The protective effects and mechanisms of human umbilical cord blood mesenchymal stem cells-derived microvesicles on the injury of rat retinal ganglion cells induced by high glucose condition_Chinese Journal of Ocular Fundus Diseases

Authors：

Liang Zeyu ,  Chen Song , Zhang Wei , He Guanghui , Wang Junhua , Gao Xiang , Wu Bin

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：

Chen Song, Email: chenson99999@126.com

Keywords：

Mesenchymal stem cells; Microvesicles; Retinal ganglion cells

DOI：

10.3760/cma.j.issn.1005-1015.2018.06.009

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ObjectiveTo observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ, CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P＜0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.

Citation： Liang Zeyu, Chen Song, Zhang Wei, He Guanghui, Wang Junhua, Gao Xiang, Wu Bin. The protective effects and mechanisms of human umbilical cord blood mesenchymal stem cells-derived microvesicles on the injury of rat retinal ganglion cells induced by high glucose condition. Chinese Journal of Ocular Fundus Diseases, 2018, 34(6): 568-574. doi: 10.3760/cma.j.issn.1005-1015.2018.06.009 Copy

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18.	Muralidharan-Chari V, Clancy J, Plou C, et al. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles[J]. Curr Biol, 2009, 19(22): 1875-1885. DOI: 10.1016/j.cub.2009.09.059.
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21.	Zhang HC, Liu XB, Huang S, et al. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo[J].Stem Cells Dev, 2012, 21(18): 3289-3297. DOI: 10.1089/scd.2012.0095.
22.	Wu S, Ju GQ, Du T, et al. Microvesicles derived from human umbilical cord Wharton’s jelly mesenchymal stem cells attenuate bladder tumor cell growth in vitro and in vivo[J/OL]. PLoS One, 2013, 8(4): 61366[2013-04-12]. http://dx.plos.org/10.1371/journal.pone.0061366. DOI: 10.1371/journal.pone.0061366.
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1. Valverde AM, Miranda S, García-Ramírez M, et al. Proapoptotic and survival signaling in the neuroretina at early stages of diabetic retinopathy[J]. Mol Vis, 2013, 19: 47-53.
2. Oshitari T, Yamamoto S, Hata N, et al. Mitochondria-and caspase-dependent cell death pathway involved in neuronal degeneration in diabetic retinopathy[J]. Br J Ophthalmol, 2008, 92(4): 552-556. DOI: 10.1136/bjo.2007.132308.
3. Khalfaoui T, Basora N, Ouertani-Meddeb A. Apoptotic factors (Bcl-2 and Bax) and diabetic retinopathy in type 2 diabetes[J]. J Mol Histol, 2010, 41(2-3): 143-152. DOI: 10.1007/s10735-010-9271-9.
4. Hsieh JY, Wang HW, Chang SJ, et al. Mesenchymal stem cells from human umbilical cord express preferentially secreted factors related to neuroprotection, neurogenesis, and angiogenesis[J/OL]. PLoS One, 2013, 8(8): 72604[2013-08-22]. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072604. DOI: 10.1371/journal.pone.0072604.
5. Kusuma GD, Carthew J, Lim R, et al. Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect[J]. Stem Cells Dev, 2017, 26(9): 617-631. DOI: 10.1089/scd.2016.0349.
6. Lamichhane TN, Sokic S, Schardt JS, et al. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine[J]. Tissue Eng Part B Rev, 2015, 21(1): 45-54. DOI: 10.1089/ten.TEB.2014.0300.
7. Mead B, Logan A, Berry M. Paracrine-mediated neuroprotection and neuritogenesis of axotomised retinal ganglion cells by human dental pulp stem cells: comparison with human bone marrow and adipose-derived mesenchymal stem cells[J/OL]. PLoS One, 2014, 9(10): 109305[2014-10-07].http://dx.plos.org/10.1371/journal.pone.0109305. DOI: 0.1371/journal.pone.0109305.
8. Xia J, Luo M, Ni N, et al. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells[J/OL]. PLoS One, 2013, 8(9): 76157[2013-09-30]. http://dx.plos.org/10.1371/journal.pone.0076157. DOI: 10.1371/journal.pone.0076157. eCollection 2013.
