Role of M2 Macrophage Exosomes in Osteogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells under High-Glucose and High-Insulin

ZHANG Cheng, BAO Li-rong, YANG Yu-tao, WANG Zhi, LI Yan

Abstract

To study the role of M2 macrophage-derived exosomes (M2-exo) in osteogenic differentiation and Hedgehog signaling pathway of mouse bone marrow mesenchymal stem cells (BMSCs) under in vitro high-glucose and high-insulin conditions.   Methods  RAW 264.7 cells were induced toward M2 macrophage polarization and then M2-exo were extracted and identified. Immunofluorescence assay was performed to detect the internalization of M2-exo by BMSCs. BMSCs were divided into the normal control group (Control group), the high-glucose and high-insulin group (HGI group), and the HGI with M2-exo intervention group (HGI+M2e group). BMSCs in the Control group were cultured in osteogenic inductive medium with 5.5 mmol/L glucose, but no insulin or M2-exo. BMSCs in the HGI group were cultured in osteogenic inductive medium with 25 mmol/L glucose and 174 nmol/L insulin. BMSCs in the HGI+M2e group were cultured in the same medium as that of the HGI group, with the additional treatment of 6, 30, 60 μg/mL M2-exo, respectively. After osteogenic induction for 7 days and 14 days, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively to assess the osteogenic differentiation potential of BMSCs from different groups. In addition, BMSCs in the Control group, HGI group, and HGI+M2e group treated with 30 μg/mL M2-exo were examined with qPCR after osteogenic induction for 14 days and Western blot after osteogenic induction for 21 days to assess the osteogenesis and the expression of Hedgehog pathway-related genes and proteins.  Results  M2 macrophage polarization was induced successfully, with highly positive expression of CD206, the M2 polarization surface marker. The M2-exo had the typical structure of round or oval-shaped bilayered-membrane vesicles. The diameter distribution of M2-exo ranged from 50 to 125 nm (accounting for 99.14% of all M2-exo). M2-exo samples showed positive expression of exosomal markers CD9, CD63 and CD81 proteins. Immunofluorescence staining showed that M2-exo were taken up and internalized by BMSCs. After osteogenic induction for 7 days, the ALP activity of BMSCs in the HGI group was lower than that of the Control group. After interventions of 6 μg/mL, 30 μg/mL, and 60 μg/mL M2-exo, the ALP activity of the HGI+M2-exo group was significantly increased compared with that of the HGI group (P<0.05). After osteogenic induction for 14 days, the number of mineralized nodules in the HGI group was lower than that in the Control group, and after intervention, only the HGI+M2e group treated with 30 μg/mL M2-exo showed higher level of mineralization than that in the HGI group (P<0.05). qPCR analysis revealed that the expression levels of the osteogenesis-related genes, including Runx2, Alp and Ocn, and Hedgehog pathway-related genes, including Gli1, Smo and Ptch1, were downregulated in the HGI group, all being lower than those of the Control group to varying degrees, while 30 μg/mL M2-exo treatment could promote the up-regulation of these genes, showing significant difference in comparison with their expression levels in the HGI group (P<0.05). In addition, Western blot analysis showed that the expression of the osteogenesis-related proteins, including RUNX2 and COL1A1, and GLI1, the Hedgehog signaling pathway protein, was down-regulated in the HGI group, while the expression of COL1A1 and GLI1 was up-regulated after 30 μg/mL M2-exo treatment, showing significant difference when compared with that of the HGI group (P<0.05).  Conclusion  High glucose and high insulin had inhibitory effect on the osteogenic differentiation potential of BMSCs. After intervention with M2-exo, the Hedgehog signaling pathway in BMSCs was activated and the osteogenic differentiation potential was enhanced, suggesting that M2-exo might have therapeutic potentials for the treatment of diabetic bone disease.

 

Keywords: M2 macrophage, Exosome, Bone marrow mesenchymal stem cells, Diabetes mellitus, Hedgehog signaling pathway

 

Full Text:

PDF


References


RODEN M， SHULMAN G I. The integrative biology of type 2 diabetes. Nature，2019，576(7785): 51–60.

LALLA E， PAPAPANOU P N. Diabetes mellitus and periodontitis: A tale of two common interrelated diseases. Nat Rev Endocrinol，2011，7(12): 738–748.

NAPOLI N， CHANDRAN M， PIERROZ D D， et al. Mechanisms of diabetes mellitus-induced bone fragility. Nat Rev Endocrinol，2017，13(4): 208–219.

RAIMONDO T M， MOONEY D J. Functional muscle recovery with nanoparticle-directed M2 macrophage polarization in mice. Proc Natl Acad Sci U S A，2018，115(42): 10648–10653.

YIN C， ZHAO Q， LI W， et al. Biomimetic anti-inflammatory nano-capsule serves as a cytokine blocker and M2 polarization inducer for bone tissue repair. Acta Biomater， 2020， 102: 416−426[2021-09-01]. https://doi.org/10.1016/j.actbio.2019.11.025.

