PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression

Wang,Xia; Liu,Xu; Ren,Yuan; Liu,Ying; Han,Shuangyu; Zhao,Jingkang; Gou,Xingchun; He,Yuan

doi:10.3892/mmr.2018.9645

PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression

Authors:
- Xia Wang
- Xu Liu
- Yuan Ren
- Ying Liu
- Shuangyu Han
- Jingkang Zhao
- Xingchun Gou
- Yuan He
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Affiliations: Department of Ophthalmology, The Second Affiliated Hospital of Xi'an Medical University, Ocular Immunology and Inflammation Institute, Shaanxi Provincial Clinical Research Center for Ophthalmology, Xi'an, Shaanxi 710038, P.R. China, Department of Neurobiology, The Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, Shaanxi 710021, P.R. China
Published online on: November 12, 2018 https://doi.org/10.3892/mmr.2018.9645
Pages: 59-74
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

To investigate the protective function of pigment epithelium‑derived factor (PEDF) against oxidative stress (OS) in ARPE‑19 cells, ARPE‑19 cells were divided into different OS groups and treated with various concentrations of H2O2 (0, 75, 150 and 200 µmol/l) for 24 h. To establish the protective group, 200 ng/ml of PEDF was administered to ARPE‑19 cells. Cell Counting Kit‑8 assays and cell growth curve experiments were performed to determine levels of cell viability; lactate dehydrogenase and propidium iodide (PI) staining assays were also performed. The expression levels of genes associated with apoptosis as well as uncoupling protein 2 (UCP2) were detected by reverse transcription‑quantitative, or semi‑quantitative polymerase chain reaction. Furthermore, an OS injury animal model was established in both C57BL/6 and BALB/c mice via injection of 5 µg of PEDF in the vitreous cavity and subsequent injection of 150 µM H2O2 following a 24 h time interval. Hematoxylin and eosin (H&E) staining, as well as UCP2 immunofluorescent labeling were also performed. One‑way analysis of variance was used to determine statistically significant differences, followed by multiple comparison analysis using the Newman Keuls method. The results of cell viability assays demonstrated that the numbers of apoptotic cells were increased following treatment with H2O2 in a dose‑dependent manner; however, this effect was reversed following treatment with PEDF. The expression levels of caspase 3 and B cell lymphoma (Bcl2) associated X genes associated with apoptosis were inhibited, whereas levels of the anti‑apoptotic gene Bcl2 were enhanced following treatment with PEDF in different passages of ARPE‑19 cells. Significant differences were demonstrated in the levels of UCP2 gene expression between the PEDF+ H2O2 treated group and cells treated with H2O2 alone. Labeling of the UCP2 detector in the confocal images demonstrated decreased UCP2 protein staining in the retinal pigment epithelium (RPE) cells and RPE layers following H2O2 injury; however, this effect was inhibited following treatment with PEDF. H&E staining was performed to investigate the thickness of the RPE layers, and the results revealed that thicknesses were significantly increased in sections treated with PEDF during OS, due to increased numbers of RPE cells. Furthermore, PEDF was demonstrated to increase UCP2 gene expression in ARPE‑19 cells and animal RPE layers under OS, which suggested that PEDF may protect RPE cells and tissues during oxidative injury.

