 2539

  Abstract

  Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

  View details for DOI 10.1038/s41467-024-46300-0

  View details for PubMedID 38570531

  View details for PubMedCentralID PMC10991410

• Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells. Developmental cell Fowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, D. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M. 2024

  Abstract

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

  View details for DOI 10.1016/j.devcel.2024.03.003

  View details for PubMedID 38569552

• CXCL12 regulates coronary artery dominance in diverse populations and links development to disease. medRxiv : the preprint server for health sciences Rios Coronado, P. E., Zanetti, D., Zhou, J., Naftaly, J. A., Prabala, P., Kho, P. F., Martínez Jaimes, A. M., Hilliard, A. T., Pyarajan, S., Dochtermann, D., Chang, K. M., Winn, V. D., Pașca, A. M., Plomondon, M. E., Waldo, S. W., Tsao, P. S., Clarke, S. L., Red-Horse, K., Assimes, T. L. 2023

  Abstract

  Mammalian cardiac muscle is supplied with blood by right and left coronary arteries that form branches covering both ventricles of the heart. Whether branches of the right or left coronary arteries wrap around to the inferior side of the left ventricle is variable in humans and termed right or left dominance. Coronary dominance is likely a heritable trait, but its genetic architecture has never been explored. Here, we present the first large-scale multi-ancestry genome-wide association study of dominance in 61,043 participants of the VA Million Veteran Program, including over 10,300 Africans and 4,400 Admixed Americans. Dominance was moderately heritable with ten loci reaching genome wide significance. The most significant mapped to the chemokine CXCL12 in both Europeans and Africans. Whole-organ imaging of human fetal hearts revealed that dominance is established during development in locations where CXCL12 is expressed. In mice, dominance involved the septal coronary artery, and its patterning was altered with Cxcl12 deficiency. Finally, we linked human dominance patterns with coronary artery disease through colocalization, genome-wide genetic correlation and Mendelian Randomization analyses. Together, our data supports CXCL12 as a primary determinant of coronary artery dominance in humans of diverse backgrounds and suggests that developmental patterning of arteries may influence one's susceptibility to ischemic heart disease.

  View details for DOI 10.1101/2023.10.27.23297507

  View details for PubMedID 37961706

  View details for PubMedCentralID PMC10635223

• Sox7-positive endothelial progenitors establish coronary arteries and govern ventricular compaction. EMBO reports Chiang, I. K., Humphrey, D., Mills, R. J., Kaltzis, P., Pachauri, S., Graus, M., Saha, D., Wu, Z., Young, P., Sim, C. B., Davidson, T., Hernandez-Garcia, A., Shaw, C. A., Renwick, A., Scott, D. A., Porrello, E. R., Wong, E. S., Hudson, J. E., Red-Horse, K., Del Monte-Nieto, G., Francois, M. 2023: e55043

  Abstract

  The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development.

  View details for DOI 10.15252/embr.202255043

  View details for PubMedID 37551717

• Dedifferentiation and Proliferation of Artery Endothelial Cells Drive Coronary Collateral Development in Mice. Arteriosclerosis, thrombosis, and vascular biology Arolkar, G., Krishna Kumar, S., Wang, H., Gonzalez, K. M., Kumar, S., Bishnoi, B., Rios Coronado, P. E., Woo, Y. J., Red-Horse, K., Das, S. 2023

  Abstract

  BACKGROUND: Collateral arteries act as natural bypasses which reroute blood flow to ischemic regions and facilitate tissue regeneration. In an injured heart, neonatal artery endothelial cells orchestrate a systematic series of cellular events, which includes their outward migration, proliferation, and coalescence into fully functional collateral arteries. This process, called artery reassembly, aids complete cardiac regeneration in neonatal hearts but is absent in adults. The reason for this age-dependent disparity in artery cell response is completely unknown. In this study, we investigated if regenerative potential of coronary arteries is dictated by their ability to dedifferentiate.METHODS: Single-cell RNA sequencing of coronary endothelial cells was performed to identify differences in molecular profiles of neonatal and adult endothelial cells in mice. Findings from this in silico analyses were confirmed with in vivo experiments using genetic lineage tracing, whole organ immunostaining, confocal imaging, and cardiac functional assays in mice.RESULTS: Upon coronary occlusion, neonates showed a significant increase in actively cycling artery cells and expressed prominent dedifferentiation markers. Data from in silico pathway analyses and in vivo experiments suggested that upon myocardial infarction, cell cycle reentry of preexisting neonatal artery cells, the subsequent collateral artery formation, and recovery of cardiac function are dependent on arterial VegfR2 (vascular endothelial growth factor receptor-2). This subpopulation of dedifferentiated and proliferating artery cells was absent in nonregenerative postnatal day 7 or adult hearts.CONCLUSIONS: These data indicate that adult artery endothelial cells fail to drive collateral artery development due to their limited ability to dedifferentiate and proliferate.

  View details for DOI 10.1161/ATVBAHA.123.319319

  View details for PubMedID 37345524

• APJ+ cells in the SHF contribute to the cells of aorta and pulmonary trunk through APJ signaling. Developmental biology Baral, K., D'amato, G., Kuschel, B., Bogan, F., Jones, B. W., Large, C. L., Whatley, J. D., Red-Horse, K., Sharma, B. 2023

  Abstract

  Outflow tract develops from cardiac progenitor cells in the second heart field (SHF) domain. APJ, a G-Protein Coupled Receptor, is expressed by cardiac progenitor cells in the SHF. By lineage tracing APJ+SHF cells, we show that these cardiac progenitor cell contribute to the cells of outflow tract (OFT), which eventually give rise to aorta and pulmonary trunk/artery upon its morphogenesis. Furthermore, we show that early APJ+cells give rise to both aorta and pulmonary cells but late APJ+cells predominantly give rise to pulmonary cells. APJ is expressed by the outflow tract progenitors but its role in the SHF is unclear. We performed knockout studies to determine the role of APJ in SHF cell proliferation and survival. Our data suggested that APJ knockout in the SHF reduced the proliferation of SHF progenitors, while there was no significant impact on survival of the SHF progenitors. In addition, we show that ectopic overexpression of WNT in these cells disrupted aorta and pulmonary morphogenesis from outflow tract. Overall, our study have identified APJ+progenitor population within the SHF that give rise to aorta and pulmonary trunk/artery cells. Furthermore, we show that APJ signaling stimulate proliferation of these cells in the SHF.

  View details for DOI 10.1016/j.ydbio.2023.04.003

  View details for PubMedID 37037405

• An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression. Nature communications Dinh, T. T., Xiang, M., Rajaraman, A., Wang, Y., Salazar, N., Zhu, Y., Roper, W., Rhee, S., Brulois, K., O'Hara, E., Kiefel, H., Dinh, T. M., Bi, Y., Gonzalez, D., Bao, E. P., Red-Horse, K., Balogh, P., Gabris, F., Gaszner, B., Berta, G., Pan, J., Butcher, E. C. 2022; 13 (1): 7448

  Abstract

  Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

  View details for DOI 10.1038/s41467-022-34991-2

  View details for PubMedID 36460642

• Identification of a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis. Science advances Sikandar, S. S., Gulati, G. S., Antony, J., Fetter, I., Kuo, A. H., Ho, W. H., Haro-Acosta, V., Das, S., Steen, C. B., Pereira, T. A., Qian, D., Beachy, P. A., Dirbas, F., Red-Horse, K., Rabbitts, T. H., Thiery, J. P., Newman, A. M., Clarke, M. F. 2022; 8 (45): eabm3548

  Abstract

  Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

  View details for DOI 10.1126/sciadv.abm3548

  View details for PubMedID 36351009

• Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4. Developmental cell D'Amato, G., Phansalkar, R., Naftaly, J. A., Fan, X., Amir, Z. A., Rios Coronado, P. E., Cowley, D. O., Quinn, K. E., Sharma, B., Caron, K. M., Vigilante, A., Red-Horse, K. 2022

  Abstract

  Endocardial cells lining the heart lumen are coronary vessel progenitors during embryogenesis. Re-igniting this developmental process in adults could regenerate blood vessels lost during cardiac injury, but this requires additional knowledge of molecular mechanisms. Here, we use mouse genetics and scRNA-seq to identify regulators of endocardial angiogenesis and precisely assess the role of CXCL12/CXCR4 signaling. Time-specific lineage tracing demonstrated that endocardial cells differentiated into coronary endothelial cells primarily at mid-gestation. A new mouse line reporting CXCR4 activity-along with cell-specific gene deletions-demonstrated it was specifically required for artery morphogenesis rather than angiogenesis. Integrating scRNA-seq data of endocardial-derived coronary vessels from mid- and late-gestation identified a Bmp2-expressing transitioning population specific to mid-gestation. Bmp2 stimulated endocardial angiogenesis in vitro and in injured neonatal mouse hearts. Our data demonstrate how understanding the molecular mechanisms underlying endocardial angiogenesis can identify new potential therapeutic targets promoting revascularization of the injured heart.

  View details for DOI 10.1016/j.devcel.2022.10.007

  View details for PubMedID 36347256

• Vascular endothelial cell development and diversity. Nature reviews. Cardiology Trimm, E., Red-Horse, K. 2022

  Abstract

  Vascular endothelial cells form the inner layer of blood vessels where they have a key role in the development and maintenance of the functional circulatory system and provide paracrine support to surrounding non-vascular cells. Technical advances in the past 5 years in single-cell genomics and in in vivo genetic labelling have facilitated greater insights into endothelial cell development, plasticity and heterogeneity. These advances have also contributed to a new understanding of the timing of endothelial cell subtype differentiation and its relationship to the cell cycle. Identification of novel tissue-specific gene expression patterns in endothelial cells has led to the discovery of crucial signalling pathways and new interactions with other cell types that have key roles in both tissue maintenance and disease pathology. In this Review, we describe the latest findings in vascular endothelial cell development and diversity, which are often supported by large-scale, single-cell studies, and discuss the implications of these findings for vascular medicine. In addition, we highlight how techniques such as single-cell multimodal omics, which have become increasingly sophisticated over the past 2 years, are being utilized to study normal vascular physiology as well as functional perturbations in disease.

  View details for DOI 10.1038/s41569-022-00770-1

  View details for PubMedID 36198871

• Targeting calcineurin induces cardiomyocyte proliferation in adult mice. Nature cardiovascular research Lam, N. T., Nguyen, N. U., Ahmed, M. S., Hsu, C. C., Rios Coronado, P. E., Li, S., Menendez-Montes, I., Thet, S., Elhelaly, W. M., Xiao, F., Wang, X., Williams, N. S., Canseco, D. C., Red-Horse, K., Rothermel, B. A., Sadek, H. A. 2022; 1 (7): 679-688

  Abstract

  The mammalian neonatal heart can regenerate for 1 week after birth, after which, the majority of cardiomyocytes exit the cell cycle. Recent studies demonstrated that calcineurin mediates cell-cycle arrest of postnatal cardiomyocytes, partly through induction of nuclear translocation of the transcription factor Hoxb13 (a cofactor of Meis1). Here we show that inducible cardiomyocyte-specific deletion of calcineurin B1 in adult cardiomyocytes markedly decreases cardiomyocyte size and promotes mitotic entry, resulting in increased total cardiomyocyte number and improved left ventricular (LV) systolic function after myocardial infarction (MI). Similarly, pharmacological inhibition of calcineurin activity using FK506 promotes cardiomyocyte proliferation in vivo and increases cardiomyocyte number; however, FK506 administration after MI in mice failed to improve LV systolic function, possibly due to inhibition of vasculogenesis and blunting of the post-MI inflammatory response. Collectively, our results demonstrate that loss of calcineurin activity in adult cardiomyocytes promotes cell cycle entry; however, the effects of the calcineurin inhibitor FK506 on other cell types preclude a significant improvement of LV systolic function after MI.

  View details for DOI 10.1038/s44161-022-00098-6

  View details for PubMedID 39196243

  View details for PubMedCentralID 4868779

• A new resource for human coronary vessel development. Cardiovascular research Phansalkar, R., Red-Horse, K. 2022

  View details for DOI 10.1093/cvr/cvac094

  View details for PubMedID 35726909

• Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell Ang, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, B. J., Fowler, J. L., Holman, A. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M. 2022

  Abstract

  Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

  View details for DOI 10.1016/j.cell.2022.05.024

  View details for PubMedID 35738284

• The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science (New York, N.Y.) Jones, R. C., Karkanias, J., Krasnow, M. A., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Pisco, A. O., Tan, S. Y., Travaglini, K. J., Xu, C., Alcántara-Hernández, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Crasta, S., Culver, R. N., Cvijović, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kolluru, S., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W. J., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Morri, M., Mrouj, K., Mukherjee, S., Muser, T., Neuhöfer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Pisco, A. O., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L. C., Subramaniam, V. R., Swarup, A., Swift, M., Travaglini, K. J., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgó, E., Martin, B. A., Ozawa, M. G., Silva, O., Tan, S. Y., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Detweiler, A. M., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., de Morree, A., Olivieri, J. E., Park, M., Pisco, A. O., Ravikumar, N., Salzman, J., Stanley, G., Swift, M., Tan, M., Tan, W., Tarashansky, A. J., Vanheusden, R., Vorperian, S. K., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Alcántara-Hernández, M., Antony, J., Chan, C. K., Chang, C. A., Colville, A., Crasta, S., Culver, R., Dethlefsen, L., Ezran, C., Gillich, A., Hang, Y., Ho, P. Y., Irwin, J. C., Jang, S., Kershner, A. M., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Liu, S., Loeb, G. B., Lu, W. J., Maltzman, J. S., Metzger, R. J., de Morree, A., Neuhöfer, P., Perez, K., Phansalkar, R., Qi, Z., Rao, P., Raquer-McKay, H., Sasagawa, K., Scott, B., Sinha, R., Song, H., Spencer, S. P., Swarup, A., Swift, M., Travaglini, K. J., Trimm, E., Veizades, S., Vijayakumar, S., Wang, B., Wang, W., Winkler, J., Xie, J., Yung, A. R., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Krasnow, M., Kuo, C. S., Nguyen, P., Quake, S. R., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wang, B., Wu, A., Wu, S. M., Wyss-Coray, T. 2022; 376 (6594): eabl4896

