Connective tissue growth factor is a Smad2 regulated amplifier of transforming growth factor beta actions in hepatocytes--but without modulating bone morphogenetic protein 7 signaling.

UNLABELLED: In vivo knockdown of connective tissue growth factor (CTGF/CCN2) was recently shown to attenuate the formation of experimental liver fibrosis. The secreted, cysteine-rich growth factor is proposed to adversely modulate the binding of profibrogenic transforming growth factor beta (TGF-beta) and its natural antagonist bone morphogenetic protein (BMP) to their cognate receptors in several cellular systems, but the functionality of CTGF in modulation of the TGF-beta/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF-beta- and BMP7-dependent transactivation of reporter gene [Ad-(CAGA)(12)-MLP-luc, Ad-hCTGF-luc, and Ad-(BRE)(2)-luc reporter gene] expression in rat hepatocytes. In this context, emphasis is also placed on the differential roles of Smad2 and Smad3 in the TGF-beta-dependent transactivation of the endogenous CTGF gene and the CTGF gene reporter, as investigated following adenoviral infection of wild-type and dominant negative Smad2/3 or treatment with the specific inhibitor of Smad3 or ALK5-specific (SB-431542) inhibitor. In this analysis, we found (1) a selective transcriptional activation of the CTGF promoter by Smad2 (but not Smad3); (2) the failure of BMP7 to inhibit the transcriptional activation of the Smad3-selective (CAGA)(12)-luc reporter by TGF-beta, as well as the failure of TGF-beta to inhibit the transcriptional activation of the Smad5-selective (BRE)(2)-luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF-beta type I receptor (ALK5)/Smad2 and Smad3-mediated TGF-beta signaling by CTGF, whereas BMP type I receptor (ALK1)/Smad5-mediated BMP7 signaling is not modulated. CONCLUSION: CTGF acts as a Smad2-dependent sensitizer of TGF-beta actions that does not influence BMP7 signaling in hepatocytes.

Identification of paraxanthine as the most potent caffeine-derived inhibitor of connective tissue growth factor expression in liver parenchymal cells.

BACKGROUND: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)-beta dependent and -independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine-alkaloid as a potential therapeutic agent. AIM: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). RESULTS: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine-derived metabolic methylxanthines with an inhibitory dosage (ID)50 of 1.15 mM, i.e. 3.84-fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water-soluble tetrazolium-1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp-adenosine 3',5-cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF-beta (0.13-1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). CONCLUSION: Our data provide an evidence-based suggestion of the caffeine-derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.

Questioning the role of actinfree Gc-Globulin as actin scavenger in neurodegenerative central nervous system disease: relationship to S-100B levels and blood-brain barrier function.

INTRODUCTION: Preliminary studies report on significantly higher levels of the major cytoskeleton protein actin in CSF of patients with neurodegenerative conditions and that the dynamics of these levels obviously correlates with disease progression and clinical disability. One of the primary functions of actinfree Gc-Globulin is to bind and neutralize extracellular monomeric actin, released into the circulation by necrotic or ruptured cells, and thus ameliorating the clinical outcome in situations of severe organ damage. AIM AND METHODS: This is the first study to investigate actinfree Gc-Globulin and S100-B levels (as reliable marker of neurodegeneration) in paired CSF and serum samples of patients with multietiological CNS diseases. RESULTS: 42% of all patients with CNS disease displayed serum concentrations of actinfree Gc-Globulin above the established reference range. CSF concentrations of actinfree Gc-Globulin and S100-B were positively correlated with the severity of blood-brain barrier (BBB) dysfunction. Furthermore, patients with severe BBB dysfunction presented a higher percentage of intrathecal synthesis of actinfree Gc-Globulin compared to patients with mild to moderate dysfunction and to patients with normal BBB function. Representative longitudinal data from selected patients demonstrated an inverse behaviour of actinfree Gc-Globulin and S100-B CSF concentrations, suggesting a consumption of the actin scavenger capacity of Gc-Globulin in times of increased neuronal damage. This presumption was supported by the fact that those conditions associated with a severe neuronal damage, in particular CNS trauma, and highest S100-B concentrations simultaneously displayed lowest actinfree Gc-Globulin levels, and thus residual actin binding capacity of Gc-Globulin. CONCLUSION: In summary, our data propose a function of actinfree Gc-Globulin also in the clearance of actin filaments from CSF of patients with neuronal damage. However, active recruitment of hepatic derived actinfree Gc-Globulin to the site of CNS injury is not observed. Much more, BBB leakage enables extraneuronally synthesized actinfree Gc-Globulin to extent its scavenger capacity for actin also to the subarachnoidal space. Furthermore, intrathecal synthesis of actinfree Gc-Globulin seems to be increased in patients with severe neurodegeneration.

