Aim Localization, expression and relevance of TGF-β2 have been documented during the course of cholestatic liver diseases from biliary liver fibrosis to CCA. TGF-β2 effects on cholangiocytes and potential regulators of TGF-β2 were analyzed and evaluated to present so far unidentified targets for intervention in cholestatic liver diseases.

Upon different types of liver injury, there are distinct patterns of hepatic fibrosis developed, such as ECM septa (fibrotic walls) connecting pericentral (CV) areas due to toxic injury or septa connecting portal (PV) compartments due to cholestatic injury. Since liver fibrosis is a multi-cellular process and difficult to capture for biological in vivo and in vitro models, computational models may shed light on exploring the mechanisms behind particular pattern formation. In this work, we present a novel computational liver model that permits to assess the potential role of biomechanics in the formation of fibrotic walls. It for the first time studies the orchestration of cell types during fibrosis development and the interaction of cell populations with the ECM network mechanics in a liver lobule. Our model results are quantitatively confronted with experimental findings. The determination of the pattern-characterizing parameters in this study e.g. the density of hepatic stellate cells (HSC) and macrophages (MC) were obtained through image analysis of 2D and 3D images from mouse experiments. Together with a model of ECM networks, these non-parenchymal cells and their intercellular signaling were integrated as new elements into computational model of basic liver micro-architecture that included hepatocytes (the main parenchymal liver cell), sinusoids, CV and PV. The new model is applied to test possible mechanisms of how the fibrotic wall may form during liver fibrosis in space and time. Using this strategy we proposed a potential scenario distinguishing regeneration after acute toxic insult and repeated toxic exposure leading to formation of characteristic fibrotic walls:

Acute-on-chronic liver failure (ACLF) is a major complication in patients with chronic liver diseases. To better understand the pathophysiology and dynamics of ACLF, we developed a mouse model based on a two-hit hypothesis. The first hit is the deletion of Abcb4 in Balb/c mice, leading to liver fibrosis and ductular reaction similar to chronic hepatobiliary injury in patients. A sublethal dose (second hit) of CCl4 to 65 week-old Abcb4KO mice recapitulates an acute event in ACLF patients, similar to drug intoxication, binge drinking, or trauma. Livers and blood were collected at time points 0, 1, 2 and 4 d after the CCl4 injection. Histologically, massive hepatic necrosis is recorded at day 1 and 2 in mice with good prognosis after CCl4 treatment, whereas almost no necrosis is present in mice with poor prognosis at day 1 after CCl4 injection. A slight increase in Tunel positive cells (reflecting apoptosis) correlates with poor prognosis of mice. Better prognosis is additionally associated with less extrahepatic injury, i.e. kidney injury as indicated by a significant increase of serum creatinine concentrations. ACLF-assigned genes, i.e. IL6, Ccl5 and Crp are significantly induced in CCl4-exposed mice. In conclusion, a single administration of CCl4 to Abcb4KO mice recapitulates several features of ACLF patients, comprising a golden window of survival, advanced liver fibrosis, ductular reaction, massive hepatic necrosis and upregulation of ACLF-related genes. Therefore, the presented mouse model is promising to investigate ACLF pathomechanisms and possible therapeutic interventions.