  Abstract

  Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

  View details for DOI 10.1126/science.abl4896

  View details for PubMedID 35549404

• Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development ELIFE Phansalkar, R., Krieger, J., Zhao, M., Kolluru, S., Jones, R. C., Quake, S. R., Weissman, I., Bernstein, D., Winn, V. D., D'Amato, G., Red-Horse, K. 2021; 10

  View details for DOI 10.7554/eLife.70246.sa2

  View details for Web of Science ID 000730934300001

• Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction. European heart journal Rhee, S., Paik, D. T., Yang, J. Y., Nagelberg, D., Williams, I., Tian, L., Roth, R., Chandy, M., Ban, J., Belbachir, N., Kim, S., Zhang, H., Phansalkar, R., Wong, K. M., King, D. A., Valdez, C., Winn, V. D., Morrison, A. J., Wu, J. C., Red-Horse, K. 2021

  Abstract

  AIMS: Non-compaction cardiomyopathy is a devastating genetic disease caused by insufficient consolidation of ventricular wall muscle that can result in inadequate cardiac performance. Despite being the third most common cardiomyopathy, the mechanisms underlying the disease, including the cell types involved, are poorly understood. We have previously shown that endothelial cell-specific deletion of the chromatin remodeller gene Ino80 results in defective coronary vessel development that leads to ventricular non-compaction in embryonic mouse hearts. We aimed to identify candidate angiocrines expressed by endocardial and ECs inwildtype and LVNC conditions in Tie2Cre;Ino80fl/fl transgenic embryonic mouse hearts, and test the effect of these candidates on cardiomyocyte proliferation and maturation.METHODS AND RESULTS: We used single-cell RNA-sequencing to characterize endothelial and endocardial defects in Ino80-deficient hearts. We observed a pathological endocardial cell population in the non-compacted hearts and identified multiple dysregulated angiocrine factors that dramatically affected cardiomyocyte behaviour. We identified Col15A1 as a coronary vessel-secreted angiocrine factor, downregulated by Ino80-deficiency, that functioned to promote cardiomyocyte proliferation. Furthermore, mutant endocardial and endothelial cells (ECs) up-regulated expression of secreted factors, such as Tgfbi, Igfbp3, Isg15, and Adm, which decreased cardiomyocyte proliferation and increased maturation.CONCLUSIONS: These findings support a model where coronary ECs normally promote myocardial compaction through secreted factors, but that endocardial and ECs can secrete factors that contribute to non-compaction under pathological conditions.

  View details for DOI 10.1093/eurheartj/ehab298

  View details for PubMedID 34279605

• Enhancing cardiovascular research with whole-organ imaging. Current opinion in hematology Rios Coronado, P. E., Red-Horse, K. 2021

  Abstract

  PURPOSE OF REVIEW: There have been tremendous advances in the tools available for surveying blood vessels within whole organs and tissues. Here, we summarize some of the recent developments in methods for immunolabeling and imaging whole organs and provide a protocol optimized for the heart.RECENT FINDINGS: Multiple protocols have been established for chemically clearing large organs and variations are compatible with cell type-specific labeling. Heart tissue can be successfully cleared to reveal the three-dimensional structure of the entire coronary vasculature in neonatal and adult mice. Obtaining vascular reconstructions requires exceptionally large imaging files and new computational methods to process the data for accurate vascular quantifications. This is a continually advancing field that has revolutionized our ability to acquire data on larger samples as a faster rate.SUMMARY: Historically, cardiovascular research has relied heavily on histological analyses that use tissue sections, which usually sample cellular phenotypes in small regions and lack information on whole tissue-level organization. This approach can be modified to survey whole organs but image acquisition and analysis time can become unreasonable. In recent years, whole-organ immunolabeling and clearing methods have emerged as a workable solution, and new microscopy modalities, such as light-sheet microscopy, significantly improve image acquisition times. These innovations make studying the vasculature in the context of the whole organ widely available and promise to reveal fascinating new cellular behaviors in adult tissues and during repair.

  View details for DOI 10.1097/MOH.0000000000000655

  View details for PubMedID 33741761

• New Research Is Shining Light on How Collateral Arteries Form in the Heart: a Future Therapeutic Direction? Current cardiology reports Red-Horse, K., Das, S. 2021; 23 (4): 30

  Abstract

  PURPOSE OF REVIEW: Collateral arteries create artery-artery anastomoses that could serve as natural bypasses that in the heart could relieve the various complications of ischemia heart disease. Recent work using animal models have begun to reveal details of how coronary collateral arteries form.RECENT FINDINGS: Mouse genetics has been used to study the cellular and molecular mechanisms of collateral artery development. Collateral arteries are not pre-existing in the mouse heart, and only form in response to injury. Myocardial infarction creates tissue hypoxia that triggers the expression of growth factors and chemokines that guide collaterogenesis. Collateral development is more robust in neonatal hearts when compared with adults, and contributes to neonatal heart regeneration. The identification of signaling pathways and cellular responses underlying coronary collateral artery development suggests potential translational strategies. Continued investigation into this subject could lead to the identification of targetable pathways that induce collateral arteries for cardiac revascularization.

  View details for DOI 10.1007/s11886-021-01460-z

  View details for PubMedID 33655379

• Dach1 Extends Artery Networks and Protects Against Cardiac Injury. Circulation research Raftrey, B., Williams, I. M., Rios Coronado, P. E., Fan, X., Chang, A. H., Zhao, M., Roth, R. K., Trimm, E., Racelis, R., D'Amato, G., Phansalkar, R., Nguyen, A., Chai, T., Gonzalez, K. M., Zhang, Y., Ang, L. T., Loh, K., Bernstein, D., Red-Horse, K. 2021

  Abstract

  Rationale: Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction (MI). Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells (ECs) in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary ECs to differentiate and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure prior to injury, but increased the number of perfused arteries in the injury zone post-MI. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of CAD.

  View details for DOI 10.1161/CIRCRESAHA.120.318271

  View details for PubMedID 34383559

• Single-Cell RNA-seq Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells. Circulation Paik, D. T., Tian, L., Williams, I. M., Rhee, S., Zhang, H., Liu, C., Mishra, R., Wu, S. M., Red-Horse, K., Wu, J. C. 2020

  Abstract

  Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. While these functional differences are presumably imprinted in the transcriptome, the pathways and networks which sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA-sequencing (scRNA-seq) data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We employed a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from scRNA-seq data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either scRNA-seq or bulk RNA-seq, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (e.g. liver, brain) whereas others (i.e. adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. In addition, we found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. Finally, we identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: In summary, we use scRNA-seq data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.

