BACKGROUND/AIMS: Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)-beta downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types. METHODS: In this study, activin A-dependent CTGF expression in hepatocytes (PC) primed by exogenous activin A and in PC maintained under complete activin-free culture conditions was analysed by Western blots, metabolic labelling, gene silencing, reverse transcriptase-polymerase chain reaction (RT-PCR) and CTGF reporter gene assays. This study was supplemented by immunocytochemical staining of activin A and CTGF in PC of injured liver. RESULTS: Using alkaline phosphatase alpha-alkaline phosphatase staining, it is demonstrated that activin A becomes increasingly detectable during the course of CCl(4)-liver damage. Addition of activin A to cultured PC induced CTGF protein expression via phosphorylation of Smad2 and Smad3. This induction can be inhibited by the antagonist follistatin and alpha-activin A antibody respectively. When PC were cultured under serum(i.e. activin A)-free culture conditions, a time-dependent increase of activin expression during the course of the culture was proven by RT-PCR. Silencing of inhibin beta(A) gene expression under serum-free conditions by small interfering RNAs greatly suppressed CTGF synthesis and the phosphorylations of Smad2 and Smad3. However, both the extracellularly acting follistatin and the alpha-activin A antibody could not inhibit spontaneous CTGF expression, which, however, was achieved by the cell-permeable TGF-beta Alk4/Alk5 receptor-kinase-inhibitor SB431542. CONCLUSIONS: In conclusion, the results point to activin A as an inducer of CTGF synthesis in PC. Intracellular activin A contributes to spontaneous CTGF expression in PC independent of exogenous activin A, which is proposed to occur via Alk4/Alk5-receptors. The findings might be important for many actions of activin A on the liver.

Host cell reactions are a crucial determinant for tumorous invasion. We analysed on a genome wide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. With this type of model the reaction of host cells on tumorous invasion can be studied without the common interference of contaminating tumor cells. Using the Gene Ontology classification we were able to determine patterns of up- and down-regulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation e.g. of the GO-terms “extracellular matrix“, “cell communication“, “response to biotic stimulus“, “structural molecule activity“ and “cell growth“, indicating a very pronounced host cell response to tumorous invasion. On the single gene level hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the non invaded liver tissue. In summary our data demonstrate the feasibility of an interspecies differential gene expression approach on a genome wide scale.

Introduction: TGF-β is the major cytokine regulating hepatic stellate cell (HSC) function in the course of liver fibrogenesis. In a previous study we could show that the reducing substance N-Acetylcysteine (NAC) is able to block TGF-β signalling in HSC by different means than modification of the cellular redox state. Analyzing the TGF-β receptor proteins ALK5 (type I receptor), RII (type II receptor) and betaglycan (type III receptor) with receptor specific antibodies revealed no differences between treated and untreated cells. In contrast, using an antibody (sc–6199), which recognizes both type III receptors, betaglycan and endoglin, we detected a redox sensitive protein, most likely endoglin. Subsequently, we cloned the rat endoglin cDNA from HSC and MFB.

Hintergrund: Das 600 kD Membranprotein Megalin wird an der apikalen Membran von Epithelien exprimiert und vermittelt die Endozytose von Vitamin A, Apolipoproteinen und multiplen weiteren Liganden (1). Wir konnten kürzlich erstmals nachweisen, dass hepatische Sternzellen (HSC)–im Gegensatz zu Hepatozyten und Sinusendothelien–Megalin exprimieren (2). Ziel dieser Studie war es, die Megalinexpression während der Leberfibrogenese in vivo und in vitro näher zu charakterisieren.

Introduction: An initial key event in liver fibrogenesis is the activation of quiescent hepatic stellate cells (HSC) and their transdifferentiation into a proliferative, myofibroblast like cell type (MFB). During this process several genes are differentially expressed and their regulatory units may be useful tools for antifibrotic strategies, which target HSC specifically. The LIM-domain protein cyteine- and glycine-rich protein 2 encoding gene (CSRP2) is within rat liver cell subpopulations solely expressed in activated HSC and downregulated in MFB. The aim of this study was the proof of the ability of human (h) CSRP2 promoter fragments to express transgenes in culture-activated rat HSC, but not in MFB or hepatocytes. Material/Methods: Culture-activated HSC from rats were infected with adenoviral vectors (Ad5) harbouring p53 or Bax encoding cDNAs under transcriptional control of a 0.9 kb hCSRP2 promoter fragment or the constitutive active CMV promoter to induce apoptosis in infected cells. Apoptosis was examined by Annexin-V-FLUOS-assay, Caspase 3-assay and cell death-ELISA. Additional reporter gene-assays were performed in the CFSC cell line, rat HSC and MFB as well as human MFB. Results: In activated HSC we found no significant p53 or Bax induced apoptosis directed by the 0.9 kb hCSRP2 promoter compared to CMV promoter driven expression of p53. Luciferase constructs transfected into CFSC showed that the activity of the CSRP2 promoter fragment is repressed by Bax, which is upregulated by p53. Beside these findings a comparison between the proximal human and rat CSRP2 promoter sequences showed only 30% homology indicating a different regulation in these species. Indeed, transgene expression after adenoviral infection of rat and human MFB with a GFP reporter gene driven by a 4.2 kb hCSRP2 promoter fragment was found to be much higher in the human system. Conclusions/Discussion: We tested a human CSRP2 promoter fragment to express apoptosis inducing proteins in rat HSC and found that overexpression of these effectors resulted in a self limitation of the chimeric expression construct. Therefore, usage of CSRP2-promoter fragments to express apoptotic acting proteins is questionable, but possible for other antifibrotic effectors. In further experiments it is important, regarding the limited homology of human and rat CSRP2 promoters, to deal with species-specific components. Therefore, we now clone rat promoter fragments to direct transgene expression.

