Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells.
Endothelial dysfunction is frequently involved in the pathogenesis of vascular disease. While nitric oxide (NO) inhibits smooth muscle cell proliferation, its effect on endothelial cell proliferation is unclear. The aim of this study was to determine if adenoviral-mediated gene transfer of endothelial NO synthase (eNOS) to human umbilical vein endothelial cells (HUVECs) would result in increased generation of NO and affect endothelial cell proliferation. HUVECs were transduced with adenoviral vectors encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta gal) or exposed to diluent (control). AdeNOS-transduced cells showed increased eNOS expression as detected by Western blot analysis, and increased concentrations of cGMP (control 0.7 +/- 0.1; Ad beta gal 0.9 +/- 0.2; AdeNOS 3.1 +/- 0.5 pmol/mg protein; p < 0.001) and nitrite (control 11.8 +/- 1.2; Ad beta gal 13.3 +/- 1.7; AdeNOS 21.1 +/- 2.2 nmol/mg protein/hour; p < 0.01). DNA synthesis as assessed by [(3)H]thymidine incorporation and cell counts were significantly reduced (by approximately 30%) in AdeNOS-transduced HUVECs. Expression of mitogen-activated protein kinase was also decreased in AdeNOS-transduced cells. This study shows that adenoviral-mediated gene transfer of eNOS to HUVECs inhibits endothelial cell proliferation.
Zanetti
M
M
Department of Endocrinology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Katusic
Z S
ZS
O'Brien
T
T
D000260:Adenoviruses, Human / Q000235:genetics; D002455:Cell Division; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D014471:Umbilical Veins; D001616:beta-Galactosidase / Q000032:analysis; D019323:omega-N-Methylarginine / Q000494:pharmacology
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009573:Nitrites; D011993:Recombinant Fusion Proteins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D020928:Mitogen-Activated Protein Kinases; D001616:beta-Galactosidase; D006152:Cyclic GMP