Adenoviral-mediated gene transfer of nitric oxide synthase isoforms and vascular cell proliferation.
OBJECTIVE
Many vascular diseases are associated with reduced nitric oxide (NO) bioavailability. Nitric oxide synthase (NOS) gene therapy to the vasculature is a possible treatment for vascular disease as a means of increasing NO bioavailability, and this may be achieved using any of the NOS isoforms. The aim of our study was to compare the effects of adenoviral-mediated overexpression of the most commonly used NOS isoforms eNOS and iNOS on vascular cell proliferation.


METHODS
Human coronary artery smooth muscle cells (HCSMCs) and human umbilical vein endothelial cells (HUVECs) were transduced with adenoviral vectors encoding eNOS or iNOS at a multiplicity of infection of 100. Control cells were exposed to AdNull (empty vector) or diluent alone. Transgene expression was sought by Western blotting. The Greiss assay was used to measure nitrite levels. Cell proliferation was assessed by cell counting on days 0, 3 and 6. Apoptosis was sought using FACS analysis. Angiogenesis was measured using a commercially available in vitro kit.


RESULTS
Expression of both isoforms was detected in transduced cells by Western blot at all three time points. NOS transduction resulted in increased nitrite levels with higher levels seen in iNOS- compared to eNOS-transduced cells. Cell proliferation was diminished in AdeNOS- and AdiNOS-transduced cells compared with non-transduced cells on days 3 and 6 in both HCSMCs and HUVECs. Apoptosis was not detected in either cell line with either of the isoforms at any timepoint studied. Both eNOS and iNOS gene transfer caused a reduction in angiogenesis.


CONCLUSIONS
NOS gene transfer to both endothelial and vascular smooth muscle cells is antiproliferative and antiangiogenic. The biological effect is identical with both isoforms and there is no evidence to support a differential effect on endothelial and vascular smooth muscle cell biology.
Cooney
Ronan
R
Regenerative Medicine Institute (REMEDI), National Centre for Biomedical Engineering Sciences (NCBES), National University of Ireland, Galway, Ireland. timothy.obrien@nuigalway.ie
Hynes
Sean O
SO
Duffy
Aoife M
AM
Sharif
Faisal
F
O'Brien
Timothy
T
D000256:Adenoviridae / Q000235:genetics*; D020533:Angiogenesis Inhibitors; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002455:Cell Division; D019976:Cloning, Organism; D003331:Coronary Vessels / Q000166:cytology / Q000502:physiology*; D003587:Cytomegalovirus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D011401:Promoter Regions, Genetic
D020533:Angiogenesis Inhibitors; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III