PITTENGER M F， DISCHER D E， PéAULT B M， et al. Mesenchymal stem cell perspective: Cell biology to clinical progress. NPJ Regen Med， 2019， 4(1): 22[2021-09-01]. https://doi.org/10.1038/s41536-019-0083-6.

LIU J， LI D， WU X， et al. Bone-derived exosomes. Curr Opin Pharmacol， 2017， 34: 64-69[2021-09-01]. https://doi.org/10.1016/j. coph.2017.08.008.

LV W T， DU D H， GAO R J， et al. Regulation of Hedgehog signaling offers a novel perspective for bone homeostasis disorder treatment. Int J Mol Sci， 2019， 20(16):3981[2021-09-01]. https://doi.org/10. 3390/ijms20163981.

GUAN C-C， YAN M， JIANG X-Q， et al. Sonic Hedgehog alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of bone marrow stromal cells. Bone，2009，45(6): 1146–1152.

TIAN Y， GONG M， HU Y， et al. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. J Extracell Vesicles， 2020， 9(1): 1697028[2021-09-01]. https://doi.org/ 10.1080/20013078.2019.1697028.

TIAN Y， MA L， GONG M， et al. Protein profiling and sizing of extracellular vesicles from colorectal cancer patients via flow cytometry. ACS Nano，2018，12(1): 671–680.

XIA Y， HE X T， XU X Y， et al. Exosomes derived from M0， M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells. Peer J， 2020， 8: e8970[2021-09-01]. https://doi.org/10.7717/peerj.8970.

ZHANG S， YANG Y， JIA S， et al. Exosome-like vesicles derived from Hertwig’s epithelial root sheath cells promote the regeneration of dentin-pulp tissue. Theranostics，2020，10(13): 5914–5931.

WEI J， SONG Y， DU Z， et al. Exosomes derived from human exfoliated deciduous teeth ameliorate adult bone loss in mice through promoting osteogenesis. J Mol Histol，2020，51(4): 455–466.

CHIARELLA E， ALOISIO A， SCICCHITANO S， et al. ZNF521 represses osteoblastic differentiation in human adipose-derived stem cells. Int J Mol Sci， 2018， 19(12): 4095[2021-09-01]. https://doi.org/10.3390/ijms 19124095.

SARGENT J. Diabetes: Functional impairment of bone marrow progenitor cells in diabetes mellitus. Nat Rev Endocrinol， 2014， 10(7): 379[2021-09-01]. https://doi.org/10.1038/nrendo.2014.66.

DENG X， XU M， SHEN M， et al. Effects of type 2 diabetic serum on proliferation and osteogenic differentiation of mesenchymal stem cells. J Diabetes Res， 2018， 2018: 5765478[2021-09-01]. https://doi.org/10. 1155/2018/5765478.

ZHANG P， ZHANG H， LIN J， et al. Insulin impedes osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence via the TGF-β1 pathway. Aging，2020，12(3): 2084–2100.

CAO B， LIU N， WANG W. High glucose prevents osteogenic differentiation of mesenchymal stem cells via lncRNA AK028326/CXCL13 pathway. Biomed Pharmacother， 2016， 84: 544−551[2021-09-01]. https://doi.org/10.1016/j.biopha.2016.09.058.

QIN X， JIANG Q， KOMORI H， et al. Runt-related transcription factor-2 (Runx2) is required for bone matrix protein gene expression in committed osteoblasts in mice. J Bone Miner Res，2021，36(10): 2081–2095.

CRESCITELLI R， LÄSSER C， LÖTVALL J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc，2021，16(3): 1548–1580.

LI Z， WANG Y， LI S， et al. Exosomes derived from M2 macrophages facilitate osteogenesis and reduce adipogenesis of BMSCs. Front Endocrinol (Lausanne)， 2021， 12: 680328[2021-09-01]. https://doi. org/10.3389/fendo.2021.680328.

XIONG Y， CHEN L， YAN C， et al. M2 macrophagy-derived exosomal miRNA-5106 induces bone mesenchymal stem cells towards osteoblastic fate by targeting salt-inducible kinase 2 and 3. J Nanobiotechnol， 2020， 18(1): 66[2021-09-01]. https://doi.org/10.1186/s12951-020-00622-5.

LACROIX R， SABATIER F， MIALHE A， et al. Activation of plasminogen into plasmin at the surface of endothelial microparticles: A mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro. Blood，2007，110(7): 2432–2439.

HELLWINKEL J E， REDZIC J S， HARLAND T A， et al. Glioma-derived extracellular vesicles selectively suppress immune responses. Neuro Oncol，2016，18(4): 497–506.

PAK E， SEGAL ROSALIND A. Hedgehog signal transduction: Key players, oncogenic drivers, and cancer therapy. Dev Cell，2016，38(4): 333–344.

BRISCOE J， THÉROND P P. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol，2013， 14(7): 416–429.


Refbacks

  • There are currently no refbacks.