Control of coronary vascular cell fate in development and regeneration.

The coronary vasculature consists of a complex hierarchal network of arteries, veins, and capillaries which collectively function to perfuse the myocardium. However, the pathways controlling the temporally and spatially restricted mechanisms underlying the formation of this vascular network remain poorly understood. In recent years, the increasing use and refinement of transgenic mouse models has played an instrumental role in offering new insights into the cellular origins of the coronary vasculature, as well as identifying a continuum of transitioning cell states preceding the full maturation of the coronary vasculature. Coupled with the emergence of single cell RNA sequencing platforms, these technologies have begun to uncover the key regulatory factors mediating the convergence of distinct cellular origins to ensure the formation of a collectively functional, yet phenotypically diverse, vascular network. Furthermore, improved understanding of the key regulatory factors governing coronary vessel formation in the embryo may provide crucial clues into future therapeutic strategies to reactivate these developmentally functional mechanisms to drive the revascularisation of the ischaemic adult heart.

Extracellular vesicles from differentiated stem cells contain novel proangiogenic miRNAs and induce angiogenic responses at low doses.

Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure in vitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs (miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure in vitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

The Influence of the LINC00961/SPAAR Locus Loss on Murine Development, Myocardial Dynamics, and Cardiac Response to Myocardial Infarction.

Long non-coding RNAs (lncRNAs) have structural and functional roles in development and disease. We have previously shown that the LINC00961/SPAAR (small regulatory polypeptide of amino acid response) locus regulates endothelial cell function, and that both the lncRNA and micropeptide counter-regulate angiogenesis. To assess human cardiac cell SPAAR expression, we mined a publicly available scRNSeq dataset and confirmed LINC00961 locus expression and hypoxic response in a murine endothelial cell line. We investigated post-natal growth and development, basal cardiac function, the cardiac functional response, and tissue-specific response to myocardial infarction. To investigate the influence of the LINC00961/SPAAR locus on longitudinal growth, cardiac function, and response to myocardial infarction, we used a novel CRISPR/Cas9 locus knockout mouse line. Data mining suggested that SPAAR is predominantly expressed in human cardiac endothelial cells and fibroblasts, while murine LINC00961 expression is hypoxia-responsive in mouse endothelial cells. LINC00961-/- mice displayed a sex-specific delay in longitudinal growth and development, smaller left ventricular systolic and diastolic areas and volumes, and greater risk area following myocardial infarction compared with wildtype littermates. These data suggest the LINC00961/SPAAR locus contributes to cardiac endothelial cell and fibroblast function and hypoxic response, growth and development, and basal cardiovascular function in adulthood.

Transcriptional regulators of arterial and venous identity in the developing mammalian embryo.

The complex and hierarchical vascular network of arteries, veins, and capillaries features considerable endothelial heterogeneity, yet the regulatory pathways directing arteriovenous specification, differentiation, and identity are still not fully understood. Recent advances in analysis of endothelial-specific gene-regulatory elements, single-cell RNA sequencing, and cell lineage tracing have both emphasized the importance of transcriptional regulation in this process and shed considerable light on the mechanism and regulation of specification within the endothelium. In this review, we discuss recent advances in our understanding of how endothelial cells acquire arterial and venous identity and the role different transcription factors play in this process.

Multi-species meta-analysis identifies transcriptional signatures associated with cardiac endothelial responses in the ischaemic heart.

AIM: Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration. METHODS AND RESULTS: We carried out a comprehensive meta-analysis of integrated single-cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularization. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1, and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularization. CONCLUSION: We present a high-resolution single-cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development.

Human and murine fibroblast single-cell transcriptomics reveals fibroblast clusters are differentially affected by ageing and serum cholesterol.

AIMS: Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. METHODS AND RESULTS: Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. CONCLUSION: We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.

Mapping the developing human cardiac endothelium at single-cell resolution identifies MECOM as a regulator of arteriovenous gene expression.

AIMS: Coronary vasculature formation is a critical event during cardiac development, essential for heart function throughout perinatal and adult life. However, current understanding of coronary vascular development has largely been derived from transgenic mouse models. The aim of this study was to characterize the transcriptome of the human foetal cardiac endothelium using single-cell RNA sequencing (scRNA-seq) to provide critical new insights into the cellular heterogeneity and transcriptional dynamics that underpin endothelial specification within the vasculature of the developing heart. METHODS AND RESULTS: We acquired scRNA-seq data of over 10 000 foetal cardiac endothelial cells (ECs), revealing divergent EC subtypes including endocardial, capillary, venous, arterial, and lymphatic populations. Gene regulatory network analyses predicted roles for SMAD1 and MECOM in determining the identity of capillary and arterial populations, respectively. Trajectory inference analysis suggested an endocardial contribution to the coronary vasculature and subsequent arterialization of capillary endothelium accompanied by increasing MECOM expression. Comparative analysis of equivalent data from murine cardiac development demonstrated that transcriptional signatures defining endothelial subpopulations are largely conserved between human and mouse. Comprehensive characterization of the transcriptional response to MECOM knockdown in human embryonic stem cell-derived EC (hESC-EC) demonstrated an increase in the expression of non-arterial markers, including those enriched in venous EC. CONCLUSIONS: scRNA-seq of the human foetal cardiac endothelium identified distinct EC populations. A predicted endocardial contribution to the developing coronary vasculature was identified, as well as subsequent arterial specification of capillary EC. Loss of MECOM in hESC-EC increased expression of non-arterial markers, suggesting a role in maintaining arterial EC identity.

Human embryonic stem cell-derived endothelial cell product injection attenuates cardiac remodeling in myocardial infarction.

Background: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium. Aim: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy. Materials and methods: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts. Results: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling. Conclusion: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction.