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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV150097	1/1	Homo sapiens	NAY	(d)(U)C	Wu M	2015	0%
Study summary Full title A novel pathway of cellular activation mediated by antiphospholipid antibody-induced extracellular vesicles All authors Wu M, Barnard J, Kundu S, McCrae KR Journal J Thromb Haemost Abstract BACKGROUND: Elevated levels of endothelial cell (EC)-derived extracellular vesicles (EVs) circulate (show more...)BACKGROUND: Elevated levels of endothelial cell (EC)-derived extracellular vesicles (EVs) circulate in patients with antiphospholipid antibodies (APLAs), and APLAs, particularly those against ?2 -glycoprotein I (?2 GPI), stimulate EV release from ECs. However, the effects of EC-derived EVs have not been characterized. OBJECTIVE: To determine the mechanism by which EVs released from ECs by anti-?2 GPI antibodies activate unstimulated ECs. PATIENTS/METHODS: We used interleukin (IL)-1 receptor inhibitors, small interfering RNA (siRNA) against Toll-like receptors (TLRs) and microRNA (miRNA) profiling to assess the mechanism(s) by which EVs released from ECs exposed to anti-?2 GPI antibodies activated unstimulated ECs. RESULTS AND CONCLUSIONS: Anti-?2 GPI antibodies caused formation of an EC inflammasome and the release of EVs that were enriched in mature IL-1?, had a distinct miRNA profile, and caused endothelial activation. However, activation was not inhibited by an IL-1? antibody, an IL-1 receptor antagonist, or IL-1 receptor siRNA. EC activation by EVs required IL-1 receptor-associated kinase 4 phosphorylation, and was inhibited by pretreatment of cells with TLR7 siRNA or RNase A, which degrades ssRNA. Profiling of miRNA in EVs released from ECs incubated with ?2 GPI and either control IgG or anti-?2 GPI antibodies revealed numerous differences in the content of specific miRNAs, including a significant decrease in mIR126. These observations demonstrate that, although anti-?2 GPI-derived endothelial EVs contain IL-1?, they activate unstimulated ECs through a TLR7-dependent and ssRNA-dependent pathway. Alterations in miRNA content may contribute to the ability of EVs derived from ECs exposed to anti-?2 GPI antibodies to activate unstimulated ECs in an autocrine or paracrine manner. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90

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EV-TRACK ID	EV150097
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	0