Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF 165 Plasmid Gene Delivery to Chronically Ischemic Skeletal Muscle

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Abstract
Current methods of gene delivery for therapeutic angiogenesis are invasive, requiring either intraarterial or intramuscular administration. A noninvasive method of gene delivery has been developed using ultrasound-mediated destruction of intravenously administered DNA-bearing carrier microbubbles during their microcirculatory transit. Here we show that chronic ischemia could be markedly improved by ultrasound-mediated destruction of microbubbles bearing vascular endothelial growth factor-165 (VEGF165) plasmid DNA. Using a model of severe chronic hindlimb ischemia in rats, we demonstrated that ultrasound mediated VEGF165/green fluorescent protein (GFP) plasmid delivery resulted in a significant improvement in microvascular blood flow by contrast-enhanced ultrasound, and an increased vessel density by fluorescent microangiography, with minimal changes in control groups. The improvement in tissue perfusion was attributed predominantly to increases in noncapillary blood volume or arteriogenesis, with perfusion peaking at 14 days after delivery, followed by a partial regression of neovascularization at 6 weeks. Transfection was localized predominantly to the vascular endothelium of arterioles in treated ischemic muscle. RT-PCR confirmed the presence of VEGF165/GFP mRNA within treated ischemic muscle, being highest at day 3 postdelivery, and subsequently decreasing, becoming almost undetectable by 6 weeks. We found a modulation of endogenous growth factor expression in VEGF-treated ischemic muscle, consistent with a biologic effect of ultrasound mediated gene delivery. The results of our study demonstrate the utility of ultrasonic destruction of plasmid-bearing microbubbles to induce therapeutic arteriogenesis in the setting of severe chronic ischemia.

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