Imaging VEGF Receptors and α v β 3 Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease

摘要

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.