VASCULAR SMOOTH MUSCLE MEMBRANE PROTEOGLYCANS

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VSM cells are subject to an array of regulatory signals that control their growth, differentiation and functional activity. Knowledge of how these regulatory signals work would provide important insights into understanding and ultimately controlling VSM cell activity in pathological conditions. The long range goal of this project is to elucidate the roles of membrane associated proteoglycans (mPGs) in the function and pathobiology of vascular smooth muscle (VSM) cells. Although mPGs are ubiquitous components of plasma membranes, and have been implicated in control of cell growth and differentiation, their specific functions are still unknown. Recent findings suggest that mPGs are involved in interactions of cells with important regulatory molecules, in particular extracellular matrix (ECM) adhesive proteins and polypeptide growth factors. Biochemical and molecular biological studies from our laboratory have identified 4 structurally distinct mPGs that are synthesized by rat aortic VSM cells. These include 3 different but structurally related members of a gene family of transmembrane proteoglycans, and a lipid-anchored mPG. A major question underlying the proposed studies is what is the functional significance of this structural diversity. Three specific aspects of VSM mPG biology are addressed in the proposal: 1) the patterns of synthesis of these mPGs by VSM cells in vivo in rats during development and in response to injury; these studies will examine levels of mPG mRNA by Northern blot analysis and ribonuclease protection assay and mPG levels by immunoblotting and immunocytochemistry; 2) analysis of the binding of these mPGs to ECM molecules and their roles in VSM cell-ECM interactions; for these experiments the mPGs will be purified from rat aortic VSM cell cultures by conventional and immunoaffinity chromatography and assayed for their ability to bind to a panel of purified ECM molecules; we will also use anti-mPG antibodies, anti-sense inhibition, and mPG overexpression to test directly their role in VSM cell attachment, spreading and migration on ECM; and 3) analysis of the role of specific mPGs in growth factor stimulated VSM cell proliferation; effects of anti-mPG antibodies, anti-sense inhibition of mPG synthesis, and specific mPG overexpression on rates of VSM cell proliferation and c-fos mRNA induction in response to growth factors will be measured, as well as growth factor binding to high affinity cell surface receptors.