NAD(P)H oxidases are broadly activated in cardiovascular diseases, including hypertension, atherosclerosis, and diabetes. Reactive oxygen species (ROS) derived from these oxidases have been implicated in impaired vascular relaxation, medial hypertrophy, and neointimal hyperplasia in various forms of hypertension. NAD(P)H oxidase-derived superoxide anion (O2-) and other ROS are believed to mediate stretch-induced signaling, leading to neointimal hyperplasia. We previously developed a cell-permeant inhibitor of gp91-phox- (nox2-) based oxidase assembly which is capable of abrogating vascular O2- production in response to angiotensin II. The current proposal stems from 3 major findings, demonstrating (a) the importance of the multi-component oxidase assembly in vascular O2- production;(b) the ability of our cell-permeant inhibitor of nox2-based oxidase assembly to inhibit O2- and attenuate neointimal proliferation of the rat carotid artery in response to balloon angioplasty;and (c) the upregulation of novel nox2 homologues, nox1 and nox4, in response to balloon injury. Since nox1 and nox4 appear to be important oxidase homologues involved differentially in vascular O2- production after stretch, we will determine the efficacy of docking sequence mimics (which inhibit nox1and nox4 assembly with other oxidase subunits) to inhibit whole-vessel and endothelial, smooth muscle cell and fibroblast O2- generation and neointimal proliferation. These studies will address the hypothesis that nox1 and nox4 are functionally involved in vascular stretch-induced oxidase assembly and O2- generation, leading to neointimal proliferation. Three specific aims will be tested: (1) to develop specific inhibitors of nox1- and nox4-based oxidases and test them in an in vitro model of hormone-induced vascular NAD(P)H oxidase activation;(2) to investigate the role of docking sequences on individual nox-based oxidases in vascular stretch-induced oxidase activity in vitro;and (3) to determine the role of nox docking sequences in balloon angioplasty-induced neointimal hyperplasia in vivo. Relevance: Therapies aimed at disrupting the various NAD(P)H oxidase systems in blood vessels should substantially improve vascular patency and function following balloon angioplasty. These inhibitors are also expected to provide broad utility in a variety of disease processes involving oxidants, including hypertension, diabetes and atherosclerosis.