As the blood vessel ages, its cells are subject to pathological changes including hypertrophy, polyploidy, proliferation, apoptosis, and remodeling, and these processes are thought to be a result of complex interactions between inflammatory events (eg. cytokines, amyloid 13 peptide) and proliferative growth factors (e.g. angiotensin (Ang) II, PDGF, EGF, etc). A long-range goal of our group is to understand how reactive oxygen (ROS) and reactive nitrogen species (RNS) derived from NADPH oxidase and nitric oxide synthase(s) regulate the outcome of the vascular cell phenotypic response to these interacting cytokines and growth factors. Our in vivo studies in young rodents indicate that Ang II, via vascular NADPH oxidase mediates smooth muscle hypertrophy, while causing inflammation, apoptosis and/or proliferation in the endothelium and adventitia. This differential cellular response may be related to the combined influence of Ang II and cytokines arising from leukocytic infiltration in the latter two cell layers. In addition, in the smooth muscle, hydrogen peroxide (H2O2) formed by dismutation of NADPH oxidase-derived superoxide (O2) is an important ROS that mediates smooth muscle cell (SMC) hypertrophy. In contrast, in the endothelium and advent/t/a, the cellular response is associated with intense nitrotyrosine (NY) staining, indicating that NO derived from eNOS and/or iNOS is inactivated by insufficiently scavenged O2 to form the RNS, peroxynitrite (ONOO). Decreased NO-bioactivity mediated by ROS is also relevant, affecting the response to growth factors and cytokines, and influencing contractility. During aging Ang II further shifts the vascular redox balance towards RNS formation. We propose to study the cellular response to Ang II and cytokines in cultured SMC as an experimental model for the more complex interactions that occur in vivo. Our primary focus will be the role of ROS/RNS in covalently modifying the GTP-binding signaling protein, Ras. In a proteomic screen, we identified Ras to be S-glutathiolated (GSS-Ras). Following up on this observation, we find that NADPH oxidase-derived H2O2, which is stimulated by Ang II, increases GSS-Ras and Ras activity. In the proposed studies, we will 1) study the effect of oxidants and Ang II on Ras function in cultured smooth muscle, and 2) determine the role of oxidants and Ras in the response to Ang II in vivo in rat and transgenic mice.
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