title: A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages.
abstract: BACKGROUND
  The cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFalpha release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-kappaB) activation.
  
  
  METHODS
  Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 muM), their respective furazan NO-free counterparts (B16, B15; 10 muM), aspirin (10 muM), existing nitroaspirin (NCX4016; 10 muM), an NO donor (DEA/NO; 10 muM) or dexamethasone (1 muM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFalpha release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-kappaB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-kappaB, IkappaBalpha (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-kappaB were determined.
  
  
  RESULTS
  B8 significantly reduced TNFalpha release from LPS-treated macrophages to 36 +/- 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFalpha release to 28 +/- 5, and 49 +/- 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 muM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IkappaBalpha and nuclear translocation of the p65 NF-kappaB subunit was inhibited by the active NO-furoxans.
  
  
  CONCLUSIONS
  Here we show that furoxan-aspirin, B8, significantly reduces TNFalpha release from both monocytes and macrophages and suggest that inhibition of NF-kappaB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, EH16 4TJ, UK. a.g.rossi@ed.ac.uk.
authors.forename: Catriona M
authors.initials: CM
authors.lastname: Turnbull
authors.forename: Paolo
authors.initials: P
authors.lastname: Marcarino
authors.forename: Tara A
authors.initials: TA
authors.lastname: Sheldrake
authors.forename: Loretta
authors.initials: L
authors.lastname: Lazzarato
authors.forename: Clara
authors.initials: C
authors.lastname: Cena
authors.forename: Roberta
authors.initials: R
authors.lastname: Fruttero
authors.forename: Alberto
authors.initials: A
authors.lastname: Gasco
authors.forename: Sarah
authors.initials: S
authors.lastname: Fox
authors.forename: Ian L
authors.initials: IL
authors.lastname: Megson
authors.forename: Adriano G
authors.initials: AG
authors.lastname: Rossi