Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization.

OBJECTIVE: Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV). RESEARCH DESIGN AND METHODS: Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF). Liquid chromatography-mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes. RESULTS: Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE-treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE. CONCLUSIONS: 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

Suppression of retinal peroxisome proliferator-activated receptor gamma in experimental diabetes and oxygen-induced retinopathy: role of NADPH oxidase.

PURPOSE: Recently, the authors have shown that NADPH oxidase is positively correlated with increased leukocyte adhesion and vascular leakage in diabetes and neovascularization in oxygen-induced retinopathy (OIR). Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been shown to prevent vascular inflammation and leakage in an experimental model of diabetes. The goal of this study was to investigate whether there is a link between NADPH oxidase and PPARgamma that leads to vascular dysfunction in diabetic retina or OIR. METHODS: Diabetes was induced with streptozotocin in wild-type mice or NOX2 knockout mice. One group of wild-type mice was treated with apocynin. Bovine retinal endothelial cells (BRECs) were treated with normal glucose (5 mM) or high glucose (25 mM) in the presence or absence of superoxide dismutase (SOD) or NADPH oxidase inhibitors (apocynin or diphenyleneiodonium [DPI]). Western blotting and immunofluorescence were used to evaluate PPARgamma expression. Activation of nuclear factor (NF)kappaB was measured using the transcription factor assay kit and Western blot analysis of phospho-NFkappaB. PPARgamma expression was also tested in OIR and lipopolysaccharide-induced retinal inflammation. RESULTS: Retinal expression of PPARgamma was suppressed in experimental models of diabetes, OIR, and retinal inflammation. This was associated with the activation of NFkappaB in the diabetic retina. These effects were prevented by apocynin or deletion of NOX2. PPARgamma expression was also suppressed in endothelial cells treated with high glucose, and this was prevented by apocynin, DPI, and SOD. CONCLUSIONS: Suppression of PPARgamma is involved in the pathogenesis of diabetic retinopathy and OIR. NADPH oxidase could be an upstream mediator of these changes.