Elevated concentrations of 15-deoxy-Delta12,14-prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor gamma-inducing drugs.

BACKGROUND/AIMS: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF-beta. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor gamma (PPARgamma) expression in hepatocytes, thus sensitizing these cells to the well-known inhibitory effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15-d-PGJ(2) is required to exert an inhibitory effect on transforming growth factor-beta (TGF-beta) target genes such as CTGF. METHODS: This study compared serum concentrations of 15-d-PGJ(2) in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non-liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARgamma-inducing (i.e. CTGF inhibitory) drugs such as caffeine. RESULTS: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2+/-5.9 microg/L) displayed strikingly higher serum concentrations of 15-d-PGJ(2) than healthy probands (mean 2.3+/-1.0) and Caucasian patients with NLD (mean 2.7+/-1.4 microg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3+/-0.7 microg/L) compared with Chinese healthy controls (mean 0.4+/-0.2 microg/L). CONCLUSIONS: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARgamma expression, such as methylxanthine derivates.

Pharmacological application of caffeine inhibits TGF-beta-stimulated connective tissue growth factor expression in hepatocytes via PPARgamma and SMAD2/3-dependent pathways.

BACKGROUND/AIMS: Epidemiological studies suggest that coffee drinking is inversely correlated with the risk of development of liver fibrosis but the molecular basis is unknown. METHODS: We investigated the pharmacological mechanisms involved in caffeine-dependent regulation of CTGF expression, an important modulator protein of fibrogenic TGF-beta, in rat hepatocytes using Western-blot, co-immunoprecipitations, reporter-gene-assays and ELISAs. RESULTS: It is demonstrated that caffeine, similar to 8-Br-cAMP, suppresses CTGF expression, decreases SMAD2 protein levels and inhibits SMAD1/3-phosphorylation. The SMAD2 level can be restored by a proteasome inhibitor. Additionally, caffeine leads to an up-regulation of PPARgamma expression, that enhances the inhibitory effect of the natural PPARgamma agonist 15-PGJ(2) on CTGF expression by inducing a dissociation of the SMAD2/3-CBP/p300-transcriptional complex. CONCLUSIONS: We show that caffeine strongly down-modulates TGF-beta-induced CTGF expression in hepatocytes by stimulation of degradation of the TGF-beta effector SMAD 2, inhibition of SMAD3 phosphorylation and up-regulation of the PPARgamma-receptor. Long-term caffeinization might be an option for anti-fibrotic trials in chronic liver diseases.

Intracrine signalling of activin A in hepatocytes upregulates connective tissue growth factor (CTGF/CCN2) expression.

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.

Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor.

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.

Gc-globulin (vitamin D binding protein) is synthesized and secreted by hepatocytes and internalized by hepatic stellate cells through Ca(2+)-dependent interaction with the megalin/gp330 receptor.

BACKGROUND: Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. METHODS: Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [(35)S]-methionine, and its intracellular binding to alpha-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and alpha-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. RESULTS: Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [(35)S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca(2+), suggesting a Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and alpha-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to alpha-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. CONCLUSION: We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to alpha-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.

Evaluation of hepatotropic targeting properties of allogenic and xenogenic erythrocyte ghosts in normal and liver-injured rats.

BACKGROUND/AIMS: Haemoglobin-depleted erythrocyte ghosts have been recommended as vesicle carriers of drugs with hepatotropic properties. However, the influence of liver injury on ghost elimination and targeting has not been reported so far. METHODS: Human and rat ghosts were prepared and loaded with model substances, and the basic parameters were characterized. Ghosts were injected intravenously into rats with acute, subacute and chronic liver injuries. Elimination from circulation, organ distribution and cellular targeting was measured. The uptake of ghosts by liver macrophages/Kupffer cells was determined in cell culture. RESULTS: Ghosts are strong hepatotropic carriers with a recovery of 90% in normal liver. Kupffer cells are the almost exclusive target cell type. Hepatotropic properties remain in rats with chronic liver diseases, but are reduced by 60-70% in acute liver damage as a result of decline of phagocytosis of macrophages/Kupffer cells. Although the uptake of ghosts per gram liver tissue in chronic liver injury was also reduced by about 40%, the increase of liver mass and of macrophages/Kupffer cells compensated for the reduced phagocytotic activity. In subacute injury, the uptake per gram liver tissue was only moderately reduced. CONCLUSION: Drug targeting with ghosts might be feasible in chronic and subacute liver injuries, e.g. fibrogenesis and tumours, because the content of ingested ghosts is released by Kupffer cells into the micro-environment, providing the uptake by and pharmacological effects on adjacent cells.

