Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis.
BACKGROUND
We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability.


METHODS
An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated.


RESULTS
AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity.


CONCLUSIONS
Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.
Tzeng
E
E
Department of Surgery, University of Pittsburgh, Pa. 15261, USA.
Kim
Y M
YM
Pitt
B R
BR
Lizonova
A
A
Kovesdi
I
I
Billiar
T R
TR
D000256:Adenoviridae; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D011651:Pulmonary Artery; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012756:Sheep; D013936:Thymidine / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis; D019323:omega-N-Methylarginine / Q000494:pharmacology
D018076:DNA, Complementary; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; D019323:omega-N-Methylarginine; D004247:DNA; D019001:Nitric Oxide Synthase; D001616:beta-Galactosidase; D013936:Thymidine