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1.  Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein 
AIM: To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation.
METHODS: Expression of CCN2/CTGF, p-STAT3, p-Smad3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reverse-transcriptase polymerase chain reaction.
RESULTS: IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80, and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients’ sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3) signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter.
CONCLUSION: CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is down-regulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.
PMCID: PMC3020368  PMID: 21245987
Hepatocytes; Interleukin-6; Connective tissue growth factor; STAT3; Liver fibrosis; Acute phase reaction
2.  Non-invasive biomarkers for monitoring the fibrogenic process in liver: A short survey 
The clinical course of chronic liver diseases is significantly dependent on the progression rate and the extent of fibrosis, i.e. the non-structured replacement of necrotic parenchyma by extracellular matrix. Fibrogenesis, i.e. the development of fibrosis can be regarded as an unlimited wound healing process, which is based on matrix (connective tissue) synthesis in activated hepatic stellate cells, fibroblasts (fibrocytes), hepatocytes and biliary epithelial cells, which are converted to matrix-producing (myo-)fibroblasts by a process defined as epithelial-mesenchymal transition. Blood (non-invasive) biomarkers of fibrogenesis and fibrosis can be divided into class I and class II analytes. Class I biomarkers are those single tests, which are based on the pathophysiology of fibrosis, whereas class II biomarkers are mostly multiparametric algorithms, which have been statistically evaluated with regard to the detection and activity of ongoing fibrosis. Currently available markers fulfil the criteria of ideal clinical-chemical tests only partially, but increased understanding of the complex pathogenesis of fibrosis offers additional ways for pathophysiologically well based serum (plasma) biomarkers. They include TGF-β-driven marker proteins, bone marrow-derived cells (fibrocytes), and cytokines, which govern pro- and anti-fibrotic activities. Proteomic and glycomic approaches of serum are under investigation to set up specific protein or carbohydrate profiles in patients with liver fibrosis. These and other novel parameters will supplement or eventually replace liver biopsy/histology, high resolution imaging analysis, and elastography for the detection and monitoring of patients at risk of developing liver fibrosis.
PMCID: PMC2686898  PMID: 19468990
Biochemical markers; Diagnostic validity; Liver fibrosis; Monitoring; Multiparametric algorithms; Non-invasive diagnostic tools
3.  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-β1 (TGF-β1) and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-β1 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-β1 and PDGF-BB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-β1. Although CysC induced an increased binding affinity of TGF-β receptor type III (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-β1, it did not modulate TGF-β1 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-β 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-β1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.
PMCID: PMC4066123  PMID: 16521186
Cystatin C; TGF-β; Hepatic stellate cells; Trans-differentiation; Beta-glycan
4.  Transforming growth factor-β1 gene polymorphisms are associated with progression of liver fibrosis in Caucasians with chronic hepatitis C infection 
AIM: Considerable attention is focused on polymorphisms in the gene encoding transforming growth factor-β1 (TGF-β1), a multifunctional cytokine that is in turn a potent growth inhibitor involved in wound healing and differentiation. In humans, it promotes the pathogenesis of organ fibrosis, atherosclerosis, cancer, autoimmune and inflammatory diseases, keloid disease, and hypertrophic scarring. For this reason, much emphasis has been placed on studies elucidating the impact of TGF-β1 and its gene variations for the susceptibility and pathogenesis of these diseases. Unfortunately, some studies have serious limitations.
METHODS: We have recently described a high-throughput method for investigation the Arg25Pro polymorphism of human TGF-β1 gene and showed that the frequency of the Pro25 allele is significantly associated with hepatic fibrogenesis. In this report, we describe two novel LightCycler (LC) techniques that facilitate the examination of the two other known alterations in the coding region of TGF-β1. We investigated whether these polymorphisms contribute to hepatitis-induced progression of fibrogenesis in Chinese and Caucasians.
RESULTS: In the Chinese ancestry, the gene polymorphisms at codons 25 and 263 were not found and the genetic variant at codon 10 is unlikely to confer susceptibility to hepatic fibrosis. Contrarily, in Caucasians TGF-β1 allelic variations are more frequent and the presence of prolines either in codon 25 or 10 is associated with the interindividual variability in developing more severe fibrosis during chronic hepatitis C infection.
CONCLUSION: In summary, these results confirm the hypothesis that TGF-β1 polymorphisms are associated with fibrosis progression in Caucasians chronically infected with hepatitis C.
PMCID: PMC4305713  PMID: 15800982
TGF-β1; Gene polymorphism; LightCycler; Viral hepatitis; Fibrosis
5.  Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options 
Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. The extension of pathogenetic concepts of fibrosis will provide new therapeutic possibilities of interference with the fibrogenic mechanism in liver and other organs.
