Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate

Objective: Overexpression of the inducible nitric oxide synthase (iNOS) gen e inhibits neointimal hyperplasia after arterial injury. The purpose of thi s study was to examine the mechanism by which nitric oxide (NO) inhibits va scular smooth muscle cell (VSMC) proliferation, specifically focusing on si gnaling pathways known to be activated by NO, including cyclic guanosine mo nophosphate (cGMP), p53, and p42/44 mitogen-activated protein kinase (MAPX) . Methods and Results: VSMCs that were subjected to iNOS gene transfer demons trated a reduction in proliferation (80%) that was associated with a marked increase in p21 expression. The antiproliferative and p21 stimulatory effe cts of NO were not suppressed by the soluble guanylate cyclase inhibitor OD Q, implicating cGMP-independent signaling The role of p53 in NO-mediated up regulation of p21 and inhibition of proliferation was evaluated using p53 - /- VSMCs. A similar reduction in cellular proliferation and upregulation of p21 expression were achieved with iNOS gene transfer as well as treatment with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrating the p53-independent nature of these NO-mediated pathways. The transfer of the iNOS gene activated the p42/44 MAPK, and inhibition of this MAPK pathway wi th PD98059 partially blocked the antiproliferative effects of NO and comple tely inhibited the p21 stimulatory effects of NO. For cofirmation that iNOS overexpression upregulated p21 in vivo, injured rat carotid arteries were infected with an adenoviral vector carrying the iNOS gene and demonstrated a marked upregulation of p21 expression at three days. However, the ability of NO to inhibit VSMC proliferation does not solely depend on p21 upregula tion since the NO-donor SNAP-inhibited VSMC proliferation in p21 -/- VSMCs. Conclusion: Nitric oxide inhibits VSMC proliferation in association with th e upregulation of p21; both occur independent of p53 and cGMP while being p artially mediated through the p42/44 MAPK signaling cascade. This represent s one potential mechanism by which NO inhibits VSMC proliferation.