  View details for DOI 10.1161/CIRCULATIONAHA.119.041433

  View details for PubMedID 32929989

• Single-cell maps of the human heart NATURE Phansalkar, R., Red-Horse, K. 2020; 577 (7792): 629–30

  View details for Web of Science ID 000510520700040

  View details for PubMedID 31988406

• Single-cell maps of the human heart NATURE Phansalkar, R., Red-Horse, K. 2020; 577 (7792): 629–30

  View details for DOI 10.1038/d41586-020-00151-z

  View details for Web of Science ID 000510520700052

• Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes. Cell stem cell Buikema, J. W., Lee, S. n., Goodyer, W. R., Maas, R. G., Chirikian, O. n., Li, G. n., Miao, Y. n., Paige, S. L., Lee, D. n., Wu, H. n., Paik, D. T., Rhee, S. n., Tian, L. n., Galdos, F. X., Puluca, N. n., Beyersdorf, B. n., Hu, J. n., Beck, A. n., Venkamatran, S. n., Swami, S. n., Wijnker, P. n., Schuldt, M. n., Dorsch, L. M., van Mil, A. n., Red-Horse, K. n., Wu, J. Y., Geisen, C. n., Hesse, M. n., Serpooshan, V. n., Jovinge, S. n., Fleischmann, B. K., Doevendans, P. A., van der Velden, J. n., Garcia, K. C., Wu, J. C., Sluijter, J. P., Wu, S. M. 2020; 27 (1): 50–63.e5

  Abstract

  Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

  View details for DOI 10.1016/j.stem.2020.06.001

  View details for PubMedID 32619518

• Whole-body tracking of single cells via positron emission tomography. Nature biomedical engineering Jung, K. O., Kim, T. J., Yu, J. H., Rhee, S. n., Zhao, W. n., Ha, B. n., Red-Horse, K. n., Gambhir, S. S., Pratx, G. n. 2020

  Abstract

  In vivo molecular imaging can measure the average kinetics and movement routes of injected cells through the body. However, owing to non-specific accumulation of the contrast agent and its efflux from the cells, most of these imaging methods inaccurately estimate the distribution of the cells. Here, we show that single human breast cancer cells loaded with mesoporous silica nanoparticles concentrating the 68Ga radioisotope and injected into immunodeficient mice can be tracked in real time from the pattern of annihilation photons detected using positron emission tomography, with respect to anatomical landmarks derived from X-ray computed tomography. The cells travelled at an average velocity of 50 mm s-1 and arrested in the lungs 2-3 s after tail-vein injection into the mice, which is consistent with the blood-flow rate. Single-cell tracking could be used to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy and the distribution of cells after transplantation.

  View details for DOI 10.1038/s41551-020-0570-5

  View details for PubMedID 32541917

• Veins and Arteries Build Hierarchical Branching Patterns Differently: Bottom-Up versus Top-Down. BioEssays : news and reviews in molecular, cellular and developmental biology Red-Horse, K., Siekmann, A. F. 2019; 41 (3): e1800198

  Abstract

  A tree-like hierarchical branching structure is present in many biological systems, such as the kidney, lung, mammary gland, and blood vessels. Most of these organs form through branching morphogenesis, where outward growth results in smaller and smaller branches. However, the blood vasculature is unique in that it exists as two trees (arterial and venous) connected at their tips. Obtaining this organization might therefore require unique developmental mechanisms. As reviewed here, recent data indicate that arterial trees often form in reverse order. Accordingly, initial arterial endothelial cell differentiation occurs outside of arterial vessels. These pre-artery cells then build trees by following a migratory path from smaller into larger arteries, a process guided by the forces imparted by blood flow. Thus, in comparison to other branched organs, arteries can obtain their structure through inward growth and coalescence. Here, new information on the underlying mechanisms is discussed, and how defects can lead to pathologies, such as hypoplastic arteries and arteriovenous malformations.

  View details for PubMedID 30805984

• Veins and Arteries Build Hierarchical Branching Patterns Differently: Bottom-Up versus Top-Down BIOESSAYS Red-Horse, K., Siekmann, A. F. 2019; 41 (3)

  View details for DOI 10.1002/bies.201800198

  View details for Web of Science ID 000459704900009

• A Unique Collateral Artery Development Program Promotes Neonatal Heart Regeneration CELL Das, S., Goldstone, A. B., Wang, H., Farry, J., D'Amato, G., Paulsen, M. J., Eskandari, A., Hironaka, C. E., Phansalkar, R., Sharma, B., Rhee, S., Shamskhou, E., Agalliu, D., Perez, V., Woo, Y., Red-Horse, K. 2019; 176 (5): 1128-+

  View details for DOI 10.1016/j.cell.2018.12.023

  View details for Web of Science ID 000459257500015

• A Unique Collateral Artery Development Program Promotes Neonatal Heart Regeneration. Cell Das, S., Goldstone, A. B., Wang, H., Farry, J., D'Amato, G., Paulsen, M. J., Eskandari, A., Hironaka, C. E., Phansalkar, R., Sharma, B., Rhee, S., Shamskhou, E. A., Agalliu, D., de Jesus Perez, V., Woo, Y. J., Red-Horse, K. 2019

  Abstract

  Collateral arteries are an uncommon vessel subtype that can provide alternate blood flow to preserve tissue following vascular occlusion. Some patients with heart disease develop collateral coronary arteries, and this correlates with increased survival. However, it is not known how these collaterals develop or how to stimulate them. We demonstrate that neonatal mouse hearts use a novel mechanism to build collateral arteries in response to injury. Arterial endothelial cells (ECs) migrated away from arteries along existing capillaries and reassembled into collateral arteries, which we termed "artery reassembly". Artery ECs expressed CXCR4, and following injury, capillary ECs induced its ligand, CXCL12. CXCL12 or CXCR4 deletion impaired collateral artery formation and neonatal heart regeneration. Artery reassembly was nearly absent in adults but was induced by exogenous CXCL12. Thus, understanding neonatal regenerative mechanisms can identify pathways that restore these processes in adults and identify potentially translatable therapeutic strategies for ischemic heart disease.

  View details for PubMedID 30686582

• Distinct origins and molecular mechanisms contribute to lymphatic formation during cardiac growth and regeneration. eLife Gancz, D. n., Raftrey, B. C., Perlmoter, G. n., Marín-Juez, R. n., Semo, J. n., Matsuoka, R. L., Karra, R. n., Raviv, H. n., Moshe, N. n., Addadi, Y. n., Golani, O. n., Poss, K. D., Red-Horse, K. n., Stainier, D. Y., Yaniv, K. n. 2019; 8

  Abstract

  In recent years there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.

