Lysine methyltransferase Set7 drives obesity-induced endothelial dysfunction by impairing mRNA splicing expression and eNOS function

Abstract Background Lysine methyltransferase Set7 is a key regulator of vascular damage in cardiometabolic diseases such as atherosclerosis and diabetes. While Set7 is well-known for its role in histone modification, i.e., monomethylation of histone H3 at lysine 4 (H3K4me1), linked to gene activation, its ability to methylate non-histone proteins has emerged as a critical mechanism influencing diverse cellular processes. Purpose To investigate the role of Set7 in obesity-induced endothelial dysfunction and identify its molecular targets. Methods Endothelium-specific Set7 knockout (Setd7EC-KO) and WT mice were fed a high-fat or standard diet for 12 weeks. Body weight and metabolic parameters were measured at study completion. Vascular function was assessed via wire myograph, and ROS production was measured by electron spin resonance (ESR) spectroscopy. Gene and protein expression were measured using RT-PCR and confocal microscopy. In vitro, human aortic endothelial cells (HAECs) were exposed to obesogenic stimuli high glucose (HG), high insulin, and lipid overload. Set7 function was modulated through overexpression, silencing, and pharmacological inhibition with cyproheptadine. Endothelial oxidative stress and inflammation were quantified through RT-PCR, ESR spectroscopy, and monocyte-adhesion assay. Chromatin immunoprecipitation (ChIP) was performed to assess H3K4me1 enrichment on target gene promoters, while LC-MS/MS identified total and Kme1-modified proteins. Site-directed mutagenesis was used to generate an eNOS variant (eNOS-K494I), substituting lysine 494 with isoleucine to prevent Set7-mediated methylation. Results Despite similar body weight and metabolic parameters indicative of hyperglycemia, hyperinsulinemia and dyslipidemia, Setd7EC-KO obese mice exhibited improved endothelial function compared to WT obese mice, along with reduced oxidative stress and inflammation markers. Among all the tested obesogenic stimuli, high glucose (HG) most strongly upregulated Set7 in HAECs, triggering pro-inflammatory and pro-oxidative responses, which were reversed by Set7 silencing or inhibition. Overexpression of an enzymatically inactive Set7 mutant demonstrated that its methyltransferase activity is essential for these pathological changes. Proteomic analysis linked Set7 to mRNA splicing regulation and a pro-inflammatory phenotype in HG-treated HAECs, with Rt-PCR and ChIP confirming H3K4me1-mediated epigenetic control. Additionally, eNOS was identified as a direct Set7 target. Notably, HAECs expressing eNOS-K494I maintained their redox balance even under HG conditions. Conclusions This study uncovers mRNA splicing and eNOS methylation as novel pathways regulated by Set7 in endothelial cells. Dysregulation of these mechanisms in obesity-induced hyperglycemia contributes to endothelial inflammation and oxidative stress. These findings provide promising avenues for mitigating endothelial dysfunction in people living with obesity.