Replacement of the aortic valve is the primary treatment for patients with symptomatic, calcific aortic valve stenosis (AVS), and is the most common valvular surgical procedure performed worldwide. Stenotic valves are histologically similar to atherosclerotic lesions, and the applicant's laboratory has shown significant ncreases in superoxide in atherosclerotic lesions and stenotic valves in hypercholesterolemic mice {Idlr-/-/ApoBIOO/100). However, while superoxide in atherosclerotic lesions is increased due to increased NAD(P)H oxidase activity, the applicant's preliminary data suggest that mechanisms contributing to increased superoxide in stenotic valves are fundamentally different, and are likely due to reductions in superoxide dismutase (SOD) enzyme activity and uncoupling of nitric oxide synthase. We will use echocardiographic and magnetic resonance imaging methods to evaluate the changes in aortic valve function in hypercholesterolemic mice over time, and confocal microscopy and laser capture microdissection to examine molecular mechanisms underlying these changes. We propose two main goals: 1) to examine the effects of mitochondria-derived oxidative stress on the progression of AVS using hyperlipidemic MnSOD-deficient and hyperlipidemic MnSOD-overexpressing mice. 2) to determine the role of nitric oxide synthase (NOS) cofactor depletion and the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) on oxidative stress in normal and stenotic human valves ex vivo. We will also examine the role of DDAH1 (an enzyme involved in ADMA degradation) in the progression of AVS using hyperlipidemic DDAH1-deficient and hyperlipidemic DDAH1-overexpressing mice. Collectively, these studies will lend novel insights into the pathophysiology of AVS as well as the effects of treatment of hypercholesterolemia, and thus will lead to new approaches to prevent and treat AVS.