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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190048	1/1	Homo sapiens	NA	DC (d)(U)C Filtration	Shaihov-Teper, Olga	2021	67%
Study summary Full title Extracellular Vesicles from Epicardial Fat Facilitate Atrial Fibrillation All authors Olga Shaihov-Teper, Eilon Ram, Nimer Ballan, Rafael Y Brzezinski, Nili Naftali-Shani, Rula Masoud, Tamar Ziv, Nir Lewis, Yeshai Schary, La-Paz Levin-Kotler, David Volvovitch, Elchanan M Zuroff, Sergei Amunts, Neta Regev-Rudzki, Leonid Sternik, Ehud Raanani, Lior Gepstein, Jonathan Leor Journal Circulation Abstract Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenes (show more...)Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. Methods: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every two days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and miRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells (MSCs) and endothelial cells (ECs) in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell-derived cardiomyocytes (iCMs). Results: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, as well as profibrotic miRNA, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human MSCs and ECs, compared to eFat-EVs from patients without AF. Finally, while eFat-EVs from patients with and without AF shortened the action potential duration of iCMs, only eFat-EVs from patients with AF induced sustained reentry (rotor) in iCMs. Conclusions: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation. (hide) EV-METRIC 67% (50th percentile 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 Epicardial fat tissue culture supernatant Sample origin cardiac disease Focus vesicles extracellular vesicle 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 DC (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ IL-10/ Alix/ TNF-A/ IL1A/ CD9 non-EV: Calreticulin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Epicardial fat tissue culture supernatant 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) 960 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 50 Wash: speed (g) 100000 Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants Calreticulin ELISA Antibody details provided? No Detected EV-associated proteins TNF-A/ IL-10/ IL1A Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 1 tablet per 10 ml extraction solution RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-170 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 30-170

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EV-TRACK ID	EV190048
species	Homo sapiens
sample type	Epicardial fat tissue culture supernatant
condition	cardiac disease
separation protocol	DC (d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	67