Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Liu Juping ^刘 , Hong Yaru ^刘 , Yao Xuyang , Zhang Zhe , Bu Shaochong , Li Hui , Cao Jingjing , Bai Xiaomei , Li Xiaorong ,  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;
Liu Juping and Hong Yaru are contributed equally to the article;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Diabetic retinopathy; Bone morphogenetic protein 4; Endothelium, vascular; Cell proliferation; Cell movement

DOI：

10.3760/cma.j.cn511434-20201109-00539

Video：

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Objective To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress. Methods The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results Compared with the control group, cell viability (t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate (t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant (t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant (t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.

Citation： Liu Juping, Hong Yaru, Yao Xuyang, Zhang Zhe, Bu Shaochong, Li Hui, Cao Jingjing, Bai Xiaomei, Li Xiaorong, Dong Lijie. Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2022, 38(4): 304-309. doi: 10.3760/cma.j.cn511434-20201109-00539 Copy

1.	Dong L, Lin T, Li W, et al. Antioxidative effects of polypyrimidine tract-binding protein-associated splicing factor against pathological retinal angiogenesis through promotion of mitochondrial function[J]. J Mol Med (Berl), 2021, 99(7): 967-980. DOI: 10.1007/s00109-021-02069-z.
2.	徐嫚鸿, 王林妮, 林婷婷, 等. 多聚嘧啶序列结合蛋白相关剪接因子对缺氧诱导人视网膜微血管内皮细胞功能的影响[J]. 中华眼底病杂志, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.Xu MH, Wang LN, Lin TT, et al. Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxia-induced human retinal microvascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.
3.	Dong LJ, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2): 233-243. DOI: 10.1007/s00441-014-2104-5.
4.	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.
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7.	Huang J, Liu Y, Oltean A, et al. BMP4 from the optic vesicle specifies murine retina formation[J]. Dev Biol, 2015, 402(1): 119-126. DOI: 10.1016/j.ydbio.2015.03.006.
8.	温德佳, 任新军, 东莉洁, 等. 应用iTRAQ蛋白组技术筛选增生性糖尿病视网膜病变防治靶点的研究[J]. 中华眼科杂志, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.Wen DJ, Ren XJ, Dong LJ, et al. New exploration of treatment target for proliferative diabetic retinopathy based on iTRAQ LC-MS/MS proteomics[J]. Chin J Ophthalmol, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.
9.	Xing X, Huang L, Lv Y, et al. DL-3-n-butylphthalide protected retinal Müller cells dysfunction from oxidative stress[J]. Curr Eye Res, 2019, 44(10): 1112-1120. DOI: 10.1080/02713683.2019.1624777.
10.	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-E452[2018-12-21]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
11.	Ding Y, Liu H, Cen M, et al. Rapamycin ameliorates cognitive impairments and Alzheimer's disease-like pathology with restoring mitochondrial abnormality in the hippocampus of streptozotocin-induced diabetic mice[J]. Neurochem Res, 2021, 46(2): 265-275. DOI: 10.1007/s11064-020-03160-6.
