Effect of Simvastatin on endothelial cells (iEC ATAC-seq)

Statins, a class of hydroxy-methylglutaryl-coenzyme A reductase inhibitors that repress the mevalonate pathway, have been increasingly recognized to reduce cardiovascular risks in a pleiotropic manner independent of their lipid-lowering effects. Yet, the precise molecular mechanisms underlying their cardiovascular protection effects remain elusive. As an unlimited alternate source of human primary cells, we sought to use human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) to tackle this question. We treated iPSC-ECs with or without statins in both baseline and diabetic conditions, and evaluated their biological functions specifically at the transcriptional and epigenetic levels using an array of state-of-the-art technologies, such as transcriptome profiling, chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), CRISPR knockout and CRISPR interference (CRISPRi). Furthermore, we validated our in vitro findings of the endothelial protective role of statin using a diabetic mouse model. We observed that, compared to vehicles, statins significantly improved endothelial functions in both baseline and diabetic conditions in iPSC-ECs. Mechanistically, statins could reduce chromatin accessibility at TEAD elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Interestingly, inhibition of geranylgeranyltransferase I (GGTase I), a mevalonate pathway intermediate, was able to block YAP nuclear translocation and thereby YAP activity by suppressing RhoA signaling. We also identified a direct target of statin-YAP signaling, a novel enhancer of SOX9 gene, which plays a critical role in EndMT process. Based on these observations, we further confirmed that inhibition of any component of the GGTase-RhoA-YAP-SOX9 signaling axis using either genetic or pharmacological approaches was effective to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions.