(Verbatim from the application): Cardiovascular diseases, including atherosclerosis, remain a leading cause of death and disability in the United States. Nitric oxide is a key regulator of vascular tone, platelet aggregation, white blood cell adhesion, and smooth muscle cell proliferation. Nitric oxide has also been recognized as a critical mediator of angiogenesis. Endothelial dysfunction due to decreased production of nitric oxide is an early event believed to play a major role in initiation and progression of atherosclerosis. Tetrahydrobiopterin (BH4) is an essential cofactor needed for enzymatic activity of nitric oxide synthase. BH4 plays a key role in the control of endothelial nitric oxide production. The general hypothesis of this proposal is that during development of atherosclerosis, up-regulation of BH4 biosynthesis is an adaptive response designed to preserve biosynthesis of nitric oxide and protect the vascular wall from oxidative stress. To test this hypothesis we propose studies with the following specific aims: (1) determine the effects of oxidative stress on vascular BH4 metabolism, (2) characterize the relationship between BH4 metabolism and endothelial dysfunction in arteries exposed to hypercholesterolemia in vivo, and (3) analyze the role of superoxide anion in BH4 metabolism and endothelial dysfunction in murine models of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice and low-density lipoprotein (LDL) receptor-deficient mice develop spontaneous hypercholesterolemia and atherosclerosis with many features that are characteristic of lesions in humans. Preliminary findings indicate that endothelial dysfunction, as reflected in impaired endothelium-dependent relaxations, is present in murine models of atherosclerosis. The exact mechanism responsible for endothelial dysfunction induced by hypercholesterolemia is not understood. Initial analysis performed on isolated aorta of ApoE-deficient mice indicated that increased production of superoxide anions in the vascular wall plays a major role in inactivation of endothelial nitric oxide. However, the effects of superoxide anions and oxidative stress on BH4 metabolism have not been studied. To characterize the role of superoxide anion in BH4 metabolism and endothelial dysfunction, arteries from superoxide dismutase (SOD) transgenic mice and SOD-deficient mice will be studied. Overexpression of SOD in ApoE-deficient mice will be used to protect BH4 from superoxide anion-induced oxidative stress and rescue endothelial dysfunction. Double knockout ApoE-SOD-deficient mice will be created to determine whether increased production of superoxide anion accelerates oxidation of BH4 and impairment of endothelium-dependent relaxation. It is anticipated that the results of the proposed experiments will provide novel and important information concerning the effect of oxidative stress on vascular BH4 metabolism and the pathogenesis of atherosclerosis. This information may help to develop new therapeutic interventions designed to prevent endothelial dysfunction and progression of atherosclerosis.
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