? This research program will test the hypothesis that ischemic myocardium can be detected and imaged by labeling angiogenic receptors, and that such an approach will permit assessment of the efficacy of angiogenesis. Vascular endothelial growth factor-121 (VEGF121) is a non-heparin binding isoform of VEGF secreted in response to hypoxia that binds to endothelial cell-specific hypoxia-inducible tyrosine kinase receptors Fit-1 and KDR. This group has recently shown that surgically induced hindlimb ischemia in rabbits can be imaged in vivo as selective uptake of intravenously injected radiolabeled VEGF121. These data suggest that labeling of hypoxia-specific angiogenic receptors using the receptors' naturally occurring ligand may be a useful approach to detect ischemically stressed tissue. Because VEGF receptors (VEGFR) are endothelial-cell specific, a VEGFR-targeted imaging agent that remains within the intravascular space would be ideal. Accordingly, this proposal will develop a targeted ultrasound imaging agent comprised of an acoustically active lipid microbubble with recombinant human VEGF121 as the targeting ligand on the bubble surface. Based on previous experience, it is expected that transient adhesion of these microbubbles to microvasculature overexpressing VEGFR in ischemic tissue can be imaged with harmonic-based echocardiographic techniques. In stepwise fashion progressing from in vitro to in vivo models, the Specific Aims of this proposal are to address the following questions: (1) Do VEGF121-conjugated microbubbles (VEGF-bubbles) bind to endothelial cells overexpressing VEGFR in vitro, and how can bubble design be manipulated to optimize binding? (2) Do VEGF-bubbles bind to microvasculature overexpressing VEGFR in vivo? (3) Can in vivo VEGF-bubble binding be ultrasonically imaged and related to the magnitude of VEGFR expression? (4) Can VEGFR-targeted echocardiographic imaging be used to identify ischemia and angiogenesis in a clinically relevant canine model of progressive coronary occlusion and collateral development? The ultimate goal of this study is to develop a non-invasive, easily available, high-resolution method for quantifying the ischemic burden of tissue. Such a non-isotope approach could have advantages over existing clinical methods for detecting atherosclerotic disease employing stress and rest nuclear perfusion imaging, and may offer a sensitive method for assessing angiogenesis. ? ?
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