NADPH Oxidase, Mitochondrial Dysfunction and Diabetic Retinopathy

描述(申请人提供)：视网膜病变仍然是糖尿病最令人畏惧的衰弱并发症之一。在糖尿病视网膜病变的发病机制中，超氧化物歧化水平显著升高，线粒体功能紊乱，DNA受损，导致超氧阴离子蓄积增加的恶性循环。新的证据表明，NADPH氧化酶(NOx)也是一种潜在的活性氧(ROS)来源，对细胞生存有不利影响。我们的初步数据显示，高血糖在线粒体失调之前激活了视网膜及其毛细血管细胞中的rac1/NOX2信号轴，这表明NOX2的激活是糖尿病诱导的线粒体功能障碍和细胞凋亡的早期事件。因此，我们的总体假设是糖尿病时NOX2衍生的ROS损伤视网膜线粒体，导致线粒体功能障碍，并加速毛细血管细胞的凋亡，从而导致糖尿病视网膜病变的发生。我们建议通过解决在三个特定目标下提出的互补性问题来系统地检验这一假设。第一个目的是研究高血糖激活视网膜中NOX2的机制(S)，并验证在高血糖中，RAC1介导的NOX2激活和ROS的产生启动线粒体损伤和细胞凋亡的假说。由于视网膜病变的严重程度与高脂血症直接相关，在第二个目标中，我们将探讨脂毒性条件促进糖尿病视网膜病变发展的机制(S)。我们的工作模型预测，脂毒条件促进了rac1介导的NOX2的激活和ROS的产生，从而启动线粒体损伤和细胞凋亡。第三个目的将确定NOX2的调节在糖尿病视网膜病变发展中的作用，并将检验抑制NOX2将减轻线粒体损伤和随后的糖尿病视网膜病变发展的假说。这些拟议的研究是基于令人信服的初步数据，这些数据是通过两个PI之间使用有效的体外和体内模型系统进行多学科合作而产生的。我们建议利用已知的Tiam1/rac1/NOX2信号通路的选择性抑制剂；这些研究的数据将通过使用该通路中关键信号蛋白的非活性突变体和siRNAs来证实。体外发现将在体内模型(STZ诱导的糖尿病大鼠和小鼠，以及Zucker糖尿病肥胖大鼠)中进一步验证，也将在患有糖尿病视网膜病变的人类供体的视网膜中得到验证。我们期望证明RAC-1介导的NOX2衍生的ROS作为线粒体功能障碍的“启动者”在视网膜病变的发病机制中的作用。这将揭示在其发展的早期阶段预防视网膜病变的新的治疗目标，并为患者提供更多的治疗手段来预防/延缓这种威胁视力的糖尿病并发症。 公共卫生相关性：糖尿病视网膜病变进展缓慢，是年轻人失明的最常见原因。尽管有尖端研究来探索疾病是如何发展的，但这种损害背后的实际分子和细胞机制仍然难以捉摸。这一建议的重点是通过系统分析特定的信号通路Tiam1-rac1-NOX2来了解导致糖尿病视网膜病变发展的潜在损害机制，Tiam1-rac1-NOX2增加氧化应激并损害线粒体。我们将测试NOX2衍生的ROS在线粒体功能障碍和糖尿病视网膜病变发展中的“启动者”作用。这项应用代表了两个成熟的实验室之间的多学科合作努力，这些实验室积极关注糖尿病视网膜病变发病机制中的G蛋白信号通路和线粒体功能障碍。这项研究的数据有望确定阻止这种失明进展的新药物靶点。 人类糖尿病中的疾病。

DESCRIPTION (provided by applicant): 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 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. PUBLIC HEALTH RELEVANCE: Diabetic retinopathy, a slow progressing, is the most frequent cause of blindness among young adults. Despite the cutting edge research to explore how the disease develops, the actual molecular and cellular mechanisms underlying this lesion remain elusive. This proposal is focused on understanding potential damaging mechanisms responsible for the development of diabetic retinopathy through systematic analysis of a specified signaling pathway, Tiam1-Rac1-Nox2, which increases oxidative stress and damages mitochondria. We will test the "initiator" roles for Nox2-derived ROS in mitochondrial dysfunction, and the development of diabetic retinopathy. The application represents a multi-disciplinary collaborative effort between two well-established laboratories actively focused on G-protein signaling pathways and mitochondrial dysfunction in the pathogenesis of diabetic retinopathy. Data from this study are expected to identify novel drug targets for halting the progression of this blinding disease in human diabetes.