Introduction: TGF-β is the major cytokine regulating hepatic stellate cell (HSC) function in the course of liver fibrogenesis [1]. In a previous study we demonstrated that the reducing substance N-Acetylcysteine (NAC) is able to block TGF-β signaling in HSC by different means than modification of the cellular redox state. As one target we could identify the type III receptor, endoglin, which is expressed in HSC and myofibroblasts (MFB) [2]. Due to its functional structure, a disulfide linked homodimer, the receptor is prone to be affected by reducing substances, and is therefore monomerized in our experimental setting. Because of the restricted expression pattern of endoglin the NAC effect is focused on a subpopulation of liver cells excluding the most prominent liver cell type, the hepatocytes, which do not express endoglin.

Introduction: Several epidemiological studies suggest that coffee drinking is an environmental risk modulator of hepatic fibrogenesis.

Transforming growth factor beta (TGF-beta) signaling is mediated by the cell surface TGF-beta type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, TbetaRII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M(r) 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-beta receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-beta receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-beta. In conclusion, we hypothesize that TGF-beta signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.

Background/Aim: Hepatic stellate cells (HSC) are prominent cellular effectors of liver fibrosis, which become activated during liver injury and transdifferentiate from quiescent, fat storing cells into a proliferative myofibroblast-like cell type (MFB) 1,2. This process is accompanied by modulations of gene expression. We examined the expression of the LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) during transdifferentiation, a protein which in smooth muscle cells is associated with the cell skeleton as well as located in the nucleus 3,4. Results: An increase of CRP2 was detectable by Western Blot in lysates from liver samples of bile duct ligated rats, an animal model of liver fibrosis. Analysis of different liver cell subpopulations revealed that activated HSC are the source of this upregulation. Cell culture experiments with primary rat HSC, which become activated by contact with uncoated plastic dishes, as well as CFSC, a rat cell line derived from MFB, and A10 cells, a rat smooth muscle cell line, demonstrated that CSRP2 (gene name of CRP2) is upregulated by TGFβ1. To localize promoter regions responsible for induction of gene expression, we generated luciferase constructs with different fragments of the rat CSRP2 promoter. Transfection experiments in CFSC and A10 cells showed that potential binding sites for Smad factors within the proximal 1100 nucleotides of the promoter are not relevant for TGFβ1 induction. Promoter region –1100 to –1600 basepairs was shown to be responsible for serum induction of reporter genes in A10 cells. Future experiments will be performed to identify promoter regions, which are necessary for response to TGFβ signals. Conclusion: Our data demonstrate that CRP2 is increased in activated HSC and that TGFβ1 is involved in upregulation of CSRP2 gene expression. These findings are similar to other known smooth muscle marker like α-smooth muscle actin or SM22α, which are upregulated in activated HSC and associated with the cell skeleton. In contrast, CRP2 is also found in the nucleus, thereby demonstrating an additional function in transition of HSC to MFB in liver fibrosis. Knowledge of CSRP2 regulation and function may help to extend our understanding of molecular mechanisms involved in hepatic fibrogenesis.

Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary -carotene, but the ability of HSCs to metabolize -carotene in situ has not been explored. To gain understanding of this, we investigated whether -carotene-15,15-monooxygensase (Bcmo1) and -carotene-9, 10-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary -carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12-carotenal levels of Bcmo1-deficient mice.

Einleitung: Der Mechanismus der durch Thioacetamid (TAA) induzierten Leberfibrose und insbesondere die Rolle der hepatischen Sternzellen (HSC) hierbei sind bislang nicht ausreichend verstanden. Das Ziel unserer Studie war eine Charakterisierung des TAA Fibrosemodells und die Untersuchung der HSC-Populationen während der Fibroseentwicklung durch TAA im Vergleich zur gut charakterisierten Fibrogenese durch CCl4 Injektion.

