Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion

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Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion Charles A. Mack, MD, Christopher J. Magovern, MD, Kurt T. Budenbender, BS, Shailen R. Patel, MD, Eric A. Schwarz, MD, Pat Zanzonico, PhD, Barbara Ferris, BS, Timothy Sanborn, MD, O.Wayne Isom, MD, Ronald G. Crystal, MD, Todd K. Rosengart, MD Journal of Vascular Surgery Volume 27, Issue 4, Pages 699-709 (April 1998) DOI: 10.1016/S0741-5214(98)70236-8 Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Schematic of experimental design. Immediately after ligation of the left common iliac artery, AdVEGF, AdNull, or phosphate-buffered saline solution was administered into the left hindlimb adipose tissue and muscle (total dose 4 × 109 pfu). An additional control group underwent left common iliac artery ligation with no injection. Twenty-one days later, the ipsilateral common femoral artery was ligated. The rats were examined for blood flow and vascularity as indicated. Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Quantification of VEGF expression in injected tissues at various time points by means of enzyme-linked immunoassay. Immediately after common iliac artery ligation, rats (n = 3 per time point) received 109 pfu of AdVEGF (closed circles) or the control vector AdNull (open circles). A. Adipose tissue. B. Muscle. Vector administration was at time zero; the first time point represents naive tissue before treatment. Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Representative 99mTc-labeled sestamibi images at 21 days for the four experimental groups of rats. A, Naive. B, Phosphate-buffered saline solution. C, Control vector AdNull. D-F, AdVEGF. Shown for each are the color images acquired from the dorsal and ventral detectors of the lower trunk, right (R) and left (L) hindlimbs, and the tail. The central abdominal regions are deleted. Yellow, highest flow. Orange, purple, and blue represent decreasing relative flow. Arrows indicate the ligated left hindlimb. There is reconstitution of flow in the left hindlimb only in the AdVEGF-treated rat. Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Relative blood flow assessed by means of 99mTc-labeled sestamibi imaging in hindlimbs immediately after ligation of left femoral artery. The experimental model is as described in Fig. 1. Relative blood flow is quantified as the ratio of left versus right geometric mean counts per pixel of dorsal and ventral views of the calf of the ligated hindlimb (left) to the same area of the contralateral hindlimb (right). Each experimental condition included a minimum of seven rats (AdVEGF versus controls p < 0.05). Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Relative blood flow assessed with color microspheres in hindlimbs. The experimental model is as described in Fig. 1. After ligation of the femoral artery, color microspheres were injected into the infrarenal aorta. Relative blood flow is expressed as microspheres per gram tissue in the ligated hindlimb (left) versus microspheres per gram tissue in the contralateral (right) hindlimb (A) and as raw number of microspheres observed in the total lower hindlimb muscles of the ligated hindlimbs (left) and contralateral hindlimb (right) (B). Each experimental condition included a minimum of eight rats (AdVEGF versus controls p < 0.004). Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Angiographic evaluation of vascularity in ischemic hindlimbs after ligation of the left femoral artery. The experimental model is as described in Fig. 1. Angiographic quantification of vascularity was performed by three blinded observers. Each experimental condition included a minimum of eight rats (AdVEGF versus controls p < 0.001). Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Results of histologic evaluation of vascularity in adipose tissue and skeletal muscle in ischemic hindlimbs after ligation of the left femoral artery. The experimental model is as described in Fig. 1. A, Adipose tissue. Histologic quantification of small-vessel (less than 80 mm diameter) count is expressed as mean number of vessels per square millimeter (AdVEGF versus controls p < 0.0003.). B. Skeletal muscle. Histologic quantification of capillary number in skeletal muscle is depicted as capillaries per muscle fiber (AdVEGF versus controls p < 0.002). Each experimental condition includes a minimum of seven rats. Journal of Vascular Surgery 1998 27, 699-709DOI: (10.1016/S0741-5214(98)70236-8) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions