The relationship between the expression of sonic hedgehog and vascular endothelial growth factor in hypoxic human retinal pigment epithelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Ji Peng ,  Lei Ningyu , Song Bin , Wang Lijun , Hou Yanan

Department of Ophthalmology, The Affiliated Hospital of Binzhou Medical College, Binzhou 256600, China;

Corresponding author：

Lei Ningyu, Email: 15105691721@163.com

Keywords：

Hedgehog proteins; Vascular endothelial growth factors; Retinal pigment epithelium; Anoxia

DOI：

10.3760/cma.j.issn.1005-1015.2018.02.014

Video：

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Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells. Methods Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl₂) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis. Results The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05). Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.

Citation： Ji Peng, Lei Ningyu, Song Bin, Wang Lijun, Hou Yanan. The relationship between the expression of sonic hedgehog and vascular endothelial growth factor in hypoxic human retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2018, 34(2): 168-171. doi: 10.3760/cma.j.issn.1005-1015.2018.02.014 Copy

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11.	Pola R, Ling LE, Aprahamian TR, et al. Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia[J]. Circulation,2003,108(4):479-485.DOI:10.1161/01.CIR.0000080338.60981.FA.
12.	Spranger J, Osterhoff M, Reimann M, et al. Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease[J].Diabetes,2001,50(12):2641-2645.
13.	Bortoloso E, Prete DD, Vestra MD, et al. Quantitave and qualitative changes in vascular endothelial growth factor gene expression in glomeruli of patients with type 2 diabetes[J].Eur J Endocrinol,2004,150(6):799-807.
14.	De-Vriese AS, Tilton RG, Stephan CC, et al.Vascular endothelial growth factor is essential for hyperglycemia-induced structural and functional alterations of the peritoneal membrane[J].J Am Soc Nephrol,2001,12(8):1734-1741.
15.	Liu H, Zhang W, Kennard S, et al. Notch3 is critical for proper angiogenesis and mural cell investment[J]. Circ Res,2010,107(7):860-870. DOI:10.1161/CIRCRESAHA.110.218271.
16.	Ola MS, Nawaz MI, Siddiquei MM, et al. Recent advances in understanding the biochemical and molecular mechanism of diabetic retinopathy[J]. J Diabetes Complications,2012,26(1):56-64. DOI:10.1016/j.jdiacomp.2011.11.004.
17.	Cain JE, Rosenblum ND. Control of mammalian kidney development by the Hedgehog signaling pathway[J].Pediatr Nephrol,2011,26(9):1365-1371. DOI: 10.1007/s00467-010-1704-x.
18.	Coorey NJ, Shen W, Chung SH, et al. The role of glia in retinal vascular disease[J]. Clin Exp Optom,2012,95(3):266-281. DOI:10.1111/j.1444-0938.2012.00741.x.
19.	Edelman JL, Castro MR. Quantitative image analysis of laser-induced choroidal neovascularization in rat[J].Exp Eye Res,2000,71(5):523-533.DOI:10.1006/exer.2000.0907.
20.	Fujita K, Miyamoto T, Saika S. Sonic hedgehog: its expression in a healing cornea and its role in neovascularization[J]. Mol Vis,2009,15:1036-1044.

1. Kraft A, Weindel K, Ochs A, et al. Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease[J]. Cancer, 1999, 85(1):178-187.
2. Qaum T, Xu Q, Joussen AM, et al. VEGF-initiated blood-retinal barrier breakdown in early diabetes[J]. Invest Ophthalmol Vis Sci, 2001,42(10):2408-2413.
3. Funatsu H, Yamashita H, Nakamura S, et al. Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor are related to diabetic macular edema[J]. Ophthalmology, 2006,113(2):294-301. DOI: 10.1016/j.ophtha.2005.10.030.
4. Simó R, Carrasco E, García-Ramírez M, et al. Angiogenic and antiangiogenic factors in proliferative diabetic retinopathy[J].Curr Diabetes Rev, 2006, 2(1):71-98. DOI:10.2174/157339906775473671.
5. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor[J]. Endocr Rev, 1997,18(1):4-25. DOI:10.1210/er.18.1.4.
6. Reinders ME, Sho M, Izawa A, et al. Proinflammatory functions of vascular endothelial growth factor in alloimmunity[J].J Clin Invest,2003,112(11):1655-1665. DOI:10.1172/JCI17712.
7. Hammerschmidt M, Brook A, McMahon AP. The world according to hedgehog[J].Trends Genet,1997,13(1):14-21.
8. Pola R, Ling LE, Silver M, et al. The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors[J].Nat Med,2001,7(6):706-711. DOI:10.1038/89083.
9. Asai J, Takenaka H, Kusano KF. Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling[J].Circulation,2006,113(20):2413-2424.DOI:10.1161/CIRCULATIONAHA.105.603167.
10. Hwang JM, Weng YJ, Lin JA, et al. Hypoxia-induced compensatory effect as related to Shh and HIF-1alpha in ischemia embryo rat heart[J].Mol Cell Biochem,2008,311(1/2):179-187. DOI:10.1007/s11010-008-9708-6.
11. Pola R, Ling LE, Aprahamian TR, et al. Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia[J]. Circulation,2003,108(4):479-485.DOI:10.1161/01.CIR.0000080338.60981.FA.
12. Spranger J, Osterhoff M, Reimann M, et al. Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease[J].Diabetes,2001,50(12):2641-2645.
13. Bortoloso E, Prete DD, Vestra MD, et al. Quantitave and qualitative changes in vascular endothelial growth factor gene expression in glomeruli of patients with type 2 diabetes[J].Eur J Endocrinol,2004,150(6):799-807.
14. De-Vriese AS, Tilton RG, Stephan CC, et al.Vascular endothelial growth factor is essential for hyperglycemia-induced structural and functional alterations of the peritoneal membrane[J].J Am Soc Nephrol,2001,12(8):1734-1741.
15. Liu H, Zhang W, Kennard S, et al. Notch3 is critical for proper angiogenesis and mural cell investment[J]. Circ Res,2010,107(7):860-870. DOI:10.1161/CIRCRESAHA.110.218271.
16. Ola MS, Nawaz MI, Siddiquei MM, et al. Recent advances in understanding the biochemical and molecular mechanism of diabetic retinopathy[J]. J Diabetes Complications,2012,26(1):56-64. DOI:10.1016/j.jdiacomp.2011.11.004.
17. Cain JE, Rosenblum ND. Control of mammalian kidney development by the Hedgehog signaling pathway[J].Pediatr Nephrol,2011,26(9):1365-1371. DOI: 10.1007/s00467-010-1704-x.
18. Coorey NJ, Shen W, Chung SH, et al. The role of glia in retinal vascular disease[J]. Clin Exp Optom,2012,95(3):266-281. DOI:10.1111/j.1444-0938.2012.00741.x.
19. Edelman JL, Castro MR. Quantitative image analysis of laser-induced choroidal neovascularization in rat[J].Exp Eye Res,2000,71(5):523-533.DOI:10.1006/exer.2000.0907.
20. Fujita K, Miyamoto T, Saika S. Sonic hedgehog: its expression in a healing cornea and its role in neovascularization[J]. Mol Vis,2009,15:1036-1044.

Chinese Journal of Ocular Fundus Diseases

The relationship between the expression of sonic hedgehog and vascular endothelial growth factor in hypoxic human retinal pigment epithelial cells

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