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


OBJECTIVE
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 (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta 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 TNFalpha release. Exposure to TNFalpha 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 TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta 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.
Fiorucci
S
S
Sezione di Gastroenterologia ed Epatologia, Dipartimento di Medicina Clinica, e Sperimentale, Universita' degli Studi di Perugia, Perugia, Italy.
Santucci
L
L
Federici
B
B
Antonelli
E
E
Distrutti
E
E
Morelli
O
O
Renzo
G D
GD
Coata
G
G
Cirino
G
G
Soldato
P D
PD
Morelli
A
A
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001241:Aspirin / Q000031:analogs & derivatives / Q000494:pharmacology; D061945:Caspase Inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005480:Flurbiprofen / Q000031:analogs & derivatives / Q000494:pharmacology; D005753:Gastric Mucosa / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D009288:Naproxen / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D000894:Anti-Inflammatory Agents, Non-Steroidal; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C111751:naproxen-n-butyl nitrate; C095631:nitroflurbiprofen; D009569:Nitric Oxide; D009288:Naproxen; D005480:Flurbiprofen; D009195:Peroxidase; C102148:nitroaspirin; D001241:Aspirin