Eph-ephrin signaling couples endothelial cell sorting and arterial specification.

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.

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

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.

CXCL12 regulates coronary artery dominance in diverse populations and links development to disease.

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.

Sox7-positive endothelial progenitors establish coronary arteries and govern ventricular compaction.

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.

Dedifferentiation and Proliferation of Artery Endothelial Cells Drive Coronary Collateral Development in Mice.

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.

APJ+ cells in the SHF contribute to the cells of aorta and pulmonary trunk through APJ signaling.

Outflow tract (OFT) develops from cardiac progenitor cells in the second heart field (SHF) domain. APJ, a G-Protein Coupled Receptor, is expressed by cardiac progenitors in the SHF. By lineage tracing APJ+SHF cells, we show that these cardiac progenitors contribute to the cells of OFT, which eventually give rise to aorta and pulmonary trunk/artery upon its morphogenesis. Furthermore, we show that early APJ â€‹+ â€‹cells give rise to both aorta and pulmonary cells but late APJ â€‹+ â€‹cells predominantly give rise to pulmonary cells. APJ is expressed by the outflow tract progenitors in the SHF but its role 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. In addition, we show that ectopic overexpression of WNT in these cells disrupted aorta and pulmonary morphogenesis from OFT. Overall, our study has identified APJ â€‹+ â€‹progenitor population within the SHF that give rise to aorta and pulmonary trunk/artery cells. Furthermore, we show that APJ signaling stimulates proliferation of these cells in the SHF.

Vascular endothelial cell development and diversity.

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.

An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression.

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.

Identification of a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis.

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.

Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4.

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.

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

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.

Targeting calcineurin induces cardiomyocyte proliferation in adult mice.

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.

Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development.

Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells 'remember' their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources-the endocardium (Endo) and sinus venosus (SV)-but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary endothelial cells (ECs) transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well established but still remodeling, capillary ECs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

Dach1 Extends Artery Networks and Protects Against Cardiac Injury.

[Figure: see text].

Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular non-compaction.

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 endothelial cells (ECs) in wildtype and LVNC conditions in Tie2Cre;Ino80fl/fltransgenic 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 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 endothelial cells normally promote myocardial compaction through secreted factors, but that endocardial and endothelial cells can secrete factors that contribute to non-compaction under pathological conditions.

Endothelial ontogeny and the establishment of vascular heterogeneity.

The establishment of distinct cellular identities was pivotal during the evolution of Metazoa, enabling the emergence of an array of specialized tissues with different functions. In most animals including vertebrates, cell specialization occurs in response to a combination of intrinsic (e.g., cellular ontogeny) and extrinsic (e.g., local environment) factors that drive the acquisition of unique characteristics at the single-cell level. The first functional organ system to form in vertebrates is the cardiovascular system, which is lined by a network of endothelial cells whose organ-specific characteristics have long been recognized. Recent genetic analyses at the single-cell level have revealed that heterogeneity exists not only at the organ level but also between neighboring endothelial cells. Thus, how endothelial heterogeneity is established has become a key question in vascular biology. Drawing upon evidence from multiple organ systems, here we will discuss the role that lineage history may play in establishing endothelial heterogeneity.

Enhancing cardiovascular research with whole-organ imaging.

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.

miR-106a-363 cluster in extracellular vesicles promotes endogenous myocardial repair via Notch3 pathway in ischemic heart injury.

Endogenous capability of the post-mitotic human heart holds great promise to restore the injured myocardium. Recent evidence indicates that the extracellular vesicles (EVs) regulate cardiac homeostasis and regeneration. Here, we investigated the molecular mechanism of EVs for self-repair. We isolated EVs from human iPSC-derived cardiomyocytes (iCMs), which were exposed to hypoxic (hEVs) and normoxic conditions (nEVs), and examined their roles in in vitro and in vivo models of cardiac injury. hEV treatment significantly improved the viability of hypoxic iCMs in vitro and cardiac function of severely injured murine myocardium in vivo. Microarray analysis of the EVs revealed significantly enriched expression of the miR-106a-363 cluster (miR cluster) in hEVs vs. nEVs. This miR cluster preserved survival and contractility of hypoxia-injured iCMs and maintained murine left-ventricular (LV) chamber size, improved LV ejection fraction, and reduced myocardial fibrosis of the injured myocardium. RNA-Seq analysis identified Jag1-Notch3-Hes1 as a target intracellular pathway of the miR cluster. Moreover, the study found that the cell cycle activator and cytokinesis genes were significantly up-regulated in the iCMs treated with miR cluster and Notch3 siRNA. Together, these results suggested that the miR cluster in the EVs stimulated cardiomyocyte cell cycle re-entry by repressing Notch3 to induce cell proliferation and augment myocardial self-repair. The miR cluster may represent an effective therapeutic approach for ischemic cardiomyopathy.

New Research Is Shining Light on How Collateral Arteries Form in the Heart: a Future Therapeutic Direction?

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.