Skip to main content

MSCs-Derived Extracellular Vesicles Carrying miR-212-5p Alleviate Myocardial Infarction-Induced Cardiac Fibrosis via NLRC5/VEGF/TGF-β1/SMAD Axis

Abstract

The purpose of the present study was to define the role of mesenchymal stem cell (MSC)–derived extracellular vesicles (EVs) in the progression of myocardial infarction (MI)–induced cardiac fibrosis. An in vitro cell model of hypoxia-induced cardiac fibrosis was constructed in cardiac fibroblasts (CFs). miR-212-5p was poorly expressed in clinical pathological samples and animal models of cardiac fibrosis caused by MI, while miR-212-5p expression was enriched in EVs released from MSCs. EVs from MSCs were isolated, evaluated, and co-cultured with CFs. Dual-luciferase reporter gene assay revealed that miR-212-5p negatively targeted NLRC5 progression of cardiac fibrosis. Following loss- and gain-function assay, EVs expressing miR-212-5p protected against cardiac fibrosis evidenced by reduced levels of α-SMA, Collagen I, TGF-β1, and IL-1β. In vivo experiments further confirmed the above research results. Collectively, EVs from MSCs expressing miR-212-5p may attenuate MI by suppressing the NLRC5/VEGF/TGF-β1/SMAD axis.

This is a preview of subscription content, access via your institution.

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

MSCs:

Mesenchymal stem cells

EVs:

Extracellular vesicles

MI:

Myocardial infarction

CFs:

Cardiac fibroblasts

MI:

Myocardial infarction

SPF:

Specific pathogen free

DMEM:

Dulbecco’s modified eagle medium

FITC:

Fluorescein isothiocyanate

PE:

Polyethylene

NC:

Negative control

BSA:

Bull serum albumin

ECG:

Echocardiogram

LVIDs:

Left ventricular inner systolic diameter

LVESV:

Left ventricular systolic volume

References

  1. 1.

    Gou, L., Xue, C., Tang, X., & Fang, Z. (2020). Inhibition of Exo-miR-19a-3p derived from cardiomyocytes promotes angiogenesis and improves heart function in mice with myocardial infarction via targeting HIF-1alpha. Aging, 12(23), 23609–23618. https://doi.org/10.18632/aging.103563(Albany NY).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Mihalko, E., Huang, K., Sproul, E., Cheng, K., & Brown, A. C. (2018). Targeted treatment of ischemic and fibrotic complications of myocardial infarction using a dual-delivery microgel therapeutic. ACS Nano, 12(8), 7826–7837. https://doi.org/10.1021/acsnano.8b01977.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Cui, S., Liu, Z., Tao, B., Fan, S., Pu, Y., Meng, X., et al. (2021). miR-145 attenuates cardiac fibrosis through the AKT/GSK-3beta/beta-catenin signaling pathway by directly targeting SOX9 in fibroblasts. Journal of Cellular Biochemistry, 122(2), 209–221. https://doi.org/10.1002/jcb.29843.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Han, C., Zhou, J., Liang, C., Liu, B., Pan, X., Zhang, Y., et al. (2019). Human umbilical cord mesenchymal stem cell derived exosomes encapsulated in functional peptide hydrogels promote cardiac repair. Biomaterials Science, 7(7), 2920–2933. https://doi.org/10.1039/c9bm00101h.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Park, S., Nguyen, N. B., Pezhouman, A., & Ardehali, R. (2019). Cardiac fibrosis: potential therapeutic targets. Translational Research, 209, 121–137. https://doi.org/10.1016/j.trsl.2019.03.001.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wang, Z., Fu, M., & Li, Y. (2020). miR-142-5p and miR-212-5p cooperatively inhibit the proliferation and collagen formation of cardiac fibroblasts by regulating c-Myc/TP53INP1. Canadian Journal of Physiology and Pharmacology, 98(5), 314–323. https://doi.org/10.1139/cjpp-2019-0495.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Xue, Y., Fan, X., Yang, R., Jiao, Y., & Li, Y. (2020). miR-29b-3p inhibits post-infarct cardiac fibrosis by targeting FOS. Biosci Rep, 40(9). https://doi.org/10.1042/BSR20201227.

  8. 8.

