Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy

Abstract

Aims/hypothesis: 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.

Methods: 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.

Results: 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.

Conclusions/interpretation: 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.

Keywords: 12-HETE; 12/15-Lipoxygenase; 15-HETE; Bioactive lipids; Calcium; Diabetic retinopathy; ER stress; Eicosanoids; NADPH oxidase; VEGFR2.

Fig. 1

Loss of 12/15-LO reduces diabetes-induced ER stress in mouse retina. (a) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas of WT non-diabetic mice (white circles), WT diabetic mice (black squares), 12/15-Lo^−/− non-diabetic mice (white triangles) and 12/15-Lo^−/− diabetic mice (black triangles). Data represent individual data points with means ± SEM. (b) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas of WT mice that received intravitreal injection of vehicle (white circles) or 12-HETE (0.1 μmol/l) (black squares). Data represent individual data points with means ± SEM. Representative western blots (c) and densitometry (d–k) of ER stress protein levels in retinas isolated from WT mice that received intravitreal injection of vehicle or 12-HETE (0.1 μmol/l). Data represent individual data points with means ± SEM; *p<0.05, **p<0.01, ***p<0.001

Fig. 2

15-HETE induces ER stress in HRECs. Representative western blots (a) and densitometry (b) for PDI protein level in HRECs treated with vehicle or 15-HETE (0.1 μmol/l) at different time points (0–24 h). Data represent individual data points with means ± SEM. Representative western blots (c) and densitometry (d–j) for ER stress proteins in HRECs treated with vehicle or 15-HETE (0.1 μmol/l) for 4 h. Data represent individual data points with means ± SEM. (k) q-RT-PCR of relative mRNA expression of ER stress markers in HRECs treated with vehicle (white circles) or 15-HETE (0.1 μmol/l; black squares) for 3 h. Data represent individual data points with means ± SEM; *p<0.05, **p<0.01 vs time 0 in (b), and as shown by brackets elsewhere

Fig. 3

ER stress induction triggers endothelial leucocyte adhesion in HRECs, while ER stress inhibition abrogates 15-HETE-induced leucocyte adhesion. (a) In vitro leucocyte adhesion assay showing fluorescently labelled leucocytes adherent to HRECs under the influence of different treatments: control (vehicle), 15-HETE, lipopolysaccharide (LPS), tunicamycin (TM), VEGF, and 15-HETE in conjunction with the ER stress inhibitor PBA. (b) Quantification of the fold change of the mean number of the adherent leucocytes in each group showing a significant increase by the ER stress inducer tunicamycin, LPS, VEGF and 15-HETE compared with the vehicle-treated group. ER stress inhibition using PBA significantly reduced 15-HETE-induced endothelial leucocyte adhesion. Data represent individual data points with means ± SEM; *p<0.05, **p<0.01, ***p<0.001; scale bar, 50 μm

Fig. 4

Crosstalk between NADPH oxidase and ER stress in 15-HETE-treated HRECs. Representative western blots (a) and densitometry (b) for PDI protein level in HRECs treated for 24 h with vehicle (control), 15-HETE (0.1 μmol/l) or 15-HETE + NADPH oxidase inhibitor (apocynin, 30 μmol/l). Data represent individual data points with means ± SEM. Representative western blots and densitometry for NOX2 protein (c, d) or P47^phox protein (e, f) in HRECs treated for 24 h with vehicle, 15-HETE (0.1 μmol/l) or 15-HETE + ER stress inhibitor (PBA, 30 μmol/l). Data represent individual data points with means ± SEM. (g) Superoxide measurement using DHE staining in HRECs treated with vehicle (white circles), 15-HETE (0.1 μmol/l) (black squares), 15-HETE (0.1 μmol/l) + VAS2870 (NADPH oxidase inhibitor, 10 μmol/l) (white triangles) or 15-HETE (0.1 μmol/l) + PBA (30 μmol/l) (black triangles). Data represent individual data points with means ± SEM

Fig. 5

Nox2 deletion has no effect on 12-HETE-induced ER stress in vivo. (a) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas isolated from Nox2^−/− mice that had received intravitreal injection of vehicle (white circles) or 12-HETE (0.1 μmol/l) (black squares). Data represent individual data points with means ± SEM. Representative western blots (b) and densitometry of ER stress proteins PDI (c) and ATF4 (d) in retinas isolated from WT or Nox2^−/− mice after intravitreal injection of 12-HETE (0.1 μmol/l). Data represent individual data points with means ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs vehicle in (c) and (d), or as shown by brackets in (a)

Fig. 6

15-HETE induces intracellular Ca²⁺ release in HRECs. (a) Real-time imaging of intracellular calcium in HRECs treated with vehicle or 15-HETE (0.1 μmol/l). HRECs were loaded with a calcium-detecting dye, and then treated with vehicle or 15-HETE under the inverted confocal microscope. Treatment of HRECs with 15-HETE caused a significant increase in the fluorescence intensity, suggesting increased intracellular cytosolic Ca²⁺ levels. (b) Quantification of normalised calcium fluorescence in HRECs treated with vehicle (blue line) or 15-HETE (0.1 μmol/l) (red line) over 30 s from the beginning of the treatment. Data represent means ± SEM. (c) Statistical analysis of the averaged normalised fluorescence intensity of at least three independent experiments shows that 15-HETE treatment significantly increases HRECs’ intracellular cytosolic calcium levels compared with the vehicle-treated HRECs (*p<0.05). Data represent individual data points with means ± SEM; scale bars, 200 μm

Fig. 7

ER stress inhibition attenuates 15-HETE-induced VEGFR2 phosphorylation. (a) ELISA of VEGF levels in supernatant of HRECs treated with vehicle or 15-HETE for 24 h showing no significant difference in the VEGF levels. Data represent individual data points with means ± SEM. (b) Immunoprecipitation (IP) of VEGFR2 in HRECs treated with 15-HETE in the presence or absence of PBA or apocynin for 5 min, followed by immunoblotting (IB) of phosphotyrosine (pTyr). Normalisation was done by re-blotting the membrane for VEGFR2. (c) Densitometry analysis shows a significant increase in VEGFR2 phosphorylation by 15-HETE compared with the control. ER stress inhibitor (PBA) and NADPH oxidase inhibitor (apocynin) reduced the effect of 15-HETE on VEGFR2 phosphorylation. Data represent individual data points with means ± SEM; *p< 0.05 vs vehicle

Fig. 8

A schematic summary. Hyperglycaemia increases the activity of 12/15-LO enzyme with a subsequent increase of its bioactive lipid products, 12- and 15-HETE, in retinal endothelial cells. 12/15-LO lipid metabolites increase endothelial intracellular calcium levels that may activate ER stress and, in turn, NADPH oxidase. These intracellular activated pathways lead to increased phosphorylation of VEGFR2 and subsequent retinal endothelial dysfunction