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.

Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts.

BACKGROUND/AIMS: The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB. METHODS: Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining. CONCLUSION: The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.

Expression and matrix deposition of latent transforming growth factor beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture.

Latent transforming growth factor beta binding protein (LTBP), a high-molecular-weight glycoprotein of the large latent TGF-beta complex is suggested to serve as an anchor for latent TGF-beta in the extracellular matrix and as a component of microfibrillar structures. Proteolytic cleavage of LTBP is supposed to be a prerequisite for the release and generation of bioactive (mature) TGF-beta. We investigated the expression of LTBP isoforms in normal and fibrotic rat liver and in cultured rat hepatic stellate cells (HSC) transdifferentiating to myofibroblasts (MFB). We further determined their interaction with the matrix and some of their basic functions. Immunostainings of normal and fibrotic livers demonstrate intense signals for LTBP-1 and -2, preferably in parenchymal, but also nonparenchymal, cells and in fibrotic extracellular matrix. However, in situ hybridization points to a restriction of transcripts to nonparenchymal cells from fibrotic livers, whereas hepatocytes were always devoid of LTBP transcripts. The findings were confirmed by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), which showed isoform-specific increases of LTBP transcripts in cultured stellate cells transdifferentiating to MFB and by Northern blot analyses showing the absence of LTBP-1 mRNA in freshly isolated hepatocytes. Using a cell enzyme-linked immunosorbent assay (ELISA), a differential increase of partly deoxycholate (DOC)-resistant, matrix-bound LTBP-1 and -2 was measured in cultured stellate cells. Treatment with plasmin generated soluble LTBP-1 and bioactive TGF-beta, which was able to induce Smad7 expression in an autocrine fashion. Our data propose (transdifferentiating) stellate cells, respectively MFB, as the major source of LTBP in normal and fibrotic liver, which here probably fulfills structural and TGF-beta-regulating functions as suggested for nonhepatic tissues.

Modulation of transforming growth factor beta response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts.

Activation of hepatic stellate cells (HSCs) is the key step in liver fibrogenesis. Increased transforming growth factor beta (TGF-beta) expression and extracellular matrix production in patients with hepatic fibrosis and experimental models of liver fibrogenesis support implication of TGF-beta in the pathogenesis of this disease. However, a causative role for TGF-beta during transdifferentiation of HSCs has not been delineated in molecular detail. Using a rat cell culture model of HSC transdifferentiation, we analyzed TGF-beta signal transduction and identified changes between stellate cells and their transdifferentiated phenotype. Fully transdifferentiated myofibroblasts, opposed to HSCs, were not inhibited in proliferation activity on treatment with TGF-beta1. Furthermore, stimulation of alpha2 (I) collagen and Smad7 messenger RNA (mRNA) expression by TGF-beta1 was achieved in stellate cells but not in myofibroblasts. Northern and Western blot analyses indicated significant expression of TGF-beta receptors I and II in both cell types. In contrast, [(125)I]-TGF-beta1 receptor affinity labeling displayed strongly reduced types I, II, and III receptor presentation at the cell surface of myofibroblasts. Moreover, myofibroblasts did not display DNA-binding SMAD proteins in electrophoretic mobility shift assays with a CAGA box. These data indicate that stellate cells are responsive to TGF-beta1 treatment and transduce a signal that may play an important role in liver fibrogenesis. Myofibroblasts display decreased availability of surface receptors for TGF-beta, which could be based on autocrine stimulation. However, lack of activated SMAD complexes with DNA-binding activity and absence of alpha2 (I) collagen transcription inhibition by latency-associated peptide (LAP)/anti-TGF-beta antibody raise the possibility of TGF-beta signaling independent receptor down-regulation in myofibroblasts.

Differential expression of monocyte chemotactic protein-1 (MCP-1) in transforming rat hepatic stellate cells.

