Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Li Hui , Hong Yaru , Liu Boshi , Dong Lijie ,  Teng He

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：

Teng He, Email: tenghe197901@163.com

Keywords：

Bone morphogenetic protein 4; Endothelium, vascular; Endothelial cells; Glycolysis; Cell proliferation; Cell movement

DOI：

10.3760/cma.j.cn511434-20210714-00379

Video：

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Objective To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). Methods A experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. Results The ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). Conclusion BMP4 induces the proliferation and migration of hRMECs through glycolysis.

Citation： Li Hui, Hong Yaru, Liu Boshi, Dong Lijie, Teng He. Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2022, 38(10): 840-845. doi: 10.3760/cma.j.cn511434-20210714-00379 Copy

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13.	Yang TT, Li H, Dong LJ. Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases[J]. Int J Ophthalmol, 2021, 14(9): 1302-1309. DOI: 10.18240/ijo.2021.09.02.
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1. 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.
2. Dong L, Li W, Lin T, et al. PSF functions as a repressor of hypoxia-induced angiogenesis by promoting mitochondrial function[J]. Cell Commun Signal, 2021, 19(1): 14. DOI: 10.1186/s12964-020-00684-w.
3. Wanet A, Arnould T, Najimi M, et al. Connecting mitochondria, metabolism, and stem cell fate[J]. Stem Cells Dev, 2015, 24(17): 1957-1971. DOI: 10.1089/scd.2015.0117.
4. 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.
5. Shao Y, Dong LJ, Takahashi Y, et al. miRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J]. Am J Physiol Endocrinol Metab, 2019, 316(3): 443-452. DOI: 10.1152/ajpendo.00360.2018.
6. Williamson KA, Fitzpatrick DR. The genetic architecture of microphthalmia, anophthalmia and coloboma[J]. Eur J Med Genet, 2014, 57(8): 369-380. DOI: 10.1016/j.ejmg.2014.05.002.
7. Modica S, Wolfrum C. The dual role of BMP4 in adipogenesis and metabolism[J]. Adipocyte, 2017, 6(2): 141-146. DOI: 10.1080/21623945.2017.1287637.
8. Watanabe R, Shirai T, Namkoong H, et al. Pyruvate controls the checkpoint inhibitor PD-L1 and suppresses T cell immunity[J]. J Clin Invest, 2017, 127(7): 2725-2738. DOI: 10.1172/jci92167.
9. Gao Q, Zhang G, Zheng Y, et al. SLC27A5 deficiency activates NRF2/TXNRD1 pathway by increased lipid peroxidation in HCC[J]. Cell Death Differ, 2020, 27(3): 1086-1104. DOI: 10.1038/s41418-019-0399-1.
10. 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.
11. De Bock K, Georgiadou M, Schoors S, et al. Role of PFKFB3-driven glycolysis in vessel sprouting[J]. Cell, 2013, 154(3): 651-663. DOI: 10.1016/j.cell.2013.06.037.
12. Li X, Kumar A, Carmeliet P. Metabolic pathways fueling the endothelial cell drive[J]. Annu Rev Physiol, 2019, 81: 483-503. DOI: 10.1146/annurev-physiol-020518-114731.
13. Yang TT, Li H, Dong LJ. Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases[J]. Int J Ophthalmol, 2021, 14(9): 1302-1309. DOI: 10.18240/ijo.2021.09.02.
14. De Bock K, Georgiadou M, Carmeliet P. Role of endothelial cell metabolism in vessel sprouting[J]. Cell Mmetab, 2013, 18(5): 634-647. DOI: 10.1016/j.cmet.2013.08.001.
15. Stapor P, Wang X, Goveia J, et al. Angiogenesis revisited-role and therapeutic potential of targeting endothelial metabolism[J]. J Cell Sci, 2014, 127(Pt 20): 4331-4341. DOI: 10.1242/jcs.153908.
16. Payen VL, Mina E, Van HéE VF, et al. Monocarboxylate transporters in cancer[J]. Mol Metab, 2020, 33: 48-66. DOI: 10.1016/j.molmet.2019.07.006.
17. Zheng Y, Pan D. The hippo signaling pathway in development and disease[J]. Dev Cell, 2019, 50(3): 264-282. DOI: 10.1016/j.devcel.2019.06.003.
18. Feng Y, Zou R, Zhang X, et al. YAP promotes ocular neovascularization by modifying PFKFB3-driven endothelial glycolysis[J]. Angiogenesis, 2021, 24(3): 489-504. DOI: 10.1007/s10456-020-09760-8.
19. Bakrania P, Efthymiou M, Klein JC, et al. Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways[J]. Am J Hum Genet, 2008, 82(2): 304-319. DOI: 10.1016/j.ajhg.2007.09.023.
20. Fischer AJ, Schmidt M, Omar G, et al. BMP4 and CNTF are neuroprotective and suppress damage-induced proliferation of Müller glia in the retina[J]. Mol Cell Neurosci, 2004, 27(4): 531-542. DOI: 10.1016/j.mcn.2004.08.007.
21. Todd L, Palazzo I, Squires N, et al. BMP-and TGFbeta-signaling regulate the formation of Muller glia-derived progenitor cells in the avian retina[J]. Glia, 2017, 65(10): 1640-1655. DOI: 10.1002/glia.23185.
22. Shin JH, Kim IY, Kim YN, et al. Obesity resistance and enhanced insulin sensitivity in ahnak-/- mice fed a high fat diet are related to impaired adipogenesis and increased energy expenditure[J/OL]. PLoS One, 2015, 10(10): e0139720[2015-10-14]. https://pubmed.ncbi.nlm.nih.gov/26466345/. DOI: 10.1371/journal.pone.0139720.

Chinese Journal of Ocular Fundus Diseases

Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells

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