Einleitung: Gentherapeutische Ansätze zur Behandlung der Leberfibrose werden zunehmend in tierexperimentellen Untersuchungen eingesetzt. Häufig werden Adenoviren genutzt, um Wirkproteine effizient in Leberzellen zu exprimieren. Ein weit verbreitetes Tiermodell zur experimentellen Induktion einer Leberfibrose ist die Gallengangsligatur in Ratten. Bisher ist aber noch wenig über das Expressionsverhalten der adenoviral eingebrachten Transgene im geschädigten Lebergewebe bekannt. Material und Methoden: Ratten wurden mit einem adenoviralen Vektor infiziert, der das Reportergen GFP unter transkriptioneller Kontrolle eines CMV-Promotors trägt. 24 Stunden nach Infektion wurden die Tiere einer Gallengangsligatur oder Scheinoperation unterzogen und nach 6 Tagen getötet. Weiterhin wurde alternativ eine Leberfibrose mittels 6-wöchiger DMN-Gabe induziert. Die Menge an GFP wurde in Leberlysaten durch Western Blot ermittelt. Zusätzlich erfolgte ein semiquantitativer Nachweis viraler DNA in den Lebern mittels PCR. Ergebnisse: Auf Proteinebene liegt in Tieren mit experimenteller Leberschädigung eine deutliche Reduktion des GFP-Gehalts vor. Die verminderte GFP-Bildung wurde nicht durch den Eingriff verursacht, da der Effekt bei scheinoperierten Tieren nicht zu beobachten war. In semiquantitativen PCR-Analysen mit isolierter DNA aus Leberzellen konnte gezeigt werden, dass die Menge viraler Partikel in Zellen von Tieren mit Leberschaden vermindert ist. Diskussion: Die experimentelle Induktion einer Leberfibrose im Rattenmodell führt zu einer verminderten adenoviral vermittelten Transgenexpression in der Leber. Die Ursache hierfür liegt anscheinend nicht auf transkriptioneller oder translationeller Ebene. Da die Infektion 24 Stunden vor der Gallengangsligatur erfolgte, kann eine verminderte Aufnahmerate der Adenoviren durch die Leberzellen ausgeschlossen werden. Möglicherweise führt die Leberschädigung an sich durch Induktion apoptotischer Prozesse zu einer Abnahme infizierter Zellen. Aufgrund der gemachten Untersuchungen kann geschlossen werden, dass die Wirksamkeit von Adenoviren als Vektoren trotz ihrer Hepatotropie zumindest in den vorgestellten experimentellen Methoden begrenzt ist.

Background/Aims Isolated rat hepatic stellate cells (HSC) are taken as a valuable in vitro model to study hepatic fibrogenesis, biotransformation of pharmaceutics, gene expression, transcription factors controlling HSC behaviour, and for the establishment of long-term cultures. Consequently, methods for the isolation and maintenance of HSC cultures are well documented. However, there is ongoing controversial discussion directed on the existence and cellular origin of different HSC subpopulations. Thus, there is a continuing need for developing methods allowing the exchange of HSC isolates between different laboratories. A practical solution to this problem is cryopreservation and banking of HSC. Methods We here describe for the first time the successful establishment of a methodology for long-term cryopreservation and recovery of primary, non-activated HSC from rats. We have optimised critical factors for HSC-banking including prefreeze processing, freezing rate, freezing medium, final cooling temperature, and thawing conditions. We found that DMSO gave far superior attachment and viability on thawing than other cryoprotectants. The viability and cellular characteristics of thawed cells was comparatively analysed by light- and electron microscopic analysis, proliferation assay, Oil Red O-staining, apoptosis testing, and evaluation of marker proteins for fibrogenic activities. Results In summary, our data reveal no significant differences in the biochemical and cellular properties between cryopreserved/thawed and freshly isolated HSC. Conclusions According to these results, we suggest that cryoprotected HSC retain functional integrity thereby allowing banking and comfortable exchange of these cells between different laboratories. Isolated rat hepatic stellate cells (HSC) are taken as a valuable in vitro model to study hepatic fibrogenesis, biotransformation of pharmaceutics, gene expression, transcription factors controlling HSC behaviour, and for the establishment of long-term cultures. Consequently, methods for the isolation and maintenance of HSC cultures are well documented. However, there is ongoing controversial discussion directed on the existence and cellular origin of different HSC subpopulations. Thus, there is a continuing need for developing methods allowing the exchange of HSC isolates between different laboratories. A practical solution to this problem is cryopreservation and banking of HSC. We here describe for the first time the successful establishment of a methodology for long-term cryopreservation and recovery of primary, non-activated HSC from rats. We have optimised critical factors for HSC-banking including prefreeze processing, freezing rate, freezing medium, final cooling temperature, and thawing conditions. We found that DMSO gave far superior attachment and viability on thawing than other cryoprotectants. The viability and cellular characteristics of thawed cells was comparatively analysed by light- and electron microscopic analysis, proliferation assay, Oil Red O-staining, apoptosis testing, and evaluation of marker proteins for fibrogenic activities. In summary, our data reveal no significant differences in the biochemical and cellular properties between cryopreserved/thawed and freshly isolated HSC. According to these results, we suggest that cryoprotected HSC retain functional integrity thereby allowing banking and comfortable exchange of these cells between different laboratories.

