Experimental study on the regulation of migration of retinal pigment epithelial cells by bone morphogenetic protein 4_Chinese Journal of Ocular Fundus Diseases

Authors：

Li Wenbo , Cao Jingjing , Zhuang Tongtong , Wang Qing ,  Dong Lijie

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Proliferative diabetic retinopathy; Bone morphogenetic protein 4; Epithelial-mesenchymal transformation; Retinal pigment epithelium cells; Proliferation; Migration; Reactive oxygen species

DOI：

10.3760/cma.j.cn511434-20230217-00069

Video：

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Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. Methods Human RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. Results Compared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). Conclusions BMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.

Citation： Li Wenbo, Cao Jingjing, Zhuang Tongtong, Wang Qing, Dong Lijie. Experimental study on the regulation of migration of retinal pigment epithelial cells by bone morphogenetic protein 4. Chinese Journal of Ocular Fundus Diseases, 2024, 40(3): 208-214. doi: 10.3760/cma.j.cn511434-20230217-00069 Copy

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2.	Shao Y, Dong LJ, Takahashi Y, et al. MiRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J/OL]. Am J Physiol Endocrinol Metab, 2019, 316(3): E443-452[2019-04-01]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
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4.	Morikawa M, Derynck R, Miyazono K. TGF-β and the TGF-β family: context-dependent roles in cell and tissue physiology[J/OL]. Cold Spring Harb Perspect Biol, 2016, 8(5): a021873[2016-05-02]. https://pubmed.ncbi.nlm.nih.gov/27141051/. DOI: 10.1101/cshperspect.a021873.
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7.	张哲, 刘竹青, 刘巨平, 等. 二甲双胍联合抗血管内皮生长因子药物治疗糖尿病视网膜病变的可能协同作用[J]. 中华眼底病杂志, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.Zhang Z, Liu ZQ, Liu JP, et al. The synergistic effect of metformin and anti-vascular endothelial growth factor in the treatment of diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.
8.	刘巨平, 洪亚茹, 姚旭阳, 等. 骨形成蛋白4促进视网膜血管内皮细胞增生和迁移[J]. 中华眼底病杂志, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.Liu JP, Hong YR, Yao XY, et al. Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.
9.	Teng H, Hong Y, Cao J, et al. Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function[J]. Bioengineered, 2022, 13(5): 12955-12971. DOI: 10.1080/21655979.2022.2079270.
10.	Steel D. Retinal detachment[J/OL]. BMJ Clin Eevid, 2014, 2014: 710[2014-04-03]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940167/.
11.	Wu F, Eliott D. Molecular targets for proliferative vitreoretinopathy[J]. Semin Ophthalmol, 2021, 36(4): 218-223. DOI: 10.1080/08820538.2021.1890791.
12.	Zou H, Shan C, Ma L, et al. Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy[J/OL]. Peer J, 2020, 8: e10136[2020-10-20]. https://pubmed.ncbi.nlm.nih.gov/33150072/. DOI: 10.7717/peerj.10136.
13.	Hu X, Wu X, Zhao B, et al. Scutellarin protects human retinal pigment epithelial cells against hydrogen peroxide (H₂O₂)-induced oxidative damage[J]. Cell Biosci, 2019, 9: 12. DOI: 10.1186/s13578-019-0276-0.
14.	邢小丽, 黄亮瑜, 张哲, 等. 丁基苯酞对H₂O₂诱导下视网膜色素上皮细胞凋亡的保护作用[J]. 中华眼底病杂志, 2019, 35(5): 480-487. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.011.Xing XL, Huang LY, Zhang Z, et al. Effects of butylphthalide on hydrogen peroxide induced retinal pigment epithelial cells injury[J]. Chin J Ocul Fundus Dis, 2019, 35(5): 480-487. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.011.
15.	Shimi T, Goldman RD. Nuclear lamins and oxidative stress in cell proliferation and longevity[J]. Adv Exp Med Biol, 2014, 773: 415-430. DOI: 10.1007/978-1-4899-8032-8_19.
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17.	Bragdon B, Moseychuk O, Saldanha S, et al. Bone morphogenetic proteins: a critical review[J]. Cell Signal, 2011, 23(4): 609-620. DOI: 10.1016/j.cellsig.2010.10.003.
18.	Church RH, Krishnakumar A, Urbanek A, et al. Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7[J]. Biochem J, 2015, 466(1): 55-68. DOI: 10.1042/BJ20140771.
19.	Wei Z, Song L, Wei J, et al. Maternal exposure to di-(2-ethylhexyl) phthalate alters kidney development through the renin-angiotensin system in offspring[J]. Toxicol Lett, 2012, 212(2): 212-221. DOI: 10.1016/j.toxlet.2012.05.023.
20.	Hollborn M, Tenckhoff S, Seifert M, et al. Human retinal epithelium produces and responds to placenta growth factor[J]. Graefe's Arch Clin Exp Ophthalmol, 2006, 244(6): 732-741. DOI: 10.1007/s00417-005-0154-9.
21.	张哲, 刘巨平, 东莉洁, 等. 高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.Zhang Z, Liu JP, Dong LJ, et al. RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.
22.	李辉, 洪亚茹, 刘勃实, 等. 骨形成蛋白4对人视网膜微血管内皮细胞糖酵解水平的影响[J]. 中华眼底病杂志, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.Li H, Hong YR, Liu BS, et al. Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.

