Changes of Circulating Extracellular Vesicles from the Liver after Roux-en-Y Bariatric Surgery

Figures

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  Figure 1. Extracellular vesicle dynamics in patients undergoing bariatric surgery. (A) Total medium extracellular vesicle content was determined by flow cytometry analysis in citrated plasma of 27 patients before and one year after surgery. Results are given as total number of extracellular vesicles per 100 µL plasma. (B) Phosphatidylserine positive medium extracellular vesicles in citrated plasma of 27 patients before and one year after surgery were identified by staining with AnnexinV (AnnV), the remaining medium extracellular vesicles were defined as AnnV–. Numbers of positive and negative medium extracellular vesicles were evaluated using flow cytometry. Results are given as total number of extracellular vesicles per 100 µL plasma. (C) Phosphatidylserine positive medium extracellular vesicles were analyzed in 26 patients using an ELISA as indicated under Materials and Methods. Values are given as AnnexinV+ extracellular vesicles in nM. For (A–C) a Wilcoxon test was used. p ≤ 0.05 was considered significant.

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  Figure 2. Extracellular hepatic vesicle dynamics in patients undergoing bariatric surgery. (A): Total hepatic medium extracellular vesicles in citrated plasma of 27 patients before and one year after surgery were defined as HepPar1+, asialoglycoprotein receptor (ASGPR)+, and HepPar1+ASGPR+ medium extracellular vesicles using flow cytometry. HepPar1+ (B) or ASGPR+ (C) medium extracellular vesicles in citrated plasma of 27 patients before and one year after surgery were evaluated for their AnnV staining pattern. Absolute numbers of extracellular vesicles positive for AnnV+ and AnnV– were determined by flow cytometry. Values are given per 100 µL of plasma. For (A–C) a Wilcoxon test was used. p ≤ 0.05 was considered significant.

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  Figure 3. Liver specific RNA patterns in patients undergoing bariatric surgery. (A) mRNA was isolated from circulating EVs from citrated plasma of 27 patients before and one year after surgery as indicated in the supplementary methods. qPCR was performed for acyl-coenzyme-A-oxidase-1 (ACOX), acyl-coenzyme-A-dehydrogenase (ACADM), hydroxyacyl-coenzyme-A-dehydrogenase (HADH) and 3-hydroxybutyrate-dehydrogenase (BDH). Patients were stratified depending on the number of detectable mRNAs. (B) RNA was isolated from circulating EVs from 27 bariatric surgery patients before and one year after surgery as indicated in Methods and the quantity was determined. (C) Relative expression levels of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and 18s from circulating EVs were determined in 27 bariatric surgery patients before and one year after surgery as indicated in Materials and Methods. Values given are relative to a spike in control. For (A) statistical significance was calculated using a chi-square test. (B, C) statistical significance was calculated using Wilcoxon test. p ≤ 0.05 was considered significant.

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  Figure 4. Histograms of extracellular vesicle staining. Histograms for cytokeratine 18 labeled with FITC (A), Annexin V labeled with PE-Cy7 (B), ASGPR labeled with APC (C), and HepPar1 labeled with PE (D) are shown. Histograms from Annexin V, ASGPR and HepPar1 are derived from the same donor.

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  Figure 5. Representative flow cytometry dot plots. Dot plots for bead-based size estimation showing a bead mix labeled with FITC and an unlabeled fraction (A). The various gates are labeled with the size of the measured beads, percentages represent the relative number of beads measured compared to the whole sample. Dot plots are further shown for Annexin V and HepPar1 (B) and ASGPR and Annexin V (C). All examples are given before and after surgery. A representative isotype control picture is included in the panel. Gating was performed according to the values obtained in the histograms, percentages display the relative amount of events in comparison to the total event number.