  View details for DOI 10.7554/eLife.44153

  View details for PubMedID 31702554

• Characterization of Brain Dysfunction Induced by Bacterial Lipopeptides That Alter Neuronal Activity and Network in Rodent Brains JOURNAL OF NEUROSCIENCE Kim, K., Zamaleeva, A. I., Lee, Y., Ahmed, M., Kim, E., Lee, H., Pothineni, V., Tao, J., Rhee, S., Jayakumar, M., Inayathullah, M., Sivanesan, S., Red-Horse, K., Palmer, T. D., Park, J., Madison, D. V., Lee, H., Rajadas, J. 2018; 38 (50): 10672–91

  View details for DOI 10.1523/JNEUROSCI.0825-17.2018

  View details for Web of Science ID 000452854200011

• Characterization of brain dysfunction induced by bacterial lipopeptides that alter neuronal activity and network in rodent brains. The Journal of neuroscience : the official journal of the Society for Neuroscience Kim, K., Zamaleeva, A. I., Woo Lee, Y., Ahmed, M. R., Kim, E., Lee, H., Raveendra Pothineni, V., Tao, J., Rhee, S., Jayakumar, M., Inayathullah, M., Sivanesan, S., Red-Horse, K., Palmer, T. D., Park, J., Madison, D. V., Lee, H., Rajadas, J. 2018

  Abstract

  The immunopathological states of the brain induced by bacterial lipoproteins have been well-characterized by employing biochemical and histological assays. However, these studies have limitations in determining functional states of damaged brains involving aberrant synaptic activity and network, which makes it difficult to diagnose brain disorders during bacterial infection. To address this, we investigated the effect of Pam3CSK4 (PAM), a synthetic bacterial lipopeptide, on synaptic dysfunction of female mice brains and cultured neurons in parallel. Our functional brain imaging using PET with [18F]-FDG and [18F]-FMZ revealed the brain dysfunction induced by PAM is closely aligned to disruption of neurotransmitter-related neuronal activity and functional correlation in the region of the limbic system rather than to decrease of metabolic activity of neurons in the injection area. This finding was verified by in vivo tissue experiments that analyzed synaptic and dendritic alterations in the regions where PET imaging showed abnormal neuronal activity and network. Recording of synaptic activity also revealed that PAM reorganized synaptic distribution and decreased synaptic plasticity in hippocampus. Further study using in vitro neuron cultures demonstrated that PAM decreased the number of presynapses and the frequency of mEPSC, which suggests PAM disrupts neuronal function by damaging presynapses exclusively. We also showed PAM caused aggregation of synapses around dendrites, which may have caused no significant change in expression level of synaptic proteins while synaptic number and function was impaired by PAM. New findings of this study could provide a useful guide for diagnosis and treatment of brain disorders specific to bacterial infection.SIGNIFICANCE STATEMENTIt is challenging to diagnose brain disorders caused by bacterial infection because neural damage induced by bacterial products involves non-specific neurological symptoms, which is rarely detected by laboratory tests with low spatiotemporal resolution. To better understand brain pathology, it is essential to detect functional abnormalities of brain over time. To this end, we investigated characteristic patterns of altered neuronal integrity and functional correlation between various regions in mice brains injected with bacterial lipopeptides by using PET with a goal to apply new findings to diagnosis of brain disorder specific to bacterial infection. In addition, we analyzed altered synaptic density and function using both in vivo and in vitro experimental models to understand how bacterial lipopeptides impair brain function and network.

  View details for PubMedID 30381406

• Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells CIRCULATION RESEARCH Paik, D. T., Tian, L., Lee, J., Sayed, N., Chen, I. Y., Rhee, S., Rhee, J., Kim, Y., Wirka, R. C., Buikema, J. W., Wu, S. M., Red-Horse, K., Quertermous, T., Wu, J. C. 2018; 123 (4): 443–50

  View details for DOI 10.1161/CIRCRESAHA.118.312913

  View details for Web of Science ID 000440670800010

• Single-cell analysis of early progenitor cells that build coronary arteries NATURE Su, T., Stanley, G., Sinha, R., D'Amato, G., Das, S., Rhee, S., Chang, A. H., Poduri, A., Raftrey, B., Thanh Theresa Dinh, Roper, W. A., Li, G., Quinn, K. E., Caron, K. M., Wu, S., Miquerol, L., Butcher, E. C., Weissman, I., Quake, S., Red-Horse, K. 2018; 559 (7714): 356-+

  View details for DOI 10.1038/s41586-018-0288-7

  View details for Web of Science ID 000439059800048

• Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells. Circulation research Paik, D. T., Tian, L., Lee, J., Sayed, N., Chen, I. Y., Rhee, S., Rhee, J., Kim, Y., Wirka, R. C., Buikema, J. W., Wu, S. M., Red-Horse, K., Quertermous, T., Wu, J. C. 2018

  Abstract

  Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

  View details for PubMedID 29986945

• Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease. Nature communications Rhee, S. n., Chung, J. I., King, D. A., D'amato, G. n., Paik, D. T., Duan, A. n., Chang, A. n., Nagelberg, D. n., Sharma, B. n., Jeong, Y. n., Diehn, M. n., Wu, J. C., Morrison, A. J., Red-Horse, K. n. 2018; 9 (1): 368

  Abstract

  During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.

  View details for PubMedID 29371594

• Single-cell analysis of early progenitor cells that build coronary arteries. Nature Su, T. n., Stanley, G. n., Sinha, R. n., D'Amato, G. n., Das, S. n., Rhee, S. n., Chang, A. H., Poduri, A. n., Raftrey, B. n., Dinh, T. T., Roper, W. A., Li, G. n., Quinn, K. E., Caron, K. M., Wu, S. n., Miquerol, L. n., Butcher, E. C., Weissman, I. n., Quake, S. n., Red-Horse, K. n. 2018

  Abstract

  Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

  View details for PubMedID 29973725

• DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis GENES & DEVELOPMENT Chang, A. H., Raftrey, B. C., D'Amato, G., Surya, V. N., Poduri, A., Chen, H. I., Goldstone, A. B., Woo, J., Fuller, G. G., Dunn, A. R., Red-Horse, K. 2017; 31 (13): 1308–24

  Abstract

  Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronary arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 had small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12 Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis but was down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically down-regulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model in which DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that down-regulates DACH1. Thus, we identified a mechanism by which blood flow quality balances artery growth and maturation.

  View details for PubMedID 28779009

• Patterning coronary artery development Red-Horse, K. WILEY. 2017

  View details for Web of Science ID 000405461400607

• Coronary Artery Development: Progenitor Cells and Differentiation Pathways. Annual review of physiology Sharma, B., Chang, A., Red-Horse, K. 2017; 79: 1-19

  Abstract

  Coronary artery disease (CAD) is the number one cause of death worldwide and involves the accumulation of plaques within the artery wall that can occlude blood flow to the heart and cause myocardial infarction. The high mortality associated with CAD makes the development of medical interventions that repair and replace diseased arteries a high priority for the cardiovascular research community. Advancements in arterial regenerative medicine could benefit from a detailed understanding of coronary artery development during embryogenesis and of how these pathways might be reignited during disease. Recent research has advanced our knowledge on how the coronary vasculature is built and revealed unexpected features of progenitor cell deployment that may have implications for organogenesis in general. Here, we highlight these recent findings and discuss how they set the stage to interrogate developmental pathways during injury and disease.

