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

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, and transgene expression from adenovirus vectors can provide in vivo delivery of proteins. On the basis of this knowledge, we hypothesized that local administration of a replication-deficient adenovirus vector expressing complementary DNA for VEGF (AdVEGF) would induce collateral vessel formation in the setting of ischemia that could protect against subsequent acute vascular occlusion. METHODS: Hindlimb ischemia was induced in Sprague-Dawley rats by means of unilateral ligation of the common iliac artery immediately followed by administration of 4 x 10(9)-plaque-forming units VEGF, the control vector AdNull, or phosphate-buffered saline solution into the iliofemoral adipose tissue and thigh muscles. Untreated rats with common iliac ligation were used as an additional control group. RESULTS: Local VEGF expression was observed for 5 days in AdVEGF-treated rats but not in controls. Three weeks after ligation and vector administration, the ipsilateral femoral artery was ligated for a model of an acute vascular occlusion in the setting of preexisting ischemia. Blood flow to the ischemic hindlimb relative to the contralateral hindlimb evaluated with color microspheres demonstrated significantly increased blood flow in the AdVEGF-treated rats compared with each control group (p < 0.0001). Relative blood flow assessed by means of 99mTc-sestamibi radionuclide scans also demonstrated increased blood flow to the ligated hindlimb of AdVEGF-treated rats compared with each control group (p < 0.002). AdVEGF-treated rats also demonstrated increased vascularity in the ligated limb compared with each control group as assessed by means of angiography (p < 0.0001) and histologic quantification of blood vessels less than 80 microm diameter in local adipose tissue and capillaries per muscle fiber (p < 0.0002). AdVEGF treatment prevented a rise in femoral venous lactate femoral venous concentrations 1 hour after femoral artery ligation in control rats (p < 0.04). CONCLUSIONS: An adenovirus vector expressing VEGF complementary DNA is capable of stimulating an angiogenic response that protects against acute vascular occlusion in the setting of preexisting ischemia, suggesting that in vivo gene transfer of VEGF complementary DNA might be useful in prophylaxis of advancing arterial occlusive disease.

Therapeutic angiogenesis: a comparative study of the angiogenic potential of acidic fibroblast growth factor and heparin.

PURPOSE: Acidic fibroblast growth factor (aFGF) is a potent mitogen for vascular and other mesenchymal cells in vitro that can induce angiogenesis in vivo. Although heparin has no mitogenic potential of its own, it is an important aFGF cofactor in vitro and may also be capable of stimulating angiogenesis. Because the development of a collateral vasculature in response to ischemia appears to be dependent on angiogenesis, we compared the abilities of aFGF with or without heparin and heparin alone to accelerate angiogenesis in a rat hind limb ischemia model. METHODS: Daily subcutaneous injections of saline solution (1 ml), heparin (0.05 mg), or human recombinant aFGF with or without heparin (1 microgram aFGF, 0.05 mg heparin) were administered into the hind limb region distal to the point of unilateral femoral artery ligation in the rat for the 10 days immediately after vascular occlusion. Angiogenicity was determined by histologic assessment of treatment outcomes. RESULTS: Histologic assessment of the number of vessels per microscopic field 10 days after vascular ligation in the fibrofatty tissues distal to the ligation point had the following results: saline solution, 10 +/- 4 vessels; heparin, 13 +/- 4 vessels (p < 0.05 vs saline solution); aFGF, 26 +/- 8 vessels; and aFGF/heparin 36 +/- 8 vessels (aFGF, aFGF/ heparin, p < 0.001 vs saline solution). Similar increases in vascularization were also noted in the skeletal muscle tissues distal to the vascular ligation point. Immunohistochemical analysis for the presence of proliferating cell nuclear antigen, a marker for mitogenic activity, demonstrated corresponding increases in proliferating cell nuclear antigen labeling for each of the treatment groups, expressed as a percentage of total vascular cell nuclei, as follows: saline solution, 7% +/- 2%; heparin, 21% +/- 8% (p < 0.05 vs saline solution); aFGF, 67% +/- 9%; and aFGF/heparin, 83% +/- 5% (aFGF, aFGF/heparin, p < 0.001 vs saline solution). CONCLUSIONS: The increased vascularization and mitogenic activity demonstrated by these respective studies suggest that angiogenesis is significantly accelerated by the administration of heparin alone and is accelerated to a greater extent by the administration of aFGF with or without heparin. The aFGF/heparin regimen may represent an optimal means of augmenting collateral vessel growth to relieve ischemia in the clinical setting.

Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis.

Omentum has been used clinically to promote wound healing and to stimulate the revascularization of ischemic tissues. The biologic mechanism responsible for these effects has, however, not yet been defined. A number of polypeptide growth factors that possess potent angiogenic properties have recently been identified, and we therefore sought to determine whether one of these growth factors might be responsible for the angiogenic properties of the omentum. The levels of vascular endothelial growth factor (VEGF) protein in a number of rat tissues and organs were analyzed by Western and enzyme immunoassay analysis. Because omentum was found to have the greatest VEGF concentrations of the tissues examined, antibody neutralization, transcription inhibition assays, and Northern blot analysis were performed under hypoxic and normoxic conditions on tissues extractions and primary tissue cultures of omentum to further characterize the functional significance of VEGF expression in these tissues. The omentum demonstrated the highest VEGF secretion rate as well as the highest concentration of VEGF protein of the various rat tissues and organs examined. Fractionation studies of the omentum furthermore demonstrated that omental adipocytes, rather than the stromal-vascular cells, were the primary source of VEGF protein. An endothelial cell mitogenic assay showed that a major portion of the mitogenic activity of heparin-binding proteins and conditioned media derived from omentum was abolished by VEGF antibody. Additional studies with the transcription inhibitor actinomycin-D furthermore demonstrated that the VEGF gene was continuously transcribed in the rat omental adipocytes. Incubation of the omental adipocytes under hypoxic conditions induced approximately a 1.7-fold increase in VEGF protein expression, which was abolished by actinomycin-D. Northern blot analysis demonstrated that hypoxia resulted in upregulation of the VEGF mRNA in the hypoxia-cultured omental adipocytes, suggesting that the augmentation of VEGF expression in omental adipocytes by hypoxia occurs at the transcriptional level. These data suggest that VEGF is the major angiogenic factor produced by omentum and possibly underlies the mechanism of omentum-induced angiogenesis. Augmented expression of VEGF by omental cells under hypoxic conditions may furthermore reflect the mechanism responsible for enhancing the angiogenic activity of omentum in the setting of ischemia.