Endothelium-derived nitric oxide (NO) is a critical mediator of diverse functions in the cardiovascular system. During the tenure of this grant, we have developed the concept that eNOS inactivation/activation can be regulated by its subcellular targeting, multi-site phosphorylation events and locale specific protein-protein interactions with several proteins including caveolin-1 (Cav-1). In exciting preliminary experiments, we have further dissected the interaction between eNOS and Cav-1 and have dissociated the binding of Cav-1 to eNOS from its inhibitory function using mutagenesis of the caveolin scaffolding domain (CSD) and have developed a novel cell permeable, eNOS activator peptide and a mutant form of Cav-1 (F92A Cav-1) that promote NO release. Indeed, a cell permeable peptide of the CSD lacking the key amino acids necessary for eNOS inhibition (called AP-Cav 3PM) prevents the binding of eNOS to Cav-1, but does not inhibit eNOS function compared to the native CSD. AP-Cav 3PM promotes NO release from cells and dose dependently lowers blood pressure in vivo. In additional data supporting the importance of the eNOS/Cav-1 interaction in vivo, we have shown that Cav-1 KO mice bred to atherosclerosis prone ApoE KO mice have a markedly enhanced proatherogenic lipid profile compared to ApoE KO mice alone, but are paradoxically protected from atherogenesis, perhaps due to elevated eNOS activation. Interestingly, breeding ApoE/ Cav-1 KO double KO (dKO) mice to EC specific Cav-1 transgenic mice (Cav-1 RC) to reconstitute Cav-1 and caveolae in the endothelium (ApoE Cav-1 RC), reduces the hyperlipidemia and promotes atherosclerosis. With this background in mind, we hypothesize that understanding the interactions between eNOS and Cav-1 will permit molecular dissection of the physiological (blood flow) and pathophysiological (atherogenesis) roles of Cav-1 as a negative regulator of eNOS and perhaps other proteins. As a corollary to this hypothesis, we predict that a mutant of Cav-1 that does not inhibit eNOS or a cell permeable eNOS activator will antagonize the inhibitory role of Cav-1 on eNOS and promote NO release and improve endothelial cell health, blood flow and reduce atherogenesis. To examine the regulation of this important interaction in more detail, the following Specific Aims are proposed: 1.Define the mechanisms by which the Cav 3PM peptide activates NO release and eNOS function in vivo. 2. Reconstitute F92A Cav -1 into wild-type and Cav-1 KO endothelial cells and vessels and generate transgenic mice that inducibly express F92A Cav-1 in the endothelium and 3. Explore the mechanistic basis of how the genetic reconstitution of Cav-1 in the endothelium of ApoE Cav-1 KO restores atherogenesis. Collectively, this work will facilitate our understanding of the molecular machinery required for eNOS regulation in EC. This research is relevant to public health since endothelial dysfunction is a common manifestation of most cardiovascular diseases. Our research has discovered the major mechanisms of how the endothelium control blood flow and atherogenesis. Research supported by this grant may help identify new drugs that reduce heart disease and improve the quality of life of people suffering with cardiovascular disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Charette, Marc F
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Yale University
Schools of Medicine
New Haven
United States
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