Differentiated, quiescent vascular smooth muscle cells (VSMC) assume a dedifferentiated, proliferative phenotype in response to injury----one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in regulating development and differentiation in various cell types. For example, deletion of the LIM protein RBTN2 by homologous recombination blocks erythrocyte development, and antisense striated muscle LIM protein (MLP) prevents differentiation of myoblasts. We have cloned a developmentally regulated LIM protein, SmLIM, that is expressed preferentially in VSMC. Like RBTN2 and MLP, SmLIM contains two LIM domains. SmLIM mRNA levels were downregulated quickly when arterial smooth muscle cells became dedifferentiated in vitro and in vivo. Thus, we hypothesize that SmLIM may have an important role in regulating VSMC development and differentiation. The goals of our proposed work are to investigate the molecular mechanisms regulating preferential expression of SmLIM in VSMC, to study the significance of SmLIM in VSMC development and differentiation (both in vitro and in vivo), and to elucidate the molecular mechanisms by which SmLIM exerts its function. We will first characterize the specific nucleotide sequences (cis-acting elements) required for VSMC- restricted expression of the SmLIM gene. The specificity of these VSMC-specific cis-acting elements will also be tested in transgenic animals in vivo. Using these cis-acting elements, we will characterize and clone the nuclear proteins (trans-acting factors) binding to them. Since SmLIM may play an important role in maintaining the differentiated VSMC phenotype, we will study the effect of sense and antisense SmLIM on VSMC phenotype in culture. In addition, we will determine the effect on VSMC development and differentiation in mice of deletion of the SmLIM gene by homologous recombination. Finally, we will elucidate the molecular mechanisms by which SmLIM exerts its biologic function by cloning its interaction partners. These experiments will provide fundamentally important information about development and differentiation in VSMC, as well as critical insights into the treatment of arteriosclerosis and tumor angiogenesis. Furthermore, identification of VSMC-specific cis- and trans-acting factors will be crucial to the future success of efforts to restrict the expression of foreign genes to the blood vessel wall, for gene therapy of vascular disease.
