Circulating Extracellular Vesicles Contain Liver-Derived RNA Species as Indicators of Severe Cholestasis-Induced Early Liver Fibrosis in Mice

Abstract

Aims: Biliary diseases represent around 10% of all chronic liver diseases and affect both adults and children. Currently available biochemical tests detect cholestasis but not early liver fibrosis. Circulating extracellular vesicles (EVs) provide a noninvasive, real-time molecular snapshot of the injured organ. We thus aimed at searching for a panel of EV-based biomarkers for cholestasis-induced early liver fibrosis using mouse models. Results: Progressive and detectable histological evidence of collagen deposition and liver fibrosis was observed from day 8 after bile duct ligation (BDL) in mice. Whole transcriptome and small RNA sequencing analyses of circulating EVs revealed differentially enriched RNA species after BDL versus sham controls. Unsupervised hierarchical clustering identified a signature that allowed for discrimination between BDL and controls. In particular, 151 microRNAs (miRNAs) enriched in BDL-derived EVs were identified, of which 66 were conserved in humans. The liver was an important source of circulating EVs in BDL animals as evidenced by the enrichment of several hepatic mRNAs, such as Albumin and Haptoglobin . Interestingly, among experimentally validated miRNAs, miR192-5p, miR194-5p, miR22-3p, and miR29a-3p showed similar enrichment patterns also in EVs derived from 3,5-diethoxycarboncyl-1,4-dihydrocollidine-treated (drug-induced severe cholestasis) but not in mice with mild phenotype or non-cholestatic liver fibrosis. Innovation: A panel of mRNAs and miRNAs contained in circulating EVs, when combined, indicates hepatic damage and fibrosis in mice and represents promising biomarkers for human severe cholestasis-induced liver fibrosis. Conclusion: Analysis of EV-based miRNAs, in combination with hepatic injury RNA markers, can detect early cholestatic liver injury and fibrosis in mice. Antioxid. Redox Signal. 36, 480–504.

Electrical Impedance-Based Characterization of Hepatic Tissue with Early-Stage Fibrosis

“…Capacitance showed the same behavior and very similar values in BDL and sham-operated samples for all time points. On the other hand, resistance decreased with time in sham-operated tissue in an almost linear way while BDL displayed an increasing trend ( Figure 4 ) in accordance with the fact that ECM accumulates with time in the fibrotic liver, as shown by both molecular and histological analyses, as we recently demonstrated [ 13 ]. With the hypothesis that capacitance reflects cellular content while resistance corresponds to the overall contribution of the ECM, we assessed whether the imbalance in the hepatic tissue architecture was caused by decreased cellularity.…”

“…BDL and sham surgical procedures were performed as previously described in [ 13 ]. Briefly, mice were subjected to double ligation of the common bile duct without dissection of the duct between the ligatures.…”

“…The well-characterized obstructive cholestasis-induced liver fibrosis model (Bile Duct Ligation, BDL) was used for this purpose. BDL induces a series of events, including acute hepatobiliary injury; proliferative response of parenchymal and non-parenchymal liver cells; and up-regulation of pro-inflammatory and pro-fibrotic cytokines and metabolic enzymes, which lead to increased deposition of ECM and scar tissue formation [ 12 , 13 , 14 ]. Liver fibrosis is histologically detected starting from 7 days after surgery [ 13 , 15 ].…”

FLVCR1a Controls Cellular Cholesterol Levels through the Regulation of Heme Biosynthesis and Tricarboxylic Acid Cycle Flux in Endothelial Cells

“…This finding was corroborated by qRT-PCR analysis conducted on ECs isolated from the liver and lung of wildtype mice (Figure 4B). Remarkably, analysis of publicly available RNA-seq data [27] on LSECs derived from normal rats and three distinct rat models of liver damage (i.e., CCl4, carbon tetrachloride model; TAA, thioacetamide model; BDL, bile duct ligation model [34,35]) revealed a significant downregulation and upregulation, respectively, of the expression of genes related to heme synthesis and cholesterol synthesis following injury ± SEM (n = 3, three independent experiments). One-way ANOVA test was performed.…”

Extracellular Vesicles as Delivery Vehicles for Non-Coding RNAs: Potential Biomarkers for Chronic Liver Diseases

“…Importantly, the underlying liver disease had a profound impact on the dynamics of EV release and uptake by the liver. In this regard, we have observed a significant increase in the number of circulating EVs in mice undergoing bile duct ligation, with respect to controls [ 53 ]. This was also observed in other liver pathologies in humans or in rodents, thus suggesting that apart from EV content, the number of circulating EVs can also be indicative of damage [ 72 , 73 ].…”