  View details for DOI 10.1146/annurev-physiol-022516-033953

  View details for PubMedID 27959616

• Cellular plasticity in cardiovascular development and disease. Developmental dynamics Das, S., Red-Horse, K. 2017

  Abstract

  Knowledge on cellular differentiation pathways is critical to understanding organ development, homeostasis, and disease. Studying cell differentiation and cell fate restrictions in these contexts can be done through lineage tracing experiments, which entail permanent labeling of a cell and all its progeny. Recent lineage experiments within the cardiovascular system have uncovered unexpected findings on cellular origins during organogenesis and cell plasticity during disease. For example, there is increasing evidence that multiple progenitor sources exist for a single cell type, and that cells have remarkable expansive capacities under disease settings. Here, we summarize some recent findings in the cardiovascular system and highlight where there is evidence that the underlying concepts are a widespread phenomenon used by other organ systems. Developmental Dynamics 246:328-335, 2017. © 2016 Wiley Periodicals, Inc.

  View details for DOI 10.1002/dvdy.24486

  View details for PubMedID 28097739

• Alternative Progenitor Cells Compensate to Rebuild the Coronary Vasculature in Elabela- and Apj-Deficient Hearts. Developmental cell Sharma, B. n., Ho, L. n., Ford, G. H., Chen, H. I., Goldstone, A. B., Woo, Y. J., Quertermous, T. n., Reversade, B. n., Red-Horse, K. n. 2017

  Abstract

  Organogenesis during embryonic development occurs through the differentiation of progenitor cells. This process is extraordinarily accurate, but the mechanisms ensuring high fidelity are poorly understood. Coronary vessels of the mouse heart derive from at least two progenitor pools, the sinus venosus and endocardium. We find that the ELABELA (ELA)-APJ signaling axis is only required for sinus venosus-derived progenitors. Because they do not depend on ELA-APJ, endocardial progenitors are able to expand and compensate for faulty sinus venosus development in Apj mutants, leading to normal adult heart function. An upregulation of endocardial SOX17 accompanied compensation in Apj mutants, which was also seen in Ccbe1 knockouts, indicating that the endocardium is activated in multiple cases where sinus venosus angiogenesis is stunted. Our data demonstrate that by diversifying their responsivity to growth cues, distinct coronary progenitor pools are able to compensate for each other during coronary development, thereby providing robustness to organ development.

  View details for PubMedID 28890073

• Endothelial cells respond to the direction of mechanical stimuli through SMAD signaling to regulate coronary artery size. Development (Cambridge, England) Poduri, A. n., Chang, A. H., Raftrey, B. n., Rhee, S. n., Van, M. n., Red-Horse, K. n. 2017; 144 (18): 3241–52

  Abstract

  How mechanotransduction intersects with chemical and transcriptional factors to shape organogenesis is an important question in developmental biology. This is particularly relevant to the cardiovascular system, which uses mechanical signals from flowing blood to stimulate cytoskeletal and transcriptional responses that form a highly efficient vascular network. Using this system, artery size and structure are tightly regulated, but the underlying mechanisms are poorly understood. Here, we demonstrate that deletion of Smad4 increased the diameter of coronary arteries during mouse embryonic development, a phenotype that followed the initiation of blood flow. At the same time, the BMP signal transducers SMAD1/5/8 were activated in developing coronary arteries. In a culture model of blood flow-induced shear stress, human coronary artery endothelial cells failed to align when either BMPs were inhibited or SMAD4 was depleted. In contrast to control cells, SMAD4-deficient cells did not migrate against the direction of shear stress and increased proliferation rates specifically under flow. Similar alterations were seen in coronary arteries in vivo Thus, endothelial cells perceive the direction of blood flow and respond through SMAD signaling to regulate artery size.

  View details for PubMedID 28760815

• Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin's glucose-lowering effects. Science translational medicine Hwangbo, C. n., Wu, J. n., Papangeli, I. n., Adachi, T. n., Sharma, B. n., Park, S. n., Zhao, L. n., Ju, H. n., Go, G. W., Cui, G. n., Inayathullah, M. n., Job, J. K., Rajadas, J. n., Kwei, S. L., Li, M. O., Morrison, A. R., Quertermous, T. n., Mani, A. n., Red-Horse, K. n., Chun, H. J. 2017; 9 (407)

  Abstract

  Treatment of type 2 diabetes mellitus continues to pose an important clinical challenge, with most existing therapies lacking demonstrable ability to improve cardiovascular outcomes. The atheroprotective peptide apelin (APLN) enhances glucose utilization and improves insulin sensitivity. However, the mechanism of these effects remains poorly defined. We demonstrate that the expression of APLNR (APJ/AGTRL1), the only known receptor for apelin, is predominantly restricted to the endothelial cells (ECs) of multiple adult metabolic organs, including skeletal muscle and adipose tissue. Conditional endothelial-specific deletion of Aplnr (Aplnr(ECKO) ) resulted in markedly impaired glucose utilization and abrogation of apelin-induced glucose lowering. Furthermore, we identified inactivation of Forkhead box protein O1 (FOXO1) and inhibition of endothelial expression of fatty acid (FA) binding protein 4 (FABP4) as key downstream signaling targets of apelin/APLNR signaling. Both the Apln(-/-) and Aplnr(ECKO) mice demonstrated increased endothelial FABP4 expression and excess tissue FA accumulation, whereas concurrent endothelial Foxo1 deletion or pharmacologic FABP4 inhibition rescued the excess FA accumulation phenotype of the Apln(-/-) mice. The impaired glucose utilization in the Aplnr(ECKO) mice was associated with excess FA accumulation in the skeletal muscle. Treatment of these mice with an FABP4 inhibitor abrogated these metabolic phenotypes. These findings provide mechanistic insights that could greatly expand the therapeutic repertoire for type 2 diabetes and related metabolic disorders.

