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

ABSTRACT: 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 characterise the transcriptome of the human fetal 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 fetal cardiac endothelial cells (EC), 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 arterialisation 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. Furthermore, we revealed that knockdown of MECOM in human embryonic stem cell-derived EC (hESC-EC) resulted in an increase in venous EC marker expression, validating our prediction of its role in arterial EC identity. Conclusions: scRNA-seq of the human fetal 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 venous EC marker expression, suggesting a role in maintaining arterial EC identity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE195911 | GEO | 2022/03/02

REPOSITORIES: GEO

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Project description:Endothelial cells (EC) lining arteries and veins have distinct molecular and functional signatures. The (epi)genetic regulatory mechanisms underlying this heterogeneity in human EC are incompletely understood. Using genome-wide microarray screening we established a specific fingerprint of freshly isolated arterial (HUAEC) and venous EC (HUVEC) from human umbilical cord comprising 64 arterial and 12 venous genes, representing distinct functions and pathways. Among the arterial genes were 8 transcription factors, including HEY2, a downstream target of Notch signaling and the current ‘golden standard’ pathway for arterial EC specification. Short-term culture of HUAEC or HUVEC abrogated differential gene expression resulting in a default state. Erasure of arterial gene expression was at least in part due to loss of canonical Notch activity and HEY2 expression. Notably, nCounter analysis revealed that restoring HEY2 expression or Delta-like 4 (Dll4)-induced Notch signaling in cultured HUVEC or HUAEC only partially reinstated the arterial EC gene signature while combined overexpression of the 8 transcription factors restored this fingerprint much more robustly. Each transcription factor had a different impact on gene regulation, with some stimulating only few and others boosting a large proportion of arterial genes. Interestingly, although there was some overlap and cross-regulation, the transcription factors largely complemented each other in regulating the arterial EC gene profile. Thus, our study showed that Notch signaling determines only part of the arterial EC signature and identified additional novel and complementary transcriptional players in the complex regulation of human arteriovenous EC identity To identify an arteriovenous (AV) fingerprint in human endothelial cells (EC) across different vascular beds, we used microarrays on RNA from 38 EC samples corresponding to 6 cultured human arterial-EC types (hepatic artery EC or HHAEC, N=3; aorta EC or HAEC, N=2; coronary artery EC or HCAEC, N=2; iliac artery EC or HIAEC, N=2; pulmonary artery EC or HPAEC, N=3; and umbilical artery EC or HUAEC-C, N=5), 4 cultured human venous-EC types (hepatic vein EC or HHVEC, N=3; iliac vein EC or HIVEC, N=3; pulmonary vein EC or HPVEC, N=2; and umbilical vein EC or HUVEC-C, N=5), freshly isolated HUAEC (HUAEC-F, N=4) and freshly isolated HUVEC (HUVEC-F, N=4). Due to the difficulty to obtain biopsies from healthy donors, we did not have access to freshly isolated aEC or vEC matched for all cultured EC types.

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

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