Blockade of endothelial to mesenchymal transition (EndMT) by an inhibitor of histone methyltransferase EZH2 attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related deaths in individuals with diabetes. Persistent endothelial cell activation caused by factors such as hyperglycaemia induces endothelial to mesenchymal transition (EndMT), which promotes both initiation as well as progression of atherosclerosis. Gene expression changes that occur during EndMT, are regulated by multiple factors including epigenetic modifiers such as the histone methyltransferase EZH2 (Enhancer of zest homolog 2). Recent studies have implicated the role of EndMT in cardiovascular complications of diabetes including atherosclerosis. However, the role of EZH2 in induction of EndMT in diabetes associated atherosclerosis is not known. Methods Aortae of atheroprone diabetic Apoe-/- mice were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced EndMT was established in human aortic endothelial cells (HAECs) using high glucose (25mM) and TNF-α containing media for 72 hrs ± a small molecule inhibitor of EZH2, GSK126. RNA and Chromatin Immunoprecipitation (ChIP) sequencing was performed to identify EZH2-mediated epigenomic and corresponding transcriptomic changes in this setting. Complementary in vitro EZH2 knockdown experiments using shRNA were also performed. Moreover, Apoe-/- mice made diabetic with streptozotocin, were treated with GSK126 (50 mg/kg BW daily for 5 weeks) in an intervention study design. Aortic sections from treated and control mice were subjected to immunofluorescent staining specific for the EndMT pathway. Results scRNA-seq analysis identified an EndMT+ve endothelial cell sub-population with a diabetes specific proatherogenic transcriptomic profile. Comparison with the ENCODE dataset for EZH2 target genes using Harmonizome showed that indeed multiple repressed genes in diabetes were targets of EZH2. Methyltransferase activity of EZH2 was elevated in in vitro settings of EndMT. Interestingly, EZH2 inhibition by GSK126 attenuated EndMT. Gene expression analysis showed that GSK126 treatment rescued 76 of 242 differentially expressed genes in HAECs exposed to EndMT conditions. Several differentially expressed genes including COL1A2, MMP2, NOS3, COL4A1, and TGFB2 (FDR <0.05, Log2 fold change (2x)) were targets of EZH2 validating integrated analysis of our scRNA-seq with the ENCODE dataset. EZH2 knockdown experiments validated experimental findings of EZH2 inhibition studies using GSK126 in HAECs. Importantly, GSK126 treatment blocked EndMT resulting in reduced atherosclerosis in diabetic Apoe-/- mice in intervention studies. Conclusion This study showed that the inhibition of EZH2 with GSK126 is a potential vasculoprotective treatment for the diabetes induced EndMT and associated atherosclerosis.