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(Circulation. 2003;108:2250.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Angiogenesis Induced by Endothelial Nitric Oxide Synthase Gene Through Vascular Endothelial Growth Factor Expression in a Rat Hindlimb Ischemia Model

Tsunetatsu Namba, MD; Hiromi Koike; Kazushi Murakami, MD, PhD; Motokuni Aoki, MD, PhD; Hirofumi Makino, MD; Naotaka Hashiya, MD; Toshio Ogihara, MD, PhD; Yasufumi Kaneda, MD, PhD; Masakazu Kohno, MD, PhD; Ryuichi Morishita, MD, PhD

From the Division of Gene Therapy Science (T.N., Y.K.), the Division of Clinical Gene Therapy (H.K., M.A., H.M., N.H., R.M.), and the Department of Geriatric Medicine (K.M., M.A., H.M., N.H., T.O.), Osaka University Medical School, Osaka, and the Second Department of Internal Medicine (T.N., K.M., M.K.), Kagawa Medical University, Kagawa, Japan.

Correspondence to Ryuichi Morishita, MD, PhD, Professor, Division of Clinical Gene Therapy, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail morishit{at}cgt.med.osaka-u.ac.jp

Received August 5, 2002; de novo received March 31, 2003; revision received July 9, 2003; accepted July 10, 2003.

Background— Because the mechanism of the angiogenic property of nitric oxide (NO) was not fully understood in vivo, we focused on the role of vascular endothelial growth factor (VEGF) in angiogenesis induced by endothelial NO synthase (eNOS) gene transfer.

Methods and Results— After intramuscular injection of eNOS DNA into a rat ischemic hindlimb, transfection of eNOS vector resulted in a significant increase in eNOS protein 1 week after transfection. In addition, tissue concentrations of nitrite and nitrate were significantly increased in rats transfected with the eNOS gene up to 2 weeks after transfection. The increase in tissue nitrite and nitrate concentrations was completely inhibited by N^G-nitro-L-arginine methyl ester (L-NAME). In contrast, serum concentrations of nitrite and nitrate and blood pressure were not changed by eNOS gene transfer. Importantly, overexpression of the eNOS gene resulted in a significant increase in peripheral blood flow, whereas L-NAME inhibited the increase in blood flow. Interestingly, basal blood flow was significantly lower in rats treated with L-NAME than in control rats. A significant increase in capillary number was consistently detected in rats transfected with the eNOS gene at 4 weeks after transfection, accompanied by a significant increase in VEGF. Moreover, administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS plasmid injection.

Conclusions— Overall, intramuscular injection of bovine eNOS plasmid induced therapeutic angiogenesis in a rat ischemic hindlimb model, a potential therapy for peripheral arterial disease. The stimulation of angiogenesis by NO might be due to upregulation of local VEGF expression.

Key Words: peripheral vascular disease • endothelium • angiogenesis • genes • nitric oxide

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