  View details for PubMedID 28904225

• MicroRNA 139-5p coordinates APLNR-CXCR4 crosstalk during vascular maturation NATURE COMMUNICATIONS Papangeli, I., Kim, J., Maier, I., Park, S., Lee, A., Kang, Y., Tanaka, K., Khan, O. F., Ju, H., Kojima, Y., Red-Horse, K., Anderson, D. G., Siekmann, A. F., Chun, H. J. 2016; 7

  Abstract

  G protein-coupled receptor (GPCR) signalling, including that involving apelin (APLN) and its receptor APLNR, is known to be important in vascular development. How this ligand-receptor pair regulates the downstream signalling cascades in this context remains poorly understood. Here, we show that mice with Apln, Aplnr or endothelial-specific Aplnr deletion develop profound retinal vascular defects, which are at least in part due to dysregulated increase in endothelial CXCR4 expression. Endothelial CXCR4 is negatively regulated by miR-139-5p, whose transcription is in turn induced by laminar flow and APLN/APLNR signalling. Inhibition of miR-139-5p in vivo partially phenocopies the retinal vascular defects of APLN/APLNR deficiency. Pharmacological inhibition of CXCR4 signalling or augmentation of the miR-139-5p-CXCR4 axis can ameliorate the vascular phenotype of APLN/APLNR deficient state. Overall, we identify an important microRNA-mediated GPCR crosstalk, which plays a key role in vascular development.

  View details for DOI 10.1038/ncomms11268

  View details for PubMedID 27068353

• Pericytes are progenitors for coronary artery smooth muscle. eLife Volz, K. S., Jacobs, A. H., Chen, H. I., Poduri, A., McKay, A. S., Riordan, D. P., Kofler, N., Kitajewski, J., Weissman, I., Red-Horse, K. 2015; 4

  Abstract

  Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.

  View details for DOI 10.7554/eLife.10036

  View details for PubMedID 26479710

  View details for PubMedCentralID PMC4728130

• Genetic targeting of sprouting angiogenesis using Apln-CreER. Nature communications Liu, Q., Hu, T., He, L., Huang, X., Tian, X., Zhang, H., He, L., Pu, W., Zhang, L., Sun, H., Fang, J., Yu, Y., Duan, S., Hu, C., Hui, L., Zhang, H., Quertermous, T., Xu, Q., Red-Horse, K., Wythe, J. D., Zhou, B. 2015; 6: 6020-?

  View details for DOI 10.1038/ncomms7020

  View details for PubMedID 25597280

• Genetic targeting of sprouting angiogenesis using Apln-CreER. Nature communications Liu, Q., Hu, T., He, L., Huang, X., Tian, X., Zhang, H., He, L., Pu, W., Zhang, L., Sun, H., Fang, J., Yu, Y., Duan, S., Hu, C., Hui, L., Zhang, H., Quertermous, T., Xu, Q., Red-Horse, K., Wythe, J. D., Zhou, B. 2015; 6: 6020-?

  Abstract

  Under pathophysiological conditions in adults, endothelial cells (ECs) sprout from pre-existing blood vessels to form new ones by a process termed angiogenesis. During embryonic development, Apelin (APLN) is robustly expressed in vascular ECs. In adult mice, however, APLN expression in the vasculature is significantly reduced. Here we show that APLN expression is reactivated in adult ECs after ischaemia insults. In models of both injury ischaemia and tumor angiogenesis, we find that Apln-CreER genetically labels sprouting but not quiescent vasculature. By leveraging this specific activity, we demonstrate that abolishment of the VEGF-VEGFR2 signalling pathway as well as ablation of sprouting ECs diminished tumour vascularization and growth without compromising vascular homeostasis in other organs. Collectively, we show that Apln-CreER distinguishes sprouting vessels from stabilized vessels in multiple pathological settings. The Apln-CreER line described here will greatly aid future mechanistic studies in both vascular developmental biology and adult vascular diseases.

  View details for DOI 10.1038/ncomms7020

  View details for PubMedID 25597280

• The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis. Development Chen, H. I., Sharma, B., Akerberg, B. N., Numi, H. J., Kivelä, R., Saharinen, P., Aghajanian, H., McKay, A. S., Bogard, P. E., Chang, A. H., Jacobs, A. H., Epstein, J. A., Stankunas, K., Alitalo, K., Red-Horse, K. 2014; 141 (23): 4500-4512

  Abstract

  Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineage-tracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardial-derived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

  View details for DOI 10.1242/dev.113639

  View details for PubMedID 25377552

• Oxygen regulates human cytotrophoblast migration by controlling chemokine and receptor expression PLACENTA Schanz, A., Red-Horse, K., Hess, A. P., Baston-Buest, D. M., Heiss, C., Kruessel, J. S. 2014; 35 (12): 1089-1094

  Abstract

  Placental development involves the variation of oxygen supply due to vascular changes and cytotrophoblast invasion. Chemokines and their receptors play an important role during placental formation. Herein, the analysis of the chemokine/receptor pair CXCL12/CXCR4 and further chemokine receptors, such as CCR1, CCR7 and CXCR6 expression in human cytotrophoblasts was conducted.Human cytotrophoblasts were examined directly after isolation or after incubation with different oxygen tensions and a chemical HIF-stimulator for 12 h with realtime PCR, immunoblot, immunohistochemistry. Conditioned media of placental villi, decidua, and endothelial cells was used for ELISA analysis of CXL12. Cytotrophoblast migration assays were conducted applying conditioned media of endothelial cells, a CXCL12 gradient, and different oxygen level. Endometrial and decidual tissue was stained for CXCL12 expression.An upregulation of CXCL12, CXCR4, CCR1, CCR7 and CXCR6 was observed after cytotrophoblast differentiation. Low oxygen supply upregulated CXCR4, CCR7 and CXCR6, but downregulated CXCL12 and CCR1. In contrast to the HIF associated upregulation of the aforementioned proteins, downregulation of CXCL12 and CCR1 seemed to be HIF independent. Cytotrophoblast migration was stimulated by low oxygen, the application of a CXCL12 gradient and endothelial cell conditioned media. CXCL12 was detected in endometrial vessels, glands and conditioned media of placental and decidual tissue, but not decidual vessels.Taken together, oxygen supply and cytotrophoblast differentiation seem to be regulators of chemokine and receptor expression and function in human cytotrophoblasts. Therefore, this system seems to be involved in placental development, directed cytotrophoblast migration in the decidual compartment and a subsequent sufficient supply of the growing fetus.

  View details for DOI 10.1016/j.placenta.2014.09.012

  View details for Web of Science ID 000346953500019

  View details for PubMedID 25293376

• The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis DEVELOPMENT Chen, H. I., Sharma, B., Akerberg, B. N., Numi, H. J., Kivela, R., Saharinen, P., Aghajanian, H., McKay, A. S., Bogard, P. E., Chang, A. H., Jacobs, A. H., Epstein, J. A., Stankunas, K., Alitalo, K., Red-Horse, K. 2014; 141 (23): 4500-4512

  Abstract

  Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineage-tracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardial-derived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

  View details for DOI 10.1242/dev.113639

  View details for Web of Science ID 000345029600009

  View details for PubMedCentralID PMC4302936

• VEGF-C and aortic cardiomyocytes guide coronary artery stem development JOURNAL OF CLINICAL INVESTIGATION Chen, H. I., Poduri, A., Numi, H., Kivela, R., Saharinen, P., McKay, A. S., Raftrey, B., Churko, J., Tian, X., Zhou, B., Wu, J. C., Alitalo, K., Red-Horse, K. 2014; 124 (11): 4899-4914

  Abstract

  Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.

