Authors J. LAMBRECHT (1), P. POORTMANS (1), H. REYNAERT (2), I. MANNAERTS (1), L. VAN GRUNSVEN (1) / [1] Vrije Universiteit Brussel, Jette, Belgium, Basic Biomedical Sciences, Liver Cell Biology Lab, [2] UZ Brussel, Jette, Belgium, Department of Gastro-enterology and Hepatology
Introduction Liver fibrosis is the pathological condition of the liver, resulting from sustained scar formation in response to chronic liver injury, such as chronic infection with hepatitis B (HBV) and hepatitis C (HCV) virus. The main effector cell in this pathology is the hepatic stellate cell (HSC) which will undergo a myofibroblastic transdifferentiation process towards an activated state, in which it will excessively produce and deposit extracellular matrix (ECM). Till date, the diagnosis of liver fibrosis occurs by several non-invasive techniques, which are however insensitive and unable to detect early disease stages, and liver biopsy, known to be invasive and associated with some minor and major complications. Novel non-invasive diagnostic scoring systems are being developed, but none of these have yet proven their sensitivity for the detection of early-stage liver fibrosis, nor their potential to discriminate between the various fibrotic stages.
Aim
Our aim was to analyze the potential of circulating miRNAs, with emphasis on extracellular vesicle (ECV)-associated miRNAs as novel biomarkers for early-stage liver fibrosis.
Methods This study included patients with liver fibrosis by chronic HBV (n=19) or HCV (n=20) infection, which were identified as early-stage fibrotic (≤ F2) by transient elastography (Fibroscan). Relative expression levels of miRNAs in circulating ECVs and total plasma were analyzed by use of qRT-PCR. MiRNAs were selected based on their significant dysregulation during HSC activation (miRNA-192, -200b, -150), or their known presence in the circulation (miRNA-21, 92a, -122). To analyze the potential of these circulating ECVs to represent the presence or absence of activated HSCs in the liver, primary murine HSCs were cultured in vitro to induce spontaneous activation. ECVs were extracted from the culture medium of quiescent- (day 0 till day 2) and activated HSCs (day 8 till day 10).
Results Analysis of miRNA levels in total plasma confirmed the up-regulation of miRNA-122 during early-stage fibrosis. With the exception of up-regulated levels of miRNA-192 in early-stage HBV-induced fibrosis, no other miRNAs showed significant changes in early-stage fibrosis by HBV or HCV infection. In contrast, miRNA-analysis of circulating ECVs identified significant changing levels of miRNA-150, -192, -200b and -92a during early-stage liver fibrosis by HBV and HCV infection. Especially the down-regulated levels of ECV-associated miRNA-192 (HBV AUC: 0.9802; HCV AUC: 0.9762) and miRNA-200b (HBV AUC: 0.9699; HCV AUC: 0.9841) seem to have an inherent diagnostic potential for early-stage fibrosis. Comparison of the miRNA levels from circulating ECVs with the ECVs extracted from in vitro activating HSCs showed a similar trend in the down-regulation of miRNA-192, suggesting that ECV-associated miRNA-192 levels might represent the activation status of HSCs in the liver.
Conclusions Circulating ECV-associated miRNAs could be used as novel tools for the diagnosis of early-stage liver fibrosis, through their potential to identify the absence or presence of activated HSCs in the liver.
