12/15-Lipoxygenase-Derived Eicosanoids Induce Vascular Inflammation and Angiogenic Effects in Human Retinal Endothelial Cells: Role of NADPH oxidase and ER Stress

Abstract

Purpose: Microvascular dysfunction during diabetic retinopathy (DR) is characterized by early inflammatory response (leukostasis and permeability) followed by retinal neovascularization. Our previous studies established 12/15-lipoxygenase as a pro-inflammatory and a pro-angiogenic mediator during DR. The current study tests the hypothesis that NADPH oxidase and endoplasmic reticulum (ER) stress contribute to the inflammatory and angiogenic effects of the 12/15LOX-derived eicosanoids (12- and 15- hydroxyeicosatetraenoic acids or HETEs).

Methods: Human retinal endothelial cells (HRECs) were used to examine the effect of 12 and 15-HETEs on leukocyte adhesion and angiogenesis. Effects of 12- or 15-HETE (0.1 μM) on HREC were tested in the presence or absence of NADPH oxidase inhibitors (Apocinin or diphenylene iodonium (DPI)) or the antioxidant N-acetyl cysteine (NAC). Human leukocytes (PMNs) were labeled with lipophilic fluorescent probe for leukostasis assay. Tube formation and migration assays were performed to evaluate the angiogenic properties of 12- and 15-HETEs. Migration assay was performed using the electrical Cell Impedance Sensor (ECIS). The levels of ER stress markers (BIP, PERK and IRE1) were detected with Western blotting. Effect of ER stress inducer, tunicamycin on leukostasis was also tested. We also tested the effect of 12- and 15-HETEs on cytokines production by Multiplex assay system.

Results: 12 and 15-HETE increased endothelial cell migration, tube formation, leukostasis and ER stress (P<0.05). These effects were inhibited by the concomitant use of NADPH oxidase inhibitor apocynin, DPI or the antioxidant NAC. Furthermore, 12- and 15-HETEs significantly increased IL6, IL17 and MCP1 production by HRECs. ER stress by tunicamycin showed significant increase in leukocyte adhesion to HRECs (P<0.05).

Conclusions: 12 and 15-HETEs promote inflammatory and angiogenic response in retinal endothelial cells via NADPH oxidase dependent mechanism which involves enhancement of ER stress and cytokine production. Targeting 12/15LOX-NADPH-ER stress signaling system represents an attractive therapeutic strategy to treat DR.

Keywords: 499 diabetic retinopathy