Cellular proliferation following vascular injury contributes to atherosclerosis and restenosis, two clinical diseases which account for considerable cardiovascular morbidity and mortality. Recent advances have been made in our understanding of growth factor gene expression and regulation in vitro, yet a major obstacle to the design of rational therapeutics is the characterization of growth factor gene expression and function in vivo. In this proposal, we use a novel approach to deliver biologically active gene products to vascular cells in the arterial circulation by direct transfer of recombinant genes in vivo. This will permit the selective introduction of a growth factor(s) whose expression and function can be characterized and the design of gene products which might regulate and inhibit such growth factor gene expression. We have recently made significant progress in this area, having shown that replication-defective retroviral vectors or liposomes can be used to introduce a reporter gene into an arterial site by direct transduction of the vessel wall. These techniques will now be optimized and used to introduce recombinant growth factor genes. Specifically, we will introduce the recombinant platelet derived growth factor (PDGF) B gene into porcine iliofemoral arteries and confirm its expression in vivo by analysis of transfer of DNA (polymerase chain reaction), expression of mRNA (in situ hybridization, S1 nuclease protection, RNA PCR), expression of protein (immunohistochemistry), and morphometric analysis. Second, we will determine whether recombinant PDGF B gene expression will stimulate cellular proliferation and formation of an atherosclerotic lesion alone or in combination with other factors, such as PDGF AA, acidic and basic FGF, and TGF beta or in association with hyperlipidemia. Third, gene products will be constructed which when expressed in vivo might inhibit cellular proliferation by antagonizing the binding of mitogen to receptor or disrupting gene regulation. Such inhibitors include the synthesis of expression vectors encoding porcine compatible monoclonal antibodies to growth factors from hybridoma lines, and soluble receptors. These potential inhibitors will be expressed in vivo, and effects on PDGF expression will be determined. In preliminary experiments, we have established a model of cellular proliferation in vivo, induced by transfection of porcine arteries with the recombinant PDGF B gene. Confirmation of recombinant PDGF gene expression is being performed, and antagonists to PDGF will be constructed. In summary, these studies will investigate the in vivo expression and function of the recombinant PDGF B gene and other recombinant growth factor genes using direct gene transfer. The results of these studies may be useful in the design of rational and novel therapeutic approaches to the treatment of atherosclerosis and restenosis.
Showing the most recent 10 out of 11 publications
