Project description:Under physiological conditions, extracellular vesicles (EVs) are present simultaneously in the extracellular compartment together with cytokines. Thus, we hypothesized that EVs in combination with cytokines induce different responses of monocyte cells compared to EVs or cytokines alone. Human monocyte U937 cells were incubated with EV-containing or EV-free CCRF human T-cell supernatant, with or without the addition of TNF. U937 cells cultured in EV-free supernatant, supernatant containing CCRF t-cell derived EVs, TNF or both. Each treatment option was measured in 3 replicates.
Project description:Under physiological conditions, extracellular vesicles (EVs) are present simultaneously in the extracellular compartment together with cytokines. Thus, we hypothesized that EVs in combination with cytokines induce different responses of monocyte cells compared to EVs or cytokines alone. Human monocyte U937 cells were incubated with EV-containing or EV-free CCRF human T-cell supernatant, with or without the addition of TNF.
Project description:Extracellular vesicles (EV) convey biological messages through their cargoes. Herein we focus on monocyte/platelet aggregates characteristic of several cardiovascular diseases with an analysis of monocyte-derived EV (mEVs) effects on the atherosclerotic plaque. Monocyte preparations were stimulated with TNF-α in presence or absence of prostacyclin and EVs isolated via centrifugation. EV physical characteristics were determined by Nanoparticle Tracking analysis while surface profile was analysed using imaging flow cytometry. Atherosclerotic plaques from 5 patients undergoing endarterectomy were cultured with or without mEVs. Cytokines and proteins released in the culture media were measured by multiplex ELISA and mass spectrometry. Proteomic of mEVs prepared in different incubation settings was also conducted. Monocyte isolation yielded ~80% platelet-monocyte aggregates. TNF-α stimulation produced CD14+ EVs as well as a subset bearing the CD41 marker for platelets (CD14+/CD41+). Prostacyclin addition did not modulate monocyte/platelet aggregates, but impacted on mEV numbers. Addition of TNF-α mEVs on atherosclerotic plaque fragments impacted on general protein release (19 upregulated and 7 downregulated) and elevated cytokine release. mEVs generated by TNF-α and prostacyclin produced minimal changes on plaque reactivity. Proteomic analysis of mEVs revealed a distinctive composition when the cell preparation was activated with TNF-α alone or with prostacyclin. In conclusion, mEVs activate the atherosclerotic plaque. Attenuating platelet activation has an effect on EV composition with downstream modulation of their pro-inflammatory actions. EV heterogeneity reflects the mode of activation of the cell of origin and may differently contribute to the development and progression of atherosclerosis.
Project description:Extracellular vesicles (EV) convey biological messages through their cargoes. Herein we focus on monocyte/platelet aggregates characteristic of several cardiovascular diseases with an analysis of monocyte-derived EV (mEVs) effects on the atherosclerotic plaque. Monocyte preparations were stimulated with TNF-α in presence or absence of prostacyclin and EVs isolated via centrifugation. EV physical characteristics were determined by Nanoparticle Tracking analysis while surface profile was analysed using imaging flow cytometry. Atherosclerotic plaques from 5 patients undergoing endarterectomy were cultured with or without mEVs. Cytokines and proteins released in the culture media were measured by multiplex ELISA and mass spectrometry. Proteomic of mEVs prepared in different incubation settings was also conducted. Monocyte isolation yielded ~80% platelet-monocyte aggregates. TNF-α stimulation produced CD14+ EVs as well as a subset bearing the CD41 marker for platelets (CD14+/CD41+). Prostacyclin addition did not modulate monocyte/platelet aggregates, but impacted on mEV numbers. Addition of TNF-α mEVs on atherosclerotic plaque fragments impacted on general protein release (19 upregulated and 7 downregulated) and elevated cytokine release. mEVs generated by TNF-α and prostacyclin produced minimal changes on plaque reactivity. Proteomic analysis of mEVs revealed a distinctive composition when the cell preparation was activated with TNF-α alone or with prostacyclin. In conclusion, mEVs activate the atherosclerotic plaque. Attenuating platelet activation has an effect on EV composition with downstream modulation of their pro-inflammatory actions. EV heterogeneity reflects the mode of activation of the cell of origin and may differently contribute to the development and progression of atherosclerosis.
Project description:The aim of this experiment was to highlight differences in expression of microRNAs transported by extracellular vesicles between two strains of the HL-60 cell line (acute promyelocytic leukemia cell line): the chemo-sensitive strain (HL-60) and an anthracyclin-resistant strain (HL-60/AR).
Project description:We evaluted the effects of extracellular vesicles of bleomycin-injured wild-type and syndecan-1 deficient mice on mouse lung epithelial cell line (MLE-12) total transcriptomic changes (bulk mRNA sequencing)
Project description:In this study, we address mRNA composition of hepatocyte-like derived extracellular vesicles (EVs), using as cellular model the mouse liver derived cell line MLP29, and primary cell culture of rat hepatocyte (RH) obtained by in vivo liver perfusion. The study shows qualitative characterization of RNA, identification of transcripts and its functional characterization through gene expression array technique. To reach a deeper nowledge in the biology of EVs, we perform RNase protection assay, density gradients matching RNA with typical exosomal protein markers, and capture assays to probe that mRNA was internalized. Aim of the project: To identify transcripts present in extracellular vesicles secreted by Rat hepatocytes primary cell culture and to identify extracellular vesicles secreted by mouse hepatocyte cell line MLP29, and in this case, compare the enrichment of transcripts respect to the cell, to know if the composition in the extracellular vesicles is similar to the cell, or if their composition is not directly determined by the abundance of transcripts in the cell.
Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Treatment with B6H12 antibody inhibited co-immunoprecipitation of EGFR with CD47 and inhibited EGF-induced EGFR tyrosine phosphorylation. B6H12 treatment of bCSC also suppressed asymmetric cell division and cell proliferation and up-regulated caspase 3/7 activity. Correspondingly, caspase-7 cleavage in human breast cancers correlated with CD47 expression. Our data shows that B6H12 specifically targets bCSCs but not differentiated cancer cells, and this CD47 signaling is independent of SIRPα. Three replicates of each condition were generated. Three replicates of each MDA-231 attached cells (differentiated), MDA-231 in suspension cells (bCSC), MDA-231 in suspension cells (bCSC) treated with Control Antibody and MDA-231 in suspension cells (bCSC) treated with B6H12 Antibody.