Role of Ras in Retinal Cell Death in Diabetes

描述（由申请人提供）：视网膜病变是糖尿病最常见和最可怕的并发症之一。虽然高血糖被认为是其发展的主要原因，但确切的机制尚不清楚。本建议的总体目的是阐明糖尿病视网膜病变发病机制的新见解。在之前的资助期间，我们已经证明了小分子量G蛋白H-Ras在视网膜毛细血管细胞加速凋亡中的重要和新颖作用。据报道，H-Ras上调基质金属蛋白酶-9 (MMP-9)，这是金属蛋白酶家族的一员，调节主要的生物功能，包括细胞凋亡，炎症和基质降解。我们的中心假设是“激活H-Ras级联激活MMP-9，加速视网膜毛细血管细胞的凋亡，最终导致糖尿病视网膜病变的发展”。我们激动人心的初步数据表明，糖尿病患者视网膜中的MMP-9被激活。H-Ras功能抑制剂可阻止葡萄糖诱导的视网膜毛细血管细胞中MMP-9的激活，而MMP-9的特异性抑制剂可改善促炎介质和细胞凋亡的增加。目的1将确定MMP-9在糖尿病视网膜微血管中激活的机制。通过分离的视网膜毛细血管细胞和糖尿病视网膜病变动物模型，我们将验证MMP-9在糖尿病中通过H-Ras介导的信号级联，Raf-1-MAPK-ERK-NF-kB被激活的假设。目的2将研究活化的MMP-9导致糖尿病视网膜病变的机制；该假说预测MMP-9的激活(a)破坏线粒体，引起视网膜微血管细胞凋亡，(b)增加炎症和通透性，导致视网膜病变的发生。这一假设将在体外和体内系统中通过药理学或遗传学操作MMP-9基因进行验证。研究表明，在良好的血糖控制被重新建立后，糖尿病视网膜病变继续发展一段时间，提示代谢记忆现象。在目标3中，我们将使用大鼠模型验证假设，即“由于H-Ras在高血糖逆转后持续激活，MMP-9在炎症介质升高和毛细血管细胞持续凋亡的情况下保持激活。”我们的初步数据显示，糖尿病大鼠在恢复正常血糖后，视网膜H-Ras激活停止。通过表征MMP-9在视网膜毛细血管细胞死亡中的作用来了解糖尿病视网膜病变发病机制的信号转导机制，将为未来的药物干预提供新的分子靶点，以抑制这种威胁视力的糖尿病并发症。这将有助于为未来的药理学干预设计特定的治疗方法（例如MMP-9和反义抑制剂），患者可以使用这些药物来补充他们的最佳血糖控制，以治疗这种威胁视力的糖尿病并发症。

DESCRIPTION (provided by applicant): Retinopathy is one of the most prevalent and feared complications of diabetes. Although hyperglycemia is considered as the major cause of its development, the exact mechanism remains unclear. The overall aim of this proposal is to elucidate new insight into the pathogenesis of diabetic retinopathy. During the previous funding period we have demonstrated an important and novel role for a small molecular weight G- protein, H-Ras, in the accelerated apoptosis of retinal capillary cells. H-Ras is reported to upregulate matrix metalloproteinases-9 (MMP-9), a member of the family of metalloproteinases that regulate major biological functions, including apoptosis, inflammation and matrix degradation. Our central hypothesis is that 'activation of H-Ras cascade activates MMP-9, accelerating apoptosis of retinal capillary cells, and this ultimately leads to the development of retinopathy in diabetes'. In support, our exciting preliminary data show that MMP-9 is activated in the retina in diabetes. Inhibitors of H-Ras function prevent glucose-induced activation of MMP-9 in retinal capillary cells, and specific inhibitors of MMP-9 ameliorate increases in proinflammatory mediators and apoptosis. Aim 1 will determine the mechanism by which MMP-9 is activated in retinal microvasculature in diabetes. Using isolated retinal capillary cells and animal models of diabetic retinopathy, we will test the hypothesis that MMP-9 is activated in diabetes via H-Ras mediated signaling cascade, Raf-1-MAPK-ERK-NF-kB. Aim 2 will investigate the mechanism by which activated MMP-9 results in the development of diabetic retinopathy; and the hypothesis predicts that the activation of MMP-9 (a) damages mitochondria provoking apoptosis of retinal microvascular cells, and (b) increases inflammation and permeability, resulting in the development of retinopathy. This hypothesis will be tested in both in vitro and in vivo systems by pharmacologically or genetically manipulating MMP-9 gene. Studies have shown that diabetic retinopathy continues to progress for sometime after good glycemic control is re-instituted, suggesting a metabolic memory phenomenon. In aim 3, using a rat model, we will test the hypothesis that 'due to the continued activation of H-Ras after reversal of hyperglycemia, MMP-9 remains activated with elevated inflammatory mediators and continued capillary cell apoptosis. This is supported by our preliminary data showing the failure of retinal H-Ras activation to halt after re-establishment of normal glucose in diabetic rats. Understanding the signal transduction mechanisms responsible for the pathogenesis of diabetic retinopathy by characterizing the role of MMP-9 in the retina capillary cell death will provide fresh insight into the novel molecular targets for future pharmacological interventions to inhibit this sight-threatening complication of diabetes. This should help design specific therapeutics (e.g. inhibitors of MMP-9 and antisense) for future pharmacological interventions which patients can use to supplement their best possible glycemic control to treat this sight-threatening complication of diabetes. PUBLIC HEALTH RELEVANCE: This proposal is aimed at understanding the putative regulatory role(s) of matrix metalloproteinases in glucose-induced alterations in metabolic functions of retinal capillary cells leading to their demise, and the development of diabetic retinopathy. The results are expected to elucidate new insight into the pathogenesis of diabetic retinopathy, and reveal novel targets for therapies to prevent the development and progression of retinopathy.