Studies with liver cells in mice have shown that even cells that are not directly attributed to the immune system can play an antigen-presenting function. The aim of this study was to investigate the behavior of these antigen-presenting hepatic stellate cells (HSC) in rat. Also changes of the antigen-presenting function in the fibrotic liver should be identified on a molecular and functional level. The antigen-presenting function of HSC is both in vitro, in the course of the transdifferentiation of HSC to myofibroblast (MFB), as well as in vivo investigated. Cultured activated HSC express mRNA for all the process of antigen presentation related genes, such as (RT1B, RT1D) (alpha- and beta-chain) of the MHC II complex, and the co-stimulatory molecules CD80 and CD86. The expression, however, decreases during the course of transdifferentiation. After stimulation with the proinflammatory cytokine IFN-gamma, the expression of the MHC II genes RT1B and RT1D on transcriptional and translational (RT1B) level is increasing, while lacking a basal protein expression of RT1B in HSC and MFB. The antigen-presenting function of HSC and MFB was also examined with antigen (gpMBP)- and MHC II (RT1B) specific 53/4 T hybridoma cells. Activated HSC are able to release a RT1B-restricted T-cell answer. They are in principle be able to process and to present antigens. The specific functionality of the activated HSC, however, decreases during the course of transdifferentiation. MFB have a lower capacity for T-cell activation. IFN-gamma stimulation causes not only increased MHC II gene expression in HSC and MFB, but also leads to increased antigen presentation along with increased T-cell activation. The change of immune status and participation of HSC was examined in vivo in two models of liver fibrosis. The one in which the bile duct was ligated (BDL) and the other, where carbon tetrachloride (CCl4) triggers a toxic cell injury. In both model systems, the protein expression of RT1B (MHC II) increases significantly. In the CCl4 model this effect could be demonstrated in addition to Western blot analysis also by immunohistochemical staining. Immunohistochemical staining against RT1B and the HSC-specific protein desmin suggests that activated HSC are the cell type which is responsible for the changed MHC II (RT1B)-cell expression. The results show that, as in mice, activated HSC can take an immunological function in healthy and fibrotic liver.

Normal cells are controlled by several exocrine factors, whereas tumor cells often lose control by antiproliferative stimuli. It is known that melanoma cells produce transforming growth factor (TGF) beta1, 2 and 3, but do not respond with growth inhibition. Recently, the Smad inhibitor Ski was found to play a role in this process. We originally analyzed Ski expression in nine melanoma cell lines, however, only one cell line (SK Mel 28) was positive. As all nine cell lines were unresponsive to TGF-beta, we continued to search for the responsible mechanism. Sequencing of the TGF-beta-receptor II, known to be mutated in other kinds of cancer, did not reveal any mutation. A family member of Ski, the proto-oncogene Sno was strongly expressed in all melanoma cell lines on RNA and protein level, but not in melanocytes. To confirm functional relevance of this observation, we used stable antisense Sno transfection for the generation of cell clones with reduced Sno expression. These cell clones displayed reduced cell proliferation, indicating participation of Sno in the escape of melanoma cells from TGF-beta dependent growth control. Searching for TGF-beta target genes that are under control of Sno interference, Id1 but not antiproliferative genes p21, p15, p57 and p27 was identified in the cell clones after antisense Sno expression. In summary, constitutive Sno expression was identified as an important mechanism to shut off antiproliferative TGF-beta signaling in malignant melanoma.

Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.

Aims: Latent transforming growth factor-β binding proteins (LTBP–1, –2, 3-, –4) are glycosylated proteins of the extracellular matrix involved in the secretion and controlling the bioavailability of active transforming growth factor-β (TGF-β). LTBP–1 and TGF-β are key factors in the pathophysiology of liver fibrogenesis. During this process, hepatic stellate cells (HSC) become activated and transdifferentiate into myofibroblasts. Because the knowledge of LTBP functionality in controlling and regulation the concentration of biological active TGF-β is crucial for the understanding of fibrogenesis in the liver we generated a knockout mice for LTBP–1. Here we present data derived from induced liver fibrosis by bile duct ligation in respective animals.

