| 11. |
- Park, Kyong-Su, et al.
(författare)
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Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles
- 2019
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Ingår i: Stem Cell Research & Therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- After the initial investigations into applications of mesenchymal stem cells (MSCs) for cell therapy, there was increased interest in their secreted soluble factors. Following studies of MSCs and their secreted factors, extracellular vesicles (EVs) released from MSCs have emerged as a new mode of intercellular crosstalk. MSC-derived EVs have been identified as essential signaling mediators under both physiological and pathological conditions, and they appear to be responsible for many of the therapeutic effects of MSCs. In several in vitro and in vivo models, EVs have been observed to have supportive functions in modulating the immune system, mainly mediated by EV-associated proteins and nucleic acids. Moreover, stimulation of MSCs with biophysical or biochemical cues, including EVs from other cells, has been shown to influence the contents and biological activities of subsequent MSC-derived EVs. This review provides on overview of the contents of MSC-derived EVs in terms of their supportive effects, and it provides different perspectives on the manipulation of MSCs to improve the secretion of EVs and subsequent EV-mediated activities. In this review, we discuss the possibilities for manipulating MSCs for EV-based cell therapy and for using EVs to affect the expression of elements of interest in MSCs. In this way, we provide a clear perspective on the state of the art of EVs in cell therapy focusing on MSCs, and we raise pertinent questions and suggestions for knowledge gaps to be filled.
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| 12. |
- Park, Kyong-Su, et al.
(författare)
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Mesenchymal stromal cell-derived nanovesicles ameliorate bacterial outer membrane vesicle-induced sepsis via IL-10.
- 2019
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Ingår i: Stem cell research & therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Sepsis remains a source of high mortality in hospitalized patients despite proper antibiotic approaches. Encouragingly, mesenchymal stromal cells (MSCs) and their produced extracellular vesicles (EVs) have been shown to elicit anti-inflammatory effects in multiple inflammatory conditions including sepsis. However, EVs are generally released from mammalian cells in relatively low amounts, and high-yield isolation of EVs is still challenging due to a complicated procedure. To get over these limitations, vesicles very similar to EVs can be produced by serial extrusions of cells, after which they are called nanovesicles (NVs). We hypothesized that MSC-derived NVs can attenuate the cytokine storm induced by bacterial outer membrane vesicles (OMVs) in mice, and we aimed to elucidate the mechanism involved.NVs were produced from MSCs by the breakdown of cells through serial extrusions and were subsequently floated in a density gradient. Morphology and the number of NVs were analyzed by transmission electron microscopy and nanoparticle tracking analysis. Mice were intraperitoneally injected with Escherichia coli-derived OMVs to establish sepsis, and then injected with 2×109 NVs. Innate inflammation was assessed in peritoneal fluid and blood through investigation of infiltration of cells and cytokine production. The biodistribution of NVs labeled with Cy7 dye was analyzed using near-infrared imaging.Electron microscopy showed that NVs have a nanometer-size spherical shape and harbor classical EV marker proteins. In mice, NVs inhibited eye exudates and hypothermia, signs of a systemic cytokine storm, induced by intraperitoneal injection of OMVs. Moreover, NVs significantly suppressed cytokine release into the systemic circulation, as well as neutrophil and monocyte infiltration in the peritoneum. The protective effect of NVs was significantly reduced by prior treatment with anti-interleukin (IL)-10 monoclonal antibody. In biodistribution study, NVs spread to the whole mouse body and localized in the lung, liver, and kidney at 6h.Taken together, these data indicate that MSC-derived NVs have beneficial effects in a mouse model of sepsis by upregulating the IL-10 production, suggesting that artificial NVs may be novel EV-mimetics clinically applicable to septic patients.
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| 13. |
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| 14. |
- Shelke, Ganesh V, 1986, et al.
(författare)
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Endosomal signalling via exosome surface TGF beta-1
- 2019
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor beta-1 (TGF beta-1) on their surfaces. The latent form of TGF beta-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGF beta-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGF beta-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGF beta-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.
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| 15. |
- Shelke, Ganesh V, 1986, et al.
(författare)
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Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum
- 2014
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.
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| 16. |
- Shelke, Ganesh V, 1986, et al.
(författare)
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TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas.
- 2018
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Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins-Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.
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| 17. |
- Svennerholm, Kristina, 1981, et al.
(författare)
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Escherichia coli outer membrane vesicles can contribute to sepsis induced cardiac dysfunction.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Sepsis induced cardiac dysfunction (SIC) is a severe complication to sepsis which significantly worsens patient outcomes. It is known that bacteria have the capacity to release outer membrane vesicles (OMVs), which are nano-sized bilayered vesicles composed of lipids and proteins, that can induce a fatal inflammatory response. The aim of this study was to determine whether OMVs from a uropathogenic Escherichia coli strain can induce cardiac dysfunction, and to elucidate any mechanisms involved. OMVs induced irregular Ca2+ oscillations with a decreased frequency in cardiomyocytes through recordings of intracellular Ca2+ dynamics. Mice were intraperitoneally injected with bacteria-free OMVs, which resulted in increased concentration of pro-inflammatory cytokine levels in blood. Cytokines were increased in heart lysates, and OMVs could be detected in the heart after OMVs injection. Troponin T was significantly increased in blood, and echocardiography showed increased heart wall thickness as well as increased heart rate. This study shows that E. coli OMVs induce cardiac injury in vitro and in vivo, in the absence of bacteria, and may be a causative microbial signal in SIC. The role of OMVs in clinical disease warrant further studies, as bacterial OMVs in addition to live bacteria may be good therapeutic targets to control sepsis.
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| 18. |
- Yin, Y., et al.
(författare)
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Extracellular vesicles from mast cells induce mesenchymal transition in airway epithelial cells
- 2020
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Ingår i: Respiratory Research. - : Springer Nature. - 1465-9921 .- 1465-993X. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the airways, mast cells are present in close vicinity to epithelial cells, and they can interact with each other via multiple factors, including extracellular vesicles (EVs). Mast cell-derived EVs have a large repertoire of cargos, including proteins and RNA, as well as surface DNA. In this study, we hypothesized that these EVs can induce epithelial to mesenchymal transition (EMT) in airway epithelial cells. Methods: In this in-vitro study we systematically determined the effects of mast cell-derived EVs on epithelial A549 cells. We determined the changes that are induced by EVs on A549 cells at both the RNA and protein levels. Moreover, we also analyzed the rapid changes in phosphorylation events in EV-recipient A549 cells using a phosphorylated protein microarray. Some of the phosphorylation-associated events associated with EMT were validated using immunoblotting. Results: Morphological and transcript analysis of epithelial A549 cells indicated that an EMT-like phenotype was induced by the EVs. Transcript analysis indicated the upregulation of genes involved in EMT, including TWIST1, MMP9, TGFB1, and BMP-7. This was accompanied by downregulation of proteins such as E-cadherin and upregulation of Slug-Snail and matrix metalloproteinases. Additionally, our phosphorylated-protein microarray analysis revealed proteins associated with the EMT cascade that were upregulated after EV treatment. We also found that transforming growth factor beta-1, a well-known EMT inducer, is associated with EVs and mediates the EMT cascade induced in the A549 cells. Conclusion: Mast cell-derived EVs mediate the induction of EMT in epithelial cells, and our evidence suggests that this is triggered through the induction of protein phosphorylation cascades.
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