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 o f vascular diseases such as atherosclerosis and restenosis. Nitric oxide (N O) donors and gene transfer of endothelial nitric oxide synthase (eNOS) hav e been shown to inhibit SMC proliferation. NO may cause this effect by dela ying cell cycle progression and/or induction of apoptosis, The aim of the c urrent study was to examine the mechanism of eNOS-mediated inhibition of SM C proliferation. In addition, the effect of eNOS expression in vascular SMC s on the expression of the cyclin dependent kinase inhibitors, p27 and p21 was examined. Methods: SMCs were transduced with an adenoviral vector encod ing eNOS (AdeNOS) or beta-galactosidase (Ad beta Gal) at a multiplicity of infection of 100. Non-transduced cells served as additional controls. Trans gene expression was sought by NADPH diaphorase staining, immunohistochemist ry and Western Blotting. Functionality of the recombinant protein was asses sed by measurement of cGMP. Cell cycle analysis was performed by flow cytom etry and p27 and p21 expression were studied by western blot analysis. Apop tosis was sought by Annexin V staining and DNA laddering. Results: eNOS exp ression 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. Conclusion: eNOS gene transfer to vascular SMCs inhibits cell prolif eration via upregulation of p27 and p21 resulting in a delay in cell cycle progression. (C) 2000 Elsevier Science B.V. All rights reserved.