Redox Regulation of p21ras in Angiogenesis

描述（由申请人提供）：本实验室的研究已经阐明了血管细胞功能中信号传导的氧化还原调节作用。在其他氧化还原敏感蛋白中，我们发现活性氮物质（RNS），包括7 NO和低浓度的过氧亚硝酸盐（OONO-），使p21 ras半胱氨酸-118（C118）的S-谷胱甘肽化，急剧增加其GTdR活性和MAP-和PI 3-激酶信号传导。组织修复过程中血管生成所需的内皮细胞（EC）的迁移也由7 NO介导，但对介导血管生成重要的7 NO的氧化还原调节蛋白靶点尚不清楚。我们的初步研究表明，野生型（WT）p21 ras的表达，而不是C118丝氨酸（C118 S）突变体诱导EC迁移。此外，谷氧还蛋白-1（GRX 1）的过表达抑制了由VEGF、7 NO和WT p21 ras刺激的EC迁移，表明p21 ras半胱氨酸-118硫醇的S-谷胱甘肽化是必需的。因此，拟议的研究将调查p21 ras的氧化还原调节，支持正常的EC迁移，这是由7 NO和HMG CoA还原酶抑制剂（他汀类药物）刺激。我们的初步研究表明，他汀类药物，通过干扰法尼基化的C-末端半胱氨酸，通常保持p21 ras拴在质膜上，促进p21 ras重新定位到线粒体，从而增加线粒体分裂，ROS/RNS的生产，和p21 ras介导的，氧化还原依赖性血管生成信号。我们还将研究EC暴露于与炎症和代谢性血管疾病相关的氧化剂和反应性脂质是否会破坏正常的p21 ras活性和/或亚细胞定位。这些研究结果在EC的文化将是相关的，我们的研究肥胖小鼠喂养高脂肪，高蔗糖（HFHS）的饮食中，我们发现血管生成受损。我们的初步研究还表明，血管生成减少在缺血后肢GRX 1转基因小鼠，这表明假设，改变GRX 1表达代谢疾病可能会抑制p21 ras和血管生成。通过了解血管生成通常由p21 ras和GRX 1氧化还原调节的分子机制，我们预期能够确定为什么代谢性疾病中血管生成受损。

DESCRIPTION (provided by applicant): Studies in this laboratory have elucidated the role of redox regulation of signaling in vascular cell function. Amongst other redox-sensitive proteins, we found that S-glutathiolation of p21ras cysteine-118 (C118) by reactive nitrogen species (RNS), including 7NO and low concentrations of peroxynitrite (OONO-), acutely increases its GTPase activity and MAP- and PI3-kinase signaling. Migration of endothelial cells (EC) which is required for angiogenesis during tissue repair is also mediated by 7NO, but the redox-regulated protein targets of 7NO that are important for mediating angiogenesis are unknown. Our preliminary studies show that expression of wild type (WT) p21ras, but not a C118 serine (C118S) mutant induces EC migration. Furthermore, overexpression of glutaredoxin-1 (GRX1), inhibits EC migration stimulated by VEGF, 7NO, and WT p21ras, suggesting that S-glutathiolation of the p21ras cysteine-118 thiol is essential. The proposed studies will therefore investigate the redox regulation of p21ras that supports normal EC migration which is stimulated by 7NO and HMG CoA reductase inhibitors (statins). Our preliminary studies suggest that statins, by interfering with farnesylation of the C-terminal cysteine which normally keeps p21ras tethered to the plasma membrane, facilitate relocation of p21ras to mitochondria, thereby increasing mitochondrial fission, ROS/RNS production, and p21ras-mediated, redox-dependent angiogenic signaling. We will also investigate whether exposure of EC to oxidants and reactive lipids associated with inflammation and metabolic vascular disease disrupts normal p21ras activity and/or subcellular localization. Results of these studies in EC in culture will be relevant for our studies in obese mice fed a high fat, high sucrose (HFHS) diet in which we find angiogenesis to be impaired. Our preliminary studies also indicate that angiogenesis is diminished in the ischemic hindlimb of GRX1 transgenic mice, suggesting the hypothesis that altered GRX1 expression in metabolic disease may inhibit p21ras and angiogenesis. By understanding the molecular mechanisms by which angiogenesis is normally redox-regulated by p21ras and GRX1, we anticipate being able to determine why angiogenesis is impaired in metabolic disease.