Published Online
on October 20, 2003

A more recent version of this article appeared on November 25, 2003

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Submitted on May 1, 2003
Revised on July 16, 2003
Accepted on July 17, 2003

Enhanced Therapeutic Angiogenesis by Cotransfection of Prostacyclin Synthase Gene or Optimization of Intramuscular Injection of Naked Plasmid DNA

Kazuya Hiraoka MD, Hiromi Koike , Seiji Yamamoto MD, Naruya Tomita MD, PhD, Chieko Yokoyama PhD, Tadashi Tanabe PhD, Takashi Aikou MD, PhD, Toshio Ogihara MD, PhD, Yasufumi Kaneda MD, PhD, and Ryuichi Morishita MD, PhD^*

From the Division of Gene Therapy Science (K.H., S.Y., Y.K.), Department of Geriatric Medicine (K.H., T.O.), Division of Clinical Gene Therapy (H.K., N.T., R.M.), Graduate School of Medicine, Osaka University; Department of Pharmacology (C.Y., T.T.), National Cardiovascular Research Center, Osaka; and First Department of Surgery (T.A.), Faculty of Medicine, Kagoshima University, Sakuragaoka, Kagoshima, Japan.

^* To whom correspondence should be addressed. E-mail: morishit{at}cgt.med.osaka-u.ac.jp.

Background--Although clinical trials of therapeutic angiogenesis by angiogenic growth factors with intramuscular injection of naked plasmid DNA have been successful, there are still unresolved problems such as low transfection efficiency. From this viewpoint, we performed the following modifications: (1) combination with vasodilation using prostacyclin and (2) changing the agents or volume of naked plasmid DNA in vivo.

Methods and Results--First, we examined cotransfection of the VEGF gene with the prostacyclin synthase gene in a mouse hindlimb ischemia model. Cotransfection of the VEGF gene with the prostacyclin synthase gene resulted in a further increase in blood flow and capillary density compared with single VEGF gene. Similar results were obtained with other angiogenic growth factors, such as hepatocyte growth factor (HGF). Alternatively, we changed the injection volume of the solution of plasmid DNA. Luciferase activity was increased in a volume-dependent manner. An increase in injection volume at 1 site rather than separate injections at multiple sites resulted in high transfection efficiency, which suggests that transfection of naked plasmid DNA is mediated by pressure. Interestingly, treatment with hyperbaric oxygen increased the transfection efficiency. Finally, we also examined the effects of different solutions. Saline and PBS, but not water, achieved high transfection efficiency. In addition, sucrose solution but not glucose solution resulted in high luciferase activity.

Conclusions--Overall, angiogenesis might be enhanced by cotransfection of prostacyclin synthase gene or an increase in injection volume and osmotic pressure. These data provide important information for the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.

Key words: peripheral vascular disease • vasodilation • angiogenesis • gene therapy

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