Quantitative proteomic analysis of hepatocyte-secreted extracellular vesicles reveals candidate markers for liver toxicity

Abstract

Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions.

Exosomes in liver pathology

“…A recent study has analyzed exosomal protein levels in urine of rats with drug-induced liver injury (DILI) and reported that exosomes in DILI rat urine contain reduced exosomal proteins, including CD26 and CD81 and candidate marker proteins compared with exosomes in control rat urine [7]. Another study has demonstrated, however, that exosomes isolated from blood serum of rats with DILI contain higher expression levels of exosomal proteins, including HSP70 and HSP90 and other candidate marker proteins although Alix, endosome-associated protein is decreased compared with exosomes from healthy rats [8]. This study also demonstrated that protein expression profiles differ between liver extracts and circulating exosomes isolated from blood serum.…”

A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes

“…Production and isolation of EVs secreted by primary rat hepatocytes were performed as described in Conde-Vancells et al (2008) and Rodríguez-Suárez et al (2014). Briefly, rat hepatocyte suspension was centrifuged at 400 rpm 3 min and seeded in 24 collagen-coated dishes (150 mm) with collagen type I from rat tail (BD Biosciences.…”

“…MB-COMT has higher affinity for catechol substrates and for S-adenosyl-L-methionine coenzyme (Lotta et al, 1995). Interestingly, this enzyme has also been detected in extracellular vesicles (EVs) secreted by hepatocytes (Conde-Vancells et al, 2008) and in serum circulating vesicles (Rodríguez-Suárez et al, 2014) suggesting a possible role of these vesicles in the metabolism of catecholamine neurotransmitters and catecholestrogens metabolisms.…”

Exosome: An Emerging Participant in the Development of Liver Disease

“…In various liver injury models induced by alcohol, drug, inflammation, or diet, the changes in the protein contents as well as mRNA levels/types in the exosomes have been proven to be satisfactory indicators for evaluating inflammation and promising biomarkers for detecting liver injury (46)(47)(48)(49)(50)(51)(52)(53).…”

“…Likewise, hepatocyte-derived exosomes showed significantly increased levels of liverspecific RNAs albumin and miR-122 to sub-toxic APAP exposure in an early step of liver injury, researchers who maintained these exosomes may be associated to early immune responses in the formation of drug-induced liver injury (56). In in vitro experiments, significant protein changes were observed in exosomes secreted by hepatotoxicant-treated hepatocytes and similar changes were also detected in the serum exosomes in a rat model of liver injury induced by galactosamine, e.g., the levels of carboxylesterase (CES) protein family members and CES3 significantly increased (51). The injection of the exosomes derived from the peripheral blood of mice that were fed a high-fat diet resulted in an accumulation of immature myeloid cells (CD11b + Ly6C + Ly6C-cells) in the liver of the recipient mice, which might be responsible for obesityrelated liver disease (57).…”