PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. METHODS: Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection. RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE. CONCLUSIONS: Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.
PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. METHODS:Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection. RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE. CONCLUSIONS:Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.
Authors: Fiona Stapleton; Carl Marfurt; Blanka Golebiowski; Mark Rosenblatt; David Bereiter; Carolyn Begley; Darlene Dartt; Juana Gallar; Carlos Belmonte; Pedram Hamrah; Mark Willcox Journal: Invest Ophthalmol Vis Sci Date: 2013-10-18 Impact factor: 4.799
Authors: Danial Roshandel; Medi Eslani; Alireza Baradaran-Rafii; Albert Y Cheung; Khaliq Kurji; Sayena Jabbehdari; Alejandra Maiz; Setareh Jalali; Ali R Djalilian; Edward J Holland Journal: Ocul Surf Date: 2018-06-20 Impact factor: 5.033
Authors: Gillian J McLellan; Zeynep Aktas; Elizabeth Hennes-Beean; Aaron W Kolb; Inna V Larsen; Emily J Schmitz; Hilary R Clausius; Jun Yang; Sung Hee Hwang; Christophe Morisseau; Bora Inceoglu; Bruce D Hammock; Curtis R Brandt Journal: J Ocul Biol Date: 2016-01-12