Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosis.

Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-beta) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-beta bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.

Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells.

Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM alpha-actin and SM22alpha are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside-outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC-the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.

Biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality.

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.

The interleukin-6 (IL6)-174 G/C promoter genotype is associated with the presence of septic shock and the ex vivo secretion of IL6.

Septic shock is associated with a high mortality and an excessive activation of immune cascades. Interleukin (IL)-6 has been found to be a key cytokine in the pathogenesis of severe sepsis, but the importance of a regulatory polymorphism within the IL6 promoter has been controversial in these patients. The aim of the study was therefore to systematically investigate the IL6-174 G/C promoter genotype with regard to the presence of shock in patients with sepsis, the IL6 serum levels, and the ex vivo secretion of IL6, respectively. Overall, 112 consecutive subjects with severe sepsis and septic shock according to consensus criteria were enrolled. The ex vivo secretion of IL6 after stimulation with lipopolysaccharide (LPS) in a whole blood assay and the IL6 serum concentrations were determined after admission of the patients. Among the 112 subjects with severe sepsis, 85 patients fulfilled the criteria of septic shock. In these patients, the frequency of the mutated C-allele of the IL6 promoter polymorphism was significantly (P = 0.04) higher compared to that in individuals without shock. IL6 serum concentrations were highest in patients with the GG genotype (mean 2209 pg mL(-1)), followed by CG genotype (mean 1113 pg mL(-1)), and lowest in individuals with the CC genotype (mean 256 pg mL(-1)). Interestingly, a significantly (P = 0.005) higher ex vivo secretion of IL6 is detected in heterozygote individuals (535 pg mL(-1)) and patients with the IL6 CC genotype (555 pg mL(-1)) compared to patients with the -174 GG genotype (276 pg mL(-1)). In conclusion, the IL6-174 G/C promoter genotype is associated with shock in patients with sepsis. Functionally, the mutated C-allele is correlated with low IL6 serum concentrations, but a high ex vivo secretion after LPS stimulation. These results further indicate a complex regulation of the expression of IL6 during infection and have implications for the design of immune intervention trials.

Elevated apoptosis-associated cytokeratin 18 fragments (CK18Asp386) in serum of patients with chronic liver diseases indicate hepatic and biliary inflammation.

OBJECTIVES: During apoptosis, intermediate filament protein cytokeratin 18 (CK18) is cleaved by caspases at Asp396 which can be specifically detected by the monoclonal antibody M30 (M30-antigen). DESIGN AND METHODS: M30-antigen serum levels were analyzed in 76 chronic liver diseases (CLD) patients and 62 healthy controls. RESULTS: M30-antigen levels were significantly elevated in CLD patients (median 296.3 U/L) compared with healthy controls (median 153.5 U/L, P<0.001) and increased with disease severity (Child-Pugh or MELD score). M30-antigen correlated with aminotransferase activities and parameters indicating cholestasis such as bile acids. Highest serum M30-antigen was associated with histologically confirmed severe intrahepatic cholestasis (median 599.1 U/L) or biliary duct inflammation (median 648.0 U/L). Furthermore, in contrast to patients with liver cirrhosis, presence of hepatocellular carcinoma was associated with elevated M30-antigen in patients without cirrhosis. CONCLUSION: Serum M30-antigen levels are elevated in CLD and correlate with hepatic inflammation as well as cholangitis and cholestasis.

Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling.

BACKGROUND/AIMS: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce. METHODS: Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting. RESULTS: Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. CONCLUSIONS: Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.

[Elevated concentrations of fecal calprotectin in patients with liver cirrhosis]. / Erhöhte Konzentrationen von fäkalem Calprotectin bei Patienten mit Leberzirrhose.

