Excessive proliferation of migration of vascular smooth muscle cells (SMC) plays a major role in the development of atherosclerotic plaques. Alpha-thrombin is of particular interest because it has been implicated in mediating vascular pathology in several different animal models. There is also compelling evidence that alpha-v-beta-3 integrins are involved in vascular healing responses; the challenge has been to understand their role in regulating SMC proliferation and migration in the injured artery. In studies from my laboratory, we found that alpha-thrombin-induced proliferation of human aortic SMC was partially inhibited by alpha-v- beta-3 antagonists and that alpha-v-beta-3 antagonists block alpha- thrombin-induced JNK1 activation. Furthermore, we have identified alphav-beta-3 integrin-dependent cytoplasmic events that occur in SMC in response to treatment with alpha-thrombin which provide insight into the integration signals from G-protein-coupled receptors and integrins. In particular, non-muscle myosin-A (NM-A), a cytoskeletal protein implicated in SMC proliferation, rapidly associates with alpha-v beta-3 integrins and with focal adhesion (FAK) following treatment of rat aortic SMC (RASMC) with alpha-thrombin. NM-A provides a unique site for regulation as an equilibrium exists between two distinct conformations: a folded state in which myosin can not assemble into filaments (i.e. 10S conformation) and an extended conformation that promotes the assembly of filaments (i.e. 6S). Equilibration between 6S and 10S is sensitive to the phosphorylation state of myosin light chain (MLC). Thus phosphorylation of MLC, which is stimulated by PAR-1-mediated signaling, could play an important regulatory role in integrin-mediate signaling in response to activation of G protein-coupled receptors. In the proposed studies, we will test the following hypothesis: The inducible association of NM-A with alpha-v beta-3 integrins, FAK and the actin cytoskeleton is required for alpha-thrombin-induced activation of c-jun NH2-terminal kinase-1 (JNK1) in SMC and is regulated by PAR-1 activation of RhoA and phosphorylation of myosin light chain. The studies we propose will provide insight into two critically important questions in vascular biology: 1) regulation of alpha-thrombin-induced SMC growth and 2) convergence of G protein-coupled receptor and integrin signaling. These studies will also contribute to the development of effective treatments for patients with vascular disease by increasing our understanding of how alpha-v beta-3 integrins regulate vascular healing.