    Hillman, Y., Mardamshina, M., Pasmanik-Chor, M., Ziporen, L., Geiger, T., Shomron, N., et al. (2019). MicroRNAs affect complement regulator expression and mitochondrial activity to modulate cell resistance to complement-dependent cytotoxicity. Cancer Immunology Research, 7(12), 1970–1983. https://doi.org/10.1158/2326-6066.CIR-18-0818.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Yang, Y., Cai, Y., Zhang, Y., Liu, J., & Xu, Z. (2018). Exosomes secreted by adipose-derived stem cells contribute to angiogenesis of brain microvascular endothelial cells following oxygen-glucose deprivation in vitro through microRNA-181b/TRPM7 axis. Journal of Molecular Neuroscience, 65(1), 74–83. https://doi.org/10.1007/s12031-018-1071-9.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Ren, N., & Wang, M. (2018). microRNA-212-induced protection of the heart against myocardial infarction occurs via the interplay between AQP9 and PI3K/Akt signaling pathway. Experimental Cell Research, 370(2), 531–541. https://doi.org/10.1016/j.yexcr.2018.07.018.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Spence, D. W., & Stewart, W. D. (1986). Proline inhibits N2-fixation in Anabaena 7120. Biochemical and Biophysical Research Communications, 139(3), 940–946. https://doi.org/10.1016/s0006-291x(86)80268-6.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    He, Y. H., Li, M. F., Zhang, X. Y., Meng, X. M., Huang, C., & Li, J. (2016). NLRC5 promotes cell proliferation via regulating the AKT/VEGF-A signaling pathway in hepatocellular carcinoma. Toxicology, 359-360, 47–57. https://doi.org/10.1016/j.tox.2016.06.012.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Melincovici, C. S., Bosca, A. B., Susman, S., Marginean, M., Mihu, C., Istrate, M., et al. (2018). Vascular endothelial growth factor (VEGF) - key factor in normal and pathological angiogenesis. Romanian Journal of Morphology and Embryology, 59(2), 455–467.

    PubMed  Google Scholar 

  14. 14.

    Wilmes, V., Lux, C., Niess, C., Gradhand, E., Verhoff, M. A., & Kauferstein, S. (2020). Changes in gene expression patterns in postmortem human myocardial infarction. International Journal of Legal Medicine, 134(5), 1753–1763. https://doi.org/10.1007/s00414-020-02311-2.

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Borger, V., Bremer, M., Ferrer-Tur, R., Gockeln, L., Stambouli, O., Becic, A., et al. (2017). Mesenchymal stem/stromal cell-derived extracellular vesicles and their potential as novel immunomodulatory therapeutic agents. International Journal of Molecular Sciences, 18(7). https://doi.org/10.3390/ijms18071450.

  16. 16.

    Zhao, Y., Sun, X., Cao, W., Ma, J., Sun, L., Qian, H., et al. (2015). Exosomes derived from human umbilical cord mesenchymal stem cells relieve acute myocardial ischemic injury. Stem Cells International, 2015, 761643. https://doi.org/10.1155/2015/761643.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Liu, Z., Liu, J., Wei, Y., Xu, J., Wang, Z., Wang, P., et al. (2020). LncRNA MALAT1 prevents the protective effects of miR-125b-5p against acute myocardial infarction through positive regulation of NLRC5. Experimental and Therapeutic Medicine, 19(2), 990–998. https://doi.org/10.3892/etm.2019.8309.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Zhou, H., Yu, X., & Zhou, G. (2017). NLRC5 silencing ameliorates cardiac fibrosis by inhibiting the TGFbeta1/Smad3 signaling pathway. Molecular Medicine Reports, 16(3), 3551–3556. https://doi.org/10.3892/mmr.2017.6990.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Park, H. Y., Kim, J. H., & Park, C. K. (2013). VEGF induces TGF-beta1 expression and myofibroblast transformation after glaucoma surgery. The American Journal of Pathology, 182(6), 2147–2154. https://doi.org/10.1016/j.ajpath.2013.02.009.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Gao, H., Bo, Z., Wang, Q., Luo, L., Zhu, H., & Ren, Y. (2019). Salvanic acid B inhibits myocardial fibrosis through regulating TGF-beta1/Smad signaling pathway. Biomedicine & Pharmacotherapy, 110, 685–691. https://doi.org/10.1016/j.biopha.2018.11.098.

