Angiogenesis induced by endothelial nitric oxide synthase gene through vascular endothelial growth factor expression in a rat hindlimb ischemia model.
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.


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 NG-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.
Division of Clinical Gene Therapy, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0871, Osaka, Japan.
Tsunetatsu
T
Namba
Hiromi
H
Koike
Kazushi
K
Murakami
Motokuni
M
Aoki
Hirofumi
H
Makino
Naotaka
N
Hashiya
Toshio
T
Ogihara
Yasufumi
Y
Kaneda
Masakazu
M
Kohno
Ryuichi
R
Morishita
D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D002417:Cattle; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006614:Hindlimb / Q000098:blood supply / Q000378:metabolism; D007511:Ischemia / Q000628:therapy*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000235:genetics*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow / Q000187:drug effects / Q000235:genetics; D013997:Time Factors; D015854:Up-Regulation / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D004791:Enzyme Inhibitors; D042461:Vascular Endothelial Growth Factor A; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019331:NG-Nitroarginine Methyl Ester