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 Ins2Akita 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-1expression 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.
Highlights
Diabetic retinopathy (DR) is the most common cause of blindness in working age Americans
Arachidonic acid is considered a target for different enzymatic pathways such as cycloxygenase (COX2), lipoxygenase (LOX), and cytochrome P450 (CYP). [8,9] Lipoxygenases are a group of closely related dioxygenases that are classified as 5, 8, 12, or 15-LOX, according to the site of oxygen insertion within arachidonic acid (AA). [10]. 12/15-LOX pathway has proven to be involved in cardiovascular complications of diabetes such diabetic nephropathy, atherosclerosis and hypertension [11,12,13,14]
The present study investigated a potential molecular mechanism of the early inflammatory response in DR and mainly focused on the interaction between 12/15-LOX and NADPH oxidase in mediating retinal endothelial cell barrier dysfunction
Summary
Diabetic retinopathy (DR) is the most common cause of blindness in working age Americans. Vision is adversely affected in clinical conditions associated with the breakdown of BRB such as DR or age related macular degeneration (AMD). Its role in the development of retinal microvascular complications has not been studied in detail [6]. Diabetic dyslipidemia is characterized by an increase in n-6 polyunsaturated fatty acids (PUFA), such as arachidonic acid (AA) [7] which is released from the cell membrane by cytosolic phospholipase A2 (cPLA2). Arachidonic acid is considered a target for different enzymatic pathways such as cycloxygenase (COX2), lipoxygenase (LOX), and cytochrome P450 (CYP). We recently demonstrated that pathological retinal neovascularization (NV) in humans with proliferative diabetic retinopathy (PDR) and mouse model of oxygen-induced retinopathy (OIR) was associated with significant increase in LOX-derived eicosanoids, 12-, 15- and 5hydroxyeicosatetreanoic acids (HETE) [10].
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