Role of Shh signal transduction pathway in vascular endothelial growth factor expression under hypoxia in cultured human retinal pigment epithelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

HouYanan ,  LeiNingyu , SongBin , WangLijun

Department of Ophthalmology, Binzhou Medical University Hospital, Binzhou 256600, China;

Corresponding author：

LeiNingyu, Email: 15105691721@163.com

Keywords：

Vascular endothelial growth factors; Retinal pigment epithelium; Cells, cultured

DOI：

10.3760/cma.j.issn.1005-1015.2016.01.015

Video：

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Objective To investigate the role of sonic hedgehog (Shh) signal transduction pathway in the expression of vascular endothelial growth factor (VEGF) under hypoxia in cultured human retinal pigment epithelial (hRPE) cells. Methods ARPE-19 were cultured and divided into normal ARPE-19 (Cont) and hypoxia group (100 μmol/L CoCl₂ Cobalt Chloride +ARPE-19); hypoxia group was further divided into CoCl₂ group, cyclopamine group (CYA) and dimethyl sulfoxide (DMSO) group. 20μmol/L cyclopamine was added to the CYA group 1 hour before hypoxia, 1‰DMSO was added into DMSO group at the same time. The hRPE cells were cultured under hypoxia for 4, 8, 12, 24 hours. The expression of Shh and VEGF were determined by Real-time fluorescent quantitate PCR (RT-PCR). The amount of VEGF in the hRPE-conditioned supernatant was measured using enzyme linked immunosorbent assay (ELISA) at 4, 8, 12, 24 hours, respectively. Results RT-PCR tests showed that the level of Shh and VEGF of hRPE was time dependently increased (Shh: F=45.260, P=0.001; VEGF: F=264.938, P=0.001). The level of Shh and VEGF of hRPE in the group treated with cyclopamine was decreased (P < 0.01). ELISA tests showed that the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner (F=3 156.676, P=0.001), and it was down-regulated by cyclopamine under hypoxia (P < 0.01). Conclusion Shh signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells.

Citation： HouYanan, LeiNingyu, SongBin, WangLijun. Role of Shh signal transduction pathway in vascular endothelial growth factor expression under hypoxia in cultured human retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2016, 32(1): 62-65. doi: 10.3760/cma.j.issn.1005-1015.2016.01.015 Copy