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1	Klein R and Klein BE: The prevalence of age-related eye diseases and visual impairment in aging: Current estimates. Invest Ophthalmol Vis Sci. 54:ORSF5–ORSF13. 2013. View Article : Google Scholar : PubMed/NCBI
2	Jermak CM, Dellacroce JT, Hefez J and Peyman GA: Triam cinolone acetonide in ocular therapeutics. Surv Ophthalmol. 52:503–522. 2007. View Article : Google Scholar : PubMed/NCBI
3	Yu CC, Nandrot EF, Ying D and Finnemann SC: Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin. Free Radic Biol Med. 52:660–670. 2012. View Article : Google Scholar : PubMed/NCBI
4	Khandhadia S and Lotery A: Oxidation and age-related macular degeneration: Insights from molecular biology. Expert Rev Mol Med. 12:e342010. View Article : Google Scholar : PubMed/NCBI
5	Jarrett S G and Boulton ME: Consequences of oxidative stress in age-related macular degeneration. Mol Aspects Med. 33:399–417. 2012. View Article : Google Scholar : PubMed/NCBI
6	Mettu PS, Wielgus AR, Ong SS and Cousins SW: Retinal pigment epithelium response to oxidant injury in the pathogenesis of early age-related macular degeneration. Mol Aspects Med. 33:376–398. 2012. View Article : Google Scholar : PubMed/NCBI
7	Golestaneh N, Chu Y, Xiao YY, Stoleru GL and Theos AC: Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration. Cell Death Dis. 8:e25372017. View Article : Google Scholar : PubMed/NCBI
8	Bailey TA, Kanuga N, Romero IA, Greenwood J, Luthert PJ and Cheetham ME: Oxidative stress affects the junctional integrity of retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 45:675–684. 2004. View Article : Google Scholar : PubMed/NCBI
9	Strunnikova N, Zhang C, Teichberg D, Cousins SW, Baffi J, Becker KG and Csaky KG: Survival of retinal pigment epithelium after exposure to prolonged oxidative injury: A detailed gene expression and cellular analysis. Invest Ophthalmol Vis Sci. 45:3767–3777. 2004. View Article : Google Scholar : PubMed/NCBI
10	Mailloux RJ and Harper ME: Uncoupling proteins and the control of mitochondrial reactive oxygen species production. Free Radic Biol Med. 51:1106–1105. 2011. View Article : Google Scholar : PubMed/NCBI
11	Collins S, Pi J and Yehuda-Shnaidman E: Uncoupling and reactive oxygen species (ROS)-A double-edged sword for β-cell function? ‘Moderation in all things’. Best Pract Res Clin Endocrinol Metab. 26:753–758. 2012. View Article : Google Scholar : PubMed/NCBI
12	Casteilla L, Rigoulet M and Pénicaud L: Mitochondrial ROS metabolism: Modulation by uncoupling proteins. IUBMB Life. 52:181–188. 2010. View Article : Google Scholar
13	Andrews ZB, Horvath B, Barnstable CJ, Elsworth J, Yang L, Beal MF, Roth RH, Matthews RT and Horvath TL: Uncoupling protein-2 is critical for nigral dopamine cell survival in a mouse model of Parkinson's disease. J Neurosci. 25:184–191. 2005. View Article : Google Scholar : PubMed/NCBI
14	Trenker M, Malli R, Fertschai I, Levakfrank S and Graier WF: Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat Cell Biol. 9:445–452. 2007. View Article : Google Scholar : PubMed/NCBI
15	Diano S and Horvath TL: Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism. Trends Mol Med. 18:52–58. 2012. View Article : Google Scholar : PubMed/NCBI
16	Sluse FE: Uncoupling proteins: Molecular, functional, regulatory, physiological and pathological aspects. Adv Exp Med Biol. 942:137–156. 2012. View Article : Google Scholar : PubMed/NCBI
17	Cardoso S, Correia S, Carvalho C, Candeias E, Plácido AI, Duarte AI, Seiça RM and Moreira PI: Perspectives on mitochondrial uncoupling proteins-mediated neuroprotection. J Bioenerg Biomembr. 47:119–131. 2015. View Article : Google Scholar : PubMed/NCBI
18	Chan SH, Wu CA, Wu KL, Ho YH, Chang AY and Chan JY: Transcriptional upregulation of mitochondrial uncoupling protein 2 protects against oxidative stress-associated neurogenic hypertension. Circ Res. 105:886–896. 2009. View Article : Google Scholar : PubMed/NCBI