Differential effects of TGF-beta on connective tissue growth factor (CTGF/CCN2) expression in hepatic stellate cells and hepatocytes.

BACKGROUND/AIMS: Connective tissue growth factor (CTGF/CCN2) has been implicated in the pathogenesis of hepatic fibrosis and suggested as a downstream mediator of the fibrogenic master cytokine TGF-beta. METHODS: We investigated the effect of TGF-beta1 on CTGF/CCN2 expression in cultured rat hepatic stellate cells and hepatocytes by means of Western and Northern blotting, immunocytochemistry, reporter gene analysis, and metabolic labelling. RESULTS: We found that the expression of CTGF/CCN2 in hepatic stellate cells is (i) only marginally (if at all) stimulated by TGF-beta and by a constitutively active type I TGF-beta receptor, (ii) independent from Smad2/3 phosphorylation, (iii) not reduced by TGF-beta1 antagonists or ALK5-receptor inhibitors and (iv) not upregulated during transdifferentiation to myofibroblasts in culture. However, expression and secretion of CTGF/CCN2 in cultured hepatocytes increased spontaneously during culture and was strongly stimulated by TGF-beta1. In bile-duct ligated and CCl(4)-treated rat livers, a strong CTGF/CCN2 expression in hepatocytes was noticed. Endothelin-1 stimulated CTGF/CCN2 expression in stellate cells but not in hepatocytes. Pathway specific signalling inhibitors point to the involvement of non-Smad signalling cascades but their contribution to CTGF/CCN2 regulation is different in both cell types. CONCLUSIONS: The results do not reveal a relevant interrelation between TGF-beta function and CTGF/CCN2 expression in hepatic stellate cells, which is in contrast to hepatocytes.

Loss of TGF-beta dependent growth control during HSC transdifferentiation.

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-beta inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-beta function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-alpha enhanced proliferation inhibition of TGF-beta, whereas interleukin-6, oncostatin M, interleukin-1alpha, and interleukin-1beta did not. Hepatocyte growth factor (HGF) counteracted TGF-beta dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-beta contributes to loss of TGF-beta dependent inhibition of DNA synthesis in HSCs.

Connective tissue growth factor in serum as a new candidate test for assessment of hepatic fibrosis.

BACKGROUND: No reliable, cost-effective serum test is available for assessment of liver fibrogenesis, the most serious complication of chronic inflammatory liver diseases (CLD). In sera of patients with CLD, we determined the concentration of connective tissue growth factor (CTGF), a secreted downstream mediator of the potent fibrogenic cytokine transforming growth factor beta (TGF-beta). PATIENTS AND METHODS: We studied 83 patients with CLD (17 with chronic hepatitis, 16 with histologically proven fibrosis, and 50 with cirrhosis) and 74 healthy individuals. Serum CTGF was measured by use of a sandwich immunoassay. RESULTS: The mean concentration of CTGF was highest in the fibrosis group (5.2-fold) and in the chronic viral hepatitis group (4.3-fold) but lower in those patients with fully developed cirrhosis. The area under the ROC curve (AUC) of CTGF for fibrosis vs control was 0.955 (95% confidence interval, 0.890-0.987). The CTGF/platelet ratio increased the detection limit for cirrhosis from 84% to 92% and the specificity from 85% to 87.5% (cutoff for CTGF was 364 microg/L, ratio 2.05). CONCLUSION: CTGF in serum is a candidate marker of ongoing fibrogenesis in chronic liver diseases.

Variable expression of cystatin C in cultured trans-differentiating rat hepatic stellate cells.

