NADPH Oxidase, Mitochondrial Dysfunction and Diabetic Retinopathy

视网膜病变仍然是糖尿病最可怕的衰弱并发症之一。的发病机制中 糖尿病视网膜病变，超氧化物水平显着升高，线粒体功能障碍，它们的DNA 被破坏，导致超氧化物积累增加的恶性循环。新出现的证据表明 NADPH氧化酶（Nox）也是活性氧（ROS）的潜在来源，对 细胞存活我们的初步数据表明，高血糖激活视网膜中的Rac 1/Nox 2信号轴 和毛细血管细胞的线粒体失调之前，这表明Nox 2激活代表了早期的 糖尿病引起的线粒体功能障碍和细胞凋亡事件。因此，我们的总体假设是， 糖尿病中Nox 2衍生的ROS损伤视网膜线粒体，导致其功能障碍和凋亡 糖尿病视网膜病变的发病机制是什么？ 我们建议通过解决根据第2.1.1条提出的补充问题来系统地检验这一假设。 三个具体目标。第一个目的是研究高血糖激活Nox 2的机制， 视网膜，并将测试假设，在高血糖Rac 1介导的Nox 2激活和ROS 第二代启动线粒体损伤和细胞凋亡。由于视网膜病变的严重程度与 在第二个目的中，研究脂毒性条件促进高脂血症的机制。 将研究糖尿病视网膜病变的发展。我们的工作模型预测脂毒性条件 促进Rac 1介导的Nox 2活化和ROS产生，从而引发线粒体损伤和细胞凋亡。 凋亡第三个目标是确定Nox 2的调节对糖尿病发展的影响。 视网膜病变，并将测试Nox 2的抑制将减弱线粒体损伤的假设， 糖尿病视网膜病变的后续发展。 这些拟议的研究是基于通过多学科产生的令人信服的初步数据， 使用有效的体外和体内模型系统，在两个PI之间进行协作。我们建议利用 Tiam 1/Rac 1/Nox 2信号通路的已知选择性抑制剂;这些研究的数据将 通过使用该途径中关键信号蛋白的失活突变体和siRNA证实了这一点。体外 这些发现将在体内模型（stz诱导的糖尿病大鼠和小鼠，以及Zucker糖尿病脂肪 大鼠），以及来自患有糖尿病视网膜病变的人类供体的视网膜。我们希望展示 Rac-1介导的Nox 2衍生的ROS作为线粒体功能障碍的“启动者”在糖尿病发病机制中的作用 视网膜病变这将揭示新的治疗目标，以防止视网膜病变的早期阶段， 发展，并为患者提供额外的治疗手段，以防止/延缓这种视力威胁 糖尿病并发症。

Retinopathy remains one of the most feared debilitating complications of diabetes. In the pathogenesis of diabetic retinopathy, superoxide levels are significantly elevated, mitochondria are dysfunctional and their DNA is damaged resulting in a vicious cycle of increased superoxide accumulation. Emerging evidence implicates NADPH oxidase (Nox) also as a potential source of reactive oxygen species (ROS) with detrimental effects on cell survival. Our preliminary data show that hyperglycemia activates Rac1/Nox2 signaling axis in the retina and its capillary cells prior to mitochondrial dysregulation, suggesting that Nox2 activation represents an early event in diabetes-induced mitochondrial dysfunction and cell apoptosis. Thus, our overall hypothesis is that Nox2-derived ROS in diabetes damage retinal mitochondria leading to their dysfunction, and apoptosis of capillary cells is accelerated resulting in the development of diabetic retinopathy. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims. The first aim will investigate the mechanism(s) by which hyperglycemia activates Nox2 in the retina, and will test the hypothesis that in hyperglycemia Rac1-mediated Nox2 activation and ROS generation initiate mitochondrial damage and cellular apoptosis. Since the severity of retinopathy is associated directly with hyperlipidemia, in the second aim, the mechanism(s) by which lipotoxic conditions promote the development of diabetic retinopathy will be investigated. Our working model predicts that lipotoxic conditions promote Rac1-mediated Nox2 activation and ROS generation to initiate mitochondrial damage and cellular apoptosis. The third aim will determine the effect of regulation of Nox2 on the development of diabetic retinopathy, and will test the hypothesis that inhibition of Nox2 will attenuate mitochondrial damage and subsequent development of diabetic retinopathy. These proposed studies are based on compelling preliminary data generated via multi-disciplinary collaborative efforts between two PIs using valid in vitro and in vivo model systems. We propose to utilize known selective inhibitors of the Tiam1/Rac1/Nox2 signaling pathways; data from these studies will be confirmed via the use of inactive mutants and siRNAs for key signaling proteins in this pathway. In vitro findings will be further validated in in vivo models (stz-induced diabetic rats and mice, and Zucker diabetic fatty rats), and also in the retina from human donors with diabetic retinopathy. We expect to demonstrate the role of Rac-1-mediated Nox2 derived ROS as the 'initiator' of mitochondrial dysfunction in the pathogenesis of retinopathy. This should reveal novel targets for therapies to prevent retinopathy in the early stages of its development, and offer patients additional therapeutic means to prevent/retard this sight-threatening complication of diabetes.