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

ABSTRACT: 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.

SUBMITTER: Borosch S

PROVIDER: S-EPMC5819478 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Characterization of extracellular vesicles derived from cardiac cells in an <i>in vitro</i> model of preconditioning.

Borosch Sebastian S Dahmen Eva E Beckers Christian C Stoppe Christian C Buhl Eva Miriam EM Denecke Bernd B Goetzenich Andreas A Kraemer Sandra S

Journal of extracellular vesicles 20171030 1

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 sh ...[more]

PMID: 29479396

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Project description:Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)-nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4-NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in ELPS-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in ELPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

Project description:BackgroundMesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia).MethodsMenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties.ResultsNo differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response.ConclusionsPriming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

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

Publications

Characterization of extracellular vesicles derived from cardiac cells in an <i>in vitro</i> model of preconditioning.

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