Deletion of p66 ^shc Gene Protects Against Age-Related Endothelial Dysfunction

Abstract

Background-Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66 shc protein controls cellular responses to oxidative stress. Mice lacking p66 shc (p66 shcϪ/Ϫ ) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. Methods and Results-Aortic rings from young and old p66shcϪ/Ϫ or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O 2 Ϫ ) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shcϪ/Ϫ mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shcϪ/Ϫ mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shcϪ/Ϫ mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O 2 Ϫ production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shcϪ/Ϫ mice. Conclusions-We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66 shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging. Key Words: aging Ⅲ endothelium Ⅲ free radicals Ⅲ nitric oxide Ⅲ genes S hc proteins are adaptor proteins that exist in 3 different isoforms with relative molecular masses of 46, 52, and 66 kDa. P52shc /p46 shc is involved in the transmission of mitogenic signals from tyrosine kinases to Ras. 1 p66 shc has the same modular structure of p52 shc /p46 shc (SH2-CH1-PTB) and contains a unique N-terminal region (CH2); however, it is not involved in Ras regulation but rather functions in the intracellular pathway that converts oxidative signals into apoptosis. Indeed, embryo fibroblasts from mice carrying a targeted mutation of p66 shc (p66 shcϪ/Ϫ ) are more resistant to oxidative stress-induced apoptosis. 2 p66 shcϪ/Ϫ mice have an approximately 30% increase in life span and reduced early atherogenesis after long-term consumption of a high-fat diet, 3 suggesting that p66shc is implicated in aging and in the pathogenesis of aging-associated diseases in mammals. The biochemical function of p66 shc remains, however, largely unknown. Recent reports demonstrated that p66 shc acts as a downstream target of the tumor suppressor p53 and is indispensable to the ability of activated p53 to induce elevation of intra...

Alteration of p66shc is associated with endothelial dysfunction in the abdominal aortic coarctation of rats

“…The pivotal role of p66shc in increasing the intracellular ROS levels is well established [7,9,10]. Especially, the genetic deletion of p66shc prevents the hyperglycemia-induced or age-related endothelial dysfunction and oxidative stress [11,29]. An important question in this study is how the changes in blood pressure affect the impaired endothelium-dependent relaxation of the vascular wall.…”

Role of the life span determinant P66shcA in ethanol-induced liver damage

“…This finding is consistent with previous reports of impaired generation of ROS in p66À/À cell lines 21 and in the vasculature of p66À/À mice. 22,33 It should also be noted that liver malondialdehyde (MDA, an index of lipid peroxidation) content was not different between normal and mutant mice, and was in either case only minimally increased by alcohol (data not shown). This finding indicates that Vitamin E depletion is a more sensitive indicator of alcohol-induced oxidative stress, at least in our experimental setting, than is MDA accumulation, and that presumably, vitamin E effectively prevents lipid peroxidation and formation of MDA in p66 þ / þ mice.…”

Ablation of the gene encoding p66Shc protects mice against AGE-induced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation

“…p66 Shc KO mice showed reduced circulating and tissue ROS levels and ROS-mediated damage in response to diabetes, ageing, hyperlipidaemia, ischaemia and ischaemia-reperfusion, as compared with the corresponding WT mice [34][35][36][37][38]. Likewise, cells from KO mice exhibited lower ROS levels and reduced cell death rate vs cells from WT animals when exposed to hydrogen peroxide or ultraviolet light [31] or conditions mimicking hyperglycaemia and ischaemia [34,37].…”