BACKGROUND:Hyperinsulinemia-induced inflammatory responses are a key pathological basis for diabetic proliferative vascular lesions. However, DOT1L impact on vascular repair following diabetic injury and the underlying mechanism remain unclear.
METHODS:Recombinant lentiviral vectors were constructed to target the upregulation or downregulation of DOT1L expression. Carotid artery balloon injury (BI) model was established in diabetic rats. In vitro experiments, an insulin (INS)-stimulated vascular smooth muscle cell (VSMC) model was used. Inflammatory factor levels, vascular intimal hyperplasia and hemodynamics, H3K79me enrichment in promoter regions were detected. ChIP-Seq was used to evaluate the distribution of proteins and genes, and the levels of proteins implicated in related pathways were analyzed.
RESULTS:We found that both in diabetic rat carotid artery tissues 28 days post-BI and in VSMCs after 12 h of insulin stimulation, DOT1L, H3K79me1, IL-6 and TNF-α levels were markedly increased. Overexpression of DOT1L enhanced the expression and release of IL-6 and TNF-α in insulin-induced VSMCs, increased the enrichment of H3K79me1 at the Acp5 gene promoter by 3.92-fold, promoted ACP5 expression, inhibited β-catenin phosphorylation, and upregulated NLRP3 levels. Conversely, downregulation of DOT1L had the opposite effects. In arteries overexpressing DOT1L, inflammatory factor expression and release were markedly enhanced, accompanied by triggering of the ACP5/β-catenin/NLRP3 signaling pathway, roughened intimal surfaces, reduced lumen diameters, decreased residual blood flow area, and increased diameter stenosis rate; greater intimal thickness, and a higher intima/media ratio. In contrast, downregulation of DOT1L exhibited opposite effects.
CONCLUSION:DOT1L aggravates the inflammatory response following diabetic vascular injury by transcriptionally activating Acp5 through H3K79me1, inhibiting β-catenin phosphorylation and inactivation, and upregulating NLRP3 expression.
