Quantitative proteomic analysis of human retinal microvascular endothelial cells stimulated with 4-Hydroxynonenal_Chinese Journal of Ocular Fundus Diseases

Authors：

Xiao Jing , Zhang Xiaomin , Shao Xianfeng , Xiao Mengran , Yang Fuhua , Zhang Hui , Ea Vicki L ,  Li Xiaorong

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin Medical University, Tianjin 300384, China;

Corresponding author：

Li Xiaorong, Email: xiaorli@163.com

Keywords：

Reitinal vessels/cytology; Endothelial; Cell cultured; Proteomics; 4- Hydroxynonenal

DOI：

10.3760/cma.j.issn.1005-1015.2019.05.012

Video：

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Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins.Results CCK-8 kits detection results showed that the A value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P＜0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and the A value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics, and 118 proteins were differentially expressed (fold change＞1.5, P＜0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.

Citation： Xiao Jing, Zhang Xiaomin, Shao Xianfeng, Xiao Mengran, Yang Fuhua, Zhang Hui, Ea Vicki L, Li Xiaorong. Quantitative proteomic analysis of human retinal microvascular endothelial cells stimulated with 4-Hydroxynonenal. Chinese Journal of Ocular Fundus Diseases, 2019, 35(5): 488-493. doi: 10.3760/cma.j.issn.1005-1015.2019.05.012 Copy

