Abstract
Background & AimsCurrently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).MethodsHuman cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.ResultsWe could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.ConclusionHere, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.
Highlights
Liver fibrosis and cirrhosis are canonical endpoint of many chronic liver diseases, including virus infections (HBV, Hepatitis C virus (HCV)), non-alcoholic steatohepatitis or damage due to alcohol consumption [1]
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking
For the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of hepatic stellate cells (HSC) leading to deposition of extracellular matrix (ECM)
Summary
Liver fibrosis and cirrhosis are canonical endpoint of many chronic liver diseases, including virus infections (HBV, HCV), non-alcoholic steatohepatitis or damage due to alcohol consumption [1]. The pathophysiology of fibrosis requires chronic liver damage (including chronic alcohol consumption, chemically-induced hepatocyte damage, and viral infections) and involves the interplay of several hepatic cell types; it requires hepatocyte injury and cell death, activation of Kupffer cells (KC), activation of hepatic stellate cells (HSC), and chronic inflammation [4,5]. Hepatic stellate cells, activated by fibrogenic cytokines (e.g. TGF-β1 and TNF-α), have been identified as the major collagen-producing cells in the injured liver. Upon hepatocyte injury, activated KC produce large amounts of reactive oxygen species (ROS) and release cytokines such as TNF-α, TGF-β1, PDGF and IL1β, leading to stellate cell activation and increased deposition of fibrillar components of the ECM [4,5,6]. In turn, produce more TGF-β1 and potentiate and perpetuate their activation in an autocrine loop [7]
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