Nitric oxide-releasing NSAIDs inhibit interleukin-1 beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNF alpha

Background: Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. Aim: To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNF alpha). In other systems, TNF alpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1 beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. Methods: Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). Results: In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated ThF alpha release. Exposure to TNF alpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNF alpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNF alpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP3 2-like proteases. NO-NSAIDs prevented IL-1 beta release from endotoxin-stimulated macrophages. Conclusions: NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.