15-hydroxy-5-8-11-13-eicosatetraenoic-acid and Diabetic-Retinopathy

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelial Cells; Ependymoglial Cells; Eye Proteins; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Intercellular Adhesion Molecule-1; Interleukin-6; Intravitreal Injections; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Nerve Growth Factors; NF-kappa B; Retina; Retinal Neovascularization; Serpins; Signal Transduction; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Zonula Occludens-1 Protein

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.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Biological Transport; Cell Membrane Permeability; Dextrans; Diabetic Retinopathy; Electric Impedance; Endothelial Cells; Enzyme Inhibitors; Flavanones; Fluorescein-5-isothiocyanate; Gene Expression; Hydroxyeicosatetraenoic Acids; Membrane Glycoproteins; Mice; Mice, Transgenic; NADPH Oxidase 2; NADPH Oxidases; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Reactive Oxygen Species; Retina; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Zonula Occludens-1 Protein

Arachidonic acid is metabolised via lipoxygenase to 15-HETE (15-hydroxyeicosatetraenoic acid) and 15-HPETE (15-hydroperoxyeicosatetraenoic acid), which are believed to influence proliferation in tissue culture. 15-HETE is the reduction product of 15-HPETE. Cell proliferation is believed to be decreased by 15-HPETE and increased by 15-HETE. The aim of this study was to investigate epiretinal membranes for the presence of these lipoxygenase products and to compare membranes from different disease processes.. Epiretinal membranes of 15 patients suffering from proliferative vitreoretinopathy (PVR, n = 7) and proliferative diabetic retinopathy (PDR; n = 8) were removed during vitrectomy and analysed by means of thin layer chromatography. The plates were evaluated by digital image analysis.. Both 15-HETE and 15-HPETE were identified in membranes from eyes of patients with PVR and PDR with HETE values significantly higher (p < 0.05) than HPETE values (HETE/HPETE ratio = 5.2).. This study demonstrates that eicosanoids are present in the epiretinal membrane tissue of patients with PVR and PDR. Considering that HETE increases cell proliferation while HPETE inhibits it, it is conceivable that eicosanoids are an additional factor contributing to the regulation of membrane growth in proliferative retinal disorders. Thus, inhibition of lipoxygenase could be a therapeutic approach in these diseases.

Topics: Biomarkers; Chromatography, Thin Layer; Diabetic Retinopathy; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Retina; Vitreoretinopathy, Proliferative