Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection.
OBJECTIVE
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 (VEGF(165)) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF(165)/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 VEGF(165) 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 VEGF(165)/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 VEGF(165) 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.
Kobulnik
Jeremy
J
Division of Cardiology, Keenan Research Centre in the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada.
Kuliszewski
Michael A
MA
Stewart
Duncan J
DJ
Lindner
Jonathan R
JR
Leong-Poi
Howard
H
D000818:Animals; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006614:Hindlimb / Q000098:blood supply; D007150:Immunohistochemistry; D007273:Injections, Intramuscular; D007511:Ischemia / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000473:pathology; D045423:Microbubbles; D008833:Microcirculation / Q000187:drug effects; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging / Q000628:therapy*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D014463:Ultrasonography; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage*
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A