BackgroundIn diabetic retinopathy, increasing evidence points to a link between the pathogenesis of retinal microangiopathy and the endothelial cell-specific factor roundabout4 (ROBO4). According to earlier research, specificity protein 1 (SP1) enhances the binding to theROBO4promoter, increasing Robo4 expression and hastening the progression of diabetic retinopathy. To determine if this is related to aberrant epigenetic modifications ofROBO4, we examined the methylation level of theROBO4promoter and the corresponding regulatory mechanism during the course of diabetic retinopathy and explored the effect of this mechanism on retinal vascular leakage and neovascularization.MethodsThe methylation level of CpG sites in theROBO4promoter was detected in human retinal endothelial cells (HRECs) cultured under hyperglycemic conditions and retinas from streptozotocin-induced diabetic mice. The effects of hyperglycemia on DNA methyltransferase 1, Tet methylcytosine dioxygenase 2 (TET2), 5-methylcytosine, 5-hydroxymethylcytosine, and the binding of TET2 and SP1 to theROBO4promoter, as well as the expression of ROBO4, zonula occludens 1 (ZO-1) and occludin were examined. Short hairpin RNA was used to suppress the expression of TET2 or ROBO4 and the structural and functional changes in the retinal microvascular system were assessed.ResultsIn HRECs cultured under hyperglycemic conditions, theROBO4promoter methylation level decreased. Hyperglycemia-induced TET2 overexpression caused active demethylation ofROBO4by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, which enhanced the binding of SP1 toROBO4, increased the expression of ROBO4, and decreased the expression of ZO-1 and occludin, leading to the abnormalities in monolayer permeability, migratory ability and angiogenesis of HRECs. The above pathway was also demonstrated in the retinas of diabetic mice, which caused leakage from retinal capillaries and neovascularization. Inhibition of TET2 or ROBO4 expression significantly ameliorated the dysfunction of HRECs and retinal vascular abnormalities.ConclusionsIn diabetes, TET2 can regulate the expression of ROBO4 and its downstream proteins by mediating active demethylation of theROBO4promoter, which accelerates the development of retinal vasculopathy. These findings suggest that TET2-inducedROBO4hypomethylation is a potential therapeutic target, and anti- TET2/ROBO4 therapy is anticipated to emerge as a novel strategy for early intervention and delayed progression of diabetic retinopathy.