Effect of SB431542 on retinal vascular endothelial cells under hypoxia_Chinese Journal of Ocular Fundus Diseases

Authors：

Cao Jingjing , Han Feifei , Kou Zhenyu , Dong Lijie , Li Wenbo ,  Liang Jingli

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：

Liang Jingli, Email: liangjingli@tmu.edu.cn

Keywords：

SB431542; Retinal vascular endothelial cells; Neovascularization; Glycolysis; Animal experiments; Cell experiment

DOI：

10.3760/cma.j.cn511434-20221107-00590

Video：

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Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. Methods In vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. Results In vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). Conclusion SB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.

Citation： Cao Jingjing, Han Feifei, Kou Zhenyu, Dong Lijie, Li Wenbo, Liang Jingli. Effect of SB431542 on retinal vascular endothelial cells under hypoxia. Chinese Journal of Ocular Fundus Diseases, 2023, 39(12): 1004-1009. doi: 10.3760/cma.j.cn511434-20221107-00590 Copy

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7.	Zhang J, Li R, Liu Q, et al. SB431542-loaded liposomes alleviate liver fibrosis by suppressing TGF-β signaling[J]. Mol Pharm, 2020, 17(11): 4152-4162. DOI: 10.1021/acs.molpharmaceut.0c00633.
8.	Li Y, Zhu H, Wei X, et al. LPS induces HUVEC angiogenesis in vitro through miR-146a-mediated TGF-β1 inhibition[J]. Am J Transl Res, 2017, 9(2): 591-600.
9.	Gong W, Sun B, Zhao X, et al. Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3 pathway[J]. Oncotarget, 2016, 7(43): 70152-70167. DOI: 10.18632/oncotarget.12161.
10.	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.
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19.	Yu WK, Hwang WL, Wang YC, et al. Curcumin suppresses TGF-β1-induced myofibroblast differentiation and attenuates angiogenic activity of orbital fibroblasts[J/OL]. Int J Mol Sci, 2021, 22(13): 6829[2021-06-25]. https://pubmed.ncbi.nlm.nih.gov/34202024/. DOI: 10.3390/ijms22136829.
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21.	Yadav H, Quijano C, Kamaraju AK, et al. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling[J]. Cell Metab, 2011, 14(1): 67-79. DOI: 10.1016/j.cmet.2011.04.013.
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23.	Shu DY, Butcher ER, Saint-Geniez M. Suppression of PGC-1α drives metabolic dysfunction in TGFβ2-induced EMT of retinal pigment epithelial cells[J/OL]. Int J Mol Sci, 2021, 22(9): 4701[2021-04-29]. https://pubmed.ncbi.nlm.nih.gov/33946753/. DOI: 10.3390/ijms22094701.

