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ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes.

Li T, Hu J, Du S, Chen Y, Wang S, Wu Q - Mol. Vis. (2014)

Bottom Line: COX-2 and VEGF mRNA were determined by quantitative RT-PCR.Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398.This study highlights the signaling pathway as a potential target for intervention in DR.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Sixth People's Hospital, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT

Purpose: Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes.

Methods: Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT-PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay.

Results: Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398.

Conclusions: Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR.

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GPR91 modulated VEGF secretion via ERK1/2/cyclooxygenase-2 (COX-2)/ Prostaglandin E2 (PGE2) signaling pathway in the retinas of STZ rats. A: Western blot analysis of the COX-2 protein in the retinas of diabetic rats in different time periods (from 1 week to 10 weeks). B: The levels of COX-2 protein were upregulated in the retinas of diabetic rats during the period of 2 weeks to 6 weeks after the induction of diabetes. Each column denotes the mean ± SD (n = 6). C: Immunofluorescence showed that COX-2 expression located in the retinal ganglion cell layer increased in the 4 week diabetic rats compared with the control. Scale bar, 100 μm. D: Changes in COX-2 in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). E: The increases in COX-2 expression were significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). Each column denotes the mean ± SD (n = 6). F: qRT-PCR showed that the levels of COX-2 mRNA were decreased obviously in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). G: Changes in vitreal PGE2 release in each group. The increase of PGE2 secretion was significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) in the retinas of 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). H: The levels of VEGF mRNA (using qRT-PCR) were downregulated in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5mM NS-398 (COX-2 inhibitor) compared with the STZ rats. Each column denotes the mean ± SD (n = 6). I: Enzyme-linked immunosorbent assay analysis of vitreal VEGF release in vitreous. The increase of VEGF secretion was significantly blocked in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). **p<0.01 versus control. #p<0.05 versus LV.shScrambled group rats. ##p<0.01 versus LV.shScrambled group rats. ψ<0.05 versus STZ group rats. ψψ<0.01 versus STZ group rats.
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f4: GPR91 modulated VEGF secretion via ERK1/2/cyclooxygenase-2 (COX-2)/ Prostaglandin E2 (PGE2) signaling pathway in the retinas of STZ rats. A: Western blot analysis of the COX-2 protein in the retinas of diabetic rats in different time periods (from 1 week to 10 weeks). B: The levels of COX-2 protein were upregulated in the retinas of diabetic rats during the period of 2 weeks to 6 weeks after the induction of diabetes. Each column denotes the mean ± SD (n = 6). C: Immunofluorescence showed that COX-2 expression located in the retinal ganglion cell layer increased in the 4 week diabetic rats compared with the control. Scale bar, 100 μm. D: Changes in COX-2 in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). E: The increases in COX-2 expression were significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). Each column denotes the mean ± SD (n = 6). F: qRT-PCR showed that the levels of COX-2 mRNA were decreased obviously in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). G: Changes in vitreal PGE2 release in each group. The increase of PGE2 secretion was significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) in the retinas of 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). H: The levels of VEGF mRNA (using qRT-PCR) were downregulated in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5mM NS-398 (COX-2 inhibitor) compared with the STZ rats. Each column denotes the mean ± SD (n = 6). I: Enzyme-linked immunosorbent assay analysis of vitreal VEGF release in vitreous. The increase of VEGF secretion was significantly blocked in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). **p<0.01 versus control. #p<0.05 versus LV.shScrambled group rats. ##p<0.01 versus LV.shScrambled group rats. ψ<0.05 versus STZ group rats. ψψ<0.01 versus STZ group rats.

Mentions: We then investigated the COX-2 and PGE2 expression and the relationship among GPR91, ERK1/2, COX-2, and PGE2 in the retinas of STZ-induced diabetic rats. The levels of COX-2 protein were increased during the period of 2 weeks to 6 weeks after the induction of diabetes (Figure 4A,B). The COX-2 expression located in RGCs was also enhanced (Figure 4C). PGE2, measured because the production of PGE2 denotes activity of COX-2, was also markedly increased in the retinas of diabetic rats at 4 weeks (p<0.01, Figure 4G). Furthermore, intravitreal injection of 0.1 mM U0126 or 0.5 mM NS-398 significantly blocked the upregulation of COX-2, PGE2, and VEGF release (p<0.01, Figure 4D-I). These findings indicate that the ERK1/2 pathway is upstream of the COX-2/PGE2 pathway, and the ERK1/2/COX-2/PGE2 pathway is associated with VEGF release.


ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes.

