Monitoring of the biological response to murine Hindlimb ischemia with 64Cu-labeled vascular endothelial growth factor-121 positron emission tomography

Background-Vascular endothelial growth factor-121 (VEGF(121)), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate Cu-64-VEGF(121) positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training. Methods and Results-64Cu-labeled VEGF(121) and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n = 58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with Cu-64-VEGF(121) positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma- counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF(121) showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF(121) uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61 +/- 0.17% ID/g (percentage of injected dose per gram) to 1.62 +/- 0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59 +/- 0.14% ID/g at day 29), and correlated with gamma-counting (R-2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF(121) uptake was increased significantly (P <= 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R-2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P < 0.001) compared with nonexercised mice. Conclusions-Cu-64-VEGF(121) positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.