1. 田芳, 东莉洁, 吉洁, 等. 多聚嘧啶序列结合蛋白相关剪接因子对视网膜血管内皮细胞IGF-1/VEGF信号通路的抑制作用[J]. 中华实验眼科杂志, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.Tian F, Dong LJ, Ji J, et al. Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling pathway in retinal vascular endothelial cells[J]. Chin J Exp Ophthalmol, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.
2. Qin Y, Zhang J, Babapoor-Farrokhran S, et al. PAI-1 is a vascular cell-specific HIF-2-dependent angiogenic factor that promotes retinal neovascularization in diabetic patients[J/OL]. Sci Adv, 2022, 8(9): 1896[2022-03-04]. https://pubmed.ncbi.nlm.nih.gov/35235351/. DOI: 10.1126/sciadv.abm1896.
3. 黄亮瑜, 柯屹峰, 林婷婷, 等. 慢病毒介导聚嘧啶束结合蛋白相关剪接因子对氧诱导视网膜病变小鼠视网膜新生血管的抑制作用[J]. 中华眼底病杂志, 2020, 36(1): 53-59. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.012.Huang LY, Ke YF, Lin TT, et al. Lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor inhibits retinal neovascularization in mice of oxygen-induced retinopathy[J]. Chin J Ocul Fundus Dis, 2020, 36(1): 53-59. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.012.
4. Yang Y, Liu Y, Li Y, et al. MicroRNA-15b targets VEGF and inhibits angiogenesis in proliferative diabetic retinopathy[J]. J Clin Endocrinol Metab, 2020, 105(11): 3404-3415. DOI: 10.1210/clinem/dgaa538.
5. Rojo Arias JE, Englmaier VE, Jászai Jl. VEGF-Trap modulates retinal inflammation in the murine oxygen-induced retinopathy (OIR) model[J]. Biomedicines, 2022, 10(2): 201. DOI: 10.3390/biomedicines10020201.
6. Gurzu S, Kobori L, Fodor D, et al. Epithelial mesenchymal and endothelial mesenchymal transitions in hepatocellular carcinoma: a review[J/OL]. Biomed Res Int, 2019, 2019: 2962580[2019-09-29]. https://pubmed.ncbi.nlm.nih.gov/31781608/. DOI: 10.1155/2019/2962580.
7. Zhang J, Li R, Liu Q, et al. SB431542-loaded liposomes alleviate liver fibrosis by suppressing TGF-β signaling[J]. Mol Pharm, 2020, 17(11): 4152-4162. DOI: 10.1021/acs.molpharmaceut.0c00633.
8. Li Y, Zhu H, Wei X, et al. LPS induces HUVEC angiogenesis in vitro through miR-146a-mediated TGF-β1 inhibition[J]. Am J Transl Res, 2017, 9(2): 591-600.
9. Gong W, Sun B, Zhao X, et al. Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3 pathway[J]. Oncotarget, 2016, 7(43): 70152-70167. DOI: 10.18632/oncotarget.12161.
10. 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.
11. 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.
12. Liu K, Gao X, Hu C, et al. Capsaicin ameliorates diabetic retinopathy by inhibiting poldip2-induced oxidative stress[J/OL]. Redox Biol, 2022, 56: 102460[2022-09-03]. https://pubmed.ncbi.nlm.nih.gov/36088760/. DOI: 10.1016/j.redox.2022.102460.
13. 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.
14. Xu H, Yang B, Ren Z, et al. MiR-429 negatively regulates the progression of hypoxia-induced retinal neovascularization by the HPSE-VEGF pathway[J/OL]. Exp Eye Res, 2022, 223: 109196[2022-07-22]. https://pubmed.ncbi.nlm.nih.gov/35872179/. DOI: 10.1016/j.exer.2022.109196.
15. 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.
16. Peng D, Fu M, Wang M, et al. Targeting TGF-β signal transduction for fibrosis and cancer therapy[J]. Mol Cancer, 2022, 21(1): 104. DOI: 10.1186/s12943-022-01569-x.
17. Zhang L, Wei W, Ai X, et al. Extracellular vesicles from hypoxia-preconditioned microglia promote angiogenesis and repress apoptosis in stroke mice via the TGF-β/Smad2/3 pathway[J/OL]. Cell Death Dis, 2021, 12(11): 1068[2021-11-09]. https://pubmed.ncbi.nlm.nih.gov/34753919/. DOI: 10.1038/s41419-021-04363-7.
18. Busse A, Keilholz U. Role of TGF-β in melanoma[J]. Curr Pharm Biotechnol, 2011, 12(12): 2165-2175. DOI: 10.2174/1389201117 98808437.
19. Yu WK, Hwang WL, Wang YC, et al. Curcumin suppresses TGF-β1-induced myofibroblast differentiation and attenuates angiogenic activity of orbital fibroblasts[J/OL]. Int J Mol Sci, 2021, 22(13): 6829[2021-06-25]. https://pubmed.ncbi.nlm.nih.gov/34202024/. DOI: 10.3390/ijms22136829.
20. 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.
21. Yadav H, Quijano C, Kamaraju AK, et al. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling[J]. Cell Metab, 2011, 14(1): 67-79. DOI: 10.1016/j.cmet.2011.04.013.
22. Hua W, Ten Dijke P, Kostidis S, et al. TGFβ-induced metabolic reprogramming during epithelial-to-mesenchymal transition in cancer[J]. Cell Mol Life Sci, 2020, 77(11): 2103-2123. DOI: 10.1007/s00018-019-03398-6.
23. Shu DY, Butcher ER, Saint-Geniez M. Suppression of PGC-1α drives metabolic dysfunction in TGFβ2-induced EMT of retinal pigment epithelial cells[J/OL]. Int J Mol Sci, 2021, 22(9): 4701[2021-04-29]. https://pubmed.ncbi.nlm.nih.gov/33946753/. DOI: 10.3390/ijms22094701.

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Chinese Journal of Ocular Fundus Diseases

Effect of SB431542 on retinal vascular endothelial cells under hypoxia

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