Imaging VEGF Receptors and αβ Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease.

Purpose: To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. αβ 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 [Tc]DOTA-PEG-scVEGF ([Tc]scV) (n = 8) or with αβ-targeting tracer [Tc]HYNIC-cycloRGD ([Tc]RGD) (n = 8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [Tc]scV uptake, six additional mice received a mixture of [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 αβ, 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 [Tc]scV compared to ligated hindlimbs in mice injected with [Tc]RGD (p = 0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [Tc]scV than for [Tc]RGD (p = 0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and αβ, 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 αβ 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, [Tc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [Tc]RGD tracer-targeting αβ. Immunofluorescent analysis suggests that uptake of [Tc]scV and [Tc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [Tc]scV may be due to the presence of VEGF receptors on additional cell types.