Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

Abstract

Background & AimNonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.DesignCholine Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.ResultsWe observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.ConclusionsThese findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

“…They have been investigated in numerous human diseases and in mouse models of disease ( 10 ), and an increase in circulating EVs is described in a mouse model of NASH ( 39 ). Our fi ndings further these observations, and suggest that the ceramide and S1P content of EVs might serve as an additional bioactive biomarker in NASH patients.…”

“…* P < 0.05 by ANOVA for comparisons involving all three groups; ** P < 0.05 for the comparisons shown across two experimental conditions. by guest, on May 7, 2018 www.jlr.org MVB fusion to the plasma membrane is a question we will address in future studies.…”

“…The ceramide-enriched vesicles also contain sphingosine-1-phosphate (S1P), a known infl ammatory mediator and activator of macrophages. We speculate that inhibition of by guest, on May 7, 2018 www.jlr.org Quantitative real-time PCR After assay medium was removed, cells were washed two times with PBS, collected in 1 ml Trizol, and the aqueous phase, after addition of 200 l chloroform and centrifugation, was applied to the G-eliminator column (RNeasy Plus; Qiagen) and RNA was extracted from the fl ow-through according to the manufacturer's instructions. Quantity and quality of RNA was measured spectrophotometrically using a NanoDrop ND1000 (Thermo Scientifi c), and RNA was reverse transcribed into cDNA by the iScript cDNA synthesis kit (Bio-Rad).…”

“…Subjects with secondary causes of steatohepatitis and other chronic liver diseases were excluded [drugs, prior gastric surgery for obesity, excessive alcohol consumption, viral hepatitis (B, C), cholestatic liver disease, hemochromatosis, Wilson disease, druginduced liver disease, and ␣ -1-antitrypsin defi ciency]. NAFLD activity score (NAS) was calculated as described, using the sum of steatosis, lobular infl ammation, and hepatocyte ballooning by guest, on May 7, 2018 www.jlr.org Downloaded from…”

Extracellular vesicles and ceramide: new mediators for macrophage chemotaxis?

“…Circulating EVs have gained much attention in recent years as potential biomarkers in NASH ( 10,11 ). Extensive characterization of serum EVs in animals with NASH led to the discovery of specifi c microRNA signatures with miR-122 and 192 ( 10 ) that was later confi rmed in studies with NASH patients ( 11,12 ).…”