Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium [RNA-seq]
Regenerative capabilities of the human endothelium are governed by vessel-resident progenitors termed endothelial colony forming cells (ECFCs). Endothelial cells acquire mesenchymal phenotypes during atherogenesis, via a process termed endothelial-to-mesenchymal transition (EndMT). This study aimed to investigate the molecular drivers of EndMT in the progenitor population under an atherogenic model. In the presence of oxidised low-density lipoprotein (oxLDL), cultured human ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Importantly, in vitro oxLDL resulted in a dramatic loss in progenitor self-renewal through single-cell colony formation assay and numbers. RNA sequencing of ECFCs exposed or not to oxLDL validated gene expression changes suggesting EndMT but identified SOX9 as one of the highly differentially expressed genes. ATACseq revealed SOX9 binding sites were associated with regions of dynamic chromosome accessibility      resulting from exposure to oxLDL. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo was reversed by an shRNA knockdown of SOX9 or endothelial specific conditional knockout of Sox9 in murine models respectively. Finally, analysis of single cell RNA sequencing of human atherosclerotic aorta identified endothelial populations with mesenchymal characteristics overexpressing SOX9 when compared to controls. Overall, our findings support that in the atherogenic process, EndMT induced by oxLDL or HFD is driven by SOX9 and strongly affects progenitor function opening new avenues for targeting this process. Overall design: RNA-sequencing of endothelial colony-forming cells (ECFCs; 4 donors per group) treated with oxLDL (25ug/ul)or vehicle for 5 days.