Effects of VEGF₁₂₁ and/or VEGF₁₆₅ gene transfection on collateral circulation development

Background and purpose: Angiogenesis is regulated by various factors, including vascular endothelial growth factor (VEGF). Five isoforms of VEGF have been discovered: VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆. The teleologic basis for the various VEGF isoforms remains unclear, but different VEGF isoforms may mediate distinct endothelial cell functions such as angiogenesis, vascular permeability, and differentiation. We sought to determine the effects of various VEGF isoforms on angiogenesis under ischemic conditions in rabbits. Methods: The effects of VEGF₁₂₁ and/or VEGF₁₆₅ gene transfection on collateral circulation development in ischemic rabbit hindlimb muscles were investigated by rising naked plasmids encoding VEGF₁₂₁ or VEGF₁₆₅ (pVEGF₁₂₁ or VEGF₁₆₅), either individually or in combination, pCMVβ was used as the control plasmid. The femoral artery on one side of New Zealand White rabbits was ligated. Ten days later, the ischemic muscles received direct intramuscular injection of pVEGF₁₂₁ (500 μg), pVEGF₁₆₅ (500 μg), or pVEGF₁₂₁ (250 μg) + pVEGF₁₆₅ (250 μg) in experimental groups, while pCMVβ (500 μg) was used in the control group. Therapeutic effects were evaluated 30 days later by anatomic and physiologic analysis. Results: Internal iliac angiography showed strong development of collateral circulation in all of the pVEGF-treatcd groups. In contrast, collateral arteries developed weakly in the control group. Combination treatment with both pVEGF₁₂₁ and pVEGF₁₆₅ did not result in additional improvement compared with pVEGF₁₂₁ or pVEGF₁₆₅ treatment alone (angiographic scores: pVEGF₁₂₁ = 0.85 ± 0.10; pVEGF₁₆₅, = 0.81 ± 0.11; pVEGF₁₂₁ + pVEGF₁₆₅ = 0.83 ± 0.09; control = 0.53 ± 0.09; p < 0.01). A favorable response in the development of circulation at the capillary level with pVEGF₁₂₁ and/or pVEGF₁₆₅ versus pCMVβ was also found. Blood pressure measurement and regional blood flow measurement using colored microspheres revealed similar results. Conclusions: Our results show that direct intramuscular injection of naked DNA encoding VEGF₁₂₁ or VEGF₁₆₅, individually or in combination, is an effective method for gene transfer in an animal model of ischemic limbs and results in augmented collateral vascular development and tissue perfusion.