Role of Ras in Retinal Cell Death in Diabetes

描述(申请人提供)：视网膜病变是糖尿病最令人衰弱的并发症之一，但导致其发展的机制尚不清楚。在糖尿病视网膜病变的发病机制中，视网膜毛细血管细胞发生凋亡，这是组织病理学发展的先导。拟议的研究旨在了解RAS(一种小分子GTP结合蛋白)在糖尿病视网膜毛细血管细胞死亡中的可能调节作用(S)。为了支持这一点，我们提供了令人兴奋的初步数据，显示在糖尿病患者的视网膜和高糖培养的视网膜内皮细胞中，RAS的蛋白表达和mRNA水平增加，并且RAS功能的特异性抑制剂也可以抑制葡萄糖诱导的毛细血管细胞死亡。这一建议的中心假设是RAS激活在视网膜细胞死亡中发挥关键作用，并最终导致糖尿病视网膜病变的发生。第一个具体目标将确定糖尿病视网膜毛细血管细胞中RAS被激活的机制。葡萄糖激活RAS的机制(S)将通过测量其表达以及在培养的视网膜毛细血管细胞和糖尿病大鼠视网膜中增加RAS上的GTP负荷来确定。RAS激活在葡萄糖诱导的视网膜毛细血管细胞死亡增加中的作用将通过使用特定的RAS功能抑制剂，以及通过涉及显性负性RAS突变体的转染途径来确定。糖尿病视网膜中RAS的激活将与毛细血管细胞死亡进行比较，并将建立与组织病理学发展的时间关系。由于RAS的激活已被证明招募其调节蛋白Raf-1，而RAS/Raf复合体启动了一系列信号级联反应，第二个特定的目的将定义高血糖诱导的视网膜毛细血管细胞死亡的依赖于RAS的细胞信号机制。了解糖尿病视网膜毛细血管细胞死亡的信号转导机制将为视网膜病变的发展提供新的见解，并将有助于阐明未来药物干预的新分子靶点，以阻止/延缓这一威胁视力的糖尿病并发症。

DESCRIPTION (provided by applicant): Retinopathy is one of the most debilitating complications of diabetes, but the mechanisms that lead to its development are poorly understood. In the pathogenesis of diabetic retinopathy, retinal capillary cells undergo apoptosis, which precedes the development of histopathology. The proposed studies are aimed at understanding the putative regulatory role(s) of Ras, a small molecular weight GTP-binding protein, in retinal capillary cell death in diabetes. In support of this, we have provided exciting preliminary data showing that the protein expression and mRNA levels of Ras are increased in the retina in diabetes and in retinal endothelial cells cultured in high glucose medium, and specific inhibitors of Ras function also inhibit glucose-induced capillary cell death. The central hypothesis of this proposal is that Ras activation plays a crucial role in retinal cell death, and ultimately the development of diabetic retinopathy. The first specific aim will determine the mechanism by which Ras is activated in retinal capillary cells in diabetes. The mechanism(s) by which glucose activates Ras will be determined by measuring its expression, and increase in GTP loading onto Ras in isolated retinal capillary cells in culture and in the retina obtained from diabetic rats. The role of Ras activation in glucose-induced increase in retinal capillary cell death will be determined by using specific inhibitors of Ras function, and via the transfection approaches involving the dominant negative Ras mutant. Activation of Ras in the retina in diabetes will be compared with the capillary cell death, and temporal relationship to the development of histopathology will be established. Since Ras activation has been shown to recruit its regulatory protein, Raf-1, and Ras/Raf complexation initiates a signaling cascade, the second specific aim will define the Ras-dependent cellular signaling mechanisms underlying hyperglycemia-induced retinal capillary cell death. Understanding the signal transduction mechanisms involved in retinal capillary cell death in diabetes will provide fresh insight into the development of retinopathy, and will help elucidate novel molecular targets for future pharmacological interventions to halt/retard this sight-threatening complication of diabetes.