Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress_Chinese Journal of Ocular Fundus Diseases

Authors：

WangJing , CaiMeng , LiJing , 田蓉 , 林少芬 , 田景毅 , 李佳 , 俞德超 ,  罗燕

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Guangzhou 510060, China;

Corresponding author：

罗燕, Email: luoyan2@mail.sysu.edu.cn

Keywords：

Retinal pigment epithelium; Receptors, complement 3b/antagonists & inhibitors; Complement activation; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2014.04.016

Video：

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Objective To observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). Conclusions CR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.

Citation： WangJing, CaiMeng, LiJing. Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress. Chinese Journal of Ocular Fundus Diseases, 2014, 30(4): 399-403. doi: 10.3760/cma.j.issn.1005-1015.2014.04.016 Copy

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12.	Bohana-Kashtan O, Ziporen L, Donin N, et al. Cell signals transduced by complement[J]. Mol Immunol, 2004, 41:583-597.
13.	Chen YW, Yang CY, Jin NS, et al. Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization[J]. Eur J Immunol, 2007, 37:167-176.
14.	Jin J, Zhou KK, Park K, et al. Anti-inflammatory and antiangiogenic effects of nanoparticle-mediated delivery of a natural angiogenic inhibitor[J]. Invest Ophthalmol Vis Sci, 2011, 52:6230-6237.
15.	Suzuki M, Tsujikawa M, Itabe H, et al. Chronic photo-oxidative stress and subsequent MCP-1 activation as causative factors for age-related macular degeneration[J].J Cell Sci, 2012, 125:2407-2415.
16.	Liu J, Jha P, Lyzogubov VV, et al. Relationship between complement membrane attack complex, chemokine (C-C motif) ligand 2(CCL2) and vascular endothelial growth factor in mouse model of laser-induced choroidal neovascularization[J]. J Biol Chem, 2011, 286:20991-21001.
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18.	Nozaki M, Raisler BJ, Sakurai E, et al. Drusen complement components C3a and C5a promote choroidal neovascularization[J]. Proc Natl Acad Sci USA, 2006, 103:2328-2333.

1. Thurman JM, Renner B, Kunchithapautham K, et al. Oxidative stress renders retinal pigment epithelial cells susceptible to complement-mediated injury[J]. J Biol Chem, 2009, 284:16939-16947.
2. Ramaglia V, Wolterman R, Kok M, et al. Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury[J]. Am J Pathol, 2008, 172:1043-1052.
3. Luo Y, Zhuo Y, Fukuhara M, et al. Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line[J]. Invest Ophthalmol Vis Sci, 2006, 47:3644-3655.
4. 徐海峰, 王宜强, 王瑶, 等.曲安奈德对人视网膜色素上皮细胞活性与屏障功能的影响[J].中华眼底病杂志, 2005, 21:237-239.
5. Ablonczy Z, Crosson CE. VEGF modulation of retinal pigment epithelium resistance[J]. Exp Eye Res, 2007, 85:762-771.
6. Joseph K, Kulik L, Coughlin B, et al. Oxidative stress sensitizes retinal pigmented epithelial (RPE) cells to complement-mediated injury in a natural antibody-, lectin pathway-, and phospholipid epitope-dependent manner[J]. J Biol Chem, 2013, 288:12753-12765.
7. Robman L, Mahdi OS, Wang JJ, et al. Exposure to chlamydia pneumoniae infection and age-related macular degeneration: the Blue Mountains Eye Study[J]. Invest Ophthalmol Vis Sci, 2007, 48:4007-4011.
8. Kannan R, Zhang N, Sreekumar PG, et al. Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells[J]. Mol Vis, 2006, 12:1649-1659.
9. Shirasawa M, Sonoda S, Terasaki H, et al. TNF-alpha disrupts morphologic and functional barrier properties of polarized retinal pigment epithelium[J]. Exp Eye Res, 2013, 110:59-69.
10. Chen Y, Yang P, Li F, et al. The effects of Th17 cytokines on the inflammatory mediator production and barrier function of ARPE-19 cells[J/OL]. PloS one, 2011, 6:18139[2011-03-30]. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018139.
11. Xie P, Kamei M, Suzuki M, et al. Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344[J/OL]. PloS one, 2011, 6:28933[2011-12-19]. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028933.
12. Bohana-Kashtan O, Ziporen L, Donin N, et al. Cell signals transduced by complement[J]. Mol Immunol, 2004, 41:583-597.
13. Chen YW, Yang CY, Jin NS, et al. Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization[J]. Eur J Immunol, 2007, 37:167-176.
14. Jin J, Zhou KK, Park K, et al. Anti-inflammatory and antiangiogenic effects of nanoparticle-mediated delivery of a natural angiogenic inhibitor[J]. Invest Ophthalmol Vis Sci, 2011, 52:6230-6237.
15. Suzuki M, Tsujikawa M, Itabe H, et al. Chronic photo-oxidative stress and subsequent MCP-1 activation as causative factors for age-related macular degeneration[J].J Cell Sci, 2012, 125:2407-2415.
16. Liu J, Jha P, Lyzogubov VV, et al. Relationship between complement membrane attack complex, chemokine (C-C motif) ligand 2(CCL2) and vascular endothelial growth factor in mouse model of laser-induced choroidal neovascularization[J]. J Biol Chem, 2011, 286:20991-21001.
17. Kunchithapautham K, Rohrer B. Sublytic membrane-attack-complex (MAC) activation alters regulated rather than constitutive vascular endothelial growth factor (VEGF) secretion in retinal pigment epithelium monolayers[J]. J Biol Chem, 2011, 286:23717-23724.
18. Nozaki M, Raisler BJ, Sakurai E, et al. Drusen complement components C3a and C5a promote choroidal neovascularization[J]. Proc Natl Acad Sci USA, 2006, 103:2328-2333.

Chinese Journal of Ocular Fundus Diseases

Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress

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