NAFLD is characterized by lipid deposition in liver cells and is progressed to NASH and HCC. Hepatocyte (HC)-derived Glycoprotein non-metastatic melanoma B (Gpnmb) was recently reported as a regulator of fat metabolism in adipose tissue, however, its role in the pathogenesis of NASH-related HCC are not clear. We aim to functionally investigate Gpnmb during liver steatogenesis and HCC. A NASH-based HCC mouse model, STAM was selected. In this model, NASH and HCC stages were analyzed. We also investigated publically available patient cohorts i.e. GSE48452 (NASH) and GSE14520 (HCC) datasets. An in vitro steatosis model was induced by oleic acid (OA) in primary mouse hepatocytes (PMHC) and AML-12. GPNMB expression was manipulated by overexpression (OE) and knockdown (KD) approaches. Steatosis induction and lipid metabolic targets was assessed upon modulation of GPNMB by PCR. Cell growth and death of Huh7 cells was analysed by MTT and caspase-3 assay, PCR, WB and time-lapse imaging. Comparative analysis of patient cohorts and STAM mouse model identifies GPNMB as a consistently upregulated gene. IHC staining shows that GPNMB protein localizes in HC in healthy and NASH, and in HC-derived tumour cells in HCC tissues. In vitro, OA induces GPNMB in PMHC and AML-12 cells. Lipid accumulation increased upon KD of Gpnmb, and decreased by OE as measured by triglyceride (TG) level in OA-treated pMHC and AML-12 cells. We analyzed GPNMB dependent gene expression alterations of critical players in hepatic lipogenesis i.e. SREBP-1c, PPAR-α, PPARγ, Fasn and Scd1, as well as targets involved in FA oxidation as Cpt1 and Acox1. In line with TG accumulation, Gpnmb KD increases SREBP-1c, PPAR-α, PPARγ, Fasn and Scd1 mRNA expression in OA-treated PMHC and AML12. However, Cpt1 and Acox1 are also upregulated. In case of GPNMB OE, we obtained complementary results. Preliminary data using Huh7 cells indicate that GPNMB OE suppresses cell proliferation and induces apoptosis. Mechanistically, GPNMB OE facilitates cell death via inhibition of AKT phosphorylation. GPNMB is a consistently upregulated target in NASH and HCC. In contrast to previous reports, GPNMB is expressed in HC and HC-derived cancer cells, instead of macrophages. In fatty liver, GPNMB is upregulated to tone down lipogenesis, therefore, it seems that GPNMB has a protective role against liver fat toxicity. In liver cancer cells, GPNMB acts as a tumor suppressor by providing cytostatic effects.

Excessive amounts of active TGF-β drive activation of hepatic stellate cells (HSCs) and promote extracellular matrix production, which results in fibrosis and cirrhosis. The mechanism on how the liver controls active TGF-β supply has not fully been understood. We investigated the role of extracellular matrix protein 1 (ECM1), a new extracellular matrix component in the liver, in TGF-β activation and fibrogenesis of mice and humans.

Liver structure is highly complex consisting of two principal cellular compartments: parenchymal cells (PCs) and non-parenchymal cells (NPCs). PCs include hepatocytes (HCs) and bile duct epithelial cells, while NPCs comprise Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs). The separation of these liver cells in high yield, optimal purity and viability from healthy and diseased mouse liver is a prerequisite to investigate their role in normal and pathological conditions in subcellular and molecular detail. Traditional isolation methods usually purify mostly single cell types to obtain high yield for subsequent analyses. Recently, we identified CD271 as a promising cell surface marker for isolation of HSCs from mouse liver using magnetic activated cell sorting (MACS). Based on this finding, we combined CD271 with other well-known NPCs markers to simultaneously purify different liver cells from healthy and diseased livers upon magnetic beads labelling.

Primär sklerosierende Cholangitis (PSC) und Primäre biliäre Cholangitis (PBC) sind seltene nicht heilbare Lebererkrankungen, die mit einer erhöhten TGF-beta2 Expression einhergehen. Das murine Mdr2- knock out Modell wird genutzt, um mechanistische Grundlagen der cholestatischen Lebererkrankungen zu untersuchen.

Reactive oxygen species (ROS) initiate several liver diseases through DNA hydroxylation, lipid peroxidation, and protein adduct formation. In addition, ROS interacts with signaling pathways such as NOTCH1 and NRF2, which may have an impact on liver regeneration. In the current study, we report that the Notch ligand Jagged-1 (JAG1) is induced by ROS in hepatocytes and functionally investigate its role in liver repair processes.

