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Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine ␤-synthase-deficient (CBS (؊/؉) ) mice and their wild-type littermates (CBS (؉/؉) ) were crossbred with mice that overexpress GPx-1 [GPx-1 (tg؉) mice]. GPx-1 activity was 28% lower in CBS (؊/؉) ͞GPx-1 (tg؊) compared with CBS (؉/؉) ͞GPx-1 (tg؊) mice (P < 0.05), and CBS (؊/؉) and CBS (؉/؉) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS (؊/؉) ͞ GPx-1 (tg؊) mice showed vasoconstriction to superfusion with ␤-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS (؉/؉) ͞GPx-1 (tg؊) and CBS (؉/؉) ͞GPx-1 Mild hyperhomocyst(e)inemia is a risk factor for atherothrombotic vascular disease (reviewed in refs. 1-5). Endothelial dysfunction appears to play a key role in homocyst(e)ine (Hcy)-mediated vascular pathophysiology. Animal models of mild hyperhomocyst(e)inemia manifest impaired endothelium-dependent vasoreactivity and regulation of blood flow, whether induced by vitamin deficiency (6), disruption of the cystathionine ␤-synthase (CBS) gene (7), or both (8). Impaired endothelium-dependent vasodilator function, but preserved endothelium-independent vasodilator response, is also a common finding in humans with either acutely elevated plasma tHcy levels after a methionine challenge (9-12) or with chronic, mild hyperhomocyst(e)inemia (13,14). In accordance with these in vivo findings, homocysteine (HcyH) has been shown to decrease the production and͞or bioactivity of NO and Snitrosothiols by cultured endothelial cells (15,16).One mechanism proposed to explain the adverse effects of Hcy on endothelial function involves oxidant stress with resulting depletion of bioavailable NO (17). HcyH undergoes autooxidation when added to plasma, leading to the formation of reactive oxygen species, including hydrogen peroxide and superoxide anion (18). Superoxide anion can react with NO to form peroxynitrite, which leads to inactivation of its biological function (19). Hydrogen peroxide decomposes to the toxic oxygen species hydroxyl radical, which is highly reactive and causes lipid peroxidation. Elevated levels of lipid peroxides lead to an increase in peroxyl radicals that can, as well, inactivate NO through the formation of lipid peroxynitrites. Homocysteineinduced vascular oxidant stress may be additionally aggravated by an Hcy-mediated, specific decrease in the expression of the cellular isoform of glutathione peroxidase (GPx-1), as recently shown in vitro and in vivo (15, 20, §). This key enzyme for the cellular defense against oxidant stress uses glutathione to reduce hydro...

Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1 Ϫ/Ϫ ). Mesenteric arterioles of GPx-1 Ϫ/Ϫ mice demonstrated paradoxical vasoconstriction to ␤-methacholine and bradykinin, whereas wildtype (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1 Ϫ/Ϫ mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1 Ϫ/Ϫ mice. We observed an increase of the isoprostane iPF 2␣-III, a marker of oxidant stress, in the plasma and aortas of GPx-1 Ϫ/Ϫ mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1 Ϫ/Ϫ mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools. nitric oxide; peroxynitrite; oxidant stress NITRIC OXIDE (NO) synthesized by the endothelium contributes to vascular tone (23), preserves endothelial integrity (13), inhibits smooth muscle cell migration and proliferation (16), and acts as an antioxidant (30). An increase in reactive oxygen species (ROS), leading to increased oxidant stress in the vasculature, promotes endothelial dysfunction (28) associated with NO insufficiency (15).The enzyme glutathione peroxidase (GPx) is a selenocysteine-containing protein that serves an important role in the cellular defense against oxidant stress (24) by utilizing reduced glutathione (GSH) to reduce hydrogen peroxide (H 2 O 2 ) and lipid peroxides to their corresponding alcohols (37). GPx exists in several isoforms, and the most abundant intracellular isoform is cellular GPx, or GPx-1. We (7, 36, 39) have previously shown that elevated homocysteine concentrations suppress GPx-1 expression in endothelial cells in vitro and in mildly hyperhomocysteinemic mice in vivo and suggested that this effect may account, in part, for the vascular oxidant stress of hyperhomocysteinemic states. Hydrogen peroxide forms the toxic oxygen species hydroxyl radical (⅐OH), which is highly reactive and causes lipid peroxidation, and hydroxide anion (OHϪ), which promotes alkaline tissue damage, a process that is offset in part by catalase and GPx-1-dependent reduction to H 2 O. Elevated levels of lipid peroxides are accompanied by an increase in peroxyl radicals, which can inactivate NO through the formation of lipid peroxynitrites (19,30), although the prec...

