Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy
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Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy.
We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12–14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy.
Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte 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. We also found that 15-HETE increases the intracellular calcium in HRECs.
ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
Keywords12/15-Lipoxygenase 12-HETE 15-HETE Bioactive lipids Calcium Diabetic retinopathy Eicosanoids ER stress NADPH oxidase VEGFR2
Activating transcription factor
Eukaryotic initiation factor 2α
Human retinal endothelial cell
Inositol-requiring enzyme 1
Protein disulfide isomerase
RNA-dependent protein kinase-like ER-regulated kinase
Quantitative real-time PCR
Unfolded protein response
Vascular endothelial growth factor
X-box binding protein 1
Authors would like to thank M. E. McGee-Lawrence (Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University) for assistance with live calcium imaging.
KE, ASI, NE and MA conceived and designed the study; KE, ASI, HS, NE, SE and KH contributed to data acquisition; KE, ASI, NE and SE analysed the data; all authors interpreted the data, drafted the article, revised it and approved the final version. MA is the guarantor of this work.
This study was funded by the National Eye Institute (National Institutes of Health) Grant 5R01EY023315 (MA) and Egyptian Cultural and Educational Bureau – Washington DC (Egyptian Ministry of Higher Education) (KE).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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