Proliferation of smooth muscle cells is a primary event in the fibroproliferative stage of atherosclerosis. This proliferation is probably induced by changes in the blood vessel chemistry which include increases in growth factors and relative decreases in inhibitory molecules. Heparin has been shown to interfere with serum and growth factor stimulated smooth muscle proliferation both in vivo and in vitro. However, the mechanism by which heparin inhibits smooth muscle proliferation is not clear. The goal of the present proposal is to investigate the hypothesis that heparin acts through specific heparin binding sites (receptors) on the smooth muscle cells. Antibodies which block the binding of heparin to porcine aortic endothelial cells have been found to block heparin binding to smooth muscle cells as well. In addition, the antibodies immunoprecipitate a smooth muscle cell protein similar to the cell surface heparin binding protein precipitated from endothelial cells. These antibodies will be used to address the question of whether the putative receptor recognized by these antibodies plays a role in heparin signaling. Specifically, the antibodies will be assessed for their ability to mimic heparin actions and to block heparin signaling. Heparin and control samples will be analyzed for each experiment. Assays will include measuring thymidine incorporation and cell proliferation in response to serum and growth factor stimulation. If, as preliminary results suggest, the antibodies mimic heparin they will be considered heparin agonists for receptor based activity. Antibodies which block heparin signaling would be considered antagonists. Either type of activity would provide a system to investigate the signaling pathway. The antibodies will also be used to investigate the heparin block in the smooth muscle cell protein kinase C signaling pathway. Phorbol esters (and serum in other assays) will be used to stimulate the signaling pathway. The ability of antibodies to interfere with heparin effects or to mimic heparin effects will be assessed by measuring mitogen activated protein kinase activity. For these experiments, kinase and phosphotyrosine specific antibodies will be employed in immunoprecipitation and western blotting experiments. C-fos and c-myc transcription will also be measured by northern blotting after activation of the protein kinase C pathway. Together the data obtained from these experiments will support or refute the hypothesis that heparin signaling in proliferation and the protein kinase C pathway is mediated through the putative heparin receptor recognized by the antibodies. This is an important step in understanding smooth muscle cell growth regulation in the vasculature. Such knowledge may lead to developing treatment strategies based on the control of smooth muscle cell proliferation in vascular disease.
