The purpose of these studies IS to analyze the molecular mechanisms by which insulin-like growth factor-1 (IGF-l) stimulates smooth muscle cell (SMC) migration and replication. SMC synthesize IGF binding protein- 4 (IGFBP-4) and an IGFBP-4 protease. IGFBP-4 inhibits IGF-l binding to receptors and the protease facilitates its release. These studies will focus on expressing pure fibulin 1-C which has IGFBP-4 protease activity, identifying the factors that regulate its synthesis and activation and determining its physiologic role in releasing IGF-l to SMC. A protease resistant form of IGFBP-4 will be used to assess the importance of release of IGF-l into the pericellular space for atherosclerotic lesion development. Several integral membrane proteins regulate the ability of target cells such as SMC to respond to IGF-l. These include the IGF-l receptor, the aV133 integrin, integrin associated protein (lAP) and SHPS-l. To study the interaction between lAP and aV133 we will prepare lAP mutants that do not bind to aVI33, express them in SMC and determine if cells expressing these mutants have an altered biologic response to IGF-l. Since truncation of aV alters aVBeta3 binding to lAP we will utilize cells expressing a truncated aV mutant to determine how this alters IGF-l stimulated binding to lAP. Since changes in lAP binding within lipid domains of cell membranes are important for controlling whether it binds to a we will determine how IGF-l facilitates this process. Atherosclerotic lesions will be analyzed to determine if IGF-l stimulates the association of lAP with avBeta3 in vivo. To determine how ligand occupancy of lAP modulates cellular responsiveness to IGF-l, we will prepare an lAP mutant that cannot bind to its principle ligand thrombospondin-1 (TSP-i) and determine if cells that express this mutant have altered biologic responses to IGF-l. We will analyze the mechanism by which TSP-i binding to lAP is altering IGF receptor function and determine if TSP-i binding to lAP is functioning through SHPS-1 to alter the rate at which the IGF-l receptor is dephosphorylated. The results of these studies should define multiple new molecular mechanisms by which IGF-l functions coordinately with extracellular matrix proteins to activate its receptor and stimulate SMC replication and migration. The results may suggest novel strategies for interfering with these processes to alter the progression of atherosclerosis.
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