    CAS  Article  Google Scholar 

  21. 21.

    Shi, Y., Lin, P., Wang, X., Zou, G., & Li, K. (2018). Sphingomyelin phosphodiesterase 1 (SMPD1) mediates the attenuation of myocardial infarction-induced cardiac fibrosis by astaxanthin. Biochemical and Biophysical Research Communications, 503(2), 637–643. https://doi.org/10.1016/j.bbrc.2018.06.054.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Weng, L., Jia, S., Xu, C., Ye, J., Cao, Y., Liu, Y., et al. (2018). Nogo-C regulates post myocardial infarction fibrosis through the interaction with ER Ca(2+) leakage channel Sec61alpha in mouse hearts. Cell Death & Disease, 9(6), 612. https://doi.org/10.1038/s41419-018-0598-6.

    CAS  Article  Google Scholar 

  23. 23.

    Hao, K., Lei, W., Wu, H., Wu, J., Yang, Z., Yan, S., et al. (2019). LncRNA-Safe contributes to cardiac fibrosis through Safe-Sfrp2-HuR complex in mouse myocardial infarction. Theranostics, 9(24), 7282–7297. https://doi.org/10.7150/thno.33920.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Kang, S. C., Sohn, E. H., & Lee, S. R. (2020). Hydrogen sulfide as a potential alternative for the treatment of myocardial fibrosis. Oxidative Medicine and Cellular Longevity, 2020, 4105382. https://doi.org/10.1155/2020/4105382.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Chistiakov, D. A., Orekhov, A. N., & Bobryshev, Y. V. (2016). Cardiac extracellular vesicles in normal and infarcted heart. International Journal of Molecular Sciences, 17(1). https://doi.org/10.3390/ijms17010063.

  26. 26.

    Sahoo, S., & Losordo, D. W. (2014). Exosomes and cardiac repair after myocardial infarction. Circulation Research, 114(2), 333–344. https://doi.org/10.1161/CIRCRESAHA.114.300639.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Gong, X. H., Liu, H., Wang, S. J., Liang, S. W., & Wang, G. G. (2019). Exosomes derived from SDF1-overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote cardiac endothelial microvascular regeneration in mice with myocardial infarction. Journal of Cellular Physiology, 234(8), 13878–13893. https://doi.org/10.1002/jcp.28070.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Harrell, C. R., Fellabaum, C., Jovicic, N., Djonov, V., Arsenijevic, N., & Volarevic, V. (2019). Molecular mechanisms responsible for therapeutic potential of mesenchymal stem cell-derived secretome. Cells, 8(5). https://doi.org/10.3390/cells8050467.

  29. 29.

    Tan, S. J. O., Floriano, J. F., Nicastro, L., Emanueli, C., & Catapano, F. (2020). Novel applications of mesenchymal stem cell-derived exosomes for myocardial infarction therapeutics. Biomolecules, 10(5). https://doi.org/10.3390/biom10050707.

  30. 30.

    Bang, C., Batkai, S., Dangwal, S., Gupta, S. K., Foinquinos, A., Holzmann, A., et al. (2014). Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. The Journal of Clinical Investigation, 124(5), 2136–2146. https://doi.org/10.1172/JCI70577.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Wang, Y., Zhang, L., Li, Y., Chen, L., Wang, X., Guo, W., et al. (2015). Exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium. International Journal of Cardiology, 192, 61–69. https://doi.org/10.1016/j.ijcard.2015.05.020.

    Article  PubMed  Google Scholar 

  32. 32.

    Chen, P., Wu, R., Zhu, W., Jiang, Z., Xu, Y., Chen, H., et al. (2014). Hypoxia preconditioned mesenchymal stem cells prevent cardiac fibroblast activation and collagen production via leptin. PLoS One, 9(8), e103587. https://doi.org/10.1371/journal.pone.0103587.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Eid, R. A., Khalil, M. A., Alkhateeb, M. A., Eleawa, S. M., Zaki, M. S. A., El-Kott, A. F., et al. (2020). Exendin-4 attenuates remodeling in the remote myocardium of rats after an acute myocardial infarction by activating beta-arrestin-2, protein phosphatase 2A, and glycogen synthase kinase-3 and inhibiting beta-catenin. Cardiovascular Drugs and Therapy. https://doi.org/10.1007/s10557-020-07006-9.