BACKGROUNDS/AIMS: Hepatic stellate cells and infiltrating leukocytes play a key role in the pathogenesis of liver fibrosis. The chronic phase of liver inflammation is characterized by immigrating mononuclear cells. To understand the underlying mechanisms responsible for the attraction of mononuclear cells in the pathogenesis of liver fibrosis, we investigated the inducible production of chemotactic activities in hepatic stellate cells. METHODS: Cultured hepatic stellate cells of different transformation grades and after in vitro transformation to myofibroblast-like cells were stimulated with tumor necrosis factor-a or bacterial lipopolysaccharide. Mononuclear cell attracting chemotactic activities were evaluated by chemotaxis assays, ELISA, and Northern blot analysis. RESULTS: We observed a transformation grade-dependent differential responsiveness of hepatic stellate cells and myofibroblast-like cells. Monocyte chemotactic protein-1 was inducible by tumor necrosis factor-alpha in non-transformed hepatic stellate cells. In contrast, monocyte chemotactic protein-1 was not inducible by bacterial lipopolysaccharide until the cells were fully transformed into myofibroblast-like cells. Despite a delayed onset, the bacterial lipopolysaccharide-inducible monocyte chemotactic protein-1 expression did not depend on an endogenous production of tumor necrosis factor-alpha. CONCLUSIONS: Our results indicate that the tumor necrosis factor-alpha and bacterial lipopolysaccharide-inducible production of chemokines plays a central role in the pathogenesis of liver fibrosis. These data suggest that when hepatic stellate cells have been transformed to a myofibroblast-like cells phenotype, e.g. by chronic injury, the cells become more sensitive to bacterial lipopolysaccharide, which may potentiate the production of chemotactic and fibrogenic mediators. A strong secretion of monocyte chemotactic protein-1 may contribute to the maintenance of an inflammatory infiltrate dominated by mononuclear cells.

Karhunen-Loeve decomposition in the presence of symmetry. I.

The Karhunen-Loève (KL) decomposition is widely used for data which very often exhibit some symmetry, afforded by a group action. For a finite group, we derive an algorithm using group representation theory to reduce the cost of determining the KL basis. We demonstrate the technique on a Lorenz-type ODE system. For a compact group such as tori or SO(3,R) the method also applies, and we extend results to these cases. As a short example, we consider the circle group S(1).

Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand.

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.

Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. Immigrating leukocytes can potentiate the progression of liver fibrosis by release of fibrogenic mediators and cytotoxic actions. The inducible production of neutrophil chemotactic activities in HSCs was investigated to understand the underlying mechanisms responsible for the attraction of leukocytes in the pathogenesis of liver fibrosis. METHODS: Cultured HSCs of different transformation grades and after transformation to myofibroblasts (MFBs) were stimulated with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS), respectively. Induced leukocyte chemotactic activities were evaluated by chemotaxis assays, enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS: A transformation grade-dependent differential responsiveness of HSCs and MFBs was observed. TNF-alpha-inducible production of chemotactic mediators increased substantially with advancing transformation. Only transformed MFBs were LPS responsive. Macrophage inflammatory protein 2 was identified as one of the inducible chemokines. CONCLUSIONS: The results suggest that chemokines play an important role in the pathogenesis of liver fibrosis. Proinflammatory cytokines can initiate the production of chemotactic activities. The more HSCs are transformed to MFBs, e.g., by chronic injury, the more sensitive the cells become to LPS, which may lead to a vicious circle of enhanced fibrogenic and chemotactic mediator production.

TGF-beta-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes.

