Advertisement

Diabetologia

, Volume 63, Issue 2, pp 431–443 | Cite as

Mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy in a mouse model of diabetes

  • Baoyan Fan
  • Chao Li
  • Alexandra Szalad
  • Lei Wang
  • Wanlong Pan
  • Ruilan Zhang
  • Michael Chopp
  • Zheng Gang Zhang
  • Xian Shuang LiuEmail author
Article
First Online:

Abstract

Aims/hypothesis

Diabetic peripheral neuropathy (DPN) is one of the major complications of diabetes, which contributes greatly to morbidity and mortality. There is currently no effective treatment for this disease. Exosomes are cell-derived nanovesicles and play an important role in intercellular communications. The present study investigated whether mesenchymal stromal cell (MSC)-derived exosomes improve neurological outcomes of DPN.

Methods

Exosomes were isolated from the medium of cultured mouse MSCs by ultracentrifugation. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at the age of 20 weeks were used as DPN models. Heterozygous mice (db/m) of the same age were used as the control. MSC-exosomes were administered weekly via the tail vein for 8 weeks. Neurological function was evaluated by testing motor and sensory nerve conduction velocities, and thermal and mechanical sensitivity. Morphometric analysis was performed by myelin sheath staining and immunohistochemistry. Macrophage markers and circulating cytokines were measured by western blot and ELISA. MicroRNA (miRNA) array and bioinformatics analyses were performed to examine the exosomal miRNA profile and miRNA putative target genes involved in DPN.

Results

Treatment of DPN with MSC-exosomes markedly decreased the threshold for thermal and mechanical stimuli and increased nerve conduction velocity in diabetic mice. Histopathological analysis showed that MSC-exosomes markedly augmented the density of FITC-dextran perfused blood vessels and increased the number of intraepidermal nerve fibres (IENFs), myelin thickness and axonal diameters of sciatic nerves. Western blot analysis revealed that MSC-exosome treatment decreased and increased M1 and M2 macrophage phenotype markers, respectively. Moreover, MSC-exosomes substantially suppressed proinflammatory cytokines. Bioinformatics analysis revealed that MSC-exosomes contained abundant miRNAs that target the Toll-like receptor (TLR)4/NF-κB signalling pathway.

Conclusions/interpretation

MSC-derived exosomes alleviate neurovascular dysfunction and improve functional recovery in mice with DPN by suppression of proinflammatory genes.

Keywords

Diabetes Diabetic peripheral neuropathy Exosomes Inflammation Mesenchymal stromal cells miRNA 

Abbreviations

BCA

Bicinchoninic acid

DPN

Diabetic peripheral neuropathy

GSK

Glycogen synthase kinase

IENF

Intraepidermal nerve fibre

iNOS

Inducible nitric oxide synthase

IPA

Ingenuity Pathway Analysis

IRAK1

Interleukin-1 receptor-associated kinase 1

MBP

Myelin basic protein

MCV

Motor nerve conduction velocity

miRNA

MicroRNA

MSC

Mesenchymal stromal cell

MSC-exosomes

MSC-derived exosomes

PGP9.5

Protein gene product 9.5

PU

Perfusion units

ROI

Region of interest

SCV

Sensory nerve conduction velocity

TLR

Toll-like receptor

This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

The authors thank J. Landschoot-Ward and Q.-e. Lu (Department of Neurology, Henry Ford Health System) for immunohistochemistry staining.

Contribution statement

XSL and BF designed the study, analysed and interpreted data, and composed the manuscript. BF, CL, AS, LW, WP, RZ, MC and ZGZ conducted the experiments, acquired and analysed data and edited the manuscript. All authors have critically reviewed and approved the manuscript. XSL and ZGZ are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Funding

This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Disease grant RO1 RDK102861A (XSL), American Heart Association Grant-in-Aid 14GRNT20460167 (XSL), and RO1 NS075156 (ZGZ).

