The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats_Chinese Journal of Ocular Fundus Diseases

Authors：

HuJianyan , LiTingting ,  WuQiang

Department of Ophthalmology, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233, China;

Corresponding author：

WuQiang, Email: wyan559@hotmail.com

Keywords：

Diabetic retinopathy/physiopathology; RNA, small interfering; Receptors, G-protein-coupled; Vascular Endothelial Growth Factors; p38 Mitogen-Activated Protein Kinases; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2015.02.012

Video：

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Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×10⁸ TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×10⁸ TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P < 0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P < 0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P < 0.05).The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45;P < 0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P < 0.05). Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.

Citation： HuJianyan, LiTingting, WuQiang. The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats. Chinese Journal of Ocular Fundus Diseases, 2015, 31(2): 157-161. doi: 10.3760/cma.j.issn.1005-1015.2015.02.012 Copy

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2.	Miller JW, Adamis AP, Shima DT, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model[J]. Am J Pathol, 1994, 145(3):574-584.
3.	Witmer AN, Blaauwgeers HG, Weich HA, et al. Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey[J].Invest Ophthalmol Vis Sci, 2002, 43(3):849-857.
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7.	李婷婷, 胡健艳, 吴强.靶向G蛋白偶联受体91的小发夹RNA慢病毒载体的构建及功能初步检测[J].眼科新进展, 2014, 34(8):705-709.
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11.	Matsumoto M, Suzuma K, Maki T, et al. Succinate increases in the vitreous fluid of patients with active proliferative diabetic retinopathy[J]. Am J Ophthalmol, 2012, 153(5):896-902.
12.	Sydorova M, Lee MS. Vascular endothelial growth factor levels in vitreous and serum of patients with either proliferative diabetic retinopathy or proliferative vitreoretinopathy[J].Ophthalmic Res, 2005, 37(4):188-190.
13.	Wang X, Wang G, Wang Y. Intravitreous vascular endothelial growth factor and hypoxia inducible factor la in patients with proliferative diabetic retinopathy[J]. Am J Ophthalmo1, 2009, 148(6):883-889.
14.	Zhang W, Liu HT.MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J].Cell Res, 2002, 12(1):9-18.
15.	李婷婷, 胡健艳, 吴强.丝裂原活化蛋白激酶信号通路与眼底新生血管性疾病相关性的研究进展[J].中华眼底病杂志, 2013, 29(4):443-447.
16.	Du Y, Tang J, Li G, et al. Effects of p38 MAPK Inhibition on early stages of diabetic retinopathy and sensory nerve function[J]. Invest Ophthalmol Vis Sci, 2010, 51(4):2158-2164.
17.	Yadav UC, Srivastava SK, Ramana KV. Prevention of VEGF-induced growth and tube formation in human retinal endothelial cells by aldose reductase inhibition[J]. J Diabetes Complications, 2012, 26(5):369-377.
18.	Vargas SL, Toma I, Kang JJ, et al. Activation of the succinate receptor GPR91 in macula densa cells causes renin release[J].J Am Soc Nephrol, 2009, 20(5):1002-1011.
19.	Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs[J]. Oncogene, 2007, 26(22):3100-3112.
20.	Lee JJ, Hsiao CC, Yang IH, et al. High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5[J]. Mol Vis, 2012, 18:838-850.
21.	Zong H, Ward M, Madden A, et al. Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)[J].Diabetologia, 2010, 53(12):2656-2666.

1. Forrester JV, Shafiee A, Schröder S, et al. The role of growth factors in proliferative diabetic retinopathy[J]. Eye(Lond), 1993, 7(Pt 2):276-287.
2. Miller JW, Adamis AP, Shima DT, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model[J]. Am J Pathol, 1994, 145(3):574-584.
3. Witmer AN, Blaauwgeers HG, Weich HA, et al. Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey[J].Invest Ophthalmol Vis Sci, 2002, 43(3):849-857.
4. Sapieha P, Sirinyan M, Hamel D, et al. The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis[J].Nat Med, 2008, 14(10):1067-1076.
5. Hu J, Wu Q, Li T, et al. Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91[J]. Exp Eye Res, 2013, 109:31-39.
6. Li T, Hu J, Du S, et al.ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes[J]. Mol Vis, 2014, 20:1109-1121.
7. 李婷婷, 胡健艳, 吴强.靶向G蛋白偶联受体91的小发夹RNA慢病毒载体的构建及功能初步检测[J].眼科新进展, 2014, 34(8):705-709.
8. Zheng Z, Chen H, Li J, et al. Sirtuin 1-mediated cellular metabolic memory of high glucose via the LKB1/AMPK/ROS pathway and therapeutic effects of metformin[J]. Diabetes, 2012, 16(1):217-228.
9. Zhang W, Yan H. Dysfunction of circulating endothelial progenitor cells in type 1 diabetic rats with diabetic retinopathy[J]. Graefe's Arch Clin Exp Ophthalmol, 2013, 251(4):1123-1131.
10. He W, Miao FJ, Lin DC, et al. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors[J].Nature, 2004, 429(6988):188-193.
11. Matsumoto M, Suzuma K, Maki T, et al. Succinate increases in the vitreous fluid of patients with active proliferative diabetic retinopathy[J]. Am J Ophthalmol, 2012, 153(5):896-902.
12. Sydorova M, Lee MS. Vascular endothelial growth factor levels in vitreous and serum of patients with either proliferative diabetic retinopathy or proliferative vitreoretinopathy[J].Ophthalmic Res, 2005, 37(4):188-190.
13. Wang X, Wang G, Wang Y. Intravitreous vascular endothelial growth factor and hypoxia inducible factor la in patients with proliferative diabetic retinopathy[J]. Am J Ophthalmo1, 2009, 148(6):883-889.
14. Zhang W, Liu HT.MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J].Cell Res, 2002, 12(1):9-18.
15. 李婷婷, 胡健艳, 吴强.丝裂原活化蛋白激酶信号通路与眼底新生血管性疾病相关性的研究进展[J].中华眼底病杂志, 2013, 29(4):443-447.
16. Du Y, Tang J, Li G, et al. Effects of p38 MAPK Inhibition on early stages of diabetic retinopathy and sensory nerve function[J]. Invest Ophthalmol Vis Sci, 2010, 51(4):2158-2164.
17. Yadav UC, Srivastava SK, Ramana KV. Prevention of VEGF-induced growth and tube formation in human retinal endothelial cells by aldose reductase inhibition[J]. J Diabetes Complications, 2012, 26(5):369-377.
18. Vargas SL, Toma I, Kang JJ, et al. Activation of the succinate receptor GPR91 in macula densa cells causes renin release[J].J Am Soc Nephrol, 2009, 20(5):1002-1011.
19. Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs[J]. Oncogene, 2007, 26(22):3100-3112.
20. Lee JJ, Hsiao CC, Yang IH, et al. High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5[J]. Mol Vis, 2012, 18:838-850.
21. Zong H, Ward M, Madden A, et al. Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)[J].Diabetologia, 2010, 53(12):2656-2666.

Chinese Journal of Ocular Fundus Diseases

The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats

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