ABSTRACT
Chronic liver injury leads to decreased liver function and increased fibrosis. Fibrosis is not only associated with the development of portal hypertension and carcinogenesis, but with the occurrence of events and a poor prognosis, highlighting the importance of non-invasive fibrosis assessment in patients. In the present study, we searched for markers related to liver fibrosis via proteomic analysis of small extracellular vesicles (sEVs). In the discovery cohort, proteomic analysis was carried out in the sEVs extracted from the sera of 5 patients with decompensated cirrhosis, 5 patients with compensated cirrhosis, and 5 controls without liver disease. Interestingly, in this cohort, fibulin-4 was significantly associated with cirrhosis while in the validation cohort [formed by 191 patients: 7 patients without disease, 16 patients without liver disease (other diseases), 38 patients with chronic liver disease (CLD), 75 patients with cirrhosis of Child-Pugh class A (36 without hepatocellular carcinoma [HCC], 29 with HCC), and 65 patients with cirrhosis of Child-Pugh class B-C (39 without HCC, 26 with HCC)], fibulin-4/CD9 levels increased with cirrhosis progression. Furthermore, the fibulin-4/CD9 ratio was significantly higher in patients with varices. Immunostaining also revealed strong fibulin-4 expression in cholangiocytes within the fibrous areas and mesothelial cells in liver tissue blood vessels. Taken together, our results suggest that fibulin-4, essential for lysyl oxidase activation, might be a new liver fibrosis marker found in the sEVs of patients with cirrhosis.
ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations' proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
