Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy

Abstract

Diabetic retinopathy is a chronic low-grade inflammatory disease; however, the mechanisms remain elusive. In the present study, we demonstrated that endoplasmic reticulum (ER) stress was activated in the retina in animal models of diabetes and oxygen-induced retinopathy (OIR). Induction of ER stress by tunicamycin resulted in significantly increased expression of inflammatory molecules in the retina. Inhibition of ER stress by chemical chaperone 4-phenyl butyric acid ameliorated inflammation in cultured human retinal endothelial cells exposed to hypoxia, and in the retinas of diabetic and OIR mice. These findings indicate that ER stress is a potential mediator of retinal inflammation in diabetic retinopathy.

Figure 1. Over-expression of pro-inflammatory factors and Up-regulation of ER stress markers in the retinas of Akita mice

A). Western blot analysis of TNF-α and VEGF in the retinas of Akita mice. The membrane was reblotted with β-actin antibody as loading control. B). Retinal levels of TNF-α and VEGF were quantified by densitometry from 4 individual animals (mean ± SD, n = 4). C). mRNA level of GRP78 in the retina determined by real-time RT-PCR and normalized by 18S (mean ± SD, n = 7 in control group and n = 4 in Akita group). D). Representative images of GRP 78 expression in the retinas from 4 Akita mice and 5 littermate controls. Note more intensive signal of GRP 78 (brown color) in the inner retina of Akita mice (C-b) when compared to control (C-a). Magnification: 200x. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. E). Western blot analysis of phospho-IRE1α, phospho-eIF2α and ATF4 in the retinas of Akita mice. E). Retinal levels of phospho-IRE1α, phospho-eIF2α and ATF4 were quantified by densitometry (mean ± SD, n = 8). * p<0.05 and **p<0.01.

Figure 2. Activation of ER stress and inflammation in the retina of OIR mice

A). mRNA level of GRP78 in the retina of OIR mice determined by real-time RT-PCR and normalized by 18S (mean ± SD, n = 5). B). Protein level of GRP78 determined by Western blot analysis. Lower panel: densitometry results (mean ± SD, n = 8). C). Western blot analysis of phospho-eIF2α and ATF4 in the retina. D). Retinal levels of phospho-eIF2α and ATF4 were quantified by densitometry (mean ± SD, n=8). **p<0.01.

Figure 3. Up-regulation of pro-inflammatory factors in the retina by tumicamycin

Adult C57BL/6J mice received periocular injection of tunicamycin in one eye and PBS as control in the contralateral eye. Twenty four hours after injection, retinas were dissected and expression of pro-inflammatory factors (TNF-α and VEGF) (A, B) and ER stress markers (GRP78 and ATF4) (C, D) were determined by Western blot analysis and quantified by densitometry (mean ± SD, n = 4). * p<0.05 and **p<0.01.

Figure 4. Inhibition of ER stress by PBA ameliorated hypoxia-induced VEGF and TNF-α expression in HREC

A). HREC were treated with hypoxia (2% O₂) for 16h and GPR78 expression was determined by immunostaining. A-a: control; A-b: hypoxia. B–E). HREC were pre-treated with PBA at the doses as indicated for 8h and then exposed to hypoxia (2% O₂) for 16h. B–C). Expression of phospho-IRE1α, phospho-eIF2α, and ATF4 was determined by Western blot analysis and quantified by densitometry (mean ± SD, n= 3) D–E). Expression of VEGF and TNF-α was measured by Western blot analysis and quantified by densitometry (mean ± SD, n = 3). The values statistically different from control were indicated as ** p<0.01; from hypoxia indicated as † p<0.05, ‡ p<0.01.

Figure 5. Local or systemic administration of PBA mitigated VEGF expression in the retina of Akita or OIR mice

A). PBA (0.4 μmol/eye) was injected periocularly into one eye and same amount of vehicle was injected into the counterlateral eye as control in the Akita mice, twice a week, for 6 weeks. Expression of VEGF in the retina was measured by Western blot analysis and quantified by densitometry (mean ± SD, n = 4). B). OIR mice received intraperitoneal injection of PBA (40 mg/kg/body weight/day) or PBS from P7 to P14. Retinal VEGF expression was measured by Western blot analysis at P15 (mean ± SD, n = 5). * p<0.05 and ** p<0.01.