This project will examine ways in which smooth muscle cells of vascular grafts are induced to proliferate. Direct platelet interaction on the graft is not the only pathway by which these cells can be initiated to proliferate since the surface is often covered with an intact endothelium thus preventing platelet interaction with the underlying cells. Despite these facts, the smooth muscle cells continue to proliferate and ultimately cause narrowing or occlusion of the graft. To understanding why these cells continue to proliferate, we have proposed studies to examine potential pathways, other than direct platelet interaction, by which these cells can be stimulated to grow.
The first aim of this proposal will be to examine what growth factors can directly stimulate those smooth muscle cells that are present in the graft. This problem will be approached using an organ culture technique. With this technique, it is possible to maintain a blood vessel in a quiescent state for several days to add known mitogens such as PDGF, FGF and TGF-beta and to quantitate their effect on the growth of both smooth muscle and endothelial cells. In addition, vascular grafts will be placed in organ culture and antibodies to the above mitogens will be added to determine if they inhibit the spontaneous smooth muscle proliferation.
A second aim will be to investigate if platelet products can be released at one site and swept downstream where they influence proliferation of smooth muscle cells. Studies are proposed to examine sites endothelialized, downstream for portions of a thrombogenic graft, for the presence of platelet products. This will be carried out with indium-111-labeled platelets and with antibodies to platelet factor 4. The proliferation rates of smooth muscle cells at these downstream sites will also be assessed using 3H-thymidine and autoradiography. If platelet released products indeed stimulate cell growth at these sites, then attempts will be made to interfere with this process by inhibiting platelets with ticlopidine. Finally, studies will be conducted to see if smooth muscle cells that populate vascular grafts can synthesize their own mitogen, in particular PDGF. These studies will harvest graft smooth muscle cells, grow them in culture, and examine their ability to secrete a PDGF-like protein. To obtain data of cells in vivo, cells will be removed from grafts, the mRNA extracted and hybridized with cDNA to PDGF, FGF and TGF-beta using both Northern and in situ hybridization techniques. The above studies are part of an ongoing theme to investigate what factors in vivo initiate smooth muscle cell proliferation.
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