Hepatic stellate cells (HSC) reside in the space of Disse and constitute 1.4% of the total liver volume and 5% of liver cell numbers [1]. Methods for isolating and maintaining HSC in culture are well documented and consistently high-quality cells have been successfully isolated from livers of many species [2]. When quiescent, HSC possess multiple intracytoplasmatic lipid droplets containing vitamin A and upon culturing on uncoated plastic these cells transdifferentiate into a myofibroblastic phenotype (MFB) representing a valuable in vitro model to investigate the processes involved in liver fibrogenesis [3]. Furthermore, it was recently demonstrated that one-third of the HSC in the liver can be replaced by new HSC derived from bone marrow and there is still an ongoing discussion directed on the existence of HSC subpopulations in different liver cell preparations [4, 5]. Thus, to respond to these scientific interrogations, there is a continuing need for the development of methods allowing interchange of HSC isolates between various experts addressing specialized issues of these cells. A pratical solution to this problem is cryopreservation and banking of HSC. We here describe for the first time the successful establishment of a methodology for long-term cryopreservation and recovery of freshly isolated primary rat HSC. We have optimized major factors for a successful protocol including prefreeze processing, freezing rate, type of cryoprotectant/freezing medium, final cooling temperature, and thawing conditions. We found that DMSO gave far superior attachment and viability on thawing than other cryoprotectants. The viability of thawed cells was comparatively analysed by light- and electronmicroscopic analysis, proliferation assay, and oil red O staining. Furthermore, apoptose testing and evaluation of fibrogenic activity reveal no significant difference between the biochemical and cellular properties of cryopreserved/thawed and freshly isolated HSC. Therefore, we suggest that cryoprotected HSC retain functional integrity thereby allowing the exchange of these cells between different laboratories addressing critical issues of HSC origin, subtypes and cellular activation.

Introduction: Adiponectin is a member of adipose tissue-secreted hormones, so-called adipokines, and has wide implications in glucose and lipid metabolism. It is also found in liver tissue and in NAFLD/NASH it provides protective features on liver tissue like antiinflammatory and antifibrotic effects. There are two specific receptors for adiponectin: AdipoR1 with abundant expression and AdipoR2 with expression especially on hepatocytes. Yet, little is known about the role of adiponectin in chronic HCV-infection. Recently, genetic studies of single nucleotide polymorphisms (SNP) of the adiponectin gene, e.g. +276G>T, have shown that genetic polymorphisms are implicated in altered protein function.

Fibrosis is a frequent, life-threatening complication of most chronic liver diseases. Despite major achievements in the understanding of its pathogenesis, the translation of this knowledge into clinical practice is still limited. In particular, non-invasive and reliable (serum-) biomarkers indicating the activity of fibrogenesis are scarce. Class I biomarkers are defined as serum components having a direct relation to the mechanism of fibrogenesis, either as secreted matrix-related components of activated hepatic stellate cells and fibroblasts or as mediators of extracellular matrix (ECM) synthesis or turnover. They reflect primarily the activity of the fibrogenic process. Many of them, however, proved to be disappointing with regard to sensitivity and specificity. Up to now hyaluronan turned out to be the relative best type I serum marker. Class II biomarkers comprise in general rather simple standard laboratory tests, which are grouped into panels. They fulfil most criteria for detection and staging of fibrosis and to a lesser extent grading of fibrogenic activity. More than 20 scores are currently available, among which Fibrotest is the most popular one. However, the diagnostic use of many of these scores is still limited and standardization of the assays is only partially realized. Combining of panel markers in sequential algorithms might increase their diagnostic validity. The translation of genetic pre-disposition biomarkers into clinical practice has not yet started, but some polymorphisms indicate a link to progression and outcome of fibrogenesis. Parallel to serum markers non-invasive physical techniques, for example, transient elastography, are developed, which can be combined with serum tests and profiling of serum proteins and glycans.

Introduction: TGF-β is regarded as the profibrogenic master cytokine. We aimed at analyzing the regulatory link between TGF-b dependent CTGF-induction and intracellular demasking of latent TGF-b in hepatocytes (PC) of healthy and injured livers.