Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (·O2 −) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented ·O2 − release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Background-Prostaglandins generated by cyclooxygenase (COX) have been implicated in hyperglycemia-induced endothelial dysfunction. However, the role of individual COX isoenzymes as well as the molecular mechanisms linking oxidative stress and endothelial dysfunction in diabetes remains to be clarified. Methods and Results-Human aortic endothelial cells were exposed to normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively increased mRNA and protein expression of COX-2. Its upregulation was associated with an increase of thromboxane A 2 and a reduction of prostacyclin (PGI 2 ) release. Glucose-induced activation of PKC resulted in the formation of peroxynitrite and tyrosine nitration of PGI 2 synthase. NO release was reduced despite 2-fold increase of endothelial NO synthase expression. Phorbol ester caused an increase of COX-2 and endothelial NO synthase expression similar to that elicited by glucose. These effects were prevented by the PKC inhibitor calphostin C. N-acetylcysteine, vitamin C, and calphostin C prevented ROS formation, restored NO release, and reduced colocalization of nitrotyrosine and PGI 2 synthase. Expression of p22 phox , a subunit of NAD(P)H oxidase, was increased, and diphenyleneiodonium inhibited ROS formation. By contrast, indomethacin did not affect glucose-induced ROS generation. Conclusions-Thus, high glucose, via PKC signaling, induces oxidative stress and upregulation of COX-2, resulting in reduced NO availability and altered prostanoid profile.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

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...

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN-and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations Ͼ1 M. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.Organic nitrates such as nitroglycerin (glyceryl trinitrate; GTN) are widely used in the therapy of cardiovascular diseases such as stable and unstable angina (Abrams, 1995).The anti-ischemic effects of organic nitrates are largely caused by venous and coronary artery dilation as well as the improvement of collateral blood flow, which all decrease myocardial oxygen consumption. Their use, however, is limited because of the rapid development of tolerance and crosstolerance characterized by decreased sensitivity of the vasculature to the organic nitrates and to endothelium-dependent vasodilators, respectively (Mangione and Glasser,

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.

The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting degradation of hypoxia-inducible HIF-alpha transcription factors1, and additional mechanisms have been proposed2. pVHL also stabilizes plant homeodomain (PHD) protein Jade-1, which is a candidate renal tumor suppressor that may correlate with renal cancer risk3-5. We show here that Jade-1 binds the oncoprotein β-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type β-catenin, but not a cancer-causing form of β-catenin. While β-TrCP ubiquitinates only phosphorylated β-catenin6, Jade-1 ubiquitinates both phosphorylated and non-phosphorylated β-catenin and therefore regulates canonical Wnt signaling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of β-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL down-regulates β-catenin in a Jade-1-dependent manner and inhibits Wnt signaling, supporting a role for Jade-1 and Wnt signaling in renal tumorigenesis. The pVHL tumor suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.