9. 董蒙, 张惟, 陈松, 等. 玻璃体腔移植人脐带间充质干细胞诱导的神经干细胞对糖尿病大鼠血-视网膜屏障的保护作用[J].中华眼科杂志, 2017, 53(1): 53-58.DOI: 10.3760/cma.j.issn.0412-4081.2017.01.011.Dong M, Zhang W, Chen S, et al. The protective effect of human umbilical cord mesenchymal stem cells-induced neural stem cells in the vitreous on the blood-retinal barrier in diabetic rats[J]. Chin J Ophthalmol, 2017, 53(1): 53-58.DOI: 10.3760/cma.j.issn.0412-4081.2017.01.011.
10. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends[J]. J Cell Biol, 2013, 200(4): 373-383.DOI: 10.1083/jcb.201211138.
11. Raisi A, Azizi S, Delirezh N, et al. The mesenchymal stem cell-derived microvesicles enhance sciatic nerve regeneration in rat: a novel approach in peripheral nerve cell therapy[J]. J Trauma Acute Care Surg, 2014, 76(4): 991-997. DOI: 10.1097/TA.0000000000000186.
12. Lin SS, Zhu B, Guo ZK, et al. Bone marrow mesenchymal stem cell-derived microvesicles protect rat pheochromocytoma PC12 cells from glutamate-induced injury via a PI3K/Akt dependent pathway[J]. Neurochem Res, 2014, 39(5): 922-931. DOI: 10.1007/s11064-014-1288-0.
13. Farinazzo A, Turano E, Marconi S, et al.Murine adipose-derived mesenchymal stromal cell vesicles: in vitro clues for neuroprotective and neuroregenerative approaches[J].Cytotherapy, 2015, 17(5): 571-578. DOI: 10.1016/j.jcyt.2015.01.005.
14. Tsutsumi T, Iwao K, Hayashi H, et al. Potential neuroprotective effects of an LSD1 inhibitor in retinal ganglion cells via p38 MAPK activity[J]. Invest Ophthalmol Vis Sci, 2016, 57(14): 6461-6473. DOI: 10.1167/iovs.16-19494.
15. Greening DW, Xu R, Ji H, et al. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods[J]. Methods Mol Biol, 2015, 1295: 179-209. DOI: 10.1007/978-1-4939-2550-6_15.
16. Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes[J]. Annu Rev Biophys, 2010, 39: 407-427. DOI: 10.1146/annurev.biophys.093008.131234.
17. Hugel B, Martínez MC, Kunzelmann C, et al.Membrane microparticles: two sides of the coin[J]. Physiology (Bethesda), 2005, 20: 22-27. DOI: 10.1152/physiol.00029.2004.
18. Muralidharan-Chari V, Clancy J, Plou C, et al. ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles[J]. Curr Biol, 2009, 19(22): 1875-1885. DOI: 10.1016/j.cub.2009.09.059.
19. Kanada M, Bachmann MH, Hardy JW, et al. Differential fates of biomolecules delivered to target cells via extracellular vesicles[J]. Proc Natl Acad Sci USA, 2015, 112(12): 1433-1442. DOI: 10.1073/pnas.1418401112.
20. Santos JM, Bárcia RN, Simões SI, et al.The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX^®) in the treatment of inflammatory arthritis[J]. J Transl Med, 2013, 17(11): 18. DOI: 10.1186/1479-5876-11-18.
21. Zhang HC, Liu XB, Huang S, et al. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo[J].Stem Cells Dev, 2012, 21(18): 3289-3297. DOI: 10.1089/scd.2012.0095.
22. Wu S, Ju GQ, Du T, et al. Microvesicles derived from human umbilical cord Wharton’s jelly mesenchymal stem cells attenuate bladder tumor cell growth in vitro and in vivo[J/OL]. PLoS One, 2013, 8(4): 61366[2013-04-12]. http://dx.plos.org/10.1371/journal.pone.0061366. DOI: 10.1371/journal.pone.0061366.
23. Baulch JE, Acharya MM, Allen BD, et al. Cranial grafting of stem cell-derived microvesicles improves cognition and reduces neuropathology in the irradiated brain[J]. Proc Natl Acad Sci USA, 2016, 113(17): 4836-4841. DOI: 10.1073/pnas.1521668113.

Chinese Journal of Ocular Fundus Diseases

The protective effects and mechanisms of human umbilical cord blood mesenchymal stem cells-derived microvesicles on the injury of rat retinal ganglion cells induced by high glucose condition

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