19	Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, et al: Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet. 26:435–439. 2000. View Article : Google Scholar : PubMed/NCBI
20	Donadelli M, Dando I, Fiorini C and Palmieri M: UCP2, a mitochondrial protein regulated at multiple levels. Cell Mol Life Sci. 71:1171–1190. 2014. View Article : Google Scholar : PubMed/NCBI
21	Duh EJ, Yang HS, Haller JA, De Juan E, Humayun MS, Gehlbach P, Melia M, Pieramici D, Harlan JB, Campochiaro PA and Zack DJ: Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor: Implications for ocular angiogenesis. Am J Ophthalmol. 137:668–674. 2004. View Article : Google Scholar : PubMed/NCBI
22	Holekamp NM, Bouck N and Volpert O: Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to age-related macular degeneration. Am J Ophthalmol. 134:220–227. 2002. View Article : Google Scholar : PubMed/NCBI
23	Ogata N, Matsuoka M, Imaizumi M, Arichi M and Matsumura M: Decreased levels of pigment epithelium-derived factor in eyes with neuroretinal dystrophic diseases. Am J Ophthalmol. 137:1129–1130. 2004. View Article : Google Scholar : PubMed/NCBI
24	Becerra SP, Dass CR, Yabe T and Crawford SE: Pigment epithelium-derived factor: Chemistry, structure, biology, and applications. J Biomed Biotechnol. 2012:8309752012. View Article : Google Scholar : PubMed/NCBI
25	Karakousis PC, John SK, Behling KC, Surace EM, Smith JE, Hendrickson A, Tang WX, Bennett J and Milam AH: Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues. Mol Vis. 7:154–163. 2001.PubMed/NCBI
26	Kozulin P, Natoli R, Bumsted O'Brien KM, Madigan MC and Provis JM: The cellular expression of antiangiogenic factors in fetal primate macula. Invest Ophthalmol Vis Sci. 51:4298–4306. 2010. View Article : Google Scholar : PubMed/NCBI
27	Becerra SP, Fariss RN, Wu YQ, Montuenga LM, Wong P and Pfeffer BA: Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: Apical secretion and distribution. Exp Eye Res. 78:223–234. 2004. View Article : Google Scholar : PubMed/NCBI
28	Perez-Mediavilla LA, Chew C, Campochiaro PA, Nickells RW, Notario V, Zack DJ and Becerra SP: Sequence and expression analysis of bovine pigment epithelium-derived factor. Biochim Biophys Acta. 1398:203–214. 1998. View Article : Google Scholar : PubMed/NCBI
29	He Y, Leung KW, Ren Y, Pei J, Ge J and Tombran-Tink J: PEDF improves mitochondrial function in RPE cells during oxidative stress. Invest Ophthalmol Vis Sci. 55:6742–6755. 2014. View Article : Google Scholar : PubMed/NCBI
30	Wankun X, Wenzhen Y, Min Z, Weiyan Z, Huan C, Wei D, Lvzhen H, Xu Y and Xiaoxin L: Protective effect of paeoniflorin against oxidative stress in human retinal pigment epithelium in vitro. Mol Vis. 17:3512–3522. 2011.PubMed/NCBI
31	Tsao YP, Ho TC, Chen SL and Cheng HC: Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells. Life Sci. 79:545–550. 2006. View Article : Google Scholar : PubMed/NCBI
32	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR a nd the 2(-Delta Delta C(T)) method. Method. 25:402–408. 2001. View Article : Google Scholar
33	Yang WQ, Sun DD, Zhao T, Ren DQ and Guo GZ: Effects of erythrocyte relative chemo-luminescence intensities induced by H2O2 in mice exposed to EMP. Environmental Electromagnetics, the 2006 Asia-Pacific Conference. Proceedings of a meeting held 1-4 August 2006 Dalian, China: pp. 91–94. 2006
34	Huang Q, Wang S, Sorenson CM and Sheibani N: PEDF-deficient mice exhibit an enhanced rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration. Exp Eye Res. 87:226–241. 2008. View Article : Google Scholar : PubMed/NCBI
35	Wu WC, Hu DN, Gao HX, Chen M, Wang D, Rosen R and McCormick SA: Subtoxic levels hydrogen peroxide-induced production of interleukin-6 by retinal pigment epithelial cells. Mol Vis. 16:1864–1873. 2010.PubMed/NCBI
36	Kaczara P, Sarna T and Burke JM: Dynamics of H2O2 availability to ARPE-19 cultures in models of oxidative stress. Free Radic Biol Med. 48:1064–1070. 2010. View Article : Google Scholar : PubMed/NCBI
