Hyperhomocyst(e)inemia is a risk factor for the development of atherotherombosis, and is believed to exert its adverse vascular effects, in part, through a mechanisms involving oxidative injury. Homocysteine is readily oxidized when added to plasma through auto-oxidative mechanisms that lead to the formation of homo-and heterodisulfides, homocysteine thiolactone, superoxide anion, and hydrogen peroxide. The central hypothesis of this proposal is that pathophysiologically relevant concentrations of homocyst(e)ine impart an oxidative stress to the vascular microenvironment that leads to a state of endothelial dysfunction manifest by reduced production of bioactive nitric oxide. This effect is achieved by the formation of reactive oxygen species that react with and inactive nitric oxide, as well as by the inactivation of enzyme(s) required for the reduction of these byproducts of homocysteine oxidation. This hypothesis will be addressed through three specific aims. Firstly, the applicants will define the molecular mechanism(s) by which pathophysiologically relevant concentrations of homocyst(e)ine induce oxidative stress, and will do so by measuring the production of reactive oxygen species and the reduction in cellular redox state in endothelial cells in culture. In addition, they will measure the activity and expression of cellular glutathione peroxidase, a selenocyteine-containing enzyme that reduces lipid and hydrogen peroxides to their corresponding alcohols. They will next define the consequences of increased concentrations of homocyst(e)ine on endothelial nitric oxide synthesis and metabolism in endothelial cells, focusing specifically on the cellular production of nitrogen oxides, the transcription of Nos 3 and Nos 2 genes, the expression of constitutive and inducible nitric oxide synthase protein and activity, the availability of substrate, and the elaboration of cofactors required for optimal enzyme activity. Lastly, they will apply these cellular studies to an animal model of hyperhomocyst(e)inemia: a murine model in which the cystathionine Beta-synthase gene has been eliminated by targeted gene disruption. In addition, they will crossbreed this animal with a murine model in which cellular glutathione peroxidase gene has been eliminated by targeted gene disruption in an effort to define the role of lipid peroxides in the endothelial dysfunction of hyperhomocyst(e)inemic states. In these animal studies, they will measure plasma and tissue markers of oxidative stress, endothelial vasodilator responses, and platelet markers of nitric oxide action. They will attempt to reverse the oxidant injury of elevated concentrations of homocyst(e)ine in cell culture and of hyperhomocyst(e)-inemia in animal models by the use of selected antioxidants and by crossbreeding the cystathionine Beta-synthase deficient mice with mice that overexpress cellular glutathione peroxidase. With these studies, the investigators hope to gain insight into the molecular mechanisms of homocyst(e)ine-induced endothelial dysfunction and potential therapies for this common metabolic abnormality.
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