Publications

The liver is frequently exposed to acute and chronic insults that disrupt its function, leading to inflammation, fibrosis, and serious conditions like cirrhosis and hepatocellular carcinoma. Fibrosis, driven by the activation of hepatic stellate cells is a key feature of chronic liver damage. Traditional 2D liver models face limitations in maintaining liver functions and accurately replicating chronic conditions like fibrosis. In response to these needs, 3D models have emerged as more physiologically relevant platforms for studying liver disease and drug testing. However, most existing 3D models still lack chronic injury features or immune elements. In this study, we developed a 3D human-liver model to investigate both acute and chronic drug-induced liver injury. The model consists of encapsulated human hepatocytes (HepaRG) and hepatic stellate cells (LX-2) within a gelatin methacryloyl (GelMA) and carboxymethyl cellulose methacrylate matrix. Over a 30-day culture period, the 3D liver constructs maintained physiologically relevant functions, including albumin secretion and cytochrome P450 activity, which are lost rapidly in conventional 2D models. The model enabled reliable simulation of low-level, long-term, and high-dose, short-term damage by administering lipopolysaccharide and Paracetamol. Chronic liver injury was characterized by progressive fibrosis, elevated expression of COL1A1, and persistent hyperammonemia. In contrast, acute models showed significant but transient injury. Moreover, dexamethasone treatment successfully reversed fibrotic markers and restored hepatocyte functionality, indicating the model's predictive power for evaluating potential therapies. Incorporating human monocytes (THP-1) allowed investigation of the immune response and macrophage activation, which are critical contributors to liver disease pathogenesis. Overall, this 3D liver model offers a physiologically relevant and versatile platform for studying complex multi-cellular interactions under acute and chronic injury. It has broad implications for drug safety screening, disease modeling, and personalized therapy. The tri-culture design extends the model's capacity to elucidate immune-driven hepatic pathology.

JTD Keywords: 3d liver model, Acute liver injury, Chronic liver injury, Culture, Drug-induced hepatotoxicity, Failure, Hepatic fibrosis, Hepatic stellate cells, Injury, Liver disease modeling

Molecular and cellular research modalities for the study of liver pathologies have been tremendously improved over the recent decades. Advanced technologies offer novel opportunities to establish cell isolation techniques with excellent purity, paving the path for 2D and 3D microscopy and high-throughput assays (e.g., bulk or single-cell RNA sequencing). The use of stem cell and organoid research will help to decipher the pathophysiology of liver diseases and the interaction between various parenchymal and non-parenchymal liver cells. Furthermore, sophisticated animal models of liver disease allow for the in vivo assessment of fibrogenesis, portal hypertension and hepatocellular carcinoma (HCC) and for the preclinical testing of therapeutic strategies. The purpose of this review is to portray in detail novel in vitro and in vivo methods for the study of liver cell biology that had been presented at the workshop of the 8th meeting of the European Club for Liver Cell Biology (ECLCB-8) in October of 2018 in Bonn, Germany.

JTD Keywords: Fibrogenesis, Hepatic stellate cells, Hepatocellular cancer, In vitro models, Steatosis

Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl4 cirrhotic rats 3 h, 6 h and 24 h after i.v. administration of Y27pPBHSA (0.5/1 mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Therefore, targeting the ROCK inhibitor Y27 to PDGFRβ decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis.

JTD Keywords: Hepatic stellate cells, Hepatorenal syndrome

Fibrosis development was initially conceived as an incessant progressive condition. Nowadays, it has become evident that fibrotic tissue undergoes a continuous two-way process: fibrogenesis and fibrinolysis, characterizing the remodeling of extracellular matrix (ECM). However, in established fibrosis, this two-way process is tipped towards fibrogenesis and this leads to a self-perpetuating accumulation of ECM, a distinct metabolic unit, together with other cells and processes promoting fibrosis deposition. Several mechanisms promote fibrosis regression, such as degradation of ECM, infiltration of restorative macrophages, prevention of the epithelial-mesenchymal transition of hepatocytes, restoration of the liver sinusoidal endothelial cells’ differentiation phenotype, and reversion to quiescence, apoptosis and senescence of hepatic stellate cells (HSC). Hence, fibrosis is the result of an unbalanced two-way process of matrix remodeling. At the late stage of the disease, antifibrotic interventions could become necessary to reverse self-perpetuating fibrogenesis and accelerate regression of fibrosis even if cause and cofactors of hepatic injury have been eliminated. This review outlines some of the important mechanisms leading towards regression of liver fibrosis.

JTD Keywords: Hepatic stellate cells, Extracellular matrix, remodeling, Rho-associated kinases, Janus kinases