37	Weigel AL, Handa JT and Hjelmeland LM: Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells. Free Radic Biol Med. 33:1419–1432. 2002. View Article : Google Scholar : PubMed/NCBI
38	Bailey TA, Kanuga N, Romero IA, Greenwood J, Luthert PJ and Cheetham ME: Oxidative stress affects the junctional integrity of retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 45:675–684. 2004. View Article : Google Scholar : PubMed/NCBI
39	Hanus J, Anderson C and Wang S: RPE necroptosis in response to oxidative stress and in AMD. Ageing Res Rev. 24:286–298. 2015. View Article : Google Scholar : PubMed/NCBI
40	Jarrett SG and Boulton ME: Consequences of oxidative stress in age-related macular degeneration. Mol Aspects Med. 33:399–417. 2012. View Article : Google Scholar : PubMed/NCBI
41	Querques G, Rosenfeld PJ, Cavallero E, Borrelli E, Corvi F, Querques L, Bandello FM and Zarbin MA: Treatment of dry age-related macular degeneration. Ophthalmic Res. 52:107–115. 2014. View Article : Google Scholar : PubMed/NCBI
42	Aksenov MY, Aksenova MV, Butterfield DA, Geddes JW and Markesbery WR: Protein oxidation in the brain in Alzheimer's disease. Neuroscience. 103:373–383. 2001. View Article : Google Scholar : PubMed/NCBI
43	Adams JD Jr, Chang ML and Klaidman L: Parkinson's disease-redox mechanisms. Curr Med Chem. 8:809–814. 2001. View Article : Google Scholar : PubMed/NCBI
44	Age-Related Eye Disease Study Research Group: A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene and zinc for age-related macular degeneration and vision loss. AREDS report no. 9. Arch Ophthalmol. 119:1439–1452. 2001. View Article : Google Scholar
45	Yuan H, Jian G, Burke JM, Myers RL, Dong ZZ and Tombran-Tink J: Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress. J Ocul Biol Dis Infor. 3:92–108. 2010. View Article : Google Scholar : PubMed/NCBI
46	He Y and Tombran-Tink J: Mitochondrial decay and impairment of antioxidant defenses in aging RPE cells. Adv Exp Med Biol. 664:165–183. 2010. View Article : Google Scholar : PubMed/NCBI
47	Barnstable CJ, Reddy R, Li H and Horvath TL: Mitochondrial uncoupling protein 2 (UCP2) regulates retinal ganglion cell number and survival. J Mol Neurosci. 58:461–469. 2016. View Article : Google Scholar : PubMed/NCBI
48	Barnstable CJ and Tombran-Tink J: Neuroprotective and antiangiogenic actions of PEDF in the eye: Molecular targets and therapeutic potential. Prog Retin Eye Res. 23:561–577. 2004. View Article : Google Scholar : PubMed/NCBI
49	Becerra SP: Focus on molecules: Pigment epithelium-derived factor (PEDF). Exp Eye Res. 82:739–740. 2006. View Article : Google Scholar : PubMed/NCBI
50	Bouck N: PEDF: Anti-angiogenic guardian of ocular function. Trends Mol Med. 8:330–334. 2002. View Article : Google Scholar : PubMed/NCBI
51	Subramanian P, Locatelli-Hoops S, Kenealey J, Desjardin J, Notari L and Becerra SP: Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF receptor (PEDF-R): Identification of a functional ligand binding site. J Biol Chem. 288:23928–23942. 2013. View Article : Google Scholar : PubMed/NCBI
52	Geiger RC, Waters CM, Kamp DW and Glucksberg MR: KGF prevents oxygen-mediated damage in ARPE-19 cells. Invest Ophthalmol Vis Sci. 46:3435–3442. 2005. View Article : Google Scholar : PubMed/NCBI
53	Subramanian P, Mendez EF and Becerra SP: A novel inhibitor of 5-Lipoxygenase (5-LOX) prevents oxidative stress-induced cell death of retinal pigment epithelium (RPE) cells. Invest Ophthalmol Vis Sci. 57:4581–4588. 2016. View Article : Google Scholar : PubMed/NCBI
54	Yao S, Zhang Y, Wang X, Zhao F, Sun M, Zheng X, Dong H and Guo K: Pigment epithelium-derived factor (PEDF) protects osteoblastic cell line from glucocorticoid-induced apoptosis via PEDF-R. Int J Mol Sci. 17(pii): E7302016. View Article : Google Scholar : PubMed/NCBI
55	Ma S, Yao S, Hua T, Jiao P, Yang N, Zhu P and Qin S: Pigment epithelium-derived factor alleviates endothelial injury by inhibiting Wnt/β-catenin pathway. Lipids Health Dis. 16:312017. View Article : Google Scholar : PubMed/NCBI