UNLABELLED: Genetic risk factors play an important role for the progression of liver fibrosis in chronic hepatitis C virus (HCV) infection, but functional data on specific alleles and their related proteins are limited. Platelet-derived growth factor BB (PDGF-BB) is one of the strongest mitogens for hepatic stellate cells and is considered as a critical soluble mediator of liver fibrosis in vitro and in vivo. The biological activity of PDGF-BB is dependent on its degradation by the factor VII-activating protease (FSAP). Here, we demonstrate that a coding polymorphism (G534E) in the gene for FSAP is significantly associated with severe HCV-induced liver fibrosis (odds ratio, 2.59; P = 0.017), which is independent of age, gender, and presence of diabetes in multivariate analysis. These genetic findings were replicated in a cohort of patients with liver transplantation due to HCV-induced cirrhosis (OR, 2.56; P = 0.011). Functional dissection of the association demonstrates that the single amino acid change encoded by G534E in the FSAP protein does not influence PDGFbeta receptor or alpha-smooth muscle actin expression but completely abrogates FSAP-mediated inhibition of PDGF-BB-induced proliferation of primary stellate cells in vitro. CONCLUSION: The G534E variant of FSAP is a risk locus for HCV-induced liver fibrosis and cirrhosis by determining PDGF-BB-mediated hepatic stellate cell proliferation through a single amino acid substitution in FSAP. FSAP G534E might be useful for risk stratification in patients with HCV infection.

Background: Cystatin C (CysC) belongs to a group of non-glycosylated, low molecular mass cysteine protease inhibitors that inactivates members of the cathepsin family. CysC is reported to control the cleavage of membrane and extracellular matrix proteins, to regulate cell proliferation and, recently, to antagonize TGF-β binding to its cell surface receptors, in particular to type II receptor (TβRII). Up to now expression, regulation, and functional significance of CysC in cultured rat and human hepatic stellate cells (HSC) and myofibroblasts (MFB) are unknown.

OBJECTIVE — Retinol-binding protein 4 (RBP4) has been identified as a novel adipokine mediating systemic insulin resistance, and elevated serum RBP4 indicates overt or impending insulin resistance in lean, obese, and type 2 diabetic subjects. As insulin resistance is present in nearly all patients with liver cirrhosis, we evaluated RBP4 in patients with chronic liver disease (CLD). RESEARCH DESIGN AND METHODS — Serum RBP4 was measured in 111 CLD patients. Ninety-nine age- and sex-matched healthy blood donors served as control subjects. RBP4 gene expression was also quantified in normal and cirrhotic rat liver. RESULTS — In CLD patients, serum RBP4 was significantly reduced compared with healthy control subjects and closely correlated with the stage of liver cirrhosis. CLD patients without cirrhosis showed normal RBP4 concentrations, which correlated with serum glucose and insulin secretion and inversely correlated with insulin sensitivity. In patients with Child A-C liver cirrhosis, however, RBP4 was not correlated with glucose metabolism or other adipokines, such as adiponectin or resistin, but closely linked to the hepatic biosynthetic capacity, fibrotic changes in liver histology, or clinical complications such as portal hypertension. In an animal model of

BACKGROUND: The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype. RESULTS: A approximately 17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as beta-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue. CONCLUSION: We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.

The pathophysiology of liver injury has attracted the interest of experimentalists and clinicians over many centuries. With the discovery of liver-specific pericytes - formerly called fat-storing cells, Ito-cells, lipocytes, and currently designated as hepatic stellate cells (HSC) - the insight into the cellular and molecular pathobiology of liver fibrosis has evolved and the pivotal role of HSC as a precursor cell-type for extracellular matrix-producing myofibroblasts has been established. Although activation and transdifferentiation of HSC to myofibroblasts is still regarded as the pathogenetic key mechanism of fibrogenesis, recent studies point to a prominent heterogeneity of the origin of myofibroblasts. Currently, the generation of matrix-synthesizing fibroblasts by epithelial-mesenchymal transition, by influx of bone marrow-derived fibrocytes into damaged liver tissue, and by differentiation of circulating monocytes to fibroblasts after homing in the injured liver are discussed as important complementary mechanisms to enlarge the pool of (myo-)fibroblasts in the fibrosing liver. Among the molecular mediators, transforming growth factor-beta (TGF-beta) plays a central role, which is controlled by the bone-morphogenetic protein (BMP)-7, an important antagonist of TGF-beta action. The newly discovered pathways supplement the linear concept of HSC activation to myofibroblasts, point to fibrosis as a systemic response involving extrahepatic organs and reactions, add further evidence to a more or less uniform concept of organ fibrosis in general (e.g. liver, lung, kidney), and offer innovative approaches for the development of non-invasive biomarkers and antifibrotic trials.