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 (VEGF121), 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 64Cu-VEGF121 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.


RESULTS
64Cu-labeled VEGF121 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 64Cu-VEGF121 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, VEGF121 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-VEGF121 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 (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 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
64Cu-VEGF121 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.
Willmann
Jürgen K
JK
Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, Calif, USA.
Chen
Kai
K
Wang
Hui
H
Paulmurugan
Ramasamy
R
Rollins
Mark
M
Cai
Weibo
W
Wang
David S
DS
Chen
Ian Y
IY
Gheysens
Olivier
O
Rodriguez-Porcel
Martin
M
Chen
Xiaoyuan
X
Gambhir
Sanjiv S
SS
D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D003097:Collateral Circulation / Q000502:physiology*; D003301:Copper Radioisotopes; D042783:Endothelial Cells / Q000166:cytology; D006614:Hindlimb / Q000098:blood supply* / Q000000981:diagnostic imaging; D006801:Humans; D007511:Ischemia / Q000000981:diagnostic imaging*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D010805:Physical Conditioning, Animal; D049268:Positron-Emission Tomography; D011485:Protein Binding; D019275:Radiopharmaceuticals; D013552:Swine; D014162:Transfection; D014463:Ultrasonography; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D003301:Copper Radioisotopes; D019275:Radiopharmaceuticals; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2