D Scholten

Experimental liver fibrosis research: update on animal models, legal issues and translational aspects.

Liver fibrosis is defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requires in vitro and in vivo experimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

Chemokine Cxcl9 attenuates liver fibrosis‐associated angiogenesis in mice

Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl4)‐induced liver fibrosis in Cxcr3−/− and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet‐inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3−/− mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF‐stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal‐regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter‐regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. Conclusion: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis. (HEPATOLOGY 2012)

The carbon tetrachloride model in mice

Recently, the need for more standardized operation procedures in experimental liver fibrosis research was suggested due to dramatic changes in European animal welfare rules. Here, we present a short series of standard operation procedures (SOPs) summarizing the most relevant and widely accepted experimental models for the induction of liver injury leading to liver fibrosis. The described procedures are based on the long-term experience of the Collaborative Research Centre ‘Organ Fibrosis: From Mechanisms of Injury to Modulation of Disease’ (http://www.sfbtrr57.rwth-aachen.de/), which is supported by the German Research Foundation (SFB/TRR57). These SOPs will help to improve standardization of fibrosis models and to increase the comparability of data between different laboratories with the aim of reducing animal experimentation according to the principle that was proposed in 1959 by Russell and Burch as an ethical framework for conducting scientific experiments with animals, namely the replacement, refinement and reduction (3R) principle. In the first section we focus on the carbon tetrachloride (CCl4) model in mice, which is the toxic model of liver fibrosis induction most commonly used worldwide.

EMT in Liver Fibrosis

Chronic liver disease is a common and epidemiologically important condition, leading to liver fibrosis and cirrhosis with many consecutive problems. In response to liver injury, activated myofibroblasts are responsible for excessive accumulation of extracellular matrix. However, the origin of myofibroblastic cells remains unclear. Myofibroblasts originate from hepatic stellate cells, portal myofibroblasts, interseptal (myo)fibroblasts, and bone marrow-derived fibrocytes. However, in other organs, numerous studies showed that myofibroblasts are derived by endothelial-to-mesenchymal transition (EMT) in response to chronic injury. In this process, mature epithelial cells lose their epithelial phenotype and gene expression characteristics and change into mesenchmyal cells. This process is well described in embryonal development, cancer progression, and metastasis; however, its existence and importance in liver fibrosis has been a topic of ongoing debates. This review summarizes the current knowledge on EMT in liver fibrosis and tries to discuss caveats and controversial results of recent studies.

Heterozygous carriage of the alpha1-antitrypsin Pi*Z variant increases the risk to develop liver cirrhosis

Objective Homozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants (‘Pi*Z’ and ‘Pi*S’), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse. Design We analysed multicentric case–control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed. Results The Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (p<0.0001). Accordingly, the Pi*Z variant increased the risk of NAFLD subjects to develop cirrhosis (adjusted OR=7.3 (95% CI 2.2 to 24.8)). Likewise, the Pi*Z variant presented in 6.2% of alcohol misusers with cirrhosis but only in 2.2% of alcohol misusers without significant liver injury (p<0.0001). Correspondingly, alcohol misusers carrying the Pi*Z variant were prone to develop cirrhosis (adjusted OR=5.8 (95% CI 2.9 to 11.7)). In contrast, the Pi*S variant was not associated with NAFLD-related cirrhosis and only borderline with alcohol-related cirrhosis (adjusted OR=1.47 (95% CI 0.99 to 2.19)). Conclusion The Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%–4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

Endoglin in human liver disease and murine models of liver fibrosis—A protective factor against liver fibrosis

Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor‐β (TGF‐β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF‐β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC‐specific endoglin deletion.

136 ENDOGLIN DEFICIENCY IN HEPATIC STELLATE CELLS HAS A DIFFERENTIAL EFFECT ON LIVER FIBROSIS AND TGF-b SIGNALLING IN TWO EXPERIMENTAL MODELS OF MURINE LIVER FIBROSIS

Background: Hepatic stellate cells (HSCs) are the major source for extracellular matrix (ECM) production in liver fibrosis. Endoglin (ENG) is a type III auxiliary receptor for TGF-b that is expressed on proliferating endothelial cells and HSCs. TGF-b is the most profibrotic cytokine expressed in response to liver injury. This study analyzes the role of ENG and TGF-b signalling in two models of experimental liver fibrosis by cell line specific Endoglin deletion in HSCs. Methods: Using the Cre-LoxP genetic recombination system we created HSC specific ENG mice by crossing GFAP to ENG mice. Mice were subjected to liver injury by CCl4 treatment (8 weeks) or bile duct ligation (BDL) (21 days). Liver fibrosis was analyzed by hydroxyproline measurement and Sirius red staining for collagen fibers. For in vitro analysis HSCs from ENG mice were isolated and treated with adenoviral CMV-RSV expressing vector. TGF-b signalling was analyzed by western blots and qPCR. Results: In response to CCl4 treatment livers of GFAP Eng mice (n =8) showed 18.2% less hydroxyproline content compared to GFAPEng /f litter mates reflecting less severe fibrosis. Sirius red stainings underlined these findings. Interestingly BDL inflicted injury showed contrasting results. Compared to GFAPEng /f mice GFAPEng litter mates (n = 8) showed 193.3% (p =0.02) more ECM deposition. Western blot analysis of TGF-b signalling in vitro using adenoviral Cre treated ENG primary HSCs demonstrated a differential effect of ENG on TGF-b signalling. In the absence of ENG activation of Smad1/5/8 was downregulated. Moreover, the phosphorylation of ERK1/2 was decreased and the expression of a-smooth muscle actin and connective tissue growth factor was downregulated. Conclusion: Endoglin deficiency in hepatic stellate cells has a differential effect on liver fibrosis depending on the cause of injury. Hepatocyte necrosis due to CCl4 treatment leads to strong TGF-b expression Smad signal transduction. Endoglin deficiency leads to amelioration of liver fibrosis, underlined by in vitro results. In contrast cholestatic liver injury is aggravated by endoglin deficiency in HSCs, reflecting a complex interplay of cholestasis, inflammation and toxic injury. Endoglin obviously modulates TGF-b signalling in this periportal-centered injury differentially, leading to significantly more fibrotic changes. 137 CXCL1 AND CXCL10 SECRETION BY FIBROCYSTIN-DEFECTIVE CHOLANGIOCYTES RECRUITS PERIBILIARY FIBROCYTES IN CONGENITAL HEPATIC FIBROSIS L. Fabris, L. Locatelli, D. Vigano, M. De Matteis, R. Fiorotto, M. Cadamuro, C. Spirli, M. Strazzabosco. Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA E-mail: luca.fabris@unipd.it