Acute-on-chronic liver failure (ACLF) is a recently recognized fraction of liver patient's entity. ACLF is characterized by decompensated cirrhosis and multi-organ failure with poor outcome (mortality rate 30 – 40%). Some patients recover without transplantation, if minimal organ functions are maintained for one week what so called “golden window”. Several histological features were described to characterize ACLF entity e.g. submassive cellular necrosis and ductular reactions upon severe chronic liver disease. Understanding of underlying mechanisms that control the pathologic decision in ACLF patients, e.g. further organ deterioration or recovery is hampered by lacking a suitable animal model. Therefore, 75 weeks old Abcb4-/- mice were treated with a sublethal dose of carbon tetrachloride (CCl4; 3.2 g/kg) intraperitoneally. Subsequently, survival rate, liver necrosis, cirrhosis and regeneration were investigated in a time-resolved manner and compared with ACLF patients. Surprisingly, survival analysis revealed that some mice died during the first 24h after CCl4, however, the mice survived if able to pass this period. Histologically, massive hepatic necrosis was recorded at days 1 and 2 after the CCl4 hit that recovered at day 4. Proliferation index was measured by Ki-67+ nuclear staining illustrating massive positivity at days 2 and 4 in surviving mice. Hepatocyte-cholangiocyte cell plasticity was investigated by CK19 immunostaining indicating that numerous CK19+ cells occur immediately after the acute insult. Molecular phenotyping of mouse livers and comparative transcriptomics analyses are currently ongoing. In conclusion, administration of a high dose of CCl4 to genetically induced cirrhotic livers induces a phenotype that recapitulates some features of ACLF patients comprising a golden window, cirrhosis, ductular reaction, submassive hepatic necrosis and parenchymal cell plasticity.

Fibrosis is a consequence of repetitive liver injuries, e.g. upon viral infection, alcohol consumption, malnutrition or hepatotoxicants. Based on the etiological factor, liver fibrosis develops in different patterns and presents as septal in toxic injuries1, biliary in cholestatic diseases2, bridging upon hepatitis virus infections or pericellular in case of alcohol consumption. The mechanism behind the generation of the different patterns is still elusive. We aim to define (a) molecular driver(s) of fibrosis pattern formation. Mice were exposed to acute or repeated doses for 6 consecutive weeks of carbon tetrachloride (CCl4). Morphologically, patterns of fibrosis and metabolizing enzymes, namely CYP2E1, were analyzed in immunostaining datasets. We found that the pattern of CYP2E1+ hepatocyte recovery after acute insult is similar to the observed toxic-induced septal fibrosis. This similarity suggested that the spatial pattern of CYP2E1 might indicate the location where the fibrosis forms. To study this hypothesis, we developed a dynamic activator-inhibitor system, where the activator is a diffusible protein released from the central vein to promote the CYP2E1 signaling while the inhibitor is a diffusible protein released from the portal vein to inhibit the CYP2E1 signaling. Currently, this model can partially capture the observed patterns of CYP2E1 and extracellular matrix (ECM) upon chronic liver injuries suggesting that the prepattern of CYP2E1 may indeed be a key factor in determining the location of fibrotic streets despite likely not the only one. We are currently extending our model by further mechanisms. These include, but are not limited to i) crosstalk between activated hepatic stellate cells and liver sinusoidal endothelial cell differentiation; ii) The dialogue between endothelial cells lining the hepatic veins and hepatocyte metabolic zonation; iii) Presence of so far unknown diffusible inhibitor in the portal compartment, i.e. a bile duct driven factor. In the next step, we target the WNT/β-Catenin pathway (CYP2E1 regulator) by monoclonal antibodies against R-spondin1, 2 or 3 in fibrosed liver, and iv) the mechanical role of ECM deposited by HSCs, all finally integrated in a spatial-temporal model established to mimic regeneration after administration of a single dose of CCl43.

Transforming growth factor (TGF)-beta influences a plethora of cellular processes such as matrix remodeling, proliferation and differentiation, and is a key player in the progression of chronic liver diseases. TGF-beta activates hepatic stellate cells (HSC)-a profibrogenic liver cell type-and triggers senescence, apoptosis of hepatocytes. We aimed to analyze cell type-specific genomic alterations (1) upon stimulating HSCs and hepatocytes with TGF-beta in vitro and (2) upon inducing active TGF-beta1 levels in healthy and diseased mouse liver.

