Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation

Background: Oxidatively modified low, density lipoprotein (OxLDL) induces apoptosis in vascular cells including macrophages, while NO exerts antiapoptotic effects. Here we studied the impact of nitric oxide (NO) on OxLDL-induced cytochrome c release, apoptosis, and expression of the proapoptotic p53 in macrophages. Methods: Human LDL was oxidised by Cu2+, and monocytes were prepared from human buffy coats. Differentiation to macrophages was achieved by culturing cells in the presence of human serum and was followed by detecting monocyte chemoattractant protein 1 (MCP-1) expression (RT-PCR). Cytochrome c release and p53 expression of macrophages were detected by immunoblotting, and apoptosis by visualisation of nuclear condensation. Results: OxLDL dose-dependently (50-200 mug/ml) induced cytochrome c release that was prevented by preincubation with the NO-donor S-nitrosoglutathione (GSNO) (100 muM) or with the cGMP analogue 8-br-cGMP (100 muM) for 15 h. In cells co-treated with GSNO and the soluble guanylate cyclase (sGC) inhibitor oxadialoquinoxalione (ODQ, 10 muM, 15 h), OxLDL-evoked cytochrome c release remained effective, indicating that NO acted via sGC-dependent cGMP formation. Parallel incubation of macrophages with 8-br-cGMP (100 muM) and ODQ (10 muM) for 15 h left the protective effect of 8-br-cGMP unaltered. Short pre-incubation (30 min) with GSNO or 8-br-cGMP was ineffective in preventing OxLDL-elicited cytochrome c release. Initiation of cytochrome c release in macrophages was paralleled by a dose-dependent accumulation of the proapoptotic factor p53, and by enhanced rate of nuclear condensation. Stabilisation of p53 was presented by preincubation with the NO-donor GSNO or 8-br-cGMP, thus implying a downmodulatory effect of cGMP on pathways that upregulate the tumor suppressor p53. Conclusions: OxLDL induces cytochrome c release and apoptosis in human macrophages in close association with p53 accumulation. NO attenuates OxLDL-induced cytochrorne c release and p53 accumulation via activation of sGC and cGMP formation. These effects may be of particular importance in arterial tissue with reduced NO activity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.