Overexpression of Exosomal Cardioprotective miRNAs Mitigates Hypoxia-Induced H9c2 Cells Apoptosis

Zhang, Jinwei; Ma, Jideng; Long, Keren; Qiu, Wanling; Wang, Yujie; Hu, Zihui; Liu, Can; Luo, Yi; Jiang, Anan; Jin, Long; Tang, Qianzi; Wang, Xun; Li, Xuewei; Li, Mingzhou

doi:10.3390/ijms18040711

Cited by 68 publications

(58 citation statements)

References 54 publications

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“…Zhang et al reported that exosomes derived from H9C2 cells carry certain cardioprotective miRNAs, which repress hypoxia-induced apoptosis. Among the hypoxia-induced exosomal miRNAs, miR-152-3p and let-7i-5p exert an anti-apoptotic function by targeting autophagy related 12 and Fas ligand, respectively ( 26 ). Cui et al confirmed that adipose-derived mesenchymal stem cell exosomes protect the ischemic myocardium from ischemia/reperfusion injury via activation of the Wnt/β-catenin signal pathway ( 27 ).…”

Section: Discussionmentioning

confidence: 99%

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation

Jiang

Yang

et al. 2018

Int J Mol Med

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Exosomes are cell-derived vesicles released from a variety of mammalian cells that are involved in cell-to-cell signalling. It has been reported that cardiac progenitor cells (CPCs) derived from an adult heart are one of the most promising stem cell types for cardioprotection and repair. The mammalian target of rapamycin (mTOR) signalling pathway is a pivotal regulator in CPCs, therefore, CPC-derived exosomes were used in the present study to investigate whether it can promote H9C2 cell growth through the protein kinase B (PKB, or Akt)/mTOR signalling pathway. The CPCs were isolated from Sprague-Dawley hearts. Following treatment with a specific medium, the exosomes were purified and identified by electron micrograph and western blot assays, using CD63 and CD81 as markers. The methyl-thiazolyl-tetrazolium and 5-ethynyl-2′-deoxyuridine methods were used to detect H9C2 cell growth. The expression of Akt and mTOR were detected by western blot analysis following treatment with 200 or 400 µg/ml of exosomes for 24 or 48 h, respectively. It was found that, compared with higher concentrations of exosomes, prolonging the duration of exposure promoted cell growth. Accordingly, CPC-derived exosomes stimulated the expression of Akt to a marked degree; groups treated with exosomes for 48 h showed higher expression of Akt than those treated for 24 h at the same concentration. mTOR was also stimulated by CPC-derived exosomes. The activation of mTOR increased in accordance with the treatment time at an exosome concentration of 200 µg/ml and decreased with treatment time at an exosome concentration of 400 µg/ml. In conclusion, the present study demonstrated that CPC-derived exosomes promoted H9C2 cell growth via the activation of Akt/mTOR in a time-dependent manner at a relatively low exosome concentration, which may provide a novel therapy for cardiovascular disease.

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Section: Discussionmentioning

confidence: 99%

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation

Jiang

Yang

et al. 2018

Int J Mol Med

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“…Adult cardiac myocytes treated with physiological concentrations of ethanol, for example, secreted more EVs [43]. Others demonstrated that stimulation with hypoxia for 48 h resulted in an elevated EV concentration but also with a higher rate of apoptosis probably due to the abiding absence of oxygen [44]. In addition, starvation of H9c2 cells also increased the release of EVs [32].…”

Section: Discussionmentioning

confidence: 99%

Characterization of extracellular vesicles derived from cardiac cells in an in vitro model of preconditioning

Borosch

Dahmen

Beckers

et al. 2017

J of Extracellular Vesicle

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Preconditioning is a promising technique to protect the heart from ischaemia-reperfusion injury. In this context, the crosstalk between different cardiac cell types and especially the exchange of cardioprotective mediators has come into the focus of current research. Recently, extracellular vesicles (EVs), nano-sized structures, emerged as possible communication mediators. They are taken up by recipient cells and can alter gene expression or activate intracellular signal cascades. It has been shown that all cardiac cell types are able to secrete EVs, but so far the influence of an in vitro preconditioning stimulus on EV concentration and composition has not been investigated. Therefore, we stimulated primary cardiac myocytes and fibroblasts from neonatal rats, as well as H9c2 cells, with two known in vitro preconditioning stimuli: hypoxia or isoflurane. EVs were isolated from cell culture supernatants 48 h after stimulation by differential centrifugation and size exclusion chromatography. They were characterized by transmission electron microscopy, tunable resistive pulse sensing, miRNA array and Western blot analysis. The detected EVs had the typical cup-shaped morphology and a size of about 150 nm. No significant differences in EV concentration were observed between the different groups. The protein and miRNA load was affected by in vitro preconditioning with isoflurane or hypoxia. EV markers like Alix, CD63, flotillin-1 and especially heat shock protein 70 were significantly up-regulated by the treatments. Several miRNAs like miR-92b-3p, miR-761 and miR-101a-5p were also significantly affected. A migration assay confirmed the physiological benefit of these EVs. Taken together, our findings show that a model of in vitro preconditioning of cardiac cells does not influence EV concentration but strongly regulates the EV cargo and affects migration. This might indicate a role for EV-mediated communication in isoflurane- and hypoxia-induced in vitro preconditioning.

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“…In this study, regarding exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. Further, we found that incubation at 37℃ for 1 h can decrease the proportion of free miRNAs in exosome pellets and exosomal miRNAs like miR-126 and miR-152 [11][12][13]18] also decreased when RNase A was used. In conclusion, our findings provide essential information on the details of serum exosome isolation methods for further studies on exosomal miRNAs.…”

mentioning

confidence: 84%

“…Recently, an increasing number of extracellular miRNAs have been detected in exosomes isolated from biological fluids and cell culture media [4]. Moreover, some exosomal miRNAs have been shown to regulate disease development and reported as biomarkers of different types of cancers, cardiovascular disease and brain injury [11][12][13]. In addition to exosomal miRNAs, there are still some circulating miRNAs free in the serum [14].…”

mentioning

confidence: 99%

Comparison of Serum Exosome Isolation Methods on Co-precipitated Free MicroRNAs

Cheng

Qu²,

Dong³

et al. 2019

Preprint

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BackgroundExosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.ResultsIn this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods. Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37℃ for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used.ConclusionsIn conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.

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Overexpression of Exosomal Cardioprotective miRNAs Mitigates Hypoxia-Induced H9c2 Cells Apoptosis

Cited by 68 publications

References 54 publications

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation

Characterization of extracellular vesicles derived from cardiac cells in an in vitro model of preconditioning

Comparison of Serum Exosome Isolation Methods on Co-precipitated Free MicroRNAs

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