Epitranscriptomic regulation of diabetic EndMT in HAECs: METTL3-dependent m6A modification promotes endothelial dysfunction.

Diabetic endothelial dysfunction involves inflammation and endothelial-to-mesenchymal transition (EndMT), however, the underlying RNA N6-methyladenosine (m6A) methylation-mediated regulatory mechanisms remain poorly defined. This study investigated high glucose (HG)-induced diabetic EndMT-like phenotype via RNA m6A methylome, transcriptome, and molecular characterization in human aortic endothelial cells (HAECs). Cell proliferation, morphology, and migration assays were performed. Stagnant cell proliferation during HG treatment began to recover after HG withdrawal. HG treatment altered cell morphology to a highly polarized spindle-like shape. HG-treated cells had a significantly lower cell migration speed. Global m6A methylation and METTL3 expression levels were significantly elevated in HG group. In MeRIP-seq analysis, we found 143 significantly differential hypermethylated peaks and 185 hypomethylated peaks, mainly enriched in pathways related to cell morphology and differentiation-typical of EndMT. In RNA-seq analysis, we found 148 significantly upregulated and 38 significantly downregulated genes implicated in various relevant pathways, including the PI3K-Akt signaling pathway. EndMT-related hub genes query confirmed its transcriptional commencement under HG, which was confirmed by upregulation of TGF-β1, Snail, Slug, mesenchymal vimentin (VIM) and smooth muscle actin (α-SMA), and downregulation of vascular endothelial cadherin (VE cadherin) and CD31. EndMT-like phenotype was significantly reversed in sh-METTL3 cells under HG treatment. Intersection analysis of methylation peak-associated genes identified IGF2 as the main METTL3-mediated m6A candidate due to its hypermethylated peak status and significantly upregulated mRNA expression under HG, and most importantly, its enrichment in the top-most enriched pathway-the PI3K-Akt pathway. Finally, we found phosphorylation-mediated activation of PI3K-Akt pathway implication in diabetic EndMT. This study identifies METTL3-mediated m6A modification in diabetic EndMT and vascular endothelial dysfunction.