  34. 34.

    Youssef, M. E., El-Mas, M. M., Abdelrazek, H. M., & El-Azab, M. F. (2021). alpha7-nAChRs-mediated therapeutic angiogenesis accounts for the advantageous effect of low nicotine doses against myocardial infarction in rats. European Journal of Pharmacology, 898, 173996. https://doi.org/10.1016/j.ejphar.2021.173996.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Wang, B., Wu, Y., Ge, Z., Zhang, X., Yan, Y., & Xie, Y. (2020). NLRC5 deficiency ameliorates cardiac fibrosis in diabetic cardiomyopathy by regulating EndMT through Smad2/3 signaling pathway. Biochemical and Biophysical Research Communications, 528(3), 545–553. https://doi.org/10.1016/j.bbrc.2020.05.151.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Wang, D. M., Jin, J. J., Tian, L. M., & Zhang, Z. (2020). MiR-195 promotes myocardial fibrosis in MI rats via targeting TGF-beta1/Smad. Journal of Biological Regulators and Homeostatic Agents, 34(4), 1325–1332. https://doi.org/10.23812/20-201-A.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Chen, G., Huang, S., Song, F., Zhou, Y., & He, X. (2020). Lnc-Ang362 is a pro-fibrotic long non-coding RNA promoting cardiac fibrosis after myocardial infarction by suppressing Smad7. Archives of Biochemistry and Biophysics, 685, 108354. https://doi.org/10.1016/j.abb.2020.108354.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Lin, Y., Zhang, F., Lian, X. F., Peng, W. Q., & Yin, C. Y. (2019). Mesenchymal stem cell-derived exosomes improve diabetes mellitus-induced myocardial injury and fibrosis via inhibition of TGF-beta1/Smad2 signaling pathway. Cellular and Molecular Biology (Noisy-le-Grand, France), 65(7), 123–126.

    Article  Google Scholar 

  39. 39.

    Pan, J., Alimujiang, M., Chen, Q., Shi, H., & Luo, X. (2019). Exosomes derived from miR-146a-modified adipose-derived stem cells attenuate acute myocardial infarction-induced myocardial damage via downregulation of early growth response factor 1. Journal of Cellular Biochemistry, 120(3), 4433–4443. https://doi.org/10.1002/jcb.27731.

    CAS  Article  PubMed  Google Scholar 

Download references

Funding

This work was supported by the Medical Scientific Research Foundation of Guangdong Province (No. A2020002) and the Science and Technology Program of Guangzhou, China (No. 202102080011).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Min Wu.

Ethics declarations

Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Associate Editor Junjie Xiao oversaw the review of this article

Supplementary Information

Figure S1
figure9

Western blots. A, NLRC5, α-SMA and VEGF protein expression in cells tested by western blot analysis. B, The expression of p-SMAD2/3, NLRC5 and VEGF in anti-miR-212-5p and GW4869 groups detected by western blot analysis.) (PNG 5043 kb)

Figure S2
figure10

MSCs-derived EVs deliver miR-212-5p to alleviate MI. A, TTC staining of the heart tissues. B, Statistics of TTC staining results. C, The serum cTnT level detected using ELISA (***p < 0.05 vs. the sham group; ###p < 0.05 vs. the MI group; &&&p < 0.05 vs. the MI-EVs-NC group). n = 8 in each group of mice. Measurement data were expressed by mean ± standard deviation. One-way ANOVA was used for multiple group comparisons, followed by Tukey's post hoc test. (PNG 2633 kb)

High resolution image (EPS 4752 kb)

High resolution image (EPS 3924 kb)

ESM 3

(DOCX 20 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wu, Y., Peng, W., Fang, M. et al. MSCs-Derived Extracellular Vesicles Carrying miR-212-5p Alleviate Myocardial Infarction-Induced Cardiac Fibrosis via NLRC5/VEGF/TGF-β1/SMAD Axis. J. of Cardiovasc. Trans. Res. (2021). https://doi.org/10.1007/s12265-021-10156-2

Download citation

Keywords

  • Mesenchymal stem cells
  • Extracellular vesicles
  • MicroRNA-212-5p
  • Cardiac fibrosis
  • Myocardial infarction