Li T, Hu J, Du S, Chen Y, Wang S, Wu Q - Mol. Vis. (2014)

GPR91 modulated VEGF secretion via ERK1/2/cyclooxygenase-2 (COX-2)/ Prostaglandin E2 (PGE2) signaling pathway in the retinas of STZ rats. A: Western blot analysis of the COX-2 protein in the retinas of diabetic rats in different time periods (from 1 week to 10 weeks). B: The levels of COX-2 protein were upregulated in the retinas of diabetic rats during the period of 2 weeks to 6 weeks after the induction of diabetes. Each column denotes the mean ± SD (n = 6). C: Immunofluorescence showed that COX-2 expression located in the retinal ganglion cell layer increased in the 4 week diabetic rats compared with the control. Scale bar, 100 μm. D: Changes in COX-2 in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). E: The increases in COX-2 expression were significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). Each column denotes the mean ± SD (n = 6). F: qRT-PCR showed that the levels of COX-2 mRNA were decreased obviously in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). G: Changes in vitreal PGE2 release in each group. The increase of PGE2 secretion was significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) in the retinas of 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). H: The levels of VEGF mRNA (using qRT-PCR) were downregulated in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5mM NS-398 (COX-2 inhibitor) compared with the STZ rats. Each column denotes the mean ± SD (n = 6). I: Enzyme-linked immunosorbent assay analysis of vitreal VEGF release in vitreous. The increase of VEGF secretion was significantly blocked in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). **p<0.01 versus control. #p<0.05 versus LV.shScrambled group rats. ##p<0.01 versus LV.shScrambled group rats. ψ<0.05 versus STZ group rats. ψψ<0.01 versus STZ group rats.
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f4: GPR91 modulated VEGF secretion via ERK1/2/cyclooxygenase-2 (COX-2)/ Prostaglandin E2 (PGE2) signaling pathway in the retinas of STZ rats. A: Western blot analysis of the COX-2 protein in the retinas of diabetic rats in different time periods (from 1 week to 10 weeks). B: The levels of COX-2 protein were upregulated in the retinas of diabetic rats during the period of 2 weeks to 6 weeks after the induction of diabetes. Each column denotes the mean ± SD (n = 6). C: Immunofluorescence showed that COX-2 expression located in the retinal ganglion cell layer increased in the 4 week diabetic rats compared with the control. Scale bar, 100 μm. D: Changes in COX-2 in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). E: The increases in COX-2 expression were significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor). Each column denotes the mean ± SD (n = 6). F: qRT-PCR showed that the levels of COX-2 mRNA were decreased obviously in the retinas of the 4 week diabetic rats treated with scrambled shRNA lentiviral particles, GPR91 shRNA lentiviral particles, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). G: Changes in vitreal PGE2 release in each group. The increase of PGE2 secretion was significantly blocked by GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) in the retinas of 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). H: The levels of VEGF mRNA (using qRT-PCR) were downregulated in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5mM NS-398 (COX-2 inhibitor) compared with the STZ rats. Each column denotes the mean ± SD (n = 6). I: Enzyme-linked immunosorbent assay analysis of vitreal VEGF release in vitreous. The increase of VEGF secretion was significantly blocked in the 4 week diabetic rats treated with GPR91 shRNA, 0.1 mM U0126 (ERK1/2 inhibitor), or 0.5 mM NS-398 (COX-2 inhibitor) compared with the 4 week diabetic rats. Each column denotes the mean ± SD (n = 6). **p<0.01 versus control. #p<0.05 versus LV.shScrambled group rats. ##p<0.01 versus LV.shScrambled group rats. ψ<0.05 versus STZ group rats. ψψ<0.01 versus STZ group rats.
Mentions: We then investigated the COX-2 and PGE2 expression and the relationship among GPR91, ERK1/2, COX-2, and PGE2 in the retinas of STZ-induced diabetic rats. The levels of COX-2 protein were increased during the period of 2 weeks to 6 weeks after the induction of diabetes (Figure 4A,B). The COX-2 expression located in RGCs was also enhanced (Figure 4C). PGE2, measured because the production of PGE2 denotes activity of COX-2, was also markedly increased in the retinas of diabetic rats at 4 weeks (p<0.01, Figure 4G). Furthermore, intravitreal injection of 0.1 mM U0126 or 0.5 mM NS-398 significantly blocked the upregulation of COX-2, PGE2, and VEGF release (p<0.01, Figure 4D-I). These findings indicate that the ERK1/2 pathway is upstream of the COX-2/PGE2 pathway, and the ERK1/2/COX-2/PGE2 pathway is associated with VEGF release.

Bottom Line: COX-2 and VEGF mRNA were determined by quantitative RT-PCR.Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398.This study highlights the signaling pathway as a potential target for intervention in DR.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, the Sixth People's Hospital, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT

Purpose: Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes.

Methods: Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT-PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay.

Results: Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398.

Conclusions: Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR.

Show MeSH
Related in: MedlinePlus