Abstract
Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.
Highlights
Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR)
In order to evaluate the potential role of bioactive lipids in mediating the edematous phenotype observed in DR, we first sought to characterize their profile under diabetic milieu
The lipidomic profile of human retinal endothelial cell (HREC) was screened for metabolites whose levels changed dramatically during hyperglycemia
Summary
All procedures with animals were performed in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare publication, National Institutes of Health 80-23) and Georgia Regents University guidelines. To avoid uncontrolled intraocular pressure increase, the volume of intravitreal injections was limited to 1 l. 12-HETE was dissolved in ethanol and a working solution of 10× was prepared by diluting 0.32 l of stock solution (312 M) to 100 l with PBS, assuming the vitreous volume of mouse eye is ف10 l [28]. By injecting 1 l of this working solution, a 0.1 M vitreal concentration of 12HETE was obtained. The volume of the injected solution apparently did not cause significant pressure-induced retinal damage, because 0.032% ethanol-PBS-injected control eyes showed normal retinal morphology with no apparent apoptosis within 7 days. The dose of 12- or 15-HETE was chosen according to what was detected previously in the vitreous of patients with DR, 50 ng/ml (ف0.1 M) [21]
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