12.	Li X, Shao S, Li H, et al. Byakangelicin protects against carbon tetrachloride-induced liver injury and fibrosis in mice[J]. J Cell Mol Med, 2020, 24(15): 8623-8635. DOI: 10.1111/jcmm.15493.
13.	O' Farrell NJ, Phelan JJ, Feighery R, et al. Differential expression profiles of oxidative stress levels, 8-oxo-dG and 4-HNE, in Barrett's esophagus compared to esophageal adenocarcinoma[J/OL]. Int J Mol Sci, 2019, 20(18): 4449[2019-09-10]. https://pubmed.ncbi.nlm.nih.gov/31509954/. DOI: 10.3390/ijms20184449.
14.	Todd L, Palazzo I, Squires N, et al. BMP-and TGFβ-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina[J]. Glia, 2017, 65(10): 1640-1655. DOI: 10.1002/glia.23185.
15.	Wu RL, Sedlmeier G, Kyjacova L, et al. Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells[J/OL]. Sci Rep, 2018, 8(1): 14913[2018-10-08]. https://pubmed.ncbi.nlm.nih.gov/30297743/. DOI: 10.1038/s41598-018-33337-7.
16.	Vogt RR, Unda R, Yeh LC, et al. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells[J]. J Cell Biochem, 2006, 98(5): 1196-1202. DOI: 10.1002/jcb.20831.
17.	Xu J, Zhu D, He S, et al. Transcriptional regulation of bone morphogenetic protein 4 by tumor necrosis factor and its relationship with age-related macular degeneration[J]. FASEB J, 2011, 25(7): 2221-2233. DOI: 10.1096/fj.10-178350.
18.	Lin S, Xie J, Gong T, et al. SMAD signal pathway regulates angiogenesis via endothelial cell in an adipose-derived stromal cell/endothelial cell co-culture, 3D gel model[J]. Mol Cell Biochem, 2016, 412(1-2): 281-288. DOI: 10.1007/s11010-015-2634-5.
19.	Dalmo E, Johansson P, Niklasson M, et al. Growth-inhibitory activity of bone morphogenetic protein 4 in human glioblastoma cell lines is heterogeneous and dependent on reduced SOX2 expression[J]. Mol Cancer Res, 2020, 18(7): 981-991. DOI: 10.1158/1541-7786.MCR-19-0638.
20.	Zhang M, Chu S, Zeng F, et al. Bevacizumab modulates the process of fibrosis in vitro[J]. Clin Exp Ophthalmol, 2015, 43(2): 173-179. DOI: 10.1111/ceo.12374.
21.	Zhang Q, Qi Y, Chen L, et al. The relationship between anti-vascular endothelial growth factor and fibrosis in proliferative retinopathy: clinical and laboratory evidence[J]. Br J Ophthalmol, 2016, 100(10): 1443-1450. DOI: 10.1136/bjophthalmol-2015-308199.
22.	Ma T, Dong LJ, Du XL, et al. Research progress on the role of connective tissue growth factor in fibrosis of diabetic retinopathy[J]. Int J Ophthalmol, 2018, 11(9): 1550-1554. DOI: 10.18240/ijo.2018.09.20.
23.	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. Synergistic effect of metformin combined with anti VEGF drugs on diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.
24.	Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
25.	Du B, Zheng JL, Huang LY, et al. Protective effect and mechanism of bone morphogenetic protein-4 on apoptosis of human lens epithelium cells under oxidative stress[J/OL]. Biomed Res Int, 2021: 8109134[2021-01-29]. https://pubmed.ncbi.nlm.nih.gov/33575344/. DOI: 10.1155/2021/8109134.

