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 Cu(2+), 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 microg/ml) induced cytochrome c release that was prevented by preincubation with the NO-donor S-nitrosoglutathione (GSNO) (100 microM) or with the cGMP analogue 8-br-cGMP (100 microM) for 15 h. In cells co-treated with GSNO and the soluble guanylate cyclase (sGC) inhibitor oxadialoquinoxalione (ODQ, 10 microM, 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 microM) and ODQ (10 microM) 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 prevented 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 cytochrome 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.
Heinloth
Alexandra
A
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Joseph-Schneider-Strasse 2, 97080 Würzburg, Germany.
Brüne
Bernhard
B
Fischer
Barbara
B
Galle
Jan
J
D017209:Apoptosis / Q000187:drug effects*; D018932:Chemokine CCL2 / Q000096:biosynthesis; D006152:Cyclic GMP / Q000008:administration & dosage; D003574:Cytochrome c Group / Q000187:drug effects* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006162:Guanylate Cyclase / Q000494:pharmacology*; D006801:Humans; D008077:Lipoproteins, LDL / Q000008:administration & dosage*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000008:administration & dosage; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026422:S-Nitrosoglutathione / Q000008:administration & dosage; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects* / Q000378:metabolism*
D018932:Chemokine CCL2; D003574:Cytochrome c Group; D008077:Lipoproteins, LDL; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D006162:Guanylate Cyclase; D006152:Cyclic GMP