The applicants proposed to test the hypothesis that transient gene therapy with G protein-coupled receptor kinases (GRKs) at the time of PTCA or saphenous vein graft placement may substantially alleviate neointimal hyperplasia. GRKs are a family of enzymes which initiate either desensitization or signal termination for many G protein-coupled receptors, several of which have been strongly implicated in the pathology of vascular smooth muscle cell proliferation and restenosis: the endothelin A and B, angiotensin II (type 1), thrombin, and thromboxane A2 receptors. Overexpression of GRK2 can drastically reduce signaling through all of these receptors in transfected cell systems. Systemically administered antagonists of the endothelin and angiotensin II (type 1) receptors significantly reduce neointimal hyperplasia in animal models of vascular injury. The applicants proposed therefore to develop recombinant adenoviruses for expressing GRKs 2,3, and 5 transiently in smooth muscle cells, and to test each of these recombinant viruses in rabbit and human smooth muscle cells for the ability to reduce cellular responses to agonists for the receptors listed above, as assessed by (a) inositol phosphate signaling, (b) cellular proliferation, and (c) stimulation of mitogen-activated protein kinase activity. Subsequently, the GRK adenoviruses will be used ex vivo to treat rabbit jugular veins, which will be grafted across the rabbit's ligated carotid artery in a well-established model of neointimal hyperplasia. Four weeks after surgery, vein grafts will be evaluated to determine the GRK's ability (a) to attenuate neointimal hyperplasia in vivo, by histology, and (b) to attenuate agonist-stimulated vein graft vasomotor function, assessed under isometric tension. Successful modulation of neointimal hyperplasia by transiently overexpressed GRKs in the rabbit model of vascular injury would have therapeutic implications for restenosis in PTCA and vein graft failure in CABG.
