title: Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation.
abstract: 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.
authors.affiliation: Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Joseph-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Heinloth
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
authors.forename: Barbara
authors.initials: B
authors.lastname: Fischer
authors.forename: Jan
authors.initials: J
authors.lastname: Galle
mesh_terms: 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*
chemical_list: 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