Backgrounde: Nitrates and nitrites are naturally found in fruits, vegetables and grains. In addition, they are used as food additives, preservatives and colour fixatives. Nitrite toxicity is induced by formation of methaemoglobin, and carcinogenic substances e.g. nitrozoamines. Materials and methods: In the current study we explored the impacts of chronic exposure of albino rats to potassium nitrate. Fourteen rats were randomly divided into 2 groups (n=7), control group and potassium nitrate treated group in dose of 20 mg/kg for 3 months. Subsequently, urine and blood samples were collected at 1, 2 and 3 months. At the end of the experiment (3 months), rats were sacrificed and thyroid glands were harvested. Results: Biochemical analysis exhibited significant decrease in the urinary iodine and blood-based thyroid hormones (T3 and T4), vitamin A compared with matched-time control group. However, calcium, nitric oxide and MDA showed significant increase in potassium nitrite treated rats. Marked thyroid injury was observed in the form of size and shape of the thyroid follicles, some follicles were enlarged and filled with colloid fluids, but other were completely empty upon sodium nitrite exposure. Moreover, in some cases hyperplasia with leukocytic infiltrations in the parafollicular cells replacing the atrophied follicles. Conclusion: Our results provide new evidences that toxic effect of potassium nitrite is possibly mediated by inhibition of iodine absorption,vitamin A level, thyroid destruction and depression of the antioxidant system.

The adult liver consists of various ploidy classes of hepatocytes, including mononuclear and binuclear cells. Nuclear division without subsequent cytokinesis is generating the different ploidy classes of hepatocytes postnatally. The exact fraction of each hepatocyte class in livers that undergo resection is still not exactly described. We developed 3D analysis protocols based on vibratome liver slices (100 µm thick) for such detailed examination. Here, liver slices are co-stained with different antibodies to visualize bile canalicular and sinusoidal networks, pericentral hepatocytes and cell nuclei1. TiQuant2 software was used to analyze and quantify nuclearity and ploidy classes in mouse livers after 2/3 partial hepatectomy (PHx) as compared to sham-operated controls in a time-resolved experiment at 1 d, 2 d, 3 d, 4 d and 7days after surgery. The results were compared with in vitro data from isolated and cultured mouse hepatocytes, as well as with such from conventional 2D analyses. We found that in adult healthy mouse liver more than 75% are binucleated hepatocytes. More than 85% of the hepatocytes are polyploid in healthy livers. Approximately 50% of binuclear hepatocytes are lost 1 d after 2/3 PHx. In regenerating livers, the number of nuclei per cell decreases, however, the DNA content is increased, indicating less binuclear and more cells. Quantification of hepatocytes in S-phase indicates no preferential DNA synthesis between mono- and binuclear cells, as revealed by BrdU incorporation. Similarly, Ki-67 immunostaining revealed no differences between mono- and binucleated hepatocytes. Time-lapse microscopy of cultured HGF stimulated mouse hepatocytes revealed that more than 95% of the binucleated hepatocytes preferentially divide into two mononuclear cells after DNA synthesis. Furthermore, the number of hepatocytes with nuclear polyploidy is increased in regenerating livers, independent of cell nuclearity. In conclusion, binuclear hepatocytes and ploidy variations represent a repository for an immediate regeneration response after liver resection.

Excessive amounts of active TGF-β drive activation of hepatic stellate cells (HSCs) and promote extracellular matrix production, which results in fibrosis and cirrhosis. The mechanism on how the liver controls active TGF-β supply has not fully been understood. We investigated the role of extracellular matrix protein 1 (ECM1), a new extracellular matrix component in the liver, in TGF-β activation and fibrogenesis of mice and humans.

Non-alcoholic fatty liver disease (NAFLD) is characterized by steatosis and in advanced stages, inflammation which is frequently accompanied by organ fibrosis (non-alcoholic steatohepatitis, NASH) and eventually hepatocellular carcinoma (HCC). Noteworthy, a considerable fraction of patients may suffer from progression of the disease towards hepatocellular carcinoma (HCC). Therefore, we aim to identify cellular, structural and molecular signatures associated with NAFLD/NASH progression to HCC.

Since the only therapy of end-stage liver disease still is transplantation, the need for an early treatment in the stage of fibrosis is becoming more important. The transforming growth factor (TGF)-β has been identified as a master regulator of liver fibrogenesis.