In recent years, reactive oxygen species (ROS) have been shown to have critical roles in normal vascular function and the pathogenesis of vascular disease. These molecules have profound effects on vascular smooth muscle cell (VSMC) growth, migration and differentiation. A major source of ROS in vascular cells is the NADPH oxidase (Nox) family of enzymes. VSMCs from large arteries express two types of NADPH oxidases, Nox1 and Nox4. These two oxidases are differentially localized within the cell, have distinct agonist specificity, and regulate specific cellular functions. What is less clear, however, is how the activity of these two enzymes is specifically regulated. While Nox1 regulatory mechanisms are similar to those of previously identified oxidases, Nox4 does not require any of the previously identified regulatory subunits for its enzymatic activity. We have identified and cloned a new protein, NoxR1, that physically and functionally interacts with Nox4. Preliminary experiments indicate that NoxR1 overexpression in VSMCs causes a significant increase in NADPH oxidase activity, an increase in focal adhesions, and an increase stress fiber formation, while knockdown of NoxR1 induces a profound alteration of the cytoskeleton and impairs VSMC migration. The overall goal of this project is thus to define the physiological function of NoxR1 regulation of Nox4 and to determine its role in an in vivo model of migration. In the first specific aim, we plan to determine the role of NoxR1 in the regulation of focal adhesion turnover and cell migration, while Aim 2 is designed to determine the role of NoxR1 in neointimal formation in vivo using a newly created NoxR1 knockout mouse. Because NoxR1 is the first known regulator of Nox4, a Nox family member that regulates such basic cellular processes as senescence, differentiation and survival, these investigations are likely to have far-reaching implications for a number of vascular and nonvascular diseases.