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2019_5043_MOESM1_ESM.pdf (512 kb)
ESM (PDF 511 kb)

References

  1. 1.
    Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414(6865):813–820.  https://doi.org/10.1038/414813a CrossRefPubMedGoogle Scholar
  2. 2.
    Said G (2007) Diabetic neuropathy--a review. Nat Clin Pract Neurol 3(6):331–340.  https://doi.org/10.1038/ncpneuro0504 CrossRefPubMedGoogle Scholar
  3. 3.
    Callaghan BC, Little AA, Feldman EL, Hughes RA (2012) Enhanced glucose control for preventing and treating diabetic neuropathy. Cochrane Database Syst Rev 6:CD007543.  https://doi.org/10.1002/14651858.CD007543.pub2 CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Malik RA, Newrick PG, Sharma AK et al (1989) Microangiopathy in human diabetic neuropathy: relationship between capillary abnormalities and the severity of neuropathy. Diabetologia 32(2):92–102.  https://doi.org/10.1007/bf00505180 CrossRefPubMedGoogle Scholar
  5. 5.
    Watkins CJ, Johnson PC, Olafsen A, Beggs J (1992) Innervation of the vasa nervorum: changes in human diabetics. J Neuropathol Exp Neurol 51(6):612–629.  https://doi.org/10.1097/00005072-199211000-00006 CrossRefPubMedGoogle Scholar
  6. 6.
    Cameron NE, Eaton SEM, Cotter MA, Tesfaye S (2001) Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy. Diabetologia 44(11):1973–1988.  https://doi.org/10.1007/s001250100001 CrossRefPubMedGoogle Scholar
  7. 7.
    Cameron NE, Cotter MA (1997) Metabolic and vascular factors in the pathogenesis of diabetic neuropathy. Diabetes 46(Suppl 2):S31–S37CrossRefGoogle Scholar
  8. 8.
    Hinder LM, Murdock BJ, Park M et al (2018) Transcriptional networks of progressive diabetic peripheral neuropathy in the db/db mouse model of type 2 diabetes: an inflammatory story. Exp Neurol 305:33–43.  https://doi.org/10.1016/j.expneurol.2018.03.011 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Austin PJ, Moalem-Taylor G (2010) The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines. J Neuroimmunol 229(1–2):26–50.  https://doi.org/10.1016/j.jneuroim.2010.08.013 CrossRefPubMedGoogle Scholar
  10. 10.
    Zhou J, Zhou S (2014) Inflammation: therapeutic targets for diabetic neuropathy. Mol Neurobiol 49(1):536–546.  https://doi.org/10.1007/s12035-013-8537-0 CrossRefPubMedGoogle Scholar
  11. 11.
    Negi G, Kumar A, Sharma SS (2011) Melatonin modulates neuroinflammation and oxidative stress in experimental diabetic neuropathy: effects on NF-κB and Nrf2 cascades. J Pineal Res 50(2):124–131.  https://doi.org/10.1111/j.1600-079X.2010.00821.x CrossRefPubMedGoogle Scholar
  12. 12.
    Katakowski M, Buller B, Zheng X et al (2013) Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett 335(1):201–204.  https://doi.org/10.1016/j.canlet.2013.02.019 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Han JW, Choi D, Lee MY, Huh YH, Yoon YS (2016) Bone marrow-derived mesenchymal stem cells improve diabetic neuropathy by direct modulation of both angiogenesis and myelination in peripheral nerves. Cell Transplant 25(2):313–326.  https://doi.org/10.3727/096368915X688209 CrossRefPubMedGoogle Scholar
  14. 14.
    Siniscalco D, Giordano C, Galderisi U et al (2010) Intra-brain microinjection of human mesenchymal stem cells decreases allodynia in neuropathic mice. Cell Mol Life Sci 67(4):655–669.  https://doi.org/10.1007/s00018-009-0202-4 CrossRefPubMedGoogle Scholar
  15. 15.
    Waterman RS, Morgenweck J, Nossaman BD, Scandurro AE, Scandurro SA, Betancourt AM (2012) Anti-inflammatory mesenchymal stem cells (MSC2) attenuate symptoms of painful diabetic peripheral neuropathy. Stem Cells Transl Med 1(7):557–565.  https://doi.org/10.5966/sctm.2012-0025 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Shibata T, Naruse K, Kamiya H et al (2008) Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats. Diabetes 57(11):3099–3107.  https://doi.org/10.2337/db08-0031 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zurita M, Vaquero J, Oya S, Bonilla C, Aguayo C (2007) Neurotrophic Schwann-cell factors induce neural differentiation of bone marrow stromal cells. Neuroreport 18(16):1713–1717.  https://doi.org/10.1097/WNR.0b013e3282f0d3b0 CrossRefPubMedGoogle Scholar
