PDF(Pubmed)
Abstract：
In 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 the ROBO4 promoter, increasing Robo4 expression and hastening the progression of diabetic retinopathy. To determine if this is related to aberrant epigenetic modifications of ROBO4, we examined the methylation level of the ROBO4 promoter and the corresponding regulatory mechanism during the course of diabetic retinopathy and explored the effect of this mechanism on retinal vascular leakage and neovascularization.
The methylation level of CpG sites in the ROBO4 promoter 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 the ROBO4 promoter, 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.
In HRECs cultured under hyperglycemic conditions, the ROBO4 promoter methylation level decreased. Hyperglycemia-induced TET2 overexpression caused active demethylation of ROBO4 by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, which enhanced the binding of SP1 to ROBO4, 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.
In diabetes, TET2 can regulate the expression of ROBO4 and its downstream proteins by mediating active demethylation of the ROBO4 promoter, which accelerates the development of retinal vasculopathy. These findings suggest that TET2-induced ROBO4 hypomethylation 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.
The methylation level of CpG sites in the ROBO4 promoter 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 the ROBO4 promoter, 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.
In HRECs cultured under hyperglycemic conditions, the ROBO4 promoter methylation level decreased. Hyperglycemia-induced TET2 overexpression caused active demethylation of ROBO4 by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, which enhanced the binding of SP1 to ROBO4, 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.
In diabetes, TET2 can regulate the expression of ROBO4 and its downstream proteins by mediating active demethylation of the ROBO4 promoter, which accelerates the development of retinal vasculopathy. These findings suggest that TET2-induced ROBO4 hypomethylation 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.
摘要：
背景：在糖尿病性视网膜病变中，越来越多的证据表明视网膜微血管病变的发病机制与内皮细胞特异性因子roundabout4(ROBO4)之间存在联系.根据之前的研究,特异性蛋白1（SP1）增强与ROBO4启动子的结合，增加Robo4表达并加速糖尿病视网膜病变的进展。为了确定这是否与ROBO4的异常表观遗传修饰有关，我们检查了糖尿病性视网膜病变过程中ROBO4启动子的甲基化水平和相应的调节机制，并探讨了该机制对视网膜血管渗漏和新生血管形成的影响。
方法：在高血糖条件下培养的人视网膜内皮细胞（HRECs）和链脲佐菌素诱导的糖尿病小鼠的视网膜中检测到ROBO4启动子中CpG位点的甲基化水平。高血糖对DNA甲基转移酶1，Tet甲基胞嘧啶双加氧酶2（TET2），5-甲基胞嘧啶，5-羟甲基胞嘧啶，以及TET2和SP1与ROBO4启动子的结合，以及ROBO4，小带闭塞1（ZO-1）和闭塞蛋白的表达。短发夹RNA用于抑制TET2或ROBO4的表达，并评估视网膜微血管系统的结构和功能变化。
结果：在高血糖条件下培养的HREC中，ROBO4启动子甲基化水平降低。高血糖诱导的TET2过表达通过将5-甲基胞嘧啶氧化为5-羟甲基胞嘧啶引起ROBO4的主动去甲基化,增强了SP1与ROBO4的结合，增加了ROBO4的表达，降低了ZO-1和occludin的表达，导致单层通透性异常，HREC的迁移能力和血管生成。上述途径也在糖尿病小鼠的视网膜中得到证实，导致视网膜毛细血管渗漏和新生血管形成。抑制TET2或ROBO4表达显著改善HREC的功能障碍和视网膜血管异常。
结论：在糖尿病中，TET2可以通过介导ROBO4启动子的主动去甲基化来调控ROBO4及其下游蛋白的表达,加速视网膜血管病变的发展.这些发现表明TET2诱导的ROBO4低甲基化是一个潜在的治疗靶点。抗TET2/ROBO4治疗有望成为糖尿病视网膜病变早期干预和延缓进展的新策略。
方法：在高血糖条件下培养的人视网膜内皮细胞（HRECs）和链脲佐菌素诱导的糖尿病小鼠的视网膜中检测到ROBO4启动子中CpG位点的甲基化水平。高血糖对DNA甲基转移酶1，Tet甲基胞嘧啶双加氧酶2（TET2），5-甲基胞嘧啶，5-羟甲基胞嘧啶，以及TET2和SP1与ROBO4启动子的结合，以及ROBO4，小带闭塞1（ZO-1）和闭塞蛋白的表达。短发夹RNA用于抑制TET2或ROBO4的表达，并评估视网膜微血管系统的结构和功能变化。
结果：在高血糖条件下培养的HREC中，ROBO4启动子甲基化水平降低。高血糖诱导的TET2过表达通过将5-甲基胞嘧啶氧化为5-羟甲基胞嘧啶引起ROBO4的主动去甲基化,增强了SP1与ROBO4的结合，增加了ROBO4的表达，降低了ZO-1和occludin的表达，导致单层通透性异常，HREC的迁移能力和血管生成。上述途径也在糖尿病小鼠的视网膜中得到证实，导致视网膜毛细血管渗漏和新生血管形成。抑制TET2或ROBO4表达显著改善HREC的功能障碍和视网膜血管异常。
结论：在糖尿病中，TET2可以通过介导ROBO4启动子的主动去甲基化来调控ROBO4及其下游蛋白的表达,加速视网膜血管病变的发展.这些发现表明TET2诱导的ROBO4低甲基化是一个潜在的治疗靶点。抗TET2/ROBO4治疗有望成为糖尿病视网膜病变早期干预和延缓进展的新策略。
