phox -gp91 phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P ϭ 0.058). In contrast, a significant reduction was achieved with Adgp91ds (P ϭ 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47 phox may be more effective than inhibition of p67 phox at attenuating neointimal growth.NADPH oxidase; p47 phox ; adventitia; restenosis NEOINTIMAL HYPERPLASIA is a major response to vascular injury (33). Proliferation and migration of vascular smooth muscle cells (VSMCs) and, more recently, fibroblasts have been implicated in narrowing of the arterial lumen in response to injury, mimicking one of the hallmark characteristics of atherosclerosis (6, 32). Smooth muscle cell migration and proliferation are involved in narrowing of the arterial lumen in response to injury and atherosclerosis, a process promoted by protooncogenes (34) and various growth factors (11,19). Recent studies have implicated superoxide anion (O 2 Ϫ ) derived from NADPH oxidase in vascular cell proliferation and neointimal growth (17,37), and processes involving reactive oxygen species (ROS) have been included in a number of growth-related signaling pathways (14).NADPH oxidase is a well-characterized ROS-generating system that catalyzes the one-electron reduction of O 2 to O 2 Ϫ , a precursor of a variety of other ROS. The classical phagocyte NADPH oxidase is known to be a multicomponent enzyme complex that includes the two membrane-spanning polypeptide subunits p22 phox and anchoring component gp91 phox , which are associated with the plasma membrane cytoskeleton (and together comprise flavocytochrome b 558 ), along with three cytoplasmic polypeptide subunits, p40phox , p47 phox , and p67 phox (8,43). The cytosolic guanine nucleotide-binding protein p21 rac , a member of the Ras family of proteins, is required for oxidase activation (20). Exposure of the cell to agonists induces interaction of cytosolic and membrane-associated components and activates the dormant oxidase (8). Numerous studies have suggested a similar process of oxidase activation in the vasculature (16,23,26,29,43).Recently, NADPH oxidases in VSMCs and adventitial fibroblasts have been implicated in the proliferation and migration of cells from the vascular wall to the neointima (13, 18). VSMCs in large arteries exp...