  18. 18.
    Jeong JO, Han JW, Kim JM et al (2011) Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ Res 108(11):1340–1347.  https://doi.org/10.1161/CIRCRESAHA.110.239848 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    van Niel G, D’Angelo G, Raposo G (2018) Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19(4):213–228.  https://doi.org/10.1038/nrm.2017.125 CrossRefPubMedGoogle Scholar
  20. 20.
    Zhang ZG, Buller B, Chopp M (2019) Exosomes – beyond stem cells for restorative therapy in stroke and neurological injury. Nat Rev Neurol 15(4):193–203.  https://doi.org/10.1038/s41582-018-0126-4 CrossRefPubMedGoogle Scholar
  21. 21.
    Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29(4):341–345.  https://doi.org/10.1038/nbt.1807 CrossRefPubMedGoogle Scholar
  22. 22.
    Furuta T, Miyaki S, Ishitobi H et al (2016) Mesenchymal stem cell-derived exosomes promote fracture healing in a mouse model. Stem Cells Transl Med 5(12):1620–1630.  https://doi.org/10.5966/sctm.2015-0285 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhang Y, Chopp M, Liu XS et al (2017) Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons. Mol Neurobiol 54(4):2659–2673.  https://doi.org/10.1007/s12035-016-9851-0 CrossRefPubMedGoogle Scholar
  24. 24.
    Obrosova IG, Ilnytska O, Lyzogubov VV et al (2007) High-fat diet induced neuropathy of pre-diabetes and obesity: effects of "healthy" diet and aldose reductase inhibition. Diabetes 56(10):2598–2608.  https://doi.org/10.2337/db06-1176 CrossRefPubMedGoogle Scholar
  25. 25.
    Liu XS, Fan B, Szalad A et al (2017) MicroRNA-146a mimics reduce the peripheral neuropathy in type II diabetic mice. Diabetes.  https://doi.org/10.2337/db16-1182 CrossRefGoogle Scholar
  26. 26.
    Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1):55–63.  https://doi.org/10.1016/0165-0270(94)90144-9 CrossRefGoogle Scholar
  27. 27.
    Lu M, Varley AW (2013) Harvest and culture of mouse peritoneal macrophages. Bio-protocol 3(22):e976.  https://doi.org/10.21769/BioProtoc.976 CrossRefGoogle Scholar
  28. 28.
    Wu X, Gao Y, Xu L et al (2017) Exosomes from high glucose-treated glomerular endothelial cells trigger the epithelial-mesenchymal transition and dysfunction of podocytes. Sci Rep 7(1):9371.  https://doi.org/10.1038/s41598-017-09907-6 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Rajendran P, Rengarajan T, Thangavel J et al (2013) The vascular endothelium and human diseases. Int J Biol Sci 9(10):1057–1069.  https://doi.org/10.7150/ijbs.7502 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Goncalves NP, Vaegter CB, Andersen H, Ostergaard L, Calcutt NA, Jensen TS (2017) Schwann cell interactions with axons and microvessels in diabetic neuropathy. Nat Rev Neurol 13(3):135–147.  https://doi.org/10.1038/nrneurol.2016.201 CrossRefPubMedGoogle Scholar
  31. 31.
    Lauria G, Cornblath DR, Johansson O et al (2005) EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy. Eur J Neurol 12(10):747–758.  https://doi.org/10.1111/j.1468-1331.2005.01260.x CrossRefPubMedGoogle Scholar
  32. 32.
    Di Scipio F, Raimondo S, Tos P, Geuna S (2008) A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation. Microsc Res Tech 71(7):497–502.  https://doi.org/10.1002/jemt.20577 CrossRefPubMedGoogle Scholar
  33. 33.
    Callaghan BC, Cheng HT, Stables CL, Smith AL, Feldman EL (2012) Diabetic neuropathy: clinical manifestations and current treatments. Lancet Neurol 11(6):521–534.  https://doi.org/10.1016/S1474-4422(12)70065-0 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Lawrence T, Fong C (2010) The resolution of inflammation: anti-inflammatory roles for NF-κB. Int J Biochem Cell Biol 42(4):519–523.  https://doi.org/10.1016/j.biocel.2009.12.016 CrossRefPubMedGoogle Scholar
