Atherosclerotic cardiovascular disease is the leading cause of mortality in the United States. Gene therapy is a rapidly expanding field with great potential for the treatment of atherosclerotic cardiovascular disease. Several specific genes, such as vascular endothelial growth factor (phVEGF'65), have been shown to be useful for preventing acute thrombosis, blocking post-angioplasty restenosis, and stimulating growth of new blood vessels. However, currently, there is no in vivo method for precise monitoring of gene expression within targeted atherosclerotic plaques. The recent discovery of green fluorescent protein (GFP), a marker gene, makes possible the use of intact living cells and organisms as experimental systems. GFP has been widely used as a sensitive reporter and the fluorescence signal emitted from GFP can be detected by optical imaging. This proposal will investigate the use of an innovative digital optical camera, developed at the Center for Medical Optical Imaging at Johns Hopkins University, to detect light signals emitted from GFP. We will test the hypothesis that digital optical imaging can be used to monitor vascular gene therapy, and in vitro fluorescent microscopic imaging of vascular GFPs may be extended to in vivo digital optical imaging of vascular GFPs. To test this hypothesis, we will develop a novel VEGF-GFP-lentiviral construct that expresses simultaneously both VEGF (to treat restenosis) and GFP (to track VEGF expression) after vascular gene transfection. In addition, we will develop two novel, extemal and intravascular digital optical imagers as in vivo molecular imaging tools to monitor vascular gene therapy, and validate the results in animal models of atherosclerosis. Since current knowledge about the biodistribution and/or in vivo pharmacokinetics of gene therapy is incomplete and relies primarily on staining of biopsied or post-mortem tissues, this innovative imaging technique will provide a unique opportunity to monitor and control vascular gene therapy in vivo and thus improve the management of atherosclerotic cardiovascular disorders.
