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(Circulation Research. 2007;101:295.)
© 2007 American Heart Association, Inc.

Integrative Physiology

Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF₁₆₅ Plasmid Gene Delivery to Chronically Ischemic Skeletal Muscle

Howard Leong-Poi, Michael A. Kuliszewski, Michael Lekas, Matthew Sibbald, Krystyna Teichert-Kuliszewska, Alexander L. Klibanov, Duncan J. Stewart, Jonathan R. Lindner

From the Division of Cardiology (H.L.P., M.A.K., M.L., M.S., K.T.K., D.J.S.), Keenan Research Centre in the Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, Ontario, Canada; the Cardiovascular Division (J.R.L.), Oregon Health and Science University, Portland; and the University of Virginia (A.L.K.), Charlottesville.

Correspondence to Howard Leong-Poi, MD, 7-052 Bond Wing, St Michael’s Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. E-mail leong-poih{at}smh.toronto.on.ca

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.

Key Words: angiogenesis • gene therapy • contrast ultrasound • peripheral vascular disease • chronic ischemia

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