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

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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 HUVEC9s 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.

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