  View details for DOI 10.1172/JCI77483

  View details for Web of Science ID 000344203300026

• The sinus venosus contributes to the coronary vasculature through VEGF-C stimulated angiogenesis Chen, H., Sharma, B., Nurmi, H. J., Kivela, R., Saharinen, P., McKay, A. S., Chang, A. H., Stankunas, K., Alitalo, K., Red-Horse, K. SPRINGER. 2014: 959

  View details for Web of Science ID 000342428100090

• Diverse functions of Apj during cardiac development Sharma, B., Jacobs, A. H., Red-Horse, K. SPRINGER. 2014: 955

  View details for Web of Science ID 000342428100074

• Aortic cardiomyocytes guide coronary artery stem formation Poduri, A., Red-Horse, K. SPRINGER. 2014: 974

  View details for Web of Science ID 000342428100136

• Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation CIRCULATION RESEARCH Ali, S. R., Ranjbarvaziri, S., Talkhabi, M., Zhao, P., Subat, A., Hojjat, A., Kamran, P., Mueller, A. M., Volz, K. S., Tang, Z., Red-Horse, K., Ardehali, R. 2014; 115 (7): 625-U81

  Abstract

  Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

  View details for DOI 10.1161/CIRCRESAHA.115.303794

  View details for Web of Science ID 000342076700010

• Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation. Circulation research Ali, S. R., Ranjbarvaziri, S., Talkhabi, M., Zhao, P., Subat, A., Hojjat, A., Kamran, P., Müller, A. M., Volz, K. S., Tang, Z., Red-Horse, K., Ardehali, R. 2014; 115 (7): 625-635

  Abstract

  Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

  View details for DOI 10.1161/CIRCRESAHA.115.303794

  View details for PubMedID 25037571

• Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform. Circulation research Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Ebert, A., Diecke, S., Liang, P., Red-Horse, K., Carette, J. E., Wu, S. M., Wu, J. C. 2014; 115 (6): 556-566

  Abstract

  Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection.This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy.hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment.This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

  View details for DOI 10.1161/CIRCRESAHA.115.303810

  View details for PubMedID 25015077

  View details for PubMedCentralID PMC4149868

• Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform. Circulation research Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Ebert, A., Diecke, S., Liang, P., Red-Horse, K., Carette, J. E., Wu, S. M., Wu, J. C. 2014; 115 (6): 556-566

  Abstract

  Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

  View details for DOI 10.1161/CIRCRESAHA.115.303810

  View details for PubMedID 25015077

• Exploring the world of human development and reproduction INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY Red-Horse, K., Drake, P. M., Fisher, S. 2014; 58 (2-4): 87-93

  Abstract

  Susan Fisher has spent her career studying human development, proteomics, and the intersection between the two. When she began studying human placentation, there had been extensive descriptive studies of this fascinating organ that intertwines with the mother's vasculature during pregnancy. Susan can be credited with numerous major findings on the mechanisms that regulate placental cytotrophoblast invasion. These include the discovery that cytotrophoblasts undergo vascular mimicry to insert themselves into uterine arteries, the finding that oxygen tension greatly effects placentation, and identifying how these responses go awry in pregnancy complications such as preeclamsia. Other important work has focused on the effect of post-translational modifications such as glycosylation on bacterial adhesion and reproduction. Susan has also forayed into the world of proteomics to identify cancer biomarkers. Because her work is truly groundbreaking, many of these findings inspire research in other laboratories around the world resulting in numerous follow up papers. Likewise, her mentoring and support inspires young scientists to go on and make their own important discoveries. In this interview, Susan shares what drove her science, how she continued to do important research while balancing other aspects of life, and provides insights for the next generation.

  View details for DOI 10.1387/ijdb.140063kr

  View details for Web of Science ID 000340702100004

  View details for PubMedID 25023674

• Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries CELL RESEARCH Tian, X., Hu, T., Zhang, H., He, L., Huang, X., Liu, Q., Yu, W., He, L., Yang, Z., Zhang, Z., Zhong, T. P., Yang, X., Yang, Z., Yan, Y., Baldini, A., Sun, Y., Lu, J., Schwartz, R. J., Evans, S. M., Gittenberger-de Groot, A. C., Red-Horse, K., Zhou, B. 2013; 23 (9): 1075-1090

  Abstract

  Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.

  View details for DOI 10.1038/cr.2013.83

  View details for Web of Science ID 000323930200007

  View details for PubMedID 23797856

  View details for PubMedCentralID PMC3760626

• The Origin Of Cardiac Fibroblasts During Normal Development And After Injury Ranjbarvaziri, S., Ali, S. R., Talkhabi, M., Subat, A., Zhao, P., Volz, K. S., Red-Horse, K., Ardehali, R. LIPPINCOTT WILLIAMS & WILKINS. 2013

  View details for Web of Science ID 000332063200170

• Aortic specific cardiomyocyte migration patterns coronary artery stem formation Red-Horse, K. SPRINGER. 2013: 249

  View details for Web of Science ID 000312658800038

• Radial Construction of an Arterial Wall DEVELOPMENTAL CELL Greif, D. M., Kumar, M., Lighthouse, J. K., Hum, J., An, A., Ding, L., Red-Horse, K., Espinoza, F. H., Olson, L., Offermanns, S., Krasnow, M. A. 2012; 23 (3): 482-493

  Abstract

  Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure.

  View details for DOI 10.1016/j.devcel.2012.07.009

  View details for Web of Science ID 000308776400007

  View details for PubMedID 22975322

  View details for PubMedCentralID PMC3500096

• Coronary arteries form by developmental reprogramming of venous cells NATURE Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. 2010; 464 (7288): 549-U100

  Abstract

  Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus-the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.

  View details for DOI 10.1038/nature08873

  View details for Web of Science ID 000275974200039

  View details for PubMedID 20336138

  View details for PubMedCentralID PMC2924433

• Lymphatic vessel dynamics in the uterine wall PLACENTA Red-Horse, K. 2008; 29: S55-S59

  Abstract

  During pregnancy, maternal uterine blood vessels undergo dramatic vascular remodeling. However, until now, little was known about whether the lymphatic circulation experiences similar changes and whether these vessels interact with placental cells that invade maternal tissue. Recent studies demonstrate that lymphatic vessels in the uterine wall are highly compartmentalized where their presence is mostly restricted to the deeper layers. In humans, this arrangement changes during pregnancy when extensive lymphangiogenesis occurs at the maternal-fetal interface. Placental cytotrophoblasts stimulate lymphatic growth in vivo and in vitro suggesting that they play a role in triggering pregnancy-induced decidual lymphangiogenesis. These data indicate that lymphatic vessels may have important functions at the implantation site during pregnancy.

  View details for DOI 10.1016/j.placenta.2007.11.011

  View details for Web of Science ID 000254629200012

  View details for PubMedID 18155143

  View details for PubMedCentralID PMC2435487