Nitric oxide modulates expression of cell cycle regulatory proteins - A cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation

Background-We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs). Methods and Results-The NO donor diethylenetriamineNONOate (10(-5) to 10(-3 ) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BE in a concentration-dependen t manner. This effect was not observed with disintegrated diethylenetriamin eNONOate or with the parent compound, diethylene-triamine, Adenoviral trans fection of endothelial NO synthase (NOS) inhibited proliferation in respons e to FCS, which was prevented with N-G-nitro-L-arginine methyl ester. NOS o verexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selecti vely enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined wit h trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas c yclin E remained unchanged. p21 expression was induced, and p27 remained un altered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% F CS was barely inhibited with 8-bromo-cCMP (10(-3) mol/L) but was blunted wi th both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects o f NO. Conclusions-NO inhibits VSMC proliferation by specifically changing the exp ression and activity of cell cycle regulatory proteins, which may occur ind ependent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.