1. Dong L, Lin T, Li W, et al. Antioxidative effects of polypyrimidine tract-binding protein-associated splicing factor against pathological retinal angiogenesis through promotion of mitochondrial function[J]. J Mol Med (Berl), 2021, 99(7): 967-980. DOI: 10.1007/s00109-021-02069-z.
2. 徐嫚鸿, 王林妮, 林婷婷, 等. 多聚嘧啶序列结合蛋白相关剪接因子对缺氧诱导人视网膜微血管内皮细胞功能的影响[J]. 中华眼底病杂志, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.Xu MH, Wang LN, Lin TT, et al. Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxia-induced human retinal microvascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.
3. Dong LJ, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2): 233-243. DOI: 10.1007/s00441-014-2104-5.
4. 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.
5. 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.
6. Kobayashi Y, Hayashi R, Shibata S, et al. Ocular surface ectoderm instigated by WNT inhibition and BMP4[J/OL]. Stem Cell Res, 2020, 46: 101868[2020-06-01]. https://pubmed.ncbi.nlm.nih.gov/32603880/. DOI: 10.1016/j.scr.2020.101868.
7. Huang J, Liu Y, Oltean A, et al. BMP4 from the optic vesicle specifies murine retina formation[J]. Dev Biol, 2015, 402(1): 119-126. DOI: 10.1016/j.ydbio.2015.03.006.
8. 温德佳, 任新军, 东莉洁, 等. 应用iTRAQ蛋白组技术筛选增生性糖尿病视网膜病变防治靶点的研究[J]. 中华眼科杂志, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.Wen DJ, Ren XJ, Dong LJ, et al. New exploration of treatment target for proliferative diabetic retinopathy based on iTRAQ LC-MS/MS proteomics[J]. Chin J Ophthalmol, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.
9. Xing X, Huang L, Lv Y, et al. DL-3-n-butylphthalide protected retinal Müller cells dysfunction from oxidative stress[J]. Curr Eye Res, 2019, 44(10): 1112-1120. DOI: 10.1080/02713683.2019.1624777.
10. 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-E452[2018-12-21]. https://pubmed.ncbi.nlm.nih.gov/30576241/. DOI: 10.1152/ajpendo.00360.2018.
11. Ding Y, Liu H, Cen M, et al. Rapamycin ameliorates cognitive impairments and Alzheimer's disease-like pathology with restoring mitochondrial abnormality in the hippocampus of streptozotocin-induced diabetic mice[J]. Neurochem Res, 2021, 46(2): 265-275. DOI: 10.1007/s11064-020-03160-6.
12. Li X, Shao S, Li H, et al. Byakangelicin protects against carbon tetrachloride-induced liver injury and fibrosis in mice[J]. J Cell Mol Med, 2020, 24(15): 8623-8635. DOI: 10.1111/jcmm.15493.
13. O' Farrell NJ, Phelan JJ, Feighery R, et al. Differential expression profiles of oxidative stress levels, 8-oxo-dG and 4-HNE, in Barrett's esophagus compared to esophageal adenocarcinoma[J/OL]. Int J Mol Sci, 2019, 20(18): 4449[2019-09-10]. https://pubmed.ncbi.nlm.nih.gov/31509954/. DOI: 10.3390/ijms20184449.
14. Todd L, Palazzo I, Squires N, et al. BMP-and TGFβ-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina[J]. Glia, 2017, 65(10): 1640-1655. DOI: 10.1002/glia.23185.
15. Wu RL, Sedlmeier G, Kyjacova L, et al. Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells[J/OL]. Sci Rep, 2018, 8(1): 14913[2018-10-08]. https://pubmed.ncbi.nlm.nih.gov/30297743/. DOI: 10.1038/s41598-018-33337-7.
16. Vogt RR, Unda R, Yeh LC, et al. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells[J]. J Cell Biochem, 2006, 98(5): 1196-1202. DOI: 10.1002/jcb.20831.
17. Xu J, Zhu D, He S, et al. Transcriptional regulation of bone morphogenetic protein 4 by tumor necrosis factor and its relationship with age-related macular degeneration[J]. FASEB J, 2011, 25(7): 2221-2233. DOI: 10.1096/fj.10-178350.
18. Lin S, Xie J, Gong T, et al. SMAD signal pathway regulates angiogenesis via endothelial cell in an adipose-derived stromal cell/endothelial cell co-culture, 3D gel model[J]. Mol Cell Biochem, 2016, 412(1-2): 281-288. DOI: 10.1007/s11010-015-2634-5.
19. Dalmo E, Johansson P, Niklasson M, et al. Growth-inhibitory activity of bone morphogenetic protein 4 in human glioblastoma cell lines is heterogeneous and dependent on reduced SOX2 expression[J]. Mol Cancer Res, 2020, 18(7): 981-991. DOI: 10.1158/1541-7786.MCR-19-0638.
20. Zhang M, Chu S, Zeng F, et al. Bevacizumab modulates the process of fibrosis in vitro[J]. Clin Exp Ophthalmol, 2015, 43(2): 173-179. DOI: 10.1111/ceo.12374.
21. Zhang Q, Qi Y, Chen L, et al. The relationship between anti-vascular endothelial growth factor and fibrosis in proliferative retinopathy: clinical and laboratory evidence[J]. Br J Ophthalmol, 2016, 100(10): 1443-1450. DOI: 10.1136/bjophthalmol-2015-308199.
22. Ma T, Dong LJ, Du XL, et al. Research progress on the role of connective tissue growth factor in fibrosis of diabetic retinopathy[J]. Int J Ophthalmol, 2018, 11(9): 1550-1554. DOI: 10.18240/ijo.2018.09.20.
23. 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. Synergistic effect of metformin combined with anti VEGF drugs on diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(5): 453-457. DOI: 10.3760/cma.j.issn.1005-1015.2018.05.008.
24. Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
25. Du B, Zheng JL, Huang LY, et al. Protective effect and mechanism of bone morphogenetic protein-4 on apoptosis of human lens epithelium cells under oxidative stress[J/OL]. Biomed Res Int, 2021: 8109134[2021-01-29]. https://pubmed.ncbi.nlm.nih.gov/33575344/. DOI: 10.1155/2021/8109134.

Chinese Journal of Ocular Fundus Diseases

Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells

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