The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells

Kovács, Árpád Ferenc; Láng, Orsolya; Turiák, Lilla; Ács, András; Kőhidai, László; Fekete, Nóra; Alasztics, Bálint; Mészáros, Tamás; Buzás, Edit I.; Rigó, János; Pállinger, Éva

doi:10.1038/s41598-018-23706-7

Cited by 24 publications

(27 citation statements)

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“…(2020) 10:4320 | https://doi.org/10.1038/s41598-020-61253-2 www.nature.com/scientificreports www.nature.com/scientificreports/ experiences in extracellular vesicle quantification [30][31][32] . Gavillet et al detected histone H3 and MPO double positive particles, we counted MPO/free DNA/histone H3 triple positive events.…”

Section: Scientific Reports |mentioning

confidence: 99%

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Plasma neutrophil extracellular trap level is modified by disease severity and inhaled corticosteroids in chronic inflammatory lung diseases

Gál

Gézsi

Pállinger

et al. 2020

Sci Rep

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www.nature.com/scientificreports www.nature.com/scientificreports/ a background. Measurements were conducted by BD FACSCalibur (BD Biosciences). Optimization of cytometer settings and gating strategy was carried out as described earlier for extracellular vesicle quantification [30][31][32] . For the quantification only the number of MPO/Sytox Red/histone triple positive events were considered.Human samples. Whole blood samples were collected to sodium citrate tubes from all individuals. During the blood collection no patients were in exacerbation. Platelet free plasma samples were prepared by centrifugation two times at 2500 RPM for 15 minutes. For in vivo NET quantification the platelet free plasma samples were processed as described above.Confocal laser-scanning microscopy. Platelet free plasma samples (10 µL) were attached to MilliCell EZ slides (Merck-Millipore), dried for 10 minutes at room temperature (RT) and were fixed with 4% 0.22 µm filtered PFA for 20 minutes at RT. Following three washes with 1% PBS, samples were blocked with 5% BSA and incubated for 30 minutes at RT. Because of NETs occurrence we used BSA instead of fetal bovine serum (FBS). Primary antibodies (mouse monoclonal anti-MPO FITC (BD Biosciences) and rabbit polyclonal anti-histone H3 antibody (ab5103, Abcam) in 5% BSA solution, both same as before) were used in 1:100 dilution for 1 hour at RT in dark. After three times washes with 5% BSA, secondary antibodies were applied such as F(ab')2-goat anti-mouse IgG (H + L) conjugated with eFluor 570 (eBioscience) in 1:200 dilution and goat anti-rabbit IgG conjugated with ATTO647N (Sigma-Aldrich) in 1:500 dilution for 1 hour at RT in dark. Washed twice with 1% PBS and twice with distillated water. Slides were mounted with DAPI containing Prolong Diamond (Invitrogen). Samples were examined by Lecia SP8 laser-confocal microscope (Leica Microsystems, Wetzlar, Germany). Statistical analysis.All statistical evaluations were performed with R statistical computing program (R Foundation for Statistical Computing, Vienna, Austria). Differences between experimental groups were analysed by one-way ANOVA. Differences between subgroups of patients were analysed by Student's t-test (unpaired, two-tailed). P-values were adjusted with Benjamini Hochberg method to correct for the false discovery rate, and adjusted p-values <0.05 were considered to be statistically significant. The relationship between the level of NET and FEV1 was assessed by calculating Pearson's correlation coefficient. R statistical computing program was used for graphic illustrations. Data availabilityAll datasets and protocols of the measurements are available in request.

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Section: Scientific Reports |mentioning

confidence: 99%

“…Measurements were conducted by BD FACSCalibur (BD Biosciences). Optimization of cytometer settings and gating strategy was carried out as described earlier for extracellular vesicle quantification [30][31][32] . For the quantification only the number of MPO/Sytox Red/histone triple positive events were considered.Human samples.…”

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confidence: 99%

Plasma neutrophil extracellular trap level is modified by disease severity and inhaled corticosteroids in chronic inflammatory lung diseases

Gál

Gézsi

Pállinger

et al. 2020

Sci Rep

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“…EV detection by flow cytometry was carried out according to [ 21 ] with minor modifications. Briefly, cells and cell fragments were removed from cell culture supernatant by serial centrifugation at 300 g for 5 min and 2000 g for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Extracellular vesicle release from intestinal organoids is modulated by Apc mutation and other colorectal cancer progression factors

Szvicsek

Oszvald

Szabó

et al. 2019

Cell. Mol. Life Sci.

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Extracellular vesicles (EVs) are membrane-surrounded structures that transmit biologically important molecules from the releasing to target cells, thus providing a novel intercellular communication mechanism. Since EVs carry their cargo in a protected form and their secretion is generally increased in tumorigenesis, EVs hold a great potential for early cancer diagnosis. By 3D culturing, we provide evidence that colorectal cancer (CRC) patient-derived organoids, representing a state-of-the-art established and essential approach for studying human CRC, is a suitable model for EV analysis. When testing the effects of major factors promoting CRC progression on EV release in the organoid model, we observed that Apc mutation, leading to uncontrolled Wnt activation and thus to tumorigenesis in the vast majority in CRC patients, critically induces EV release by activating the Wnt pathway. Furthermore, the extracellular matrix component collagen, known to accumulate in tumorigenesis, enhances EV secretion as well. Importantly, we show that fibroblast-derived EVs induce colony formation of CRC organoid cells under hypoxia. In contrast, there was no major effect of tumor cell-derived EVs on the activation of fibroblasts. Collectively, our results with CRC and Apc -mutant adenoma organoids identify Apc mutation and collagen deposition as critical factors for increasing EV release from tumors. Furthermore, we provide evidence that stromal fibroblast-derived EVs contribute to tumorigenesis under unfavorable conditions in CRC. Electronic supplementary material The online version of this article (10.1007/s00018-019-03052-1) contains supplementary material, which is available to authorized users.

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“…The proteomic, miRNA and dsDNA content of EV fractions were assessed with Qubit 4 Fluorometer using the Qubit™ Protein Assay Kit, Qubit microRNA Assay Kit and Qubit 1X dsDNA HS Assay Kit, respectively. The iEV number was quantified by annexin V staining, by flow cytometry as previously described by us [32] (Supplementary Figure S5).…”

Section: Methodsmentioning

confidence: 99%

Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation

Kovács

Fekete

Turiák

et al. 2019

IJMS

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Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto–maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization.

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The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells

Cited by 24 publications

References 53 publications

Plasma neutrophil extracellular trap level is modified by disease severity and inhaled corticosteroids in chronic inflammatory lung diseases

Plasma neutrophil extracellular trap level is modified by disease severity and inhaled corticosteroids in chronic inflammatory lung diseases

Extracellular vesicle release from intestinal organoids is modulated by Apc mutation and other colorectal cancer progression factors

Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation

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