12/15-lipoxygenase contributes to retinal inflammation in diabetic retinopathy via activation of ER stress/NADPH oxidase signaling pathway

Abstract

Purpose : Our previous studies have established the role of 12/15-lipoxygenase (LO) in facilitating inflammation during diabetic retinopathy (DR). However, the exact mechanism is still unclear. The goal of the current study was to identify the possible role of endoplasmic reticulum (ER) stress as a key cellular stress response in 12/15-LO-induced retinal changes in DR.

Methods : Wild-type (WT) and 12/15-lipoxygenase knockout (LO^-/-) mice were rendered diabetic via repeated injection of STZ, followed by ER stress evaluation after 12-14 week of diabetes. Retinal ER stress was assessed at RNA/protein levels after Intravitreal injection of 12- hydroxyeicosatetraenoic acid (HETE) (0.1µM) into eyes of WT mice and in mice lacking the catalytic subunit of NADPH oxidase, NOX2 (NOX2^-/-). Human retinal endothelial cells (HRECs) were treated with 15-HETE (0.1µM) or vehicle with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress (RNA/proteins), NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, confocal microscopy was used to perform real-time imaging of intracellular calcium (Ca⁺²) release in HRECs treated with or without 15-HETE.

Results : Retinas of diabetic LO^-/- mice showed a significant decrease in diabetes-induced ER stress mRNAs such as XBP1, ATF6, PDI, and CHOP. Also, HRECs treated with 15-HETE showed an increase in ER stress markers such as p-PERK, ATF6, and PDI. Furthermore, ER stress inhibition reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation, and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of NOX2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. Interestingly, we found that 15-HETE increases the intracellular calcium release in HRECs.

Conclusions : 12/15-LO contributes to retinal inflammatory response in DR via ER stress-dependent pathway that involves activation of NADPH oxidase and VEGF signaling. Inhibition of ER stress could be beneficial in blocking bioactive lipids mediated vascular abnormalities in DR.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.