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PRE-CLINICAL RESEARCH

Comparison of Gene Delivery Techniques for Therapeutic Angiogenesis

Ultrasound-Mediated Destruction of Carrier Microbubbles Versus Direct Intramuscular Injection

Jeremy Kobulnik, MD^_*, Michael A. Kuliszewski, BSc^_*, Duncan J. Stewart, MD^_*, Jonathan R. Lindner, MD and Howard Leong-Poi, MD^_*,_*

^_* Division of Cardiology, Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada
Cardiovascular Division, Oregon Health & Science University, Portland, Oregon

Manuscript received May 10, 2009; revised manuscript received July 6, 2009, accepted July 6, 2009.

^_* Reprint requests and correspondence: Dr. Howard Leong-Poi, 7-052 Bond Wing, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada (Email: leong-poih{at}smh.toronto.on.ca).

Objectives: This study was designed to compare the efficacy of angiogenic gene delivery by ultrasound-mediated (UM) destruction of intravenous carrier microbubbles to direct intramuscular (IM) injections.

Background: Current trials of gene therapy for angiogenesis remain limited by suboptimal, invasive delivery techniques.

Methods: Hind-limb ischemia was produced by iliac artery ligation in 99 rats. In 32 rats, UM delivery of green fluorescent protein (GFP)/vascular endothelial growth factor-165 (VEGF₁₆₅) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF₁₆₅/GFP plasmid. Remaining rats received no treatment. Before delivery (day 14 after ligation) and at days 17, 21, and 28 and week 8 after ligation, microvascular blood volume and microvascular blood flow to the proximal hind limbs were assessed by contrast-enhanced ultrasound (n = 8 per group). Total transfection was assessed by reverse transcriptase–polymerase chain reaction, and localization of transfection was determined by immunohistochemistry.

Results: By day 28, both IM and UM delivery of VEGF₁₆₅ produced significant increases in microvascular blood volume and microvascular blood flow. Whereas increases in microvascular blood volume were similar between treatment groups, microvascular blood flow was greater (p < 0.005) in UM-treated animals as compared with IM-treated animals, persisting to week 8. The VEGF₁₆₅/GFP messenger ribonucleic acid expression was greater (p < 0.05) for IM-treated animals. A strong GFP signal was detected for both groups and was localized to focal perivascular regions and myocytes around injection sites for IM and to the vascular endothelium of arterioles/capillaries in a wider distribution for UM delivery.

Conclusions: Despite lower transfection levels, UM delivery of VEGF₁₆₅ is as effective as IM injections. The UM delivery results in directed vascular transfection over a wider distribution, which may account for the more efficient angiogenesis.

Key Words: angiogenesis • gene therapy • chronic ischemia

Abbreviations and Acronyms   CEU = contrast-enhanced ultrasound   DNA = deoxyribonucleic acid   GFP = green fluorescent protein   FMA = fluorescent microangiography   IM = intramuscular   MBF = microvascular blood flow   MBV = microvascular blood volume   PBS = phosphate buffered saline   RNA = ribonucleic acid   UM = ultrasound-mediated   VEGF = vascular endothelial growth factor

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J. Am. Coll. Cardiol. 2009 54: A26. [Full Text] [PDF]