Abstract-Previous in vitro experiments have shown that hyperhomocysteinemia leads to oxidative inactivation of nitric oxide, in part by inhibiting the expression of cellular glutathione peroxidase (GPx-1). To elucidate the role of intracellular redox status on homocysteine-induced endothelial dysfunction and oxidant stress, heterozygous cystathionine ␤-synthase-deficient (CBS -/ϩ ) and wild-type (CBS ϩ/ϩ ) mice were treated with the cysteine donor L-2-oxothiazolidine-4-carboxylic acid (OTC). CBS -/ϩ mice had significantly lower GPx-1 activity compared with their CBS ϩ/ϩ littermates, and OTC treatment led to a modest increase in tissue GPx-1 activity and significant increases in total thiols and in reduced glutathione levels in both CBS ϩ/ϩ and CBS -/ϩ mice. Superfusion of the mesentery with ␤-methacholine or bradykinin produced dose-dependent vasodilation of mesenteric arterioles in CBS ϩ/ϩ mice and in CBS ϩ/ϩ mice treated with OTC. In contrast, mesenteric arterioles from CBS -/ϩ mice manifested dose-dependent vasoconstriction in response to both agonists. OTC treatment of CBS -/ϩ mice restored normal microvascular vasodilator reactivity to ␤-methacholine and bradykinin. These findings demonstrate that mild hyperhomocysteinemia leads to endothelial dysfunction in association with decreased bioavailable nitric oxide. Increasing the cellular thiol and reduced glutathione pools and increasing GPx-1 activity restores endothelial function. These findings emphasize the importance of intracellular redox balance for nitric oxide bioactivity and endothelial function. Key Words: homocysteine Ⅲ endothelial function Ⅲ oxidant stress Ⅲ nitric oxide Ⅲ P-selectin M ild hyperhomocysteinemia, ie, an elevation of the plasma levels of homocysteine, homocystine, or homocysteine-mixed disulfides, has been shown to be a risk factor for atherosclerotic vascular disease and its thrombotic complications. Population-based, nested case-control studies demonstrate that a 5 mol/L increase in plasma homocysteine concentrations leads to a 30% increase in cardiovascular risk (reviewed in the first 5 references). [1][2][3][4][5] In addition, several prospective studies indicate that the increased risk is higher during short-term follow-up and declines after 3 to 4 years. Hyperhomocysteinemia is an even stronger predictor of cardiovascular risk in patients with preexisting conditions, such as chronic renal failure, 6 coronary heart disease, 7 diabetes mellitus, 8 peripheral arterial occlusive disease, 9 systemic lupus erythematosus, 10 or venous thromboembolism. 11 In accordance with these observations, elevated homocysteine levels are more strongly associated with recurrent cardiovascular events than with first stroke or myocardial infarction. 12 These data suggest that homocysteine promotes acute thrombotic events in the presence of preexisting vascular lesions rather than induces atherosclerotic lesions de novo.The mechanisms by which elevated homocysteine levels alter the vascular environment to facilitate the development and progression of ...

Lysophosphatidic acid (LPA) is the smallest and structurally simplest of all the glycerophospholipids. It occurs normally in serum and binds with high affinity to albumin, while retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. We show a novel role for LPA: as a macrophage survival factor with potency equivalent to serum. Administration of LPA protects macrophages from apoptosis induced by serum deprivation, and protection is equivalent to that with conventional survival factors such as macrophage colony stimulating factor. The ability of LPA to act as a survival factor is mediated by the lipid kinase phosphatidylinositol 3-kinase (PI3K), since LPA activated both the p85-p110 and p110 ␥ isoforms of PI3K and macrophage survival was blocked completely by wortmannin or LY294002, two mechanistically dissimilar inhibitors of PI3K.