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.
Cardiac and limb ischemia caused by diabetic vascular disease is due in part to impaired angiogenesis, the formation of new blood vessels. We propose that angiogenesis is impaired because oxidants and lipids prevent the normal chemical reactions between nitric oxide and the signaling protein, p21ras which controls angiogenesis. Our studies will not only help explain why angiogenesis is impaired in diabetes, but also provide insight into the therapeutic mechanism of HMG CoA reductase inhibitors (statins) and novel strategies to improve diabetic vascular disease.
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