eNOS gene transfer to vascular smooth muscle cells inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis.
OBJECTIVE
Smooth muscle cell (SMC) proliferation is a critical component of vascular diseases such as atherosclerosis and restenosis. Nitric oxide (NO) donors and gene transfer of endothelial nitric oxide synthase (eNOS) have been shown to inhibit SMC proliferation. NO may cause this effect by delaying cell cycle progression and/or induction of apoptosis. The aim of the current study was to examine the mechanism of eNOS-mediated inhibition of SMC proliferation. In addition, the effect of eNOS expression in vascular SMCs on the expression of the cyclin dependent kinase inhibitors, p27 and p21 was examined.


METHODS
SMCs were transduced with an adenoviral vector encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta Gal) at a multiplicity of infection of 100. Non-transduced cells served as additional controls. Transgene expression was sought by NADPH diaphorase staining, immunohistochemistry and Western Blotting. Functionality of the recombinant protein was assessed by measurement of cGMP. Cell cycle analysis was performed by flow cytometry and p27 and p21 expression were studied by western blot analysis. Apoptosis was sought by Annexin V staining and DNA laddering.


RESULTS
eNOS expression was detected in transduced SMCs. cGMP levels were increased in eNOS-transduced compared to control cells. Expression of eNOS in SMCs resulted in a delay in cell cycle progression and upregulation of p27 and p21. There was no increase in apoptosis detected in eNOS transduced cells after 24 or 72 h.


CONCLUSIONS
eNOS gene transfer to vascular SMCs inhibits cell proliferation via upregulation of p27 and p21 resulting in a delay in cell cycle progression.
Sato
J
J
Department of Endocrinology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Nair
K
K
Hiddinga
J
J
Eberhardt
N L
NL
Fitzpatrick
L A
LA
Katusic
Z S
ZS
O'Brien
T
T
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000032:analysis / Q000378:metabolism*; D005434:Flow Cytometry; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D013552:Swine; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D018844:Cyclin-Dependent Kinases