Cooperation between Aspirin-Triggered Lipoxin and Nitric Oxide (NO) Mediates Antiadhesive Properties of 2-(Acetyloxy)benzoic Acid 3-(Nitrooxymethyl)phenyl Ester (NCX-4016) (NO-Aspirin) on Neutrophil-Endothelial Cell Adherence
- Stefano Fiorucci,
- Eleonora Distrutti,
- Andrea Mencarelli,
- Giovanni Rizzo,
- Anna Rita Di Lorenzo,
- Monia Baldoni,
- Piero del Soldato,
- Antonio Morelli and
- John L. Wallace
- Clinica di Gastroenterologia ed Epatologia, Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Perugia, Perugia, Italy (S.F., E.D., A.Me., G.R., A.R.D.L., M.B., A.Mo.); Nicox SA, Sophia Antipolis, France (P.d.S.); and Mucosal Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada (J.L.W.)
- Address correspondence to:
Dr. Stefano Fiorucci, Clinica di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluce, 06100 Perugia, Italy. E-mail: fiorucci{at}unipg.it
Abstract
2-(Acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester (NCX-4016) is a nitric oxide (NO)-releasing derivative of aspirin that inhibits cyclooxygenase (COX) activity and releases NO. Acetylation of COX-2 by aspirin activates a transcellular biosynthetic pathway that switches eicosanoid biosynthesis from prostaglandin E2 to 15-epi-lipoxin (LX)A4 or aspirin-triggered lipoxin (ATL). Here, we demonstrate that exposure of neutrophil (PMN)/human umbilical vein endothelial cell (HUVEC) cocultures to aspirin and NCX-4016 triggers ATL formation and inhibits cell-to-cell adhesion induced by endotoxin (LPS) and interleukin (IL)-1β by 70 to 90%. However, although selective and nonselective COX-2 inhibitors (celecoxib, rofecoxib, and naproxen) or N-t-butoxycarbonyl-methionine-leucine-phenylalanine (Boc-1), an LXA4 receptor antagonist, reduced the antiadhesive properties of aspirin by ≈70%, antiadhesive effects of NCX-4016 were only marginally affected (≈30%) by COX inhibitors and Boc-1, implying that COX-independent mechanisms mediate the antiadhesive properties of NCX-4016. Indeed, NCX-4016 causes a long-lasting (up to 12 h) release of NO and cGMP accumulation in HUVEC. Scavenging NO with 10 mM hemoglobin, in the presence of celecoxib, reduced the antiadhesive properties of NCX-4016 by ≈80%. Confirming a role for NO, the NO donor diethylenetriamine-NO also inhibited PMN/HUVEC adhesion by ≈80%. NCX-4016, but not aspirin, decreased DNA binding of nuclear factor-κB (NF-κB) on gel shift analysis and HUVEC's overexpression of CD54 and CD62E induced by LPS/IL-1β. Reduction of binding of the two NF-κB subunits p50-p50 and p50-p65 was reversed by dithiothreitol, implying S-nitrosylation as mechanism of inhibition. In summary, our results support that ATL and NO are formed at the PMN/HUVEC interface after exposure to NCX-4016 and mediate the antiadhesive properties of this compound.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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DOI: 10.1124/jpet.103.063651.
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ABBREVIATIONS: PMN, polymorphonuclear neutrophils; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin; LX, lipoxin; LXA4, lipoxin A4; ATL, aspirin-triggered lipoxin or 15-epi-LXA4; NO, nitric oxide; ICAM, intercellular adhesion molecule; IL, interleukin; NF, nuclear factor; DTT, 1,4-dithio-dl-threitol; LPS, lipopolysaccharide; l-NAME, Nω-nitro-l-arginine methyl ester; Boc-1, N-t-butoxycarbonyl-methionine-leucine-phenylalanine; DETA-NO, diethylenetriamine-nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; Hb, hemoglobin; PMSF, phenylmethylsulfonyl fluoride; EMSA, electrophoretic mobility shift analysis; RT-PCR, reverse transcription-polymerase chain reaction.
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- Received November 29, 2003.
- Accepted February 3, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