AIM: To study the expression of cystatin C (CysC), its regulation by transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-beta1 signaling in this special cell type. METHODS: We evaluated the CysC expression in cultured, profibrogenic hepatic stellate cells and trans-differentiated myofibroblasts by Northern and Western blotting and confocal laser scanning microscopy. RESULTS: CysC was increased significantly in the course of trans-differentiation. Both TGF-beta1 and PDGF-BB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-beta1. Although CysC induced an increased binding affinity of TGF-beta receptor type III (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-beta1, it did not modulate TGF-beta1 signal transduction as shown by evaluating the Smad2/3 phosphorylation status and [CAGA]-MLP-luciferase reporter gene assay. Interestingly, the shedding of type III TGF-beta receptor beta-glycan was reduced in CysC-treated cells. Our data indicated that CysC expression was upregulated during trans-differentiation. CONCLUSION: Increased CysC levels in the serum of patients suffering from liver diseases are at least partially due to a higher expression in activated hepatic stellate cells. Furthermore, TGF-beta1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.

Elevation of Nepsilon-(carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis.

OBJECTIVES: Progression of liver fibrosis to cirrhosis is a dire consequence of chronic liver diseases (CLD). Nepsilon-(carboxymethyl)lysine (CML)-modified advanced glycation end products (AGEs) in patients with CLD could reflect the degree of severity of the disease. DESIGN AND METHODS: In 110 patients with CLD and 124 healthy controls, CML serum levels and their diagnostic sensitivity and specificity were determined and compared to hyaluronan (HA). RESULTS: Serum levels of CML were significantly affected by the stage of liver cirrhosis and were closely associated with liver function capacity. CML correlated positively with HA (r = 0.639, P < 0.0001). In ROC analysis, the diagnostic sensitivity and specificity in distinguishing healthy controls from liver disease patients for CML (AUC 0.908; 95%-CI 0.863-0.942, cut-off 640 ng/mL, sensitivity 74.5% and specificity 97.6%) resembled HA (AUC 0.948; 95%-CI 0.907-0.974; cut-off 50 ng/mL, sensitivity 80.7% and specificity 97.9%). The combination of CML and HA shows an AUC of 0.932; 95%-CI 0.888-0.962; sensitivity 82.6%; and specificity 95.8%. CONCLUSIONS: Our data suggest that serum levels of CML could provide a supplementary diagnostic marker for advanced stages of liver cirrhosis. However, the quality of interaction needs further investigation.

N-acetyl-L-cysteine suppresses TGF-beta signaling at distinct molecular steps: the biochemical and biological efficacy of a multifunctional, antifibrotic drug.

The interrelated signaling via TGF-beta1 and reactive oxygen species has a profound impact on fibrogenesis and is therefore selected as target for antifibrotic therapies. This prompted us to investigate the influence of the antioxidant N-acetyl-L-cysteine on TGF-beta signaling in culture-activated hepatic stellate cells, the most relevant pro-fibrogenic cell type in liver. Dissection of the molecular steps involved in TGF-beta signaling revealed that N-acetyl-L-cysteine dose-dependently abrogated the induction of the TGF-beta1 signaling reporter gene activation, the phosphorylation of Smad2 and Smad3, and the up-regulation of Smad7 mRNA. By means of Western blot analysis and cross-linking experiments, it was demonstrated that these effects are based on disintegration of TGF-beta1 and the TGF-beta receptor endoglin, as well as a reduced ligand binding capacity of betaglycan. We conclude that N-acetyl-L-cysteine is a specific inhibitor of TGF-beta signaling targeting different components of the TGF-beta signaling machinery. In conclusion, these findings suggest that this non-toxic aminothiol downregulates TGF-beta signal transduction thereby mediating beneficial effects on experimental liver fibrosis characterized by TGF-beta hyperactivity.

Identification of endoglin in rat hepatic stellate cells: new insights into transforming growth factor beta receptor signaling.

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.

Expression of cytosolic and membrane associated tissue transglutaminase in rat hepatic stellate cells and its upregulation during transdifferentiation to myofibroblasts in culture.

Transdifferentiation of hepatic stellate cells (HSC) to collagen producing myofibroblasts (MFB) is a principal event in liver fibrogenesis. In our studies we investigated if tissue transglutaminase (tTG) from these cell types may play a role in liver fibrosis. Separation of cytosol and membrane components showed membrane associated tTG and during transdifferentiation an upregulation of total tTG on mRNA and protein level was found, but no modulation during stimulation with TGF-beta1. In HSC and fully differentiated MFB a significant amount of the total tTG synthesised during transdifferentiation is found to be membrane-associated whereas the remaining portion is cytosol-associated and only very little is found within the extracellular matrix (ECM). The data implicate that tTG in this cell type seems to play an important role in liver fibrogenesis.

Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-beta1 inhibits fibrogenic activities of hepatic stellate cells.

Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.