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Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70.

ABSTRACT: Myocardial reperfusion injury (MRI) induced by cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. New MRI treatments involving stem cells are currently being developed because these cells may exert their therapeutic effects primarily through paracrine mechanisms. Microvesicles (MVs) are small extracellular vesicles that have become the key mediators of intercellular communication. MVs derived from stem cells have been reported to play an important role in MRI. In this article, we attempted to explore the mechanisms by which MVs derived from human embryonic neural stem cells (hESC-NSC-derived MVs) rescue MRI. hESCs were differentiated into NSCs, and MVs were isolated from their supernatants by ultracentrifugation. H2O2 was used to induce apoptosis in HL-1 cardiomyocytes. Cell viability was detected by using the CCK-8 assay, apoptosis was detected by Annexin V-FITC/PI staining, and apoptosis-related proteins and signalling pathway-related proteins were detected by western blot analysis. Autophagic flux was measured using the tandem fluorescent mRFG-GFP-LC3 assay. Transmission electron microscopy and western blot analysis were adopted to evaluate autophagy levels. hESC-NSC-derived MVs increased the autophagy and inhibited the apoptosis of HL-1 cells exposed to H2O2 for 3 h in a dose-dependent manner. Additionally, hESC-NSC-derived MVs contained high levels of heat shock protein 70 (HSP-70), which can increase the level of HSP-70 in cells. Moreover, the same effect could be achieved by heat shock preconditioning of HL-1 cells overexpressing HSP-70. The benefits of NSC-MVs may be due to the involvement of AKT and mTOR signalling pathways. Importantly, hESC-NSC-derived MVs stimulated the activation of the AKTand mTOR signalling pathway in those cells by transporting HSP-70. Our results suggest that hESC-NSC-derived MVs inhibit the apoptosis of HL-1 cardiomyocytes by promoting autophagy and regulating AKT and mTOR via transporting HSP-70. However, this hypothesis requires in vivo confirmation.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6854932 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundRecombinant human erythropoietin (rhuEPO) and asialoerythropoietin (asialo-rhuEPO) are cardioprotective. However, the protective effects of rhuEPO could not be translated into clinical practice because of its hematopoiesis-associated side effects while non-erythropoietic asialo-rhuEPO is unavailable in large quantities for clinical studies. This study was designed to investigate the cardiomyocyte protective potential of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine (STS)-induced injury in HL-1 murine cardiomyocytes and identify cellular pathway(s) responsible for its cardioprotection.MethodsHL-1 cardiomyocytes were simultaneously treated with STS and asialo-rhuEPOP. Cellular injury, apoptosis, and cell viabilities were measured by LDH assay, Hoechst staining and trypan blue exclusion method, respectively while western blotting was used to study its effects on apoptosis and autophagy hallmarks.ResultsOur results showed that 20 IU/ml asialo-rhuEPOP provided 39% protection to cardiomyocytes compared to STS-treated cells, which is 2-fold better than that of mammalian cell-produce rhuEPO (rhuEPOM). Asialo-rhuEPOP was found to suppress activation of proapoptotic kinase Mst1 (mammalian Sterile-20-like kinase 1) and FOXO3, leading to inhibition of apoptotic pathway and restoration of autophagy as indicated by the reduction of fragmented/condensed nuclei, altered ratios of Bax/Bcl2, p-Bad/Bad, cytosol/mitochondrial cyt c and caspase-3 activation, and the restored levels of autophagy markers Beclin1, p62 and LC3B-II. Additionally, Akt was found to be activated and FOXO3 was phosphorylated on Ser253, suggesting inhibition of FOXO3 transcriptional function.ConclusionsAsialo-rhuEPOP-mediated cardioprotection occurs through activation of PI3K/Akt pathway leading to suppression of Mst1 activation and promoting cardiomyocyte survival.General significanceAsialo-rhuEPOP could be used to modulate Mst1 activity elevated under numerous pathological states.

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Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70.

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