EXPRESSION/FUNCTION OF RECOMBINANT TGF-BETA IN ARTERIES

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Atherosclerosis and restenosis are two cardiovascular diseases which alter cardiac function and produce considerable morbidity and mortality. Gene therapy treatments to these diseases hold considerable promise, but a major obstacle to their implementation is the development of appropriate animal models and the investigation of molecular mechanisms of vascular pathophysiology. A major interest of my laboratory is to develop animal models of vascular disease based upon direct gene transfer of recombinant growth factor genes which will facilitate the understanding the treatment of cardiovascular diseases. The purpose of this proposal is to study the basic biology of growth factor gene expression and to develop gene therapy approaches to treat cardiovascular diseases. Transforming growth factor beta (TGF-beta) genes will be examined since this growth factor regulates cellular proliferation and connective tissue production following vascular injury. We will use direct gene transfer to transfect recombinant TGF-beta genes into vascular cells in porcine arteries in vivo. Expression of TGF-beta genes in normal and injured arteries will permit characterization of gene expression and function, and the design of gene products which might regulate and modify TGF-beta induced changes. We have previously developed methods for cell-mediated and direct gene transfer of recombinant genes into the vasculature. These studies have established the feasibility for the proposed experiments by demonstrating that recombinant genes can be expressed in the vasculature. Gene expression can be confirmed at the DNA, mRNA and protein level, and the effect of the recombinant gene product on vascular cells can be analyzed. In this proposal, we plan to develop an animal model of recombinant TGF-beta gene expression (TGF -beta1, -beta2 and -beta3) in normal porcine arteries following direct gene transfer, to confirm gene expression, and to investigate the effects of TGF-beta on these arteries. Second, we will investigate the mechanism of recombinant TGF-beta regulation of vascular growth in transduced arteries by characterizing collagen synthesis, cell proliferation, and release of growth factors and cytokines. Third, the effect of TGF-beta gene expression on intimal proliferation and connective tissue production in injured arteries will be investigated. Preliminary data suggest that expression of TGF-beta could help to modify the response to injury. These systems can also be used to develop antagonists to TGF-beta which can further define and/or treat vascular diseases. Such agents, including dominant negative inhibitors, antisense oligonucleotides or other antagonists, will be analyzed as part of future studies beyond the scope of this proposal. In summary, these studies will develop animal models of vascular disease from direct gene transfer of recombinant TGF-beta into normal and injured porcine arteries. Studies of basic biology of gene expression will be conducted and molecular genetic interventions which target TGF-beta will be developed to treat cardiovascular diseases.