Prevention of gastric damage by no-releasing aspirin (NCX-4016) is mediated by inhibition of ICE/-like proteases in gastric mucosa and endothelial cells

Abstract

Background. Tumor necrosis factor-ct (TNFtx), a proinflammatory cytokine, is released in vivo and in vitro after exposure to non-steroidal anti-inflammatory drugs (NSAIDs). TNFct receptor binding leads to activation of multiple interleukin-1 converting enzyme (ICE)-like proteases (caspases) and endothelial cell death. Endothelial cell dysfunction is an early step in the process of NSAID-gastropathy. Nilric oxide(NO)-releasing NSAIDs, are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. NO-releasing agents (sodium nitroprusside [SNP] and L-Arginine) prevent TNF-induced ICE/caspase 1 activation in human endothelial ceils (HUVECs). Aims. To assess whether NO-NSAIDs modulate ICE-like proteases in vivo and in vitro and protect endothelial cells from TNFct-induced apoptosis. Methods. Macrophages, prepared from mouse spleen, were used to investigate TNFet release in vitro. Gastric damage was induced in rat by oral administration of 150 mg/kg aspirin or an equimolar dose (249 mg/kg) of NCX-4016 (NicOx, Milan, Italy). ICE-like activity was measured in cell Iysates and gastric homogenates using YVAD.AMC as substrate in a fluorimetric assay. HUVEC apoptosis was assessed by FACS-scan analysis. Results. In vitro studies demonstrated that aspirin, but not NCX-4016, released TNFct from mouse macrophages. Co-incubation with 1-100 laM SNP resulted in a concentrationdependent inhibition of aspirin-induced TNFct-release. In viw) aspirin administration caused a time-dependent increase in gastric mucosal damage and gastric mucosa ICE/caspase activity. Pretreating rats with 0.1 mM L-Arginine, SNP and Z.VAD.fmk, a pan-ICE/caspase inhibitor, prevented both gastric mucosal damage and ICE/caspase upregulation induced by aspirin. No gastric damage was detectable with any dose of NCX-4016 tested. Like aspirin NCX-4016 was largely metabolized to salicylate. In contrast to aspirin NCX-416 significantly increased plasma nitrite/nitrate concentrations and inhibited the aspirin-induced upregulation of ICE/caspase activity into the gastric mucosa. Although aspirin had no effect on HUVEC apoptosis induced by TNFcq NCX-4016 and SNP caused a concentrationdependent inhibition of apoptosis and prevented ICEqike pmteases activation as demonstrated by Westem blot analysis and fluorimetric assay. NCX-4016 inhibited IL-113 release from endotoxin-stimulated macrophages. Conclusions. 1) Activation of ICE/caspase pmteases is a key step in the process of NSAID-gastropathy; 2) NCX-4016 spares the gastric mucosa and protects endothelial cells from TNFetinduced apoptosis. The potential of NO-releasing NSAIDs to modulate ICE-like pmteases and IL-113 release might help to explain the anti-inflammatory activity of these compounds.