oa Role of lipoxygenase pathway in diabetic retinopathy

Background: Features of diabetic retinopathy (DR) include leukocyte adhesion, hyperpermeability and retinal neovascularization (RNV). Our previous studies demonstrated that reactive oxygen species (ROS) derived from the NADPH oxidase activity play crucial role in the pathogenesis of retinal vascular injury during DR. Recently we also demonstrated that upregulation of 12/15 lipoxygenase (12/15-LOX) and its lipid metabolites, 12- and 15-HETEs during DR contributes to RNV via disrupting the delicate balance in the levels of vascular endothelial growth factor and pigment epithelium derived factor (VEGF/PEDF). The goal of our study is to investigate whether 12/15-LOX also contributes to retinal inflammation during DR via activation of NADPH oxidase, endoplasmic reticulum (ER) stress response, and VEGF-receptor2 (KDR). Methods: We used cultured human retinal endothelial cells (HRECs) to test the effect of 12/15-LOX derived lipid metabolites on barrier function, leukostasis and tube formation. The amount of HETEs product of 12/15-LOX in the vitreous of patients with or without DR was measured by LC/MS. HETEs were also tested in the retinas of diabetic mice and in mice with oxygen-induced retinopathy (OIR). The direct effect of 12- and 15-HETE on HREC barrier was examined in the presence or absence of NADPH oxidase inhibitors diphenylene iodonium (DPI) and apocynin using FITC-dextran flux assay and electrical cell-substrate impedance sensing (ECIS). The impact of HETEs on leukocyte/endothelial cell interaction and tube formation was also tested. Production of reactive oxygen species in response to HETEs treatment was measured by dihydroethedium (DHE) and dichlorofluorescein (DCF) reactions. Western blotting (WB) was used to evaluate the changes in the protein levels of the catalytic subunit of the NADPH oxidase (NOX2), ER stress proteins, phospho-VEGF-R2 and the protein tyrosine phosphatase (SHP1). In vivo studies were performed using a mouse model of type 1 diabetes, the akita mice (Ins2Akita) treated with or without the 12/15-LOX inhibitor baicalein (75 mg/kg in drinking water) for ~10 weeks. This was followed by analysis of ROS, HETEs, leukostasis and inflammatory mediators. Multiplex Immunoassay was used to measure the levels of inflammatory mediators such as the adhesion molecules (ICAM-1 and VCAM-1) and IL-6. Results: Our experiments showed significant increase in the REC permeability and reduction in the transcellular electrical resistance (TER) by 12- and 15- HETEs compared to the control suggesting pro-permeability role of 12/15-LOX. Leukocyte adhesion and tube formation were also increased by 12- and 15-HETEs. This was associated with significant increases in ROS generation, levels of NOX2, ER stress response proteins and p-VEGF-R2.There was also a significant decrease in the levels of p-SHP1. These effects of HETEs were prevented by NADPH oxidase or VEGF-R2 inhibitors. In vivo studies demonstrated significant abrogation of the retinal HETEs, adhesion molecules (ICAM-1 and VCAM-1), IL6, ROS generation and NOX2 expression in diabetic mice treated with baicalein. Furthermore, the number of adherent leukocytes was reduced in 12/15-LOX-deficient and baicalein-treated mice. Conclusion: 12/15-LOX contributes to DR via NADPH oxidase-dependent mechanism which involves activation of ER stress response and VEGF-R2. Thus, 12/15-LOX is a potential therapeutic target to prevent the development of vascular dysfunction during DR.