Nitric oxide‐releasing NSAIDs inhibit interleukin‐1β converting enzyme‐like cysteine proteases and protect endothelial cells from apoptosis induced by TNFα

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Abstract
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‐α (TNFα). In other systems, TNFα‐induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL‐1β 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 TNFα release. Exposure to TNFα 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 TNFα‐induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFα‐induced cysteine protease activation. Inhibition of caspase activity was due to S‐nitrosylation of ICE/CPP32‐like proteases. NO‐NSAIDs prevented IL‐1β 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.