Glycemic Control and Progression of Diabetic Retinopathy

 描述(申请人提供)：糖尿病患者的视网膜病变在高血糖终止后很长一段时间内仍在发展，在疾病早期强化控制的好处不仅存在于其机构，提示了一种“新陈代谢记忆”现象。糖尿病大鼠重新建立良好的血糖控制未能逆转视网膜氧化应激的增加，线粒体仍然肿胀，线粒体(MtDNA)和转录受损，电子传输链(ETC)继续功能障碍。DNA甲基化是由DNA甲基转移酶(DNMT)促进的一种强大的表观遗传修饰，在调控基因转录方面发挥着重要作用。糖尿病患者视网膜Dnmts被激活，核DNA高甲基化与线粒体DNA复制受损有关。我们的初步数据显示，Dnmt1在视网膜线粒体中增加，线粒体DNA的D-loop区域，即具有必要的转录和复制元件的区域，被高度甲基化。高血糖的逆转并不能阻止dnmt的增加，mtDNA仍处于高甲基化状态。基于这些，我们的总体假设是：由于dnmt的增加，(A)mtDNA高甲基化，其转录减少，(B)在线粒体动态平衡中重要的nDNA编码基因受到损害。停止高血糖并不能逆转DNA高甲基化，线粒体继续受到破坏，导致早期糖尿病视网膜病变抵抗停止。这一假设将得到系统的检验。 通过评估线粒体DNA和核DNA的甲基化，将在三个具体目标中加以解决。目的研究线粒体DNA甲基化在糖尿病视网膜病变发展过程中线粒体持续损伤中的作用，并验证线粒体DNA高甲基化损害其转录等功能障碍；终止高血糖不能逆转高甲基化的假说。由于线粒体动态平衡所需的大部分蛋白质都是由核DNA编码的，Aim 2将研究核DNA甲基化在线粒体持续损伤中的作用，并假设“由于核DNMT增加，线粒体基因组稳定性和结构/功能仍受到损害，进一步加剧了线粒体损伤”。在目标3中，将研究直接抑制DNMT在高血糖逆转后糖尿病视网膜病变抵抗停止中的作用，该假说预测，在高血糖后，在正常血糖期间直接抑制DNMT，将抑制持续的DNA甲基化(mtDNA和nDNA)，并抑制视网膜病变的进展。这些研究是基于使用有效的体外和体内模型产生的令人信服的数据。中心假说将使用siRNAs和药物抑制剂在分离的细胞中进行测试，体外结果将得到验证。 在体内模型中，使用来自大鼠和基因操纵小鼠的视网膜微血管，以及来自患有糖尿病视网膜病变的人类捐赠者的视网膜微血管。我们新的表观遗传学方法有望为糖尿病视网膜病变在高血糖终止后未能停止提供新的见解，并有望证明线粒体DNA和核DNA的DNA甲基化在线粒体动态平衡中的关键作用。这将有助于揭示抑制DNA甲基化和防止线粒体损伤的治疗新靶点，并将为患者提供一个机会，以辅助治疗来补充他们可能最好的血糖控制，以预防/延缓这种威胁视力的糖尿病并发症的进展。

 DESCRIPTION (provided by applicant): Retinopathy continues to develop in diabetic patients long after termination of hyperglycemia, and the benefits of intensive control during early stages of the disease persist beyond its institution, suggesting a `metabolic memory' phenomenon. Re-institution of good glycemic control in diabetic rats fails to reverse increase in retinal oxidativ stress and mitochondria remain swollen with damaged (mtDNA) and transcription, and the electron transport chain (ETC) continues to be dysfunctional. DNA methylation, a robust epigenetic modification facilitated by DNA methyltransferases (Dnmts), plays an important role in regulating gene transcription. Retinal Dnmts are activated in diabetes, and nuclear DNA hypermethylation is implicated in the impaired mtDNA replication. Our preliminary data show that Dnmt1 is increased in the retinal mitochondria and the D-loop region of the mtDNA, the region with essential transcription and replication elements, is hypermethylated. Reversal of hyperglycemia does not prevent increase in Dnmt, and mtDNA remains hypermethylated. Based on these, our overall hypothesis is that `due to increased Dnmt, (a) mtDNA is hypermethylated and its transcription is decreased, and (b) nDNA-encoded genes, important in mitochondria homeostasis, are compromised. Cessation of hyperglycemia does not reverse DNA hypermethylation, and mitochondria continue to be damaged, contributing to the resistance of incipient diabetic retinopathy to arrest'. The hypothesis will be tested methodically by evaluating methylation of both mtDNA and nuclear DNA, and will be addressed in three specific aims. Aim will investigate the role of mtDNA methylation in the continued damage of mitochondria in the progression of diabetic retinopathy, and will test the hypothesis that `hypermethylation of mtDNA impairs its transcription and ETC becomes dysfunctional; termination of hyperglycemia fails to reverse hypermethylation. Since majority of the proteins required for mitochondrial homeostasis are encoded by nuclear DNA, aim 2 will examine the role of nuclear DNA methylation in the continued mitochondrial damage, and the hypothesis is that `due to increased nuclear Dnmt, mitochondrial genomic stability and structure/ function remain compromised, further fueling into the mitochondrial damage'. In aim 3, the effect of direct inhibition of Dnmt in the resistance of diabetic retinopathy to halt after reversal of hyperglycemi will be investigated, and the hypothesis predicts that `direct inhibition of Dnmt during normal glycemia, that has followed hyperglycemia, will inhibit continued DNA methylation (mtDNA and nDNA), and the progression of retinopathy'. These studies are based on compelling data generated using valid in vitro and in vivo models. The central hypothesis will be tested in isolated cells using siRNAs and pharmacological inhibitors, and in vitro findings will be validated in in vivo models using retinal microvessels from rats and genetically manipulated mice and also in retinal microvessels from human donors with diabetic retinopathy. Our novel epigenetic approach is anticipated to yield fresh insights into the failure of diabetic retinopathy to arrest after hyperglycemia is terminated, and is expected to demonstrate a critical role of DNA methylation of both mtDNA and nuclear DNA in mitochondrial homeostasis. This should help reveal novel targets for therapies to inhibit DNA methylation and prevent mitochondrial damage, and will offer patients an opportunity to supplement their best possible glycemic control with adjunct therapies to prevent/retard the progression of this sight-threatening complication of diabetes.