ABSTRACT
The purpose of the current study was to evaluate the effect of 12/15-lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2(Akita) mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1 expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.
Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/pharmacology , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Enzyme Inhibitors/pharmacology , Flavanones/pharmacology , Hydroxyeicosatetraenoic Acids/pharmacology , Membrane Glycoproteins/genetics , NADPH Oxidases/genetics , Animals , Biological Transport/drug effects , Cell Membrane Permeability/drug effects , Dextrans , Diabetic Retinopathy/drug therapy , Diabetic Retinopathy/genetics , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Electric Impedance , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Fluorescein-5-isothiocyanate/analogs & derivatives , Gene Expression/drug effects , Membrane Glycoproteins/metabolism , Mice , Mice, Transgenic , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Reactive Oxygen Species/metabolism , Retina/drug effects , Retina/metabolism , Retina/pathology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor Receptor-2/agonists , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolismABSTRACT
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.