Vascular smooth muscle cell (VSMC) migration is critically important in neointimal formation following angioplasty and atherosclerotic lesion formation. During the last grant period, we showed that VSMC migration in response to platelet-derived growth factor (PDGF) requires the production of reactive oxygen species (ROS), which mediate the tyrosine phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), a serine/threonine kinase that mediates actin cytoskeletal reorganization. PDK1, in turn, phosphorylates p21- activated kinase-1 (PAK1), and both kinases are required for migration. However, cell migration is a multistep process that involves formation of lamellipodia, creation of focal adhesions at the front of the cell, contraction of the cell body, and dissolution of focal adhesions at the trailing edge. The role of ROS and PDK1 in each of these steps remains unclear. In this grant period, we propose to investigate the mechanisms leading to ROS formation and the downstream molecular targets that regulate the early steps in migration. 4 specific aims will be accomplished. First, we will investigate the sources of ROS in PDGF-stimulated cells, focusing on the NAD(P)H oxidases Nox1 and Nox4. We will use RNA silencing techniques to investigate the separate and joint roles of these 2 important oxidases in formation of lamellipodia and focal adhesions. Second, we will investigate the signaling processes leading to Nox1 activation and PDK1- mediated lamellipodia formation. Third, we will investigate the role of Nox4 and PDK1 in mediating focal adhesion kinase activation and phosphorylation 'of paxillin, which in turn regulate the conversion of focal contacts to focal adhesions. Finally, we will use unique transgenic and knockout animals (smooth muscle specific nox1- or catalase-overexpressors and nox1-/- mice) to investigate the role of Nox1 and ROS in an in vivo model of VSMC migration. Migration will be induced by carotid ligation or femoral wire injury and followed using morphometric techniques. Together, these Aims will provide new insight into how NAD(P)H oxidases mediate VSMC migration and therefore lesion formation. Such information may lead to the development of new therapeutic strategies that can be carefully and specifically targeted to the critically important events in disease initiation.