Abnormalities of vascular smooth muscle cell (VSMC) growth and migration play an important role in hypertension and atherosclerosis and are cardinal features of restenosis seen after balloon angioplasty of human coronary arteries. We have employed several strategies to elucidate the genetic program associated with VSMC growth in culture and have identified growth factor-responsive genes that encode a broad range of proteins, including mediators of inflammation and thrombosis, components of the extracellular matrix, components of the cytoskeleton, and intracellular enzymes. This proposal will extend investigations of two genes: osteopontin (OPN), a secreted glycoprotein involved in matrix organization, and SM-20, a novel """"""""immediate early"""""""" gene. The primary goal is to establish the biologic role of these genes and to determine whether they are involved in regulating VSMG growth or migration. A recently developed antibody to SM20 will be used to determine the intra- and extra- cellular distribution of SM-20 protein in rat and human VSMC and study its expression in response to growth factors and vasoactive agonists. The cellular distribution of SM-20 mRNA and protein in adult rat tissues, during mouse development, and in balloon injured aorta will be examined using in situ hybridization and immunohistochemistry. To elucidate the biologic role of SM-20, VSMC will be transfected with expression vectors containing the full length SM20 mRNA in either the sense or antisense orientations. Permanent cell lines will be isolated which either over- or under-express SM-20 protein and used to determine the effect of varying levels of SM-20 expression on VSMC growth, migration, and shape. Transfections performed in parallel (Aim 3) will test the hypothesis that through its effects on matrix organization, OPN plays an important role in regulating VSMC growth and migration. These studies will also employ exogenous administration of expressed OPN protein. In the latter stages of the project, the permanent cell lines will be used to elucidate the mechanism(s) by which SM-20 and OPN regulate VSMC growth and migration. Experiments will include identification of mRNA species which are altered in normal vs. growth- inhibited cells using differential display of mRNA. In addition, mutations will be made in the SM-20 and OPN proteins to determine which areas are necessary for their effects.
In Aim 4, studies will characterize the induction of OPN mRNA and protein in the vessel wall in response to balloon injury and will test the hypothesis that blocking the induction will inhibit the development of intimal hyperplasia. Two approaches will be used to block OPN induction in vivo: antisense oligonucleotides and transferrin-coupled ribozymes, both delivered to the vessel wall using a porous balloon catheter. The effect of OPN inhibition on VSMC growth and migration and on intimal hyperplasia will be assayed by light microscopy and by 3H-thymidine incorporation. In addition to providing new insights into the regulation of VSMC growth, these studies will evaluate intravascular approaches to blocking gene induction in the vessel wall.
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