Over growth of vascular smooth muscle cells is a hallmark of the late stages of vascular disease and contributes to the eventual blocking of affected arteries. Heparin has been shown to slow smooth muscle cell growth in vitro and in selected in vivo animal and human studies. Heparin effects on vascular smooth muscle cells have been explained by a variety of theories of theories including heparin receptors, heparin interaction with growth factors, and endocytosis of heparin. Data show that heparin treatment resulted in decreased levels of active MAPK. This research proposal aims: to test hypothesis that heparin modifies the behavior of sensitive vascular smooth muscle cells by causing an increase in intracellular cyclic GMP levels through the interaction of heparin with a heparin-binding site on the cell membrane. This work will involve cultured porcine aortic smooth muscle cells that are serum starved and then activated with phorbol myristate or PDGF. The ability of heparin to induce rises in cGMP concentration and decrease MAPK activity will be monitored using western blotting of proteins from cells treated with or without heparin or cGMP analogs. Commercially available ELISA assays will be used to monitor cellular cGMP levels. antibodies to a putative heparin receptor that were developed in the PI's laboratory and have been shown to act as heparin agonists in previous studies will be compared with heparin for their ability to induce cGMP increases. The ability of cGMP to cause increases in MKP-1 synthesis, as shown in the PI's laboratory for heparin, will also be determined by western blotting. Finally, the current protocol used in the PI's laboratory for partial purification of the putative heparin receptor will be improved. the resultant receptor preparations will be used in the future to obtain sequence for cloning the putative receptor and to allow further characterization of characterization of the receptor-ligand interactions. Together this work will confirm the importance of the putative heparin receptor in heparin-induced changes in vascular smooth muscle cells and provide a significant advancement in understanding of how heparin alters sensitive VSMC.
