The proposed research is a study of the molecular mechanisms involved in atherogenesis and restenosis. Gene transfer provides a means to examine these mechanisms by genetic modification of vascular cell biology. I will examine the feasibility of modifying vascular cells within atheromatous plaque: (1) by resurfacing with genetically altered endothelial cells and (2) by direct retroviral infection in vivo. Heterozygote Watanabe Heritable Hyperlipemic rabbits fed a high cholesterol and high fat diet will serve as the atherosclerotic model. Atheromatous plaques within the iliac arteries will be treated with an atherectomy device. Tissue removal will be performed to varying depths within the vessel wall to examine how this influences endothelial cell adhesion and the efficiency of direct retroviral infection in vivo. The preferred source of autologous rabbit endothelial cells (large vessel vs. microvascular) will be determined. The exposed vascular cells of the atherectomy-treated iliac artery segments will be resurfaced with genetically modified endothelial cells. In parallel experiments the atherectomy-treated iliac artery segments will undergo direct retroviral infection in vivo. Replication-defective retroviruses with amphotropic host range will be used. Initial experiments will use a retroviral construct containing the E. coli lacZ reporter gene to assess the resurfacing efficiency with the modified endothelial cells and the transduction efficiency of vascular cells by direct in vivo infection. Enzymatic histochemical studies will allow determination of the in vivo survival of modified endothelial cells, the vascular cell types involved in direct infection, and the persistence of retroviral gene expression. Retroviral constructs encoding genes for thrombolytic agents, thrombin and platelet-aggregation inhibitors, growth factor antagonists and cytokine receptor antagonists will also be transfected into endothelial cells. Application of these gene transfer methods in an experimental atherosclerotic animal model will permit examination of the role of specific gene products in the pathogenesis of atherosclerosis and restenosis.
| Ory, D S; Neugeboren, B A; Mulligan, R C (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A 93:11400-6 |
