SECOND PRIZE, COS AWARDS FOR EXCELLENCE IN OPHTHALMIC RESEARCH

Author Block: James J. Armstrong, Liu Hong, Cindy M. L. Hutnik
Author Disclosure Block: J.J. Armstrong: None. L. Hong: None. C.M.L. Hutnik: None.

Abstract Body:

Purpose: Lipid autacoids derived from n-3 and n-6 polyunsaturated fatty acids are some of the earliest inflammatory signals. Phospholipase A2 (PLA2) generates substrates for the biosynthesis of these mediators, and is upstream of the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. The COX pathway’s products, the prostaglandins, are responsible the cardinal signs of inflammation. The LOX pathway’s products, the lipoxins and specialized pro-resolving mediators, are essential endogenous signals mediating the resolution of inflammation. Acetylsalicylic acid (ASA) can acetylate the cyclooxygenase-2 isoform, such that its enzymatic activity becomes lipoxygenase-like. Unfortunately, the amount of LOX-derived mediators produced by acetylated-COX invivo is limited by the salicylate ion from ASA competitively inhibiting the acetylated enzyme. We hypothesized that by acetylating COX-2 with ASA in the presence of a PLA2 agonist, increased upstream metabolites will out compete salicylate for the active site, and more acetylated-COX2 derived products will be produced than with ASA alone.
Study Design: In vitro cell-based model of inflammation
Methods: Human ocular fibroblasts were induced with 1ug/ml each of INFy, TNFa and IL-1B for 24 hours before being treated with one of three experimental treatments: 1) vehicle (DMEM), 2) ASA 200ug/ml, 3) Melittin 5ug/ml (a PLA2 agonst) or 4) ASA 200ug/ml and Melittin 5ug/ml combined. Supernatant was collected at 6, 12, 24 and 48 hours after experimental treatment and analyzed for lipid mediators of inflammation / resolution using denutered internal standards and liquid chromatography tandem mass spectrometry.
Results: ASA alone did not cause a significant increase in acetylated-COX2 products compared to control (p > 0.05), however it did significantly inhibit prostaglandin production. The PLA2 agonist alone significantly increased the abundance of PLA2 products arachadnic acid, eicosapentaenoic acid and docosahexaenoic acid relative to control (p < 0.001) and a corresponding increase in all COX and LOX derived downstream metabolites was also observed. The combination ASA and PLA2 agonist resulted in a 200 to 400 times increase in acetylated-COX2 derived products compared to ASA only treated replicates (p < 0.001), further there was only a 20 times increase versus control in the production of prostaglandins.
Conclusions: Taken together these data support the competitive inhibition of acetylated-COX2 by salicylate. We have shown that this inhibition can be “out competed” by agonizing production of COX2 substrates. To our knowledge, this is the first treatment strategy that modulates autacoid signalling to promote early engagement of the resolution axis of inflammation.