Influence of oxidative stress-induced exosomes on Akt and vascular endothelial growth factor-A of retinal pigment epithelium cells_Chinese Journal of Ocular Fundus Diseases

Authors：

ZhangWei , YangJing ,  ChenSong , HeGuanghui , MaYingxue , ChenLi , JiangJianhong , SongJian

Tianjin Eye Hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin Eye Institute, Clinical College of Ophthalmology, Tianjin Medical University, Tianjin 300020, China;

Corresponding author：

ChenSong, Email: chensong20@hotmail.com

Keywords：

Vascular endothelial growth factor A; Protein-serine-threonine kinases; Exosomes; Retinal pigment epithelium cell; Cells, cultured; Oxidative stress

DOI：

10.3760/cma.j.issn.1005-1015.2017.01.015

Video：

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Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.

Citation： ZhangWei, YangJing, ChenSong, HeGuanghui, MaYingxue, ChenLi, JiangJianhong, SongJian. Influence of oxidative stress-induced exosomes on Akt and vascular endothelial growth factor-A of retinal pigment epithelium cells. Chinese Journal of Ocular Fundus Diseases, 2017, 33(1): 57-61. doi: 10.3760/cma.j.issn.1005-1015.2017.01.015 Copy

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1. Mitter SK, Song C, Qi X, et al. Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD[J]. Autophagy, 2014, 10(11): 1989-2005. DOI: 10.4161/auto.36184.
2. Lässer C. Exosomes in diagnostic and therapeutic applications: biomarker, vaccine and RNA interference delivery vehicle[J]. Expert Opin Biol Ther, 2015, 15(1): 103-117. DOI: 10.1517/ 14712598.2015.977250.
3. Stamer WD, Hoffman EA, Luther JM, et al. Protein profile of exosomes from trabecular meshwork cells[J]. J proteomics, 2011, 74(6): 796-804. DOI: 10.1016/j.jprot.2011.02.024.
4. Lee JK, Park SR, Jung BK, et al. Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells[J/OL]. PLoS One, 2013, 8(12): 84256[2013-12-31]. http://dx.plos.org/10.1371/journal. pone.0084256. DOI: 10.1371/journal.pone.0084256.
5. Lai RC, Yeo RW, Padmanabhan J, et al. Isolation and characterization of exosome from human embryonic stem cell-derived C-myc-immortalized mesenchymal stem cells[J]. Methods Mol Biol, 2016, 1416: 477-494.DOI: 10.1007/978-1-4939-3584-0_29.
6. Yamagishi S, Matsui T. Advanced glycation end products (AGEs), oxidative stress and diabetic retinopathy[J]. Curr Pharm Biotechnol, 2011, 12(3): 362-368.
7. Michael A, Bajracharya SD, Yuen PS, et al. Exosomes from human saliva as a source of microRNA biomarkers[J]. Oral Dis, 2010, 16(1): 34-38. DOI: 10.1111/j.1601-0825.2009.01604.x.
8. Hajrasouliha AR, Jiang G, Lu Q, et al. Exosomes from retinal astrocytes contain antiangiogenic components that inhibit laser-induced choroidal neovascularization[J]. J Biol Chem, 2013, 288(39): 28058-28067. DOI: 10.1074/jbc.M113.470765.
9. Pant S, Hilton H, Burczynski ME. The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities[J]. Biochem Pharmacol, 2012, 83(11): 1484-1494. DOI:10.1016/ j.bcp.2011.12.037.
10. Bhat SP, Gangalum RK. Secretion of alphaB-crystallin via exosomes: new clues to the function of human retinal pigment epithelium[J]. Commun Integr Biol, 2011, 4(6): 739-741.
11. Grassmann F, Schoenberger PG, Brandl C, et al. A circulating microRNA profile is associated with late-stage neovascular age-related macular degeneration[J/OL]. PLoS One, 2014, 9(9): 107461[2014-09-09]. http://dx.plos.org/10.1371/journal. pone.0107461. DOI: 10.1371/journal.pone.0107461.
12. Melo SA, Luecke LB, Kahlert C, et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer[J]. Nature, 2015, 523(7559): 177-182. DOI: 10.1038/nature14581.
13. Kinnunen K, Petrovski G, Moe MC, et al. Molecular mechanisms of retinal pigment epitelium damage and development of age-related macular degeneration[J]. Acta Ophthalmhol, 2012, 90(4): 299-309. DOI: 10.1111/j.1755-3768.2011.02179.
14. Saxena K, Rutar MV, Provis JM, et al. Identification of miRNAs in a model of retinal degenerations[J]. Invest Ophthalmol Vis Sci, 2015, 56(3): 1820-1829. DOI: 10.1167/iovs.14-15449.
15. Rabin DM, Rabin RL, Blenkinsop TA, et al. Chronic oxidative stress upregulates drusen-related protein expression in adult human RPE stem cell-derived RPE cells: a novel culture model for dry AMD[J]. Aging (Albany NY), 2013, 5(1): 51-66.
16. Abdelsalam RM, Safar MM. Neuroprotective effects of vildagliptin in rat rotenone Parkinson's disease model: role of RAGE-NFκB and Nrf2-antioxidant signaling pathways[J]. J Neurochem, 2015, 133(5): 700-707. DOI: 10.1111/jnc.13087.
17. Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004, 1697(1-2): 3-16. DOI: 10.1016/j.bbapap. 2003.11.009.
18. Yang P, Peairs JJ, Tano R, et al. Oxidant-mediated Akt activation in human RPE cells[J]. Invest Ophthalmol Vis Sci, 2006, 47(10): 4598-4606. DOI: 10.1167/iovs.06-0140.
19. Pechan P, Rubin H, Lukason M, et al. Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization[J]. Gene Ther, 2009, 16(1): 10-16. DOI: 10.1038/gt.2008.115.
20. Stahl A, Paschek L, Martin G, et al. Combinatory inhibition of VEGF and FGF2 is superior to solitary VEGF inhibition in an in vitro model of RPE-induced angiogenesis[J]. Graefe’s Arch Clin Exp Ophthalmol, 2009, 247(6): 767-773. DOI: 10.1007/s00417-009-1058-x.
21. Sengupta N, Afzal A, Caballero S, et al. Paracrine modulation of CXCR4 by IGF-1 and VEGF: implications for choroidal neovascularization[J]. Invest Ophthalmol Vis Sci, 2010, 51(5): 2697-2704. DOI: 10.1167/iovs.09-4137.
22. Quittet MS, Touzani O, Sindji L, et al. Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat[J]. Acta Biomater, 2015, 15(3): 77-88. DOI: 10.1016/j. actbio. 2014.12.017.

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