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Extracellular vesicles derived from hypoxia-preconditioned bone marrow mesenchymal stem cells ameliorate lower limb ischemia by delivering miR-34c

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Abstract

Hypoxic mesenchymal stem cell-derived extracellular vesicles (EVs) have been suggested as a promising therapy for various diseases. This study aims to determine the effect of EVs derived from bone marrow mesenchymal stem cells (BMMSCs) under hypoxia on lower limb ischemia and the underlying mechanism. Human BMMSCs were subjected to hypoxia or normoxia followed by the isolation of EVs. Nanoparticle trafficking analysis (NTA), transmission electron microscopy (TEM), and Western Blotting using corresponding markers were performed to confirm the EVs. The EVs from BMMSCs under hypoxia condition (Hyp-EVs) or normoxia condition (Nor-EVs) were subjected to hindlimb ischemia (HI) mice. MiR-34c expression in BMMSCs and BMMSC-EVs was detected. The role of miR-34c in regulating M2 macrophage polarization, as well as the target of miR-34c, were explored. HI mice with Hyp-EV treatment, as compared to the Nor-EV or the PBS group, had better blood flow and higher capillary density. MiR-34c expression was increased in BMMSCs, BMMSC-EVs, and the adductor muscle of HI mice. Hyp-EVs promoted the M2 macrophage polarization and anti-inflammatory cytokine production, and enhanced the blood flow and capillary density in HI mice, while the knockdown of miR-34c partly reversed these effects. PTEN is a target of miR-34c, and the PTEN silencing facilitated M2 macrophage polarization, whereas the inhibition of AKT signaling partly abolished the effect. Hyp-EVs promoted M2 macrophage polarization by delivering miR-34c via PTEN/AKT pathway, which could be a promising therapeutic strategy to ameliorate lower limb ischemia.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

Not applicable.

Funding

This study was supported by Henan provincial joint construction project of medical science and technology of China (No. LHGJ20190857 to QY, No. LHGJ20190858 to XP).

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Authors and Affiliations

  1. Department of Vascular Surgery, The Third Provincial People’s Hospital of Henan Province, No.198 Funiu Road, Zhengzhou, 450000, Henan Province, China

    Xitao Peng, Junhua Liu, Bing Liang, Haisheng Wang, Jingyuan Hou & Qidong Yuan

  2. Department of Endocrinology, The Third Provincial People’s Hospital of Henan Province, Zhengzhou, 450000, China

    Lijun Ren

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  1. Xitao Peng
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Contributions

XP and QY: conceptualization, methodology,writing—original draft. JL, LR and BL: formal analysis. HW and JH: data curation. All authors have read and approved the final manuscript.

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Correspondence to Qidong Yuan.

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11010_2023_4666_MOESM1_ESM.tif

Supplementary file1 (TIF 9392 KB) Supplemental Figure 1. (A) The expression of miR-34c in M0, M1, and M2 macrophages (n = 3, one-way ANOVA with Tukey’s post hoc test). The THP-1 cells were differentiated into M0 macrophages by incubating in 320 nM PMA for 24 h. THP-1 cells were differentiated into M1 macrophages by first being treated with 320 nM PMA for 6 h, followed by culturing by the addition of LPS. THP-1 cells were differentiated into M2 macrophages by first being treated with 320 nM PMA for 6 h, followed by cultured with IL-4 and IL-13 (20 ng/ml) for another 18 h. (B) The co-localization of miR-34c and the F4/80 antibody in Hyp-EV-treated mouse gastrocnemius muscle tissues.

11010_2023_4666_MOESM2_ESM.pdf

Supplementary file2 (PDF 449 KB) Supplemental Figure 2. Human BMMSCs transfected with LV-miR-34c inhibitor or LV-inhibitor-NC were treated with hypoxia or normoxia followed by the isolation of EVs. HUVECs were co-cultured with 30 μg/ml Nor-EVs or Hyp-EVs for 24 h. Cells were grouped as follows: control, Nor-EV, Hyp-EV, LV-in-NC-Hyp-EV, and LV-miR-34c in-Hyp-EV. (A) HUVEC viability was determined using the MTT cell proliferation and cytotoxicity assay kit (n = 3, one-way ANOVA with Tukey’s post hoc test). (B) Cell migration was observed using the Transwell assay (n = 3, one-way ANOVA with Tukey’s post hoc test). (C) Tube formation assay (n = 3, one-way ANOVA with Tukey’s post hoc test). *p < 0.05, **p<0.01, ***p < 0.001.

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Peng, X., Liu, J., Ren, L. et al. Extracellular vesicles derived from hypoxia-preconditioned bone marrow mesenchymal stem cells ameliorate lower limb ischemia by delivering miR-34c. Mol Cell Biochem 478, 1645–1658 (2023). https://doi.org/10.1007/s11010-023-04666-7

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