1.	Di Y, Zhang Y, Yang H, et al. The mechanism of CCN1-enhanced retinal neovascularization in oxygen-induced retinopathy through PI3K/Akt-VEGF signaling pathway[J]. Drug Des Devel Ther, 2015, 30(9):2463-2473.DOI: 10.2147/DDDT.S79782.
2.	Lamoke F, Labazi M, Montemari A, et al.Trans-Chalcone prevents VEGF expression and retinal neovascularization in the ischemic retina[J].Exp Eye Res, 2011, 93(4): 350-354.DOI: 10.1016/j.exer.2011.02.007.
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4.	Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products[J]. J Biol Chem, 2001, 276(47):43836-43841.
5.	Roberg-Larsen H, Strand MF, Krauss S, et al.Metabolites in vertebrate Hedgehog signaling[J]. Biochem Biophys Res Commun, 2014, 446(3):669-674.DOI: 10.1016/j.bbrc.2014.01.087.
6.	Yao Q, Renault MA, Chapouly C, et al.Sonic hedgehog mediates a novel pathway of PDGF-BB-dependent vessel maturation[J].Blood, 2014, 123(15):2429-2437.DOI: 10.1182/blood-2013-06-508689.
7.	Satoh K, Kanno A, Hamada S, et al.Expression of Sonic hedgehog signaling pathway correlates with the tumorigenesis of intraductal papillary mucinous neoplasm of the pancreas[J].Oncol Rep, 2008, 19(5):1185-1190.
8.	Marsh JL, Jackman CP, Tang SN, et al.Embelin suppresses pancreatic cancer growth by modulating tumor immune microenvironment[J].Front Biosci (Landmark Ed), 2014, 1(19):113-125.
9.	Jiang WG, Ye L, Ruge F, et al.Expression of Sonic Hedgehog (SHH) in human lung cancer and the impact of YangZheng XiaoJi on SHH-mediated biological function of lung cancer cells and tumor growth[J].Anticancer Res, 2015, 35(3):1321-1331.
10.	Shigemura K, Fujisawa M. Hedgehog signaling and urological cancers[J].Curr Drug, Targets, 2015, 16(3):258-271.
11.	Efroni S, Meerzaman D, Schaefer CF, et al.Systems analysis utilising pathway interactions identifies sonic hedgehog pathway as a primary biomarker and oncogenic target in hepatocellular carcinoma[J].IET Syst Biol, 2013, 7(6):243-251.
12.	Miyazaki K, Saika S, Yamanaka O, et al.Treatment of eyelid epithelial neoplasm by targeting sonic hedgehog signaling: an experimental study[J].Jpn J Ophthalmol, 2006, 50(4):305-311.
13.	Banerjee J, Papu John AM, Schuller HM, Regulation of nonsmall-cell lung cancer stem cell like cells by neurotransmitters and opioid peptides[J]. Int J Cancer, 2015, 137(12):2815-2824. DOI: 10.1002/ijc.29646.
14.	Popov TM, Goranova T, Stancheva G, et al.Relative quantitative expression of hypoxia-inducible factor-1α, -2αand-3α, and vascular endothelial growth factor A in laryngeal carcinoma[J].Oncol Lett, 2015, 9(6):2879-2885.
15.	Campochiaro PA.Molecular pathogenesis of retinal and choroidal vascular diseases[J].Prog Retin Eye Res, 2015, 9462(15):67-81.DOI: 10.1016/j.Preteyeres.2015.06.002.
16.	Srinivasan S, Chitalia V, Meyer RD, et al.Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis[J]. Angiogenesis, 2015, 18(4):449-462.DOI:10.1007/s10456-015-9468-3.
17.	Wang J, Cao J, Dickson AL, Poss KD, Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signaling[J]. Nature, 2015, 522(7555):226-230. DOI: 10.1038/nature14325.
18.	Walshe TE, Connell P, Cryan L, et al. Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling[J].Invest Ophthalmol Vis Sci, 2011, 52(7):4472-4483. DOI:10.1167/iovs.10-7061.
19.	van Tuyl M, Groenman F, Wang J, et al.Angiogenic factors stimulate tubular branching morphogenesis of sonic hedgehog-deficient lungs[J].Dev Biol, 2007, 303 (2):514-526.
20.	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.
21.	Chinchilla P, Xiao L, Kazanietz MG, et al.Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways[J].Cell Cycle, 2010, 9(3):570-579.
22.	Renault MA, Roncalli J, Tongers J, et al.Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells[J].J Mol Cell Cardiol, 2010, 49(3):490-498.DOI:10.1016/j.yjmcc.2010.05.003.
23.	Heretsch P, Tzagkaroulaki L, Giannis A.Cyclopamine and hedgehog signaling: chemistry, biology, medical perspectives[J].Angew Chem Int Ed Engl, 2010, 49(20): 3418-3427.DOI:10.1002/anie.200906967.
24.	Liu NN, Zhao N, Cai N.Suppression of the proliferation of hypoxia-Induced retinal pigment epithelial cell by rapamycin through the /mTOR/HIF-1α/VEGF/ signaling[J].IUBMB Life, 2015, 67(6):446-452.DOI: 10.1002/iub.1382.
25.	Paeng SH, Jung WK, Park WS, et al.Caffeic acid phenethyl ester reduces the secretion of vascular endothelial growth factor through the inhibition of the ROS, PI3K and HIF-1αsignaling pathways in human retinal pigment epithelial cells under hypoxic conditions[J].Int J Mol Med, 2015, 35(5):1419-1426.DOI: 10.3892/ijmm.2015.2116.
26.	Wang Y, Tang Z, Xue R, et al.Diff erential response to CoCl₂-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells[J]. Mol Cell Biochem, 2012, 360:235-242.DOI:10.1007/s11010-011-1061-5.
27.	Cervellati F, Cervellati C, Romani A.Hypoxia induces cell damage via oxidative stress in retinal epithelial cells[J].Free Radical Research, 2014, 48(3):303-312. DOI:10.3109/10715762.2013.867484.
28.	Berra E, Ginouvès A, Pouysségur J.The hypoxia-induciblefactor hydroxylases bring fresh air into hypoxia signalling[J].EMBO Rep, 2006, 7(1):41-45.