UNLABELLED: BACKGROUND AND OBJECTION: Bacterial translocation from the gut lumen is considered to play an important role in the development of infectious complications in patients with liver cirrhosis. This translocation might be increased by inflammation of the gut mucosa. Calprotectin is a cytoplasmatic protein of neutrophilic granulocytes and is an established marker for the assessment of localized intestinal inflammation. It was the aim of the current study to systematically evaluate a localized intestinal inflammation in patients with liver cirrhosis by means of fecal calprotectin concentrations. PATIENTS AND METHODS: Fecal calprotectin concentrations were determined in 53 consecutive patients with liver cirrhosis and in 18 subjects without intestinal or liver diseases, who were comparable with respect to age and gender. Patients with diarrhoea, inflammatory bowel disease and a positive stool test for occult blood were excluded from the study. Fecal calprotectin concentrations were measured by a sandwich ELISA. The systemic inflammatory reaction of the patients was assessed by C-reactive protein, white blood cells counts and the serum concentrations of the cytokines IL-6, IL-8 and IL-10. RESULTS: Fecal calprotectin concentrations were significantly increased in patients with liver cirrhosis (median 37.0 mg/kg) compared to controls patients (median 2.2, P < 0.0001). There were no significant correlations of calprotectin concentrations with systemic inflammatory parameters, like CRP, white blood cell count or serum cytokines. However, fecal calprotectin concentrations were significantly associated with the stage of liver cirrhosis as expressed by the Child-Pugh score ( P < 0.001). A trend towards higher concentrations of calprotectin was found in patients with alcoholic liver cirrhosis ( P = 0.1). CONCLUSIONS: Patients with liver cirrhosis display elevated fecal calprotectin concentrations as a potential sign of intestinal inflammation. Further studies are warranted to establish a role of calprotectin for the risk assessment of infectious complications secondary to bacterial translocation in patients with liver cirrhosis.

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation.

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.

Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells.

Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.

Hepatic fibrosis is a scarring process that is associated with an increased and altered deposition of extracellular matrix in liver. At the cellular and molecular level, this progressive process is mainly characterized by cellular activation of hepatic stellate cells and aberrant activity of transforming growth factor-beta1 and its downstream cellular mediators. Although the cellular responses to this cytokine are complex, the signalling pathways of this pivotal cytokine during the fibrogenic response and its connection to other signal cascades are now understood in some detail. Based on the current advances in understanding the pleiotropic reactions during fibrogenesis, various inhibitors of transforming growth factor-beta were developed and are now being investigated as potential drug candidates in experimental models of hepatic injury. Although it is too early to favour one of these antagonists for the treatment of hepatic fibrogenesis in human, the experimental results obtained yet provide stimulatory impulses for the development of an effective treatment of choice in the not too distant future. The present review summarises the actual knowledge on the pathogenesis of hepatic fibrogenesis, the role of transforming growth factor-beta and its signalling pathways in promoting the fibrogenic response, and the therapeutic modalities that are presently in the spotlight of many investigations and are already on the way to take the plunge into clinical studies.

Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis.

Pleiotrophin (PTN) is a secretory heparin binding protein with various biological activities including mitogenesis, angiogenesis, and tissue repair after injury. Recent studies have shown that PTN is a strong mitogen of hepatocytes and involved in liver regeneration. In adult liver cells Ptn gene is mainly expressed by quiescent hepatic stellate cells (HSCs). Although we have been able to demonstrate mRNA and protein expression of the anaplastic lymphoma kinase-the receptor tyrosine kinase for PTN-on HSCs, PTN did not act as a mitogen of HSCs in contrast to hepatocytes. PTN immunoreactivity was markedly increased in experimental fibrogenesis by common bile duct ligation and observed in sinusoidal HSCs. In primary HSC cultures, Ptn transcription was significantly increased by PDGF-BB, and under hypoxic atmosphere. Mechanistically, hypoxia and PDGF mediated induction of PTN expression in sinusoidal HSCs may provide a strong mitogenic signal for hepatocytes to limit the damage to the parenchymal cells in biliary-type liver fibrogenesis.

Identification of an unconventional nuclear localization signal in human ribosomal protein S2.

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells.

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

Thrombospondin 1 acts as a strong promoter of transforming growth factor beta effects via two distinct mechanisms in hepatic stellate cells.

BACKGROUND AND AIMS: Thrombospondin 1 (TSP-1) is an important activator of latent transforming growth factor beta (TGF-beta) but little is known of the expression patterns and functions of TSP-1 in liver cells. We therefore analysed if and how TSP-1 acts on TGF-beta during fibrogenesis. METHODS AND RESULTS: Using reverse transcription-polymerase chain reaction, we demonstrated that hepatocytes from normal liver expressed no TSP-1 mRNA whereas Kupffer cells and sinusoidal endothelial cells did. TSP-1 mRNA and protein were detected in quiescent and activated cultured hepatic stellate cells (HSC) and TSP-1 expression was highly inducible by platelet derived growth factor BB (PDGF-BB) and, to a lesser extent, by tumour necrosis factor alpha in activated HSC. Furthermore, addition of PDGF-BB directly led to enhanced TGF-beta mRNA expression and a TSP-1 dependent increase in TGF-beta/Smad signalling. Using either a peptide specifically blocking the interaction of TSP-1 with latent TGF-beta or antibodies against TSP-1 not only abrogated activation of latent TGF-beta but also reduced the effects of the active dimer itself. CONCLUSIONS: Our data suggest that TSP-1 expression is important for TGF-beta effects and that it is regulated by the profibrogenic mediator PDGF-BB in HSC. Furthermore, the presence of TSP-1 seems to be a prerequisite for effective signal transduction by active TGF-beta not only in rat HSC but also in other cell types such as human dermal fibroblasts.

