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...

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66 Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66 Shc (p66 Shc؊/؊ ) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66 Shc؊/؊ mouse. p66 Shc؊/؊ and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66 Shc؊/؊ and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endotheliumdependent relaxations, increased peroxynitrite (ONOO ؊ ) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66 Shc؊/؊ diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66 Shc؊/؊ but not in WT mice. We report that p66 Shc؊/؊ mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66 Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.

Abstract-Oxidative stress affects the availability of key-regulators of vascular homeostasis and controls a number of signaling pathways relevant to myocardial and vascular disease. Reactive oxygen species are generated by different intracellular molecular pathways principally located in mitochondria. The notion that mice carrying a targeted mutation of the p66 Shc gene display prolonged lifespan, reduced production of intracellular oxidants, and increased resistance to oxidative stress-induced apoptosis prompted a series of studies aimed at defining the biochemical function of p66Shc and its possible implication in cardiovascular diseases. Indeed, p66ShcϪ/Ϫ mice are protected against vascular, cardiac, and renal impairment attributable to hypercholesterolemia, aging, diabetes, and ischemia/reperfusion. The present review focuses on the biochemical and physiological function of the p66Shc adaptor protein as well as on the mechanisms linking p66Shc -associated generation of free radicals to the pathophysiology of aging and cardiovascular disease. On the whole, the evidence so far reported and here discussed supports the concept that pharmacological modulation of p66

The losartan-unmasked AT2R-vasorelaxation could significantly contribute to the beneficial hemodynamic effects of AT1R blockade. In view of this, our study highlights the importance of the integrated Ang II receptor network, which may help to define further the mechanisms of the well-established vascular protective effects of AT1R blockers.