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Imaging VEGF Receptors and αvβ3 Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease

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Abstract

Purpose

To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. αvβ3 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 [99mTc]DOTA-PEG-scVEGF ([99mTc]scV) (n = 8) or with αvβ3-targeting tracer [99mTc]HYNIC-cycloRGD ([99mTc]RGD) (n = 8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [99mTc]scV uptake, six additional mice received a mixture of [99mTc]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 αvβ3, 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 [99mTc]scV compared to ligated hindlimbs in mice injected with [99mTc]RGD (p = 0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [99mTc]scV than for [99mTc]RGD (p = 0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and αvβ3, 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 αvβ3, 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, [99mTc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [99mTc]RGD tracer-targeting αvβ3. Immunofluorescent analysis suggests that uptake of [99mTc]scV and [99mTc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [99mTc]scV may be due to the presence of VEGF receptors on additional cell types.

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Financial Support

5 P01 HL060901-12 (Schmidt, PI) NIH/NHLBI, Title: RAGE and mechanisms of Vascular Dysfunction.

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Authors and Affiliations

  1. Department of Medicine, Columbia University Medical Center, 650 W. 168th St. BB 10, room 1019, New York, NY, 10032, USA

    Yared Tekabe, Qing Li, Geping Zhang, Jordan Johnson & Lynne L. Johnson

  2. Department of Medicine, New York University Medical Center, New York, NY, USA

    Ann Marie Schmidt

  3. SibTech, Inc. 115A Commerce Drive, Brookfield, CT, 06804, USA

    Marina Backer & Joseph Backer

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  1. Yared Tekabe
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  2. Qing Li
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Correspondence to Yared Tekabe.

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Tekabe, Y., Li, Q., Zhang, G. et al. Imaging VEGF Receptors and αvβ3 Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease. Mol Imaging Biol 20, 963–972 (2018). https://doi.org/10.1007/s11307-018-1191-1

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