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1. Polak M, Zagorski Z. Lipid peroxidation in diabetic retinopathy[J]. Ann Univ Mariae Curie Sklodowska Med, 2004, 59(1): 434-437.
2. Poli G, Schaur RJ, Siems WG, et al. 4-hydroxynonenal: a membrane lipid oxidation product of medicinal interest[J]. Med Res Rev, 2008, 28(4): 569-631. DOI: 10.1002/med.20117.
3. Liu Q, Li J, Cheng R, et al. Nitrosative stress plays an important role in Wnt pathway activation in diabetic retinopathy[J]. Antioxid Redox Signal, 2013, 18(10): 1141-1153. DOI: 10.1089/ars.2012.4583.
4. Zhou T, Zhou KK, Lee K, et al. The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy[J]. Diabetologia, 2011, 54(2): 459-468. DOI: 10.1007/s00125-010-1943-1.
5. 肖静, 张晓敏, 李筱荣. 糖尿病视网膜病变的蛋白质组学研究进展[J]. 中华眼底病杂志, 2018, 34(4): 407-411. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.024.Xiao J,Zhang XM,Li XR. Research progress of proteomic in diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 407-411. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.024.
6. Yang Y, Sharma R, Sharma A, et al. Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling[J]. Acta Biochim Pol, 2003, 50(2): 319-336.
7. Sharma R, Sharma A, Dwivedi S, et al. 4-Hydroxynonenal self-limits fas-mediated DISC-independent apoptosis by promoting export of Daxx from the nucleus to the cytosol and its binding to Fas[J]. Biochemistry, 2008, 47(1): 143-156. DOI: 10.1021/bi701559f.
8. Schaur RJ. Basic aspects of the biochemical reactivity of 4-hydroxynonenal[J]. Mol Aspects Med, 2003, 24(4-5): 149-159. DOI: 10.1016/S0098-2997(03)00009-8.
9. Wenzel P, Rossmann H, Muller C, et al. Heme oxygenase-1 suppresses a pro-inflammatory phenotype in monocytes and determines endothelial function and arterial hypertension in mice and humans[J]. Eur Heart J, 2015, 36(48): 3437-3446. DOI: 10.1093/eurheartj/ehv544.
10. Abraham NG, Rezzani R, Rodella L, et al. Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes[J]. Am J Physiol Heart Circ Physiol, 2004, 287(6): 2468-2477. DOI: 10.1152/ajpheart.01187.2003.
11. Li X, He P, Wang XL, et al. Sulfiredoxin-1 enhances cardiac progenitor cell survival against oxidative stress via the upregulation of the ERK/NRF2 signal pathway[J]. Free Radic Biol Med, 2018, 123: 8-19. DOI: 10.1016/j.freeradbiomed.2018.05.060.
12. Wu J, Chen Y, Yu S, et al. Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/reperfusion oxidative stress injury in rats[J]. Brain Res Bull, 2017, 132: 99-108. DOI: 10.1016/j.brainresbull.2017.05.012.
13. Zhang J, Li X, Han X, et al. Targeting the thioredoxin system for cancer therapy[J]. Trends Pharmacol Sci, 2017, 38(9): 794-808. DOI: 10.1016/j.tips.2017.06.001.
14. Sennoune SR, Bakunts K, Martinez GM, et al. Vacuolar H⁺-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity[J]. Am J Physiol Cell Physiol, 2004, 286(6): 1443-1452. DOI: 10.1152/ajpcell.00407.2003.
15. Morimura T, Fujita K, Akita M, et al. The proton pump inhibitor inhibits cell growth and induces apoptosis in human hepatoblastoma[J]. Pediatr Surg Int, 2008, 24(10): 1087-1094. DOI: 10.1007/s00383-008-2229-2.
16. Zoncu R, Bar-Peled L, Efeyan A, et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase[J]. Science, 2011, 334(6056): 678-683. DOI: 10.1126/science.1207056.
17. Zucker S, Mirza H, Conner CE, et al. Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation[J]. Int J Cancer, 1998, 75(5): 780-786. DOI: 10.1002/(ISSN)1097-0215.
18. Balaiya S, Zhou Z, Chalam KV. Characterization of vitreous and aqueous proteome in humans with proliferative diabetic retinopathy and its clinical correlation[J/OL]. Proteomics Insights, 2017, 8: 1178641816686078[2017-03-15]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398322/. DOI: 10.1177/1178641816686078.
19. Casaroli Marano RP, Preissner KT, Vilaró S. Fibronectin, laminin, vitronectin and their receptors at newly-formed capillaries in proliferative diabetic retinopathy[J]. Exp Eye Res, 1995, 60(1): 5-17. DOI: 10.1016/S0014-4835(05)80079-X.
20. Esser P, Bresgen M, Weller M, et al. The significance of vitronectin in proliferative diabetic retinopathy[J]. Graefe's Arch Clin Exp Ophthalmol, 1994, 232(8): 477-481. DOI: 10.1007/BF00195357.
21. Hammes HP, Brownlee M, Jonczyk A, et al. Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization[J]. Nat Med, 1996, 2(5): 529-533. DOI: 10.1038/nm0596-529.
22. Zamanian-Daryoush M, Lindner D, Tallant TC, et al. The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects[J]. J Biol Chem, 2013, 288(29): 21237-21252. DOI: 10.1074/jbc.M113.468967.
23. Zhang Q, Hu J, Hu Y, et al. Relationship between serum apolipoproteins levels and retinopathy risk in subjects with type 2 diabetes mellitus[J]. Acta Diabetol, 2018, 55(7): 681-689. DOI: 10.1007/s00592-018-1136-9.
24. Sasongko MB, Wong TY, Nguyen TT, et al. Novel versus traditional risk markers for diabetic retinopathy[J]. Diabetologia, 2012, 55(3): 666-670. DOI: 10.1007/s00125-011-2424-x.
25. Hu A, Luo Y, Li T, et al. Low serum apolipoprotein A1/B ratio is associated with proliferative diabetic retinopathy in type 2 diabetes[J]. Graefe's Arch Clin Exp Ophthalmol, 2012, 250(7): 957-962. DOI: 10.1007/s00417-011-1855-x.
26. Wang D, Sun X, Wei Y, et al. Nuclear miR-122 directly regulates the biogenesis of cell survival oncomiR miR-21 at the posttranscriptional level[J]. Nucleic Acids Res, 2018, 46(4): 2012-2029. DOI: 10.1093/nar/gkx1254.
27. Krug S, Huth J, Goke F, et al. Knock-down of Pdcd4 stimulates angiogenesis via up-regulation of angiopoietin-2[J]. Biochim Biophys Acta, 2012, 1823(4): 789-799. DOI: 10.1016/j.bbamcr.2012.01.006.

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Quantitative proteomic analysis of human retinal microvascular endothelial cells stimulated with 4-Hydroxynonenal

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