PMCID: PMC1994681  PMID: 17663771
6.  Glucocorticoids activate TGF-β induced PAI-1 and CTGF expression in rat hepatocytes 
In addition to the activation of hepatic stellate cells TGF-β govern apoptosis and growth control of hepatocytes in liver injury. In non-parenchymal cells, TGF-β induces plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF) expression, which are involved in extra cellular matrix formation. Both genes were also regulated by glucocorticoids, which in certain cases showed antagonistic effects to the TGF-β-Smad 3 pathway. The purpose of our work was to investigate the influence of TGF-β and dexamethasone on PAI-1 and CTGF expression and secretion in primary hepatocytes.
By examining PAI-1 and CTGF mRNA and protein expression in cell lysates and cell-conditioned media under the influence of TGF-β and dexamethasone, we analysed signalling pathways controlling their expression. TGF-β and dexamethasone significantly co-induce PAI-1 and CTGF protein expression. On the other hand, we showed that TGF-β diminished a glucocorticoid receptor dependent luciferase reporter signal in Hep-G2. Inhibition of Erk downstream activation decreased TGF-β induced CTGF and PAI-1 expression to a basal level. PAI-1 was directly secreted by hepatocytes, whereas secretion of CTGF was retarded.
The data provide evidence that beside the TGF-β-Smad 3 pathway CTGF and PAI-1 expression is additionally dependent on Erk activity in hepatocytes giving new insights into regulation of the profibrogenic proteins.
PMCID: PMC1868771  PMID: 17474984
7.  Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults 
BMC Medical Genetics  2004;5:15.
Clefts of the lip, alveolus, and palate (CLPs) rank among the most frequent and significant congenital malformations. Leu10Pro and Arg25Pro polymorphisms in the precursor region and Thr263Ile polymorphism in the prodomain of the transforming growth factor β1 (TGF-β1) gene have proved to be crucial to predisposition of several disorders.
In this study, polymorphism analysis was performed by real-time polymerase chain reaction (LightCycler) and TGF-β1 levels determined by enzyme-linked immunosorbent assay.
Only 2/60 Caucasian non-syndromic patients with CLP (3.3%) carried the Arg25Pro and another 2/60 patients (3.3%) the Thr263Ile genotypes, whereas, in a control group of 60 healthy Caucasian blood donors, these heterozygous genotypes were more frequent 16.7% having Arg25Pro (10/60; p < 0.035) and 10,0% having Thr263Ile (6/60), respectively. TGF-β1 levels in platelet-poor plasma of heterozygous Arg25Pro individuals were lower than those of homozygous members (Arg25Arg) in the latter group, but this discrepancy narrowly failed to be significant. Although polymorphisms in codon 10 and 25 were associated with each other, no difference was found between patients and controls concerning the Leu10Pro polymorphism.
The genetic differences in codons 25 and 263 suggest that TGF-β1 could play an important role in occurrence of CLP, however, functional experiments will be required to confirm the mechanisms of disturbed development.
PMCID: PMC441379  PMID: 15212689
8.  Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats 
BMC Gastroenterology  2003;3:29.
Transforming growth factor-β (TGF-β) is a key mediator in establishing liver fibrosis. Therefore, TGF-β as a causative agent may serve as a primary target for antifibrotic gene therapy approaches. We have previously shown that the adenoviral delivery of a transgene constitutively expressing a TGF-β1 antisense mRNA blocks TGF-β synthesis in culture-activated hepatic stellate cells and effectively abolishes ongoing fibrogenesis in vitro.
Ligature of the common bile duct was used to induce liver fibrosis in rats. The effect of the TGF-β1 antisense on fibrogenesis was analyzed in this model of liver injury.
In the present study, we demonstrate that the adenoviral vector directs the synthesis of mRNA quantities that are approximately 8000-fold more abundant than endogenous TGF-β1 mRNA. In experimentally injured rat livers induced by ligature of the common bile duct, a model for persistent fibrogenesis and cirrhosis, administration of the adenoviral vector abrogates TGF-β-enhanced production of collagen and α-smooth muscle actin. Furthermore, the number of cells positive for α-smooth muscle actin resulting from active recruitment of activated hepatic stellate cells around the bile ductular structures was significantly reduced in animals after application of Ad5-CMV-AS-TGF-β1. However, the observed elevated serum levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin induced in this obstructive liver injury model were not significantly altered in the presence of the TGF-β antagonist.
Taken together, our data provides in vivo evidence that the delivery of TGF-β1 antisense mRNA specifically abolishes the diverse effects of direct TGF-β function in ongoing liver fibrogenesis. Therefore, we conclude that the expressed transgene is therapeutically useful for inhibition of TGF-β effects in diverse applications, ranging from clarification of TGF-β function in the course of liver injury to the development of novel gene therapeutic approaches.
PMCID: PMC270053  PMID: 14565855
9.  Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts 
BMC Cell Biology  2000;1:4.
The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.
With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.
Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.
PMCID: PMC29065  PMID: 11178102

Results 1-9 (9)