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Purpose: To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. α v β 3 integrins in a mouse hindlimb ischemia model of peripheral artery disease., Procedures: Male wild-type (WT) C57BL/6 mice (8- to 10-week old) (n = 24) underwent left femoral artery ligation. The right leg served as control. Five days later, mice were injected with either VEGF receptor targeting [ 99m Tc]DOTA-PEG-scVEGF ([ 99m Tc]scV) (n = 8) or with α v β 3 -targeting tracer [ 99m Tc]HYNIC-cycloRGD ([ 99m Tc]RGD) (n = 8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [ 99m Tc]scV uptake, six additional mice received a mixture of [ 99m Tc]scV and 30-fold excess of targeting protein, scVEGF. Tracer uptake as %ID was measured using volumetric regions encompassing the hindlimb muscles and as %ID/g from harvested limb muscles. Double and triple immunofluorescent analysis on tissue sections established localization of α v β 3 , VEGFR-1, VEGFR-2, as well as certain cell lineage markers., Results: Tracer uptake, as %ID/g, was higher in ligated limbs of mice injected with [ 99m Tc]scV compared to ligated hindlimbs in mice injected with [ 99m Tc]RGD (p = 0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [ 99m Tc]scV than for [ 99m Tc]RGD (p = 0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and α v β 3 , in damaged vs. undamaged hindlimb tissue, but with little co-localization of these markers. Double immunofluorescent staining showed partial co-localization of VEGFR-1, VEGFR-2, and α v β 3, with endothelial cell marker FVIII, but not with CD31. Immunostaining for VEGFR-1 and VEGFR-2 additionally co-localized with lineage markers for endothelial progenitor cell and monocytes/macrophages, with a more diverse pattern of co-localization for VEGFR-2., Conclusion: In a mouse hindlimb ischemia model of peripheral artery disease, [ 99m Tc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [ 99m Tc]RGD tracer-targeting α v β 3 . Immunofluorescent analysis suggests that uptake of [ 99m Tc]scV and [ 99m Tc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [ 99m Tc]scV may be due to the presence of VEGF receptors on additional cell types.

We investigated treatment with a receptor for advanced glycation endproduct (RAGE) blocking antibody on angiogenic response to hind limb ischemia in diabetic mice. Streptozotocin treated C57BL/6 mice received either murine monoclonal anti-RAGE F(ab')2 intraperitoneally (n=10) or saline (n=9) for 9 weeks. Diabetic plus 10 non-diabetic C57BL/6 mice underwent left femoral artery ligation and 5 days later angiogenesis imaging with (99m)Tc-Arg-Gly-Asp (RGD) nanoSPECT/CT. Twenty-four days later, hind limb blood flow was measured with ultrasound, the mice were euthanized, and tissue was taken for immunohistochemistry. The angiogenic imaging signal in ischemic limbs was higher in RAGE-ab treated versus saline treated mice at day 5 (3.1±1.4 vs 1.68±0.35, p=0.02) and blood flow was higher at day 24 (1.49±0.5 vs 0.61±0.39, p=0.04). Immunohistochemistry of ischemic muscles showed greater capillary density in the RAGE-ab treated group versus the vehicle-treated group (p<0.001) (NS from non-diabetic mice). In conclusion, treatment with anti-RAGE F(ab')2 in diabetic mice improves neovascularization in the ischemic leg., (© The Author(s) 2015.)

Optical agents targeting α(v)β₃ are potential tools to image the angiogenic response to limb ischemia. The left (L) femoral artery was ligated in 17 mice and sham surgery performed on the contralateral right (R) hindlimb. Seven days later, IntegriSense (2 nmol) was injected into 11 mice and 6 were probe controls. Six hours later, mice underwent optical imaging. Ratios of photon flux in the L/R limbs were calculated. Tissue was stained for α(v) , CD31, and lectin. The signal was increased in the ischemic limbs compared to contralateral legs and ratio of photon flux in L/R limb averaged 2.37. Control probe showed no hindlimb signal. IntegriSense colocalized with CD31 by dual fluorescent staining. Ratios for L/R hindlimbs correlated with quantitative lectin staining (r = 0.88, p = 0.003). Optical imaging can identify and quantify angiogenic response to hindlimb ischemia., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)