1. Idrees S, Sridhar J, Kuriyan AE. Proliferative vitreoretinopathy: a review[J]. Int Ophthalmol Clin, 2019, 59(1): 221-240. DOI: 10.1097/IIO.0000000000000258.
2. Shao Y, Dong LJ, Takahashi Y, et al. MiRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J/OL]. Am J Physiol Endocrinol Metab, 2019, 316(3): E443-452[2019-04-01]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
3. Simão S, Santos DF, Silva GA. Aliskiren decreases oxidative stress and angiogenic markers in retinal pigment epithelium cells[J]. Angiogenesis, 2017, 20(1): 175-181. DOI: 10.1007/s10456-016-9526-5.
4. Morikawa M, Derynck R, Miyazono K. TGF-β and the TGF-β family: context-dependent roles in cell and tissue physiology[J/OL]. Cold Spring Harb Perspect Biol, 2016, 8(5): a021873[2016-05-02]. https://pubmed.ncbi.nlm.nih.gov/27141051/. DOI: 10.1101/cshperspect.a021873.
5. Hirsch L, Nazari H, Sreekumar PG, et al. TGF-β2 secretion from RPE decreases with polarization and becomes apically oriented[J]. Cytokine, 2015, 71(2): 394-396. DOI: 10.1016/j.cyto.2012.11.014.
6. Wang Y, Li H, Cao J, et al. BMP4 aggravates mitochondrial dysfunction of HRMECs[J/OL]. Heliyon, 2023, 9(3): e13824[2023-02-18]. https://pubmed.ncbi.nlm.nih.gov/36895361/. DOI: 10.1016/j.heliyon.2023.e13824.
7. 张哲, 刘竹青, 刘巨平, 等. 二甲双胍联合抗血管内皮生长因子药物治疗糖尿病视网膜病变的可能协同作用[J]. 中华眼底病杂志, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.Zhang Z, Liu ZQ, Liu JP, et al. The synergistic effect of metformin and anti-vascular endothelial growth factor in the treatment of diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.
8. 刘巨平, 洪亚茹, 姚旭阳, 等. 骨形成蛋白4促进视网膜血管内皮细胞增生和迁移[J]. 中华眼底病杂志, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.Liu JP, Hong YR, Yao XY, et al. Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(4): 304-309. DOI: 10.3760/cma.j.cn511434-20201109-00539.
9. Teng H, Hong Y, Cao J, et al. Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function[J]. Bioengineered, 2022, 13(5): 12955-12971. DOI: 10.1080/21655979.2022.2079270.
10. Steel D. Retinal detachment[J/OL]. BMJ Clin Eevid, 2014, 2014: 710[2014-04-03]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940167/.
11. Wu F, Eliott D. Molecular targets for proliferative vitreoretinopathy[J]. Semin Ophthalmol, 2021, 36(4): 218-223. DOI: 10.1080/08820538.2021.1890791.
12. Zou H, Shan C, Ma L, et al. Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy[J/OL]. Peer J, 2020, 8: e10136[2020-10-20]. https://pubmed.ncbi.nlm.nih.gov/33150072/. DOI: 10.7717/peerj.10136.
13. Hu X, Wu X, Zhao B, et al. Scutellarin protects human retinal pigment epithelial cells against hydrogen peroxide (H₂O₂)-induced oxidative damage[J]. Cell Biosci, 2019, 9: 12. DOI: 10.1186/s13578-019-0276-0.
14. 邢小丽, 黄亮瑜, 张哲, 等. 丁基苯酞对H₂O₂诱导下视网膜色素上皮细胞凋亡的保护作用[J]. 中华眼底病杂志, 2019, 35(5): 480-487. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.011.Xing XL, Huang LY, Zhang Z, et al. Effects of butylphthalide on hydrogen peroxide induced retinal pigment epithelial cells injury[J]. Chin J Ocul Fundus Dis, 2019, 35(5): 480-487. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.011.
15. Shimi T, Goldman RD. Nuclear lamins and oxidative stress in cell proliferation and longevity[J]. Adv Exp Med Biol, 2014, 773: 415-430. DOI: 10.1007/978-1-4899-8032-8_19.
16. Ma X, Long C, Wang F, et al. METTL3 attenuates proliferative vitreoretinopathy and epithelial-mesenchymal transition of retinal pigment epithelial cells via wnt/β-catenin pathway[J]. J Cell Mol Med, 2021, 25(9): 4220-4234. DOI: 10.1111/jcmm.16476.
17. Bragdon B, Moseychuk O, Saldanha S, et al. Bone morphogenetic proteins: a critical review[J]. Cell Signal, 2011, 23(4): 609-620. DOI: 10.1016/j.cellsig.2010.10.003.
18. Church RH, Krishnakumar A, Urbanek A, et al. Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7[J]. Biochem J, 2015, 466(1): 55-68. DOI: 10.1042/BJ20140771.
19. Wei Z, Song L, Wei J, et al. Maternal exposure to di-(2-ethylhexyl) phthalate alters kidney development through the renin-angiotensin system in offspring[J]. Toxicol Lett, 2012, 212(2): 212-221. DOI: 10.1016/j.toxlet.2012.05.023.
20. Hollborn M, Tenckhoff S, Seifert M, et al. Human retinal epithelium produces and responds to placenta growth factor[J]. Graefe's Arch Clin Exp Ophthalmol, 2006, 244(6): 732-741. DOI: 10.1007/s00417-005-0154-9.
21. 张哲, 刘巨平, 东莉洁, 等. 高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.Zhang Z, Liu JP, Dong LJ, et al. RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.
22. 李辉, 洪亚茹, 刘勃实, 等. 骨形成蛋白4对人视网膜微血管内皮细胞糖酵解水平的影响[J]. 中华眼底病杂志, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.Li H, Hong YR, Liu BS, et al. Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2022, 38(10): 840-845. DOI: 10.3760/cma.j.cn511434-20210714-00379.

Chinese Journal of Ocular Fundus Diseases

Experimental study on the regulation of migration of retinal pigment epithelial cells by bone morphogenetic protein 4

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