15-lipoxygenase A Resident Protective Pathway in the Cornea Suppresses Inflammatory Neovascularization

Abstract

Purpose:: Execution of an acute inflammatory/reparative response in the cornea is essential to maintain vision. This vital response is regulated by balanced formation of chemical mediators, of which lipid autacoids hold a key role as inflammatory and immune modulators. We recently identified a lipid autacoid circuit in the cornea, 15-lipoxygenase, which generates anti-inflammatory signals including lipoxin A₄ and docosahexaenoic acid-derived NPD1. We set out to investigate if this endogenous protective circuit has a role in suppressing inflammatory neovascularization in the cornea.

Methods:: A silk suture (8.0) was placed in the apex of the cornea of 15-LOX knockout mice and matched congenic controls for 7 days. Neovascularization was quantitated by vital microscopy and immuno-fluorescence using CD31 as an endothelial marker. Inflammation was assessed by measuring MPO activity as a quantitative PMN marker. Expression of 15-LOX, heme-oxygenase, and selected inflammatory markers was assessed by Western Blot and Real-Time PCR analyses. Lipid autacoid formation was analyzed by LC/MS/MS-based lipidomics employing a triple quadruple linear ion trap LC/MS/MS system.

Results:: Lipidomic analyses demonstrated significant formation of inflammatory and angiogenic eicosanoids, PGE₂, 5-HETE, 15-HETE, 12-HETE, 12-HETrE. Moreover, docosahexaenoic acid-derived NPD1 was a prominent product in mouse corneas. In vivo deletion of 15-LOX, in Alox15^-/- mice, correlated with a shift of the lipid autacoid profile towards predominant PGE₂ formation and a loss of NPD1 formation, which was associated with a significant increase (2 fold) in neovascularization and neutrophils content in the cornea. Moreover, Alox15^-/- mice demonstrated impaired induction of the cytoprotective heme-oxygenase system in macrophages and injured corneas.

Conclusions:: Collectively these results provide the first direct evidence for formation of potent lipid inflammatory/immune and angiogenic mediators in inflammatory neovascularization and demonstrate that the 15-LOX system has a key role in counter-regulating inflammation and suppressing neovascularization. These protective actions are in part mediated by regulating expression of the cytoprotective heme-oxygenase system. In view of the high expression of the 15-LOX pathway in human and mouse corneas, this protective circuit is a promising therapeutic target for limiting the sequelae of corneal injury.

Keywords: neovascularization • cornea: epithelium • inflammation