Gene transfer of human endothelial nitric oxide synthase inhibits proliferation of human vascular smooth muscle cells and the mechanism

Abstract

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis. Key words: Endothelial nitric oxide synthase; Cell cycle; p27