BACKGROUND/AIMS: Primary cultures of rat hepatocytes, rat (FAO) and human (HepG2) hepatoma cells were studied by immunocytochemistry for expression of transforming growth factor (TGF)-beta, for the release of TGF-beta into the medium, and generation of hepatocellular apoptosis by the respective cell-conditioned media. METHODS/RESULTS: Using the alkaline-phosphatase anti-alkaline-phosphatase technique, intense TGF-beta immunostaining was shown in all cell types. The cytokine is released almost entirely in the latent form into the culture medium; only the FAO-cells had a substantial fraction of bioactive TGF-beta in the native (unacidified) culture fluid. Exposure of hepatocytes with the respective cell-conditioned media in the activated, but not in the native form (except for FAO-cell media), induced severe detrimental effects as evidenced by: (i) gross morphological alterations, (ii) functional impairment (reduction of WST-1 test, detachment of cells, lactate dehydrogenase increase in the medium), and (iii) generation of apoptosis. The latter phenomenon was confirmed by an increase of internucleosomal DNA fragments, positive TUNEL reaction, and intense binding of the fluorochrome Hoechst 33342 to fragmented nuclei. All these effects, which were mimicked by addition of recombinant human TGF-beta 1, were almost entirely antagonized by pre-incubation of the conditioned media with latency associated peptide. In contrast to hepatocytes, both types of hepatoma cells were completely resistant to the multiple actions of TGF-beta and activated conditioned media. CONCLUSIONS: It is concluded that hepatocytes might have the ability to induce autocrine, TGF-beta-mediated apoptosis, whereas hepatoma cells, because of their TGF-beta resistance, might generate TGF-beta-mediated peritumorous apoptosis of hepatocytes in a paracrine way, which could facilitate their expansion in situ. Both mechanisms, however, are critically dependent on extracellular TGF-beta activation.

Attenuation of TGF-beta-induced apoptosis in primary cultures of hepatocytes by calpain inhibitors.

Apoptosis induced in primary cultures of rat hepatocytes by transforming growth factor beta 1 (TGF-beta 1) was greatly attenuated by inhibitors (5 microM) of calpain I and calpain II, respectively. Both inhibitors prevented the TGF-beta-elicited increase of nucleosomal DNA fragments and the occurrence of DNA-breaks in the TUNEL reaction. The detrimental effect of TGF-beta on cell viability measured by the WST-1 test was strongly reduced by calpain inhibitors. Calpain II > I inhibitors suppressed spontaneous DNA cleavage in hepatocytes during culture and prevented the appearance of immunocytochemically visible TGF-beta (APAAP staining), which occurs in untreated parenchymal cell cultures. The data show that inactivation of calpains attenuates both the TGF-beta-elicited and the spontaneous apoptosis of cultured hepatocytes; the latter effect is likely due to the suppression of endogenous TGF-beta activation. It is suggested that calpains participate in these processes.

Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta.

The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor beta (TGF-beta)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-beta1. MFB were obtained by subculture of FSC. The TGF-beta concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-beta was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-beta1. The exclusive role of active TGF-beta in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-beta receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-beta.

Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells.

The activation of proliferation of rat liver hepatic stellate cells (HSC) in cooperation with hepatocytes (PC) was studied using a coculture system and cell-conditioned media, respectively. The proliferation of HSC was followed by incorporation of [3H] thymidine and BrdU into DNA and by DNA content per culture. Strong stimulation of HSC proliferation was noticed under reduced fetal calf serum (FCS) conditions (0.2%) during a 48-hour coculture with PC, rat hepatoma, human hepatoma, and transforming growth factor (TGF)-alpha-transgenic mouse PC, respectively. The extent of stimulation was frequently higher than that observed by the addition of 10% FCS. Transformed HSC (myofibroblasts) could also be stimulated by cocultured PC, but the magnitude of activation was lower than that of (untransformed) HSC. Using radioreceptor assays, we could demonstrate significant concentrations of insulinlike growth factor (IGF)-1 (300 ng/10(6) cells x 48 hours) and quite lower concentrations of bFGF and TGF-alpha in the hepatocyte-conditioned media (PCcM), whereas IGF-2 was not detectable. With anti-IGF-1 neutralizing antibody, the stimulatory activity of PCcM could be reduced by approximately 50%. PCcM, which mimics the effects of cocultures and supports strongly the action of exogenous IGF-1 on HSC proliferation, leaving that of other cytokines (TGF-alpha, IL-1 alpha, bFGF, aFGF, TNF-alpha), added either separately or in various combinations, uninfluenced. The latter cytokines were without significant effects on HSC proliferation. The mitogenic activity of cytokine combinations containing IGF-1 could be enhanced severalfold by limiting amounts of PCcM. Maximum stimulation of cell proliferation of 40-fold above control cultures was reached by IGF-1 in combination with TGF-alpha and bFGF in presence of diluted PCcM, which is approximately 6-fold higher than in the absence of PCcM. [125I] IGF-1 added to PCcM was bound by more than 90% to carrier proteins. The results confirm in cocultures strong mitogenic activation of HSC by PC. It is suggested that IGF-1 and respective IGF-binding proteins are of great importance in the mitogenic signal transfer between hepatocytes and hepatic stellate cells.

Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells (parasinusoidal lipocytes).

The molecular and cellular mechanisms of activation of fat-storing cells (Ito cells or parasinusoidal lipocytes), a prerequisite of the fibrogenic response of injured liver, were studied by analysis in vitro of some aspects of the intercellular communication between parenchymal liver cells and fat-storing cells. Conditioned medium harvested from early serum-free monolayer cultures of hepatocytes isolated from normal rat liver stimulated strongly, reproducibly and dose-dependently the proliferation of nonconfluent fat-storing cells maintained under serum-reduced conditions. During exposure of fat-storing cells for 48 hr to the conditioned medium, the incorporation of [3H]thymidine into DNA was stimulated four to six times over control values, the DNA content per culture well was elevated by 40% above control values and the immunocytochemical detection of bromodeoxyuridine-labeled cell nuclei was increased from 13% stained nuclei in controls to 70% stained nuclei in treated fat-storing cells. The mitogenic effects of hepatocyte-conditioned medium were similar to or even higher than those of 10% fetal calf serum. No mitoinhibitory activity could be detected in the hepatocyte-conditioned medium when arginase, as a potential inhibitor, was excluded. Rat skin fibroblasts could not be stimulated under conditions where the proliferation activity of fat-storing cells was greatly enhanced. The occurrence of the mitogenic activity in the medium is not dependent on de novo synthesis or secretion because the media of hepatocytes cultured under anoxic conditions in the presence of cycloheximide, brefeldin A or ethylenediaminetetraacetate were highly active in promoting fat-storing cell proliferation, although hepatocyte viability was greatly reduced under some of these conditions. A significant positive correlation (r = 0.95, p < 0.01) was found between lactate dehydrogenase activity and the mitogenic potency of the conditioned medium. The proliferation factor for fat-storing cells could also be demonstrated in the lysate of freshly isolated hepatocytes from normal liver. The stimulatory activity in the medium was partially enriched by a combination of gel permeation and anion exchange fast protein liquid chromatography and characterized as a protein with an apparent molecular weight of about 60 kD that is heat and pH sensitive but insensitive to reducing agents. It does not bind to immobilized heparin; nor does soluble heparin or proteinase inhibitor affect the mitogenic activity of the factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Frequency and clinical significance of DNA aneuploidy in acute leukemia.

Analyses of the cellular DNA content were carried out in 446 patients with newly diagnosed acute myeloid (AML) and lymphoblastic (ALL) leukemias in order to assess the frequency of DNA aneuploidies and its relation to immunologic and morphologic subtypes as well as its prognostic relevance. Based on high resolution FCM analyses and standardized reference measurements, DNA aneuploidies were identified at a similar frequency in children and adults with AML (38.0% and 40.0%) and ALL (40.0% and 37.4%). In AML aneuploid DNA stemlines were significantly less frequent in FAB-M 1 cases as compared to the other morphologic subtypes (p less than 0.05), whereas the degree of DNA aneuploidy was significantly lower in M 1 and M 2 leukemias as compared to the M 4 and M 5 subgroups (p less than 0.05). In ALL non-T/non-B and C-ALL revealed a higher frequency of DNA aneuploidies than T- and Null-ALL cases (p less than 0.05). No differences in the response to induction therapy were found between patients with and without DNA aneuploidy in children or adults with AML or ALL. In the childhood ALL trial BFM 79/81, however, a significantly higher frequency of long term remissions was observed in children with DNA aneuploidy (0.91 versus 0.66 at five years, p = 0.053). A similar though not significant tendency was also revealed from the AML studies BFM 78 in children and 78/81 in adults. In the subsequent studies these differences could not be confirmed at present, possibly because of the considerably shorter observation time.