Could protein content of Urinary Extracellular Vesicles be useful to detect Cirrhosis in Alcoholic Liver Disease?

Figure 1. Characterization of uEVs in the cohort 1. A, B) Representative NTA profiles and Cryo-EM images corresponding to a representative non-cirrhotic (Control) individual and a cirrhotic patient. NTA profiles show the average values of 5 measurements and the associated standard deviation for each size in grey shadow. Cryo-EM images show illustrative uEVs. Scale bar 100 nm. C) WB for TSG101, Flotillin1, CD10 and Syntenin-1 on 15 µL of sample from 1:1000Volo.

Figure 2. NTA analysis of uEVs in the cohort 1. A, B) Analysis of uEVs in terms of concentration and size (mode), respectively: AA, BA) Non-cirrhotic (Controls) vs cirrhotic patients (t-Student, p>0.05); AB, BB) Comparison among Controls, A, B and C Child-Pugh categories; AC, BC) Comparison among three age ranges from both Controls and patients; AD, BD) Comparison among male and female groups from both Controls and patients (ANOVA, p>0.05 in all cases).

Figure 3. Western Blot analysis of uEVs in the cohort 1. A, B, C, D) Analysis of abundance of TSG101, Flotillin-1, CD10 and Syntenin-1, respectively: AA, BA, CA, DA) Non-cirrhotic (Controls) vs cirrhotic patients (Student's test, p>0.05); AB, BB, CB, DB) Comparison among Controls, A, B and C Child-Pugh categories; AC, BC, CC, DC) Comparison among three age ranges from both Controls and patients; AD, BD, CD, DD) Comparison among male and female groups from both Controls and patients (ANOVA, p>0.05 in all cases).

Figure 4. Venn diagrams of proteins detected in uEVs from non-cirrhotic (Controls) and cirrhotic individuals by Synapt and Orbitrat acquisition strategies. Diagrams represent the number of Uniprot/Swissprot entries identified by Mascot search engine (Matrix Science Ltd.) through Proteome Discoverer software 1.4 (Thermo).

Figure 5. Heat maps for differential proteins between controls and cirrhotic patients in Synapt and Orbitrap methodologies. Hierarchical clustering of the relative abundance of A) 56 (Synapt) and B) 44 (Orbitrap) identified proteins. For a clearer representation, protein abundances were normalized against Control average values. The vertical dendrogram represents the correlation distances between protein abundance levels (Perseus program). Colors represent the relative abundance (upregulation in red, downregulation in green). Relative abundance and raw data of each protein are included in Table S2. Only proteins identified with at least two peptides at FDR<1% were considered in the analysis.

Figure 6. GO enrichment analysis. GO enrichment analysis of differentially regulated proteins between controls and patients found by Synapt and Orbitrap methodologies was performed by DAVID. The GO term or categories Molecular Function (MF), Cellular Component (CC) and Biological Process (BP) were assessed. Additionally, Interpro (IP), KEGG Pathways (KP), sequences (UPSF) and keywords (UPK) were also analyzed, considering terms with an enrichment p-value<0.05.