Elucidation of the mechanisms that regulate differentiation and maturation of smooth muscle cells (SMC) will be critical to understanding both developmental growth, as well as the accelerated growth of SMC associated with cardiovascular disease. The long-term goals of our research are to elucidate the cellular and molecular mechanisms that regulate determination, differentiation, and maturation of SMC, and to determine the relationships between growth and differentiation in this cell. We have developed methods for culturing SMC that express multiple SM contractile proteins, and have developed (or acquired) various antibodies, cDNA probes, as well as biochemical, and recombinant DNA techniques to study SM contractile protein expression at the transcriptional and posttranscriptional levels. We have shown that PDGF BB and platelet PDGF suppress SM alpha-actin expression suggesting that they act as negative regulators of SMC differentiation. In contrast, PDGF AA, angiotensin II, and arginine vasopressin increased SM alpha-actin expression suggesting that they act as positive differentiation factors.
Aim 1 of this proposal will be to determine the role of PDGF isoforms in control of SMC growth, differentiation, and maturation both in vivo as well as in cultured cells. Studies will include: determination as to whether PDGF isoforms influence expression of multiple SM contractile proteins indicative of an overall effect on SMC differentiation; exploration of the hypothesis that PDGF AA and contractile agonists promote SMC differentiation while PDGF BB inhibits it determination of the role of PDGF in regulation of growth and differentiation during vascular development; examination of the effect of SMC differentiation on growth responsiveness;and elucidation of the role of PDGF isoforms in mediation of SMC proliferation and altered SMC contractile protein expression following vascular injury.
Aim 2 will be to identify molecular mechanisms that control expression of SM contractile proteins at both the transcriptional and post-transcriptional levels. Studies will include identification of tissue specific and PDGF-responsive regulatory elements in the SM alpha-actin gene, and identification of the molecular mechanisms whereby PDGF BB destabilizes SM alpha-actin mRNA.
Aim 3 will be to identify genes that control lineage determination and/or regulate expression of the differentiation program in SMC. These studies will utilize subtractive hybridization techniques similar to those used to clone the skeletal muscle lineage gene,Myo D 1, and will make use of an inducible SM lineage system developed in the multipotential embryonal carcinoma cell, P19.
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