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

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 E(2) to 15-epi-lipoxin (LX)A(4) 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)-1beta by 70 to 90%. However, although selective and nonselective COX-2 inhibitors (celecoxib, rofecoxib, and naproxen) or N-t-butoxycarbonylmethionine-leucine-phenylalanine (Boc-1), an LXA(4) receptor antagonist, reduced the antiadhesive properties of aspirin by approximately 70%, antiadhesive effects of NCX-4016 were only marginally affected ( approximately 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 approximately 80%. Confirming a role for NO, the NO donor diethylenetriamine-NO also inhibited PMN/HUVEC adhesion by approximately 80%. NCX-4016, but not aspirin, decreased DNA binding of nuclear factor-kappaB (NF-kappaB) on gel shift analysis and HUVEC's overexpression of CD54 and CD62E induced by LPS/IL-1beta. Reduction of binding of the two NF-kappaB 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.