Extracellular matrix-mediated signaling in the regulation of vascular smooth muscle cell phenotype and function

Abstract: Cardiovascular disease is the leading cause of mortality in the world. Even though death rates are today dropping in the developed countries, disability due to the disease is rising worldwide. The major portion of cardiovascular disease associated deaths is due to occlusive atherosclerotic lesions. A major drawback to the surgical treatment of atherosclerosis is the high rate of restenosis or stenosis of bypass grafts that occurs within six months of the procedure. Despite years of research and multiple clinical trials, no effective pharmacological therapy against restenosis or graft stenosis is yet available. This clearly motivates a thorough analysis of the cellular mechanisms involved in these processes. In this thesis, I have focused on the role of the extracellular matrix, and specifically fibronectin (FN), in the regulation of vascular smooth muscle cell (SMC) differentiation and function. Restenosis is characterized by intimal hyperplasia, which is largely due to SMC migration and proliferation, and deposition of extracellular matrix. SMCs in the normal arterial media, whose primary function is contraction, must first undergo a change in phenotype before they can migrate and proliferate. This modification includes loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. SMCs undergo a similar change in phenotype when established in culture either in serum-containing medium or on a substrate of FN under serum-free conditions. Using an in vitro system where freshly isolated rat aortic SMCs a re cultured on extracellular matrix components, we have analyzed the intracellular signaling pathways that mediate the effects of FN on SMC phenotype. The results demonstrate that, in addition to the above-mentioned structural reorganization, the transition from a contractile to a synthetic state includes a decrease in caveolae numbers and internalization of caveolin. Caveolae are specialized plasma membrane invaginations with caveolin as the major coat protein and have been implicated in cholesterol transport and signaling. Culture of freshly isolated SMCs on a substrate of FN was further shown to be associated with sustained activation of several signaling molecules, including the small GTPbinding proteins of the Rho family, the extracellular regulated kinases ERK1 and 2, and tyrosine kinases such as focal adhesion kinase (FAK). By using specific inhibitors, we show that activation of integrin-linked tyrosine kinases, ERK1/2 and Rho are necessary for phenotypic modulation. In addition, cyclin DI was found to be induced as the cells were grown on FN. The induction of this cyclin was also dependent on integrin-linked tyrosine kinase, ERK1/2 and Rho activities. Phenotypic modulation of SMCs in vitro is strongly coupled to cell spreading. Studies in other cell systems have indicated that the Rho proteins regulate the actin cytoskeleton. We show here that this is also the case in SMCs in primary culture and that inhibition of cell spreading using different drugs suppressed the shift in differentiated properties of the cells. Mevinolin, a member of the statin family of lipidlowering drugs, strongly blocked phenotypic modulation and mitogen-induced DNA synthesis and increased apoptosis. These results suggest that statins, in addition to other described and beneficial effects on the vascular wall, may also function via an effect on SMC phenotypic modulation. In summary, this thesis has investigated the effects of FN on the differentiated state of SMCs in primary culture. We have demonstrated that caveolae decrease in number concomitant with an internalization of caveolin. This is coupled with activation of signaling molecules such as Rho, ERK1/2 and FAK. In addition, we have targeted cell spreading, an essential process for the shift in phenotype, using a drug that in addition to reducing blood cholesterol also inhibits the activation of Rho proteins. It is our hope that the knowledge gained in studying the cellular signaling mechanisms in control of SMC function will help to develop new strategies to combat restenosis and graft stenosis.