  35. 35.
    Toth C, Martinez J, Zochodne DW (2007) RAGE, diabetes, and the nervous system. Curr Mol Med 7(8):766–776.  https://doi.org/10.2174/156652407783220705 CrossRefPubMedGoogle Scholar
  36. 36.
    Powell HC, Rosoff J, Myers RR (1985) Microangiopathy in human diabetic neuropathy. Acta Neuropathol 68(4):295–305.  https://doi.org/10.1007/bf00690832 CrossRefPubMedGoogle Scholar
  37. 37.
    Shoelson SE, Lee J, Goldfine AB (2006) Inflammation and insulin resistance. J Clin Invest 116(7):1793–1801.  https://doi.org/10.1172/JCI29069 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Reddy MA, Jin W, Villeneuve L et al (2012) Pro-inflammatory role of microrna-200 in vascular smooth muscle cells from diabetic mice. Arterioscler Thromb Vasc Biol 32(3):721–729.  https://doi.org/10.1161/ATVBAHA.111.241109 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Gong M, Yu B, Wang J et al (2017) Mesenchymal stem cells release exosomes that transfer miRNAs to endothelial cells and promote angiogenesis. Oncotarget 8(28):45200–45212.  https://doi.org/10.18632/oncotarget.16778 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Peters CM, Jimenez-Andrade JM, Jonas BM et al (2007) Intravenous paclitaxel administration in the rat induces a peripheral sensory neuropathy characterized by macrophage infiltration and injury to sensory neurons and their supporting cells. Exp Neurol 203(1):42–54.  https://doi.org/10.1016/j.expneurol.2006.07.022 CrossRefPubMedGoogle Scholar
  41. 41.
    Zhu T, Meng Q, Ji J, Lou X, Zhang L (2015) Toll-like receptor 4 and tumor necrosis factor-α as diagnostic biomarkers for diabetic peripheral neuropathy. Neurosci Lett 585:28–32.  https://doi.org/10.1016/j.neulet.2014.11.020 CrossRefPubMedGoogle Scholar
  42. 42.
    Elzinga S, Murdock BJ, Guo K et al (2019) Toll-like receptors and inflammation in metabolic neuropathy; a role in early versus late disease? Exp Neurol 320:112967.  https://doi.org/10.1016/j.expneurol.2019.112967 CrossRefPubMedGoogle Scholar
  43. 43.
    Phinney DG, Di Giuseppe M, Njah J et al (2015) Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat Commun 6:8472.  https://doi.org/10.1038/ncomms9472 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Zhang J, Li S, Li L et al (2015) Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics 13(1):17–24.  https://doi.org/10.1016/j.gpb.2015.02.001 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Xin H, Katakowski M, Wang F et al (2017) MicroRNA cluster miR-17-92 cluster in exosomes enhance neuroplasticity and functional recovery after stroke in rats. Stroke 48(3):747–753.  https://doi.org/10.1161/STROKEAHA.116.015204 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Monaco F, Gaetani S, Alessandrini F et al (2019) Exosomal transfer of miR-126 promotes the anti-tumour response in malignant mesothelioma: role of miR-126 in cancer–stroma communication. Cancer Lett 463:27–36.  https://doi.org/10.1016/j.canlet.2019.08.001 CrossRefPubMedGoogle Scholar
  47. 47.
    Saravanan PB, Vasu S, Yoshimatsu G et al (2019) Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress. Diabetologia. 62(10):1901–1914.  https://doi.org/10.1007/s00125-019-4950-x CrossRefPubMedGoogle Scholar
  48. 48.
    Yuan Z, Petree JR, Lee FE et al (2019) Macrophages exposed to HIV viral protein disrupt lung epithelial cell integrity and mitochondrial bioenergetics via exosomal microRNA shuttling. Cell Death Dis 10(8):580.  https://doi.org/10.1038/s41419-019-1803-y CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Baoyan Fan
    • 1
  • Chao Li
    • 1
  • Alexandra Szalad
    • 1
  • Lei Wang
    • 1
  • Wanlong Pan
    • 1
  • Ruilan Zhang
    • 1
  • Michael Chopp
    • 1
    • 2
  • Zheng Gang Zhang
    • 1
  • Xian Shuang Liu
    • 1
    Email author
  1. 1.Department of NeurologyHenry Ford Health SystemDetroitUSA
  2. 2.Department of PhysicsOakland UniversityRochesterUSA

Personalised recommendations

Cite article

Buy options