Comparative analysis of adenoviral transgene delivery via tail or portal vein into rat liver.

The potential indications for gene therapy are expanding continuously. Currently, hepatotropic adenoviruses are useful vector systems for targeting liver in experimental animal models. Although this gene delivery technique is widely distributed, there is no common sense about how these viruses should be applied. In general, the local delivery into portal vein and the systemic application via tail vein induces above all substantial transgene expression. We here comparatively analysed both methods and found that the systemic administration of an adeno-virus expressing the green fluorescent protein resulted in a stronger infiltration, a more homogenous distribution, and a higher inter-individual reproducibility of reporter gene expression in rat liver than organ-specific administration via the portal vein.

The influence of HES on the filtration properties of capillary membrane plasmaseparation.

Plasmaseparation is a treatment under discussion for critically ill patients, especially in sepsis and multiorgan failure. These patients receive a variety of different fluid substitutes, including hydroxyethylstarch (HES). HES is known to influence rheological properties, but nothing is known about the possible interactions between HES and the plamaseparation procedure. We used an in vitro plasmaseparation circuit with heparinized porcine blood. Before priming the system, 2 liters of blood were supplemented by adding 100 ml of either NaCl 0.9% or HES (n=6 in each group). We monitored the transmembrane (TMP) and the filtration pressure (PF) and measured free plasma hemoglobin (free Hb) and platelet counts before and after the two hours plasmaseparation procedure. The final transmembrane pressure was significantly higher with HES substitution. In the HES group we found negative filtration pressures from the very beginning with a significant further decrease toward the end of the experiments. A significant increase in free Hb and decrease in platelet counts were noted only in the HES group. Volume substitution with HES leads to impaired filtration properties and deteriorated hemocompatibility in in vitro plasmaseparation. Further studies have yet to evaluate whether or not the effects described also occur under clinical conditions.

Quantitative monitoring of the mRNA expression pattern of the TGF-beta-isoforms (beta 1, beta 2, beta 3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR.

Current methods to determine the mRNA of the TGF-beta-isoforms, beta 1, beta 2, and beta 3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-beta 1-mRNA was found to be the predominant isoform expressed followed by TGF-beta 3 and low amounts of TGF-beta 2-mRNA. An alteration of the TGF-beta 1,-beta 2, and -beta 3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.

LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1.

Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.

Up-regulated expression of the receptor for advanced glycation end products in cultured rat hepatic stellate cells during transdifferentiation to myofibroblasts.

Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell-surface molecules. Blockade of RAGE has been reported to considerably improve liver function and accelerate regeneration after hepatectomy. The aim of this study was to investigate the cell type-specific expression of RAGE, and to examine whether transdifferentiation of hepatic stellate cells (HSC) into myofibroblasts (MFB) is associated with changes in RAGE expression. Northern blot analysis revealed that RAGE mRNA was exclusively expressed by HSC isolated from rat liver, while no transcripts were seen in hepatocytes, Kupffer cells, or sinusoidal endothelial cells. Expression of RAGE mRNA was up-regulated during transdifferentiation of HSC into MFB. Concomitantly, expression of RAGE protein was increased as confirmed by Western blotting and immunohistochemistry. As assessed by radioactive labeling, transforming growth factor beta(1) (TGF-beta(1)) induced a time-dependent 2- to 15-fold increase in the de novo synthesis of RAGE protein, which was completely abolished using PD098059, a specific inhibitor of the mitogen-activated protein kinase (MAPK) kinase. As shown by double-immunofluorescence staining, RAGE colocalized with alpha-smooth muscle actin, and immunoelectron microscopy demonstrated the most prominent labeling for RAGE at filopodial membranes of MFB. In conclusion, this study demonstrates that expression of RAGE is restricted to rat HSC, and that expression is up-regulated during activation of HSC and transition to MFB. The preferential immunogold labeling of RAGE to focal membrane areas of filopodia of MFB is suggestive of a role of RAGE in the spreading and migration of activated HSC/MFB, major players in liver fibrogenesis.