12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

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.

Vascular Endothelial Tight Junctions and Barrier Function Are Disrupted by 15(S)-Hydroxyeicosatetraenoic Acid Partly via Protein Kinase Cϵ-mediated Zona Occludens-1 Phosphorylation at Threonine 770/772

“…A large body of literature points out a link between oxidative stress and vascular diseases (62). A recent study showed that 12(S)-HETE increases retinal endothelial barrier permeability by NADPH oxidase-dependent reactive oxygen species production leading to phosphorylation and activation of VEGF receptor-2 (26). We have previously shown that 15(S)-HETE by activating nonreceptor tyrosine kinases, Src and Pyk2, stimulates ZO-2 tyrosine phosphorylation and its dissociation from claudins 1/5 and thereby disrupts aortic endothelial TJs in response to HFD feeding (27).…”

“…In this context, it should be noted that studies from other laboratories have reported that deletion of the 12/15-LO gene reduces vascular permeability in a mouse model of acute lung injury (25). In addition, a recent report showed that 12(S)-HETE by NADPH oxidase-dependent reactive oxygen species production and VEGF receptor-2 tyrosine phosphorylation increases retinal endothelial cell permeability (26). Previously, we have demonstrated that 15(S)-HETE by stimulating ZO-2 tyrosine phosphorylation and its dissociation from claudins 1/5 disrupts endothelial TJs and its barrier function.…”

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

“…Moreover, we have demonstrated in both human patients and animal models that retinal expression of 12/15-LOX is robustly induced during diabetes ( 21 ). At the same time, its pharmacological inhibition dampened the levels of infl ammatory cytokines, reactive oxygen species (ROS) gener ation, and phosphorylated VEGF receptor 2 expression in the retinas of diabetic mice ( 22 ). However, the cellular source of these lipid metabolites is unknown and could be derived from retinal tissues, including the retinal vascular endothelial, glial, and pigmented epithelial cells, as well as from infi ltrated infl ammatory cells.…”

“…Our recent study showed that inhibiting 12/15-LOX by baicalein reduced diabetes-induced ROS generation and NOX2 expression in mouse retina, suggesting the existence of an interconnected signaling between LOXs and NOX ( 22 ). However, the cross-talk between NOX and infl ammatory bioactive lipids remains ill-defi ned in DR.…”

The Blood Brain Barrier — Regulation of Fatty Acid and Drug Transport

“…These authors concluded that free radicals generated by either COX or LOX pathways were responsible for the permeability response to AA. In a mouse model of diabetic retinopathy 12-HETE and 15-HETE, products of the lipoxygenase pathway, were shown to be responsible for increasing the permeability of retinal endothelial cell barrier via an NADPH oxidase-dependent mechanism [59]. Interestingly, AA inhibited the cytokine-induced up-regulation of several genes involved in endothelial cell inflammation [60].…”