1. Di Y, Zhang Y, Yang H, et al. The mechanism of CCN1-enhanced retinal neovascularization in oxygen-induced retinopathy through PI3K/Akt-VEGF signaling pathway[J]. Drug Des Devel Ther, 2015, 30(9):2463-2473.DOI: 10.2147/DDDT.S79782.
2. Lamoke F, Labazi M, Montemari A, et al.Trans-Chalcone prevents VEGF expression and retinal neovascularization in the ischemic retina[J].Exp Eye Res, 2011, 93(4): 350-354.DOI: 10.1016/j.exer.2011.02.007.
3. Li Z, He T, Du K, et al.Overexpression of 15-lipoxygenase-1 in oxygen-induced ischemic retinopathy inhibits retinal neovascularization via downregulation of vascular endothelial growth factor-A expression[J].Mol Vis, 2012, 18:2847-2859.
4. Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Regulation of vascular endothelial growth factor expression by advanced glycation end products[J]. J Biol Chem, 2001, 276(47):43836-43841.
5. Roberg-Larsen H, Strand MF, Krauss S, et al.Metabolites in vertebrate Hedgehog signaling[J]. Biochem Biophys Res Commun, 2014, 446(3):669-674.DOI: 10.1016/j.bbrc.2014.01.087.
6. Yao Q, Renault MA, Chapouly C, et al.Sonic hedgehog mediates a novel pathway of PDGF-BB-dependent vessel maturation[J].Blood, 2014, 123(15):2429-2437.DOI: 10.1182/blood-2013-06-508689.
7. Satoh K, Kanno A, Hamada S, et al.Expression of Sonic hedgehog signaling pathway correlates with the tumorigenesis of intraductal papillary mucinous neoplasm of the pancreas[J].Oncol Rep, 2008, 19(5):1185-1190.
8. Marsh JL, Jackman CP, Tang SN, et al.Embelin suppresses pancreatic cancer growth by modulating tumor immune microenvironment[J].Front Biosci (Landmark Ed), 2014, 1(19):113-125.
9. Jiang WG, Ye L, Ruge F, et al.Expression of Sonic Hedgehog (SHH) in human lung cancer and the impact of YangZheng XiaoJi on SHH-mediated biological function of lung cancer cells and tumor growth[J].Anticancer Res, 2015, 35(3):1321-1331.
10. Shigemura K, Fujisawa M. Hedgehog signaling and urological cancers[J].Curr Drug, Targets, 2015, 16(3):258-271.
11. Efroni S, Meerzaman D, Schaefer CF, et al.Systems analysis utilising pathway interactions identifies sonic hedgehog pathway as a primary biomarker and oncogenic target in hepatocellular carcinoma[J].IET Syst Biol, 2013, 7(6):243-251.
12. Miyazaki K, Saika S, Yamanaka O, et al.Treatment of eyelid epithelial neoplasm by targeting sonic hedgehog signaling: an experimental study[J].Jpn J Ophthalmol, 2006, 50(4):305-311.
13. Banerjee J, Papu John AM, Schuller HM, Regulation of nonsmall-cell lung cancer stem cell like cells by neurotransmitters and opioid peptides[J]. Int J Cancer, 2015, 137(12):2815-2824. DOI: 10.1002/ijc.29646.
14. Popov TM, Goranova T, Stancheva G, et al.Relative quantitative expression of hypoxia-inducible factor-1α, -2αand-3α, and vascular endothelial growth factor A in laryngeal carcinoma[J].Oncol Lett, 2015, 9(6):2879-2885.
15. Campochiaro PA.Molecular pathogenesis of retinal and choroidal vascular diseases[J].Prog Retin Eye Res, 2015, 9462(15):67-81.DOI: 10.1016/j.Preteyeres.2015.06.002.
16. Srinivasan S, Chitalia V, Meyer RD, et al.Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis[J]. Angiogenesis, 2015, 18(4):449-462.DOI:10.1007/s10456-015-9468-3.
17. Wang J, Cao J, Dickson AL, Poss KD, Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signaling[J]. Nature, 2015, 522(7555):226-230. DOI: 10.1038/nature14325.
18. Walshe TE, Connell P, Cryan L, et al. Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling[J].Invest Ophthalmol Vis Sci, 2011, 52(7):4472-4483. DOI:10.1167/iovs.10-7061.
19. van Tuyl M, Groenman F, Wang J, et al.Angiogenic factors stimulate tubular branching morphogenesis of sonic hedgehog-deficient lungs[J].Dev Biol, 2007, 303 (2):514-526.
20. 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.
21. Chinchilla P, Xiao L, Kazanietz MG, et al.Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways[J].Cell Cycle, 2010, 9(3):570-579.
22. Renault MA, Roncalli J, Tongers J, et al.Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells[J].J Mol Cell Cardiol, 2010, 49(3):490-498.DOI:10.1016/j.yjmcc.2010.05.003.
23. Heretsch P, Tzagkaroulaki L, Giannis A.Cyclopamine and hedgehog signaling: chemistry, biology, medical perspectives[J].Angew Chem Int Ed Engl, 2010, 49(20): 3418-3427.DOI:10.1002/anie.200906967.
24. Liu NN, Zhao N, Cai N.Suppression of the proliferation of hypoxia-Induced retinal pigment epithelial cell by rapamycin through the /mTOR/HIF-1α/VEGF/ signaling[J].IUBMB Life, 2015, 67(6):446-452.DOI: 10.1002/iub.1382.
25. Paeng SH, Jung WK, Park WS, et al.Caffeic acid phenethyl ester reduces the secretion of vascular endothelial growth factor through the inhibition of the ROS, PI3K and HIF-1αsignaling pathways in human retinal pigment epithelial cells under hypoxic conditions[J].Int J Mol Med, 2015, 35(5):1419-1426.DOI: 10.3892/ijmm.2015.2116.
26. Wang Y, Tang Z, Xue R, et al.Diff erential response to CoCl₂-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells[J]. Mol Cell Biochem, 2012, 360:235-242.DOI:10.1007/s11010-011-1061-5.
27. Cervellati F, Cervellati C, Romani A.Hypoxia induces cell damage via oxidative stress in retinal epithelial cells[J].Free Radical Research, 2014, 48(3):303-312. DOI:10.3109/10715762.2013.867484.
28. Berra E, Ginouvès A, Pouysségur J.The hypoxia-induciblefactor hydroxylases bring fresh air into hypoxia signalling[J].EMBO Rep, 2006, 7(1):41-45.

Chinese Journal of Ocular Fundus Diseases

Role of Shh signal transduction pathway in vascular endothelial growth factor expression under hypoxia in cultured human retinal pigment epithelial cells

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