Nitric oxide-dependent cytoskeletal changes and inhibition of endothelial cell migration contribute to the suppression of angiogenesis by RAD50 gene transfer.
Previous reports showed that human RAD50 (hRAD50) gene delivery induced regression of an experimental rat tumor and porcine neointimal hyperplasia. In this study, we examined the effects of hRAD50 on the morphological changes and migration of endothelial cells (EC) as possible mechanisms by which hRAD50 might block angiogenesis. Quantitative image analysis revealed significant inhibition of the number and total area of blood vessels in rat tumor tissues following hRAD50 gene delivery. hRAD50 distorted actin and tubulin arrangements, and significantly reduced the F/G-actin ratio and increased the nitric oxide (NO) production in the primary cultured human EC. These effects were blocked by pretreatment with L-NAME (N(G)-nitro-L-arginine-methyl ester), a NO synthase inhibitor. FACScan analysis showed that NO was involved in the necrosis and apoptosis of EC by hRAD50. hRAD50 also inhibited EC migration in an in vitro wound-healing model. These results indicate that NO-dependent cytoskeletal changes and inhibition of EC migration contribute to the suppression of angiogenesis by hRAD50 delivery in vivo.
Kook
Hyun
H
Research Institute of Medical Sciences and Medical Research Center for Gene Regulation, Chonnam National University Medical School, Kwangju, South Korea.
Ahn
Kyu Youn
KY
Lee
Song Eun
SE
Na
Hee Sam
HS
Kim
Kyung Keun
KK
D017766:Acid Anhydride Hydrolases; D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002465:Cell Movement; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004195:Disease Models, Animal; D004727:Endothelium / Q000166:cytology*; D006801:Humans; D009336:Necrosis; D009374:Neoplasms, Experimental / Q000098:blood supply; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured; D014945:Wound Healing
D000199:Actins; D004268:DNA-Binding Proteins; D009569:Nitric Oxide; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes