Effect of exosomes derived from human umbilical cord mesenchymal stem cells on the expression of vascular endothelial growth factor A in blue light injured human retinal pigment epithelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

HeGuanghui ,  ChenSong , MaYingxue , YangJing , WangJian , ChenLi , WangYuchuan , HaoPeng

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

Corresponding author：

ChenSong, Email: chensong9999@126.com

Keywords：

Mesenchymal stem cells; Exosomes; Retinal pigment epithelium; Vascular endothelial growth factor A; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2016.06.010

Video：

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Objective To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSC) on the expression of vascular endothelial growth factor (VEGF) A in blue light injured human retinal pigment epithelial (RPE) cells. Methods hUCMSC were cultured with exo-free fetal bovine serum for 48 hours, and then the supernatants were collected to isolate and purify exosomes by gradient ultracentrifugation method. Transmission electron microscopy was used to identify the morphology of exosomes. Surface specific maker protein CD63 and CD90 were detected via Western blot. Cultured ARPE-19 cells were divided into normal control group, blue light injured group and hUCMSC exosomes treated group. Cells were exposed to the blue light at the intensity of (2000±500) Lux for 12 hours to establish the light injured models. The cells of hUCMSC exosomes treated group were treated by different concentrations of exosomes for 8, 16, 24 hours. The mRNA and protein of VEGF-A were determined by real time-polymerase chain reaction and Western blot. Immunofluorescence assay were used to detect the expression levels of VEGF-A. Results hUCMSC exosomes were successfully isolated, they exhibited round or oval shape and their diameter ranged from 50 to 100 nm with membrane structure through electron microscope. hUCMSC exosomes expressed the common surface marker protein CD63 and the surface marker protein CD90 of hUCMSC. The protein and mRNA level of VEGF A in the blue light injured group increased significantly compared to that in normal control group (t=-16.553, -19.456; P < 0.05). After treating with low, middle and high concentration of hUCMSC exosomes for 8, 16 and 24 hours, the protein and mRNA level of VEGF A of injured RPE were significantly decreased (P < 0.05). With the treated time and concentration of hUCMSC exosomes improved, the protein and mRNA level of VEGF A of injured RPE gradually decreased (P < 0.05). Immunofluorescence assay showed the protein level of VEGF-A of injured RPE gradually decreased with the same concentration of hUCMSC exosomes treated over time. Conclusion hUCMSC exosomes can effectively down-regulate the mRNA and protein level of VEGF-A in blue light injured RPE, the effect depends on the concentration and treated time of hUCMSC exosomes.

Citation： HeGuanghui, ChenSong, MaYingxue, YangJing, WangJian, ChenLi, WangYuchuan, HaoPeng. Effect of exosomes derived from human umbilical cord mesenchymal stem cells on the expression of vascular endothelial growth factor A in blue light injured human retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2016, 32(6): 605-610. doi: 10.3760/cma.j.issn.1005-1015.2016.06.010 Copy

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1. Kinnunen K, Petrovski G, Moe MC, et al. Molecular mechanisms of retinal pigment epithelium damage and development of age-related macular degeneration[J]. Acta Ophthalmol, 2012, 90(4):299-309. DOI:10.1111/j.1755-3768.2011.02179.
2. Blaauwgeers HG, Holtkamp GM, Rutten H, et al. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation[J]. Am J Pathol, 1999, 155(2):421-428. DOI:10.1016/S0002-9440(10)65138-3.
3. Lykke-Andersen S, Brodersen DE, Jensen TH. Origins and activities of the eukaryotic exosome[J].J Cell Sci, 2009, 122(10):1487-1494.DOI:10.1242/jcs.047399.
4. Rani S, Ritter T. The exosome-a naturally secreted nanoparticle and its application to wound healing[J].Adv Mater, 2016, 28(27):5542-5552. DOI:10.1002/adma.201504009.
5. 毕雪, 陈松, 林锦镛, 等.不同途径移植人脐带间充质干细胞对糖尿病大鼠视网膜病变的影响[J].中华眼底病杂志, 2014, 30(2):166-170. DOI:10.3760/cma.j.issn.1005-1015.2014.02.012.Bi X, Chen S, Lin JY, et al. Systemic and ocular transplantation of human umbilical cord mesenchymal stem cells into rats with diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2014, 30(2):166-170.DOI:10.3760/cma.j.issn.1005-1015.2014.02.012.
6. Wang S, Lu B, Girman S, et al. Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology[J/OL]. PLoS One, 2010, 5(2):9200[2010-02-15]. http://dx.plos.org/10.1371/journal.pone.0009200. DOI:10.1371/journal.pone.0009200.
7. 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.
8. 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.
9. Kernt M, Neubauer AS, Liegl RG, et al. Sorafenib prevents human retinal pigment epithelium cells from light-induced overexpression of VEGF, PDGF and PlGF[J].Br J Ophthalmol, 2010, 94(11):1533-1539. DOI:10.1136/bjo.2010.182162.
10. Crabb JW. The proteomics of drusen[J/OL].Cold Spring Harb Perspect Med, 2014, 4(7):017194[2014-05-05]. http://perspectivesinmedicine.cshlp.org/cgi/pmidlookup?view=long&pmid=24799364. DOI:10.1101/cshperspect.a017194.
11. Marazita MC, Dugour A, Marquioni-Ramella MD, et al. Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression inretinal pigment epithelial cells:implications for age-related macular degeneration[J]. Redox Biol, 2016, 4(7):78-87. DOI:10.1016/j.redox.2015.11.011.
12. 潘颖喆, 邢怡桥, 陈长征, 等.抑制光损伤人视网膜色素上皮细胞血管内皮生长因子A的表达对趋化因子受体3配体表达的影响[J].中华眼底病杂志, 2015, 31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016.Pan YZ, Xing YQ, Chen CZ, et al. Down-regulated expression of CC chemokine receptor 3 ligands in ranibizumab treated light-injured human retinal pigment epithelium cells[J]. Chin J Ocul Fundus Dis, 2015, 31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016.
13. 柯屹峰, 陈松, 郝瑞.蓝光诱导大鼠视网膜色素上皮细胞复制衰老[J].中华眼底病杂志, 2009, 25(6):462-465. DOI:10.3760/cma.j.issn.1015-1005.2009.06.15.Ke YF, Chen S, Hao R. Blue light-induced replicative senescence of rat retinal pigment epithelial cells[J].Chin J Ocul Fundus Dis, 2009, 25(6):462-465. DOI:10.3760/cma.j.issn. 1015-1005.2009.06.15.
14. Ho IA, Toh HC, Ng WH, et al. Human bone marrow-derived mesenchymal stem cells suppress human glioma growth through inhibition of angiogenesis[J].Stem Cells, 2013, 31(1):146-155.DOI:10.1002/stem.1247.
15. Otsu K, Das S, Houser SD, et al. Concentration-dependent inhibition of angiogenesis by mesenchymal stem cells[J].Blood, 2009, 113(18):4197-4205. DOI:10.1182/blood-2008-09-176198.
16. Nakano M, Nagaishi K, Konari N, et al. Bone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by exosome transfer into damaged neurons and astrocytes[J/OL]. Sci Rep, 2016, 22(6):24805[2016-04-22]. http://dx.doi.org/10.1038/srep24805. DOI:10.1038/srep24805.
17. Lai RC, Yeo RW, Tan KH, et al. Mesenchymal stem cell exosome ameliorates reperfusion injury through proteomic complementation[J].Regen Med, 2013, 8(2):197-209. DOI:10.2217/rme.13.4.
18. Gnecchi M, Zhang Z, Ni A, et al. Paracrine mechanisms in adult stem cell signaling and therapy[J].Circ Res, 2008, 103(11):1204-1219. DOI:10.1161/CIRCRESAHA.108.176826.
19. Batrakova EV, Kim MS. Development and regulation of exosome-based therapy products[J].Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2016, 8(5):744-757.DOI:10.1002/wnan.1395.
20. Stickney Z, Losacco J, McDevitt S, et al.Development of exosome surface display technology in living human cells[J]. Biochem Biophys Res Commun, 2016, 472(1):53-59.DOI:10.1016/j.bbrc.2016.02.058.
21. 张娟, 刘峰, 张薇, 等.人脐血间充质干细胞来源的外泌体:分离鉴定及生物学特性[J].中国组织工程研究, 2014, 18(37):5955-5960.DOI:10.3969/j.issn.2095-4344.2014.37.009.Zhang J, Liu F, Zhang W, et al. Exosomes derived from human umbilical cord blood mesenchymal stem cells:isolation, identification and biological characteristics[J]. Journal of Clinical Rehabilitative Tissue Engineering Research, 2014, 18(37):5955-5960.DOI:10.3969/j.issn.2095-4344.2014.37.009.
22. Hua Z, Lv Q, Ye W, et al. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia[J/OL].PLoS One, 2006, 27(1):116[2006-12-27]. http://dx.plos.org/10.1371/journal.pone.0000116. DOI:10.1371/journal.pone.0000116.
23. 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.

Chinese Journal of Ocular Fundus Diseases

Effect of exosomes derived from human umbilical cord mesenchymal stem cells on the expression of vascular endothelial growth factor A in blue light injured human retinal pigment epithelial cells

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