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

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. Comprehensive characterisation 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 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 expression of non-arterial markers, suggesting a role in maintaining arterial EC identity. Translational Perspective Endogenous blood vessel formation in the adult heart following myocardial infarction is insufficient to support adequate survival of the remaining myocardium, often ultimately leading to heart failure. Improved understanding of the mechanisms regulating human coronary vessel formation is required to inform therapeutic strategies to reactivate developmental pathways promoting therapeutic angiogenesis in patients. We applied scRNA-seq to map the transcriptome of the endothelium of the developing human heart. We identified novel transcriptional signatures underlying the cellular heterogeneity and dynamic changes occurring within the developing cardiac endothelium. This included identifying and validating MECOM as a novel regulator of arterial EC identity which may serve as a target for therapeutic neovascularization.

Vascular endothelial cell development and diversity

“…Techniques such as reference mapping, as well as comparisons of mouse and human scRNA-seq datasets through robust integration, provide information about the transcriptional similarities and differences between species. These analyses of developing mouse and human hearts revealed that the developmental environment and endothelial cell subtype transcriptional states are similar in both species 66 , 129 , validating the use of mice as a model for many aspects of human coronary development (Fig. 2a ).…”

“…Follow-up functional experiments used these data in an induced pluripotent stem cell culture model to determine that the histone–lysine N -methyltransferase MECOM is crucial for human artery endothelial cell differentiation 129 . Furthermore, trajectory analyses of human data predicted a transitory cell population from the endocardium 129 , which had a gene expression pattern validated using lineage tracing that overlapped with that of the transitory cell population in mice 123 . Therefore, despite the dearth of lineage-tracing techniques for human studies, overlapping human and mouse datasets provide some evidence that coronary vessels from humans also arise from the endocardium.…”

Dynamic chromatin organization and regulatory interactions in human endothelial cell differentiation

“…Several genes with well-established roles in endothelial cell biology associate with PPIs as their transcription increases in development. A prominent example is MECOM (MDS1 and EVI1 complex locus), a transcription factor that promotes arterial EC identity 50 , MECOM is expressed in EP and EC (Figure 4F), and comes to overlap increasing numbers of PPIs in the course of differentiation (Figure 4G). Additional examples include VEGFC (vascular endothelial growth factor C; Supplemental Figure 5C, D), which codes for a protein critical for angiogenesis, endothelial cell growth, and blood vessel permeability in vascular and lymphatic vessels 51,52 ; KDR (kinase insert domain receptor; Supplemental Figure 5E, F), which encodes a VEGF receptor 53 ; and TFPI (tissue factor pathway inhibitor; Supplemental Figure 5G, H), a gene that encodes a serine protease inhibitor with anti-coagulative effects 54–56 .…”

Identification of biomarkers and therapeutic targets in the peripheral immune landscape from coronary artery disease

“…4A). Since the immune status of patients with CAD would change signi cantly, resulting in changes in the proportion of corresponding immune cell subsets, identifying changes in the classi cation proportion of cell subsets was also a potential diagnostic basis [25]. To this end, we examined the proportion of cell subsets in these chips and found seven kinds of cell groups different between CAD and healthy people.…”