Biomedical subjects

Jean Rosenbaum

Publications and source records attributed to Jean Rosenbaum.

At least 19 recordsLinked to original sources

PubMed2006

The ARE-associated factor AUF1 binds poly(A) in vitro in competition with PABP.

The ARE (AU-rich element) is a post-transcriptional element controlling both mRNA turnover and translation initiation by primarily inducing poly(A) tail shortening. The mechanisms by which the ARE-associated proteins induce deadenylation are still obscure. One possibility among others would be that an ARE-ARE-BP (ARE-binding protein) complex intervenes in the PABP [poly(A)-binding protein]-poly(A) tail association and facilitates poly(A) tail accessibility to deadenylases. Here, we show by several experimental approaches that AUF1 (AU-rich element RNA-binding protein 1)/hnRNP (heterogeneous nuclear ribonucleoprotein) D, an mRNA-destabilizing ARE-BP, can bind poly(A) sequence in vitro. First, endogenous AUF1 proteins from HeLa cells specifically bound poly(A), independently of PABP. Secondly, using polyadenylated RNA probes, we showed that (i) the four recombinant AUF1 isoforms bind poly(A) as efficiently as PABP, (ii) the AUF1 binding to poly(A) does not change when the polyadenylated probe contains the GM-CSF (granulocyte/macrophage-colony stimulating factor) ARE, suggesting that, in vitro, the AUF1-poly(A) association was independent of the ARE sequence itself. In vitro, the binding of AUF1 isoforms to poly(A) displayed oligomeric and co-operative properties and AUF1 efficiently displaced PABP from the poly(A). Finally, the AUF1 molar concentration in HeLa cytoplasm was only 2-fold lower than that of PABP, whereas in the nucleus, its molar concentration was similar to that of PABP. These in vitro results suggest that, in vivo, AUF1 could compete with PABP for the binding to poly(A). Altogether, our results may suggest a role for AUF1 in controlling PABP-poly(A) tail association.

Binding, Competitive↗

PubMed2006

Pravastatin reduces lung metastasis of rat hepatocellular carcinoma via a coordinated decrease of MMP expression and activity.

BACKGROUND/AIMS: Statins have beneficial effects in early pre-clinical models of hepatocellular carcinoma (HCC). Our aim was to test the efficacy of pravastatin on the progression of established HCC in rat, and to study its mechanisms. METHODS: HCC was induced with diethylnitrosamine and N-nitrosomorpholine. After 14 weeks, all rats developed HCC and then received pravastatin or its solvent for 10 weeks (10 rats/group). RESULTS: Liver tumor mass was lower in pravastatin group (PG) than control group (CG), as estimated from the number of liver tumors (p<0.004) and the liver weight/body weight ratio (p<0.04). Every CG rat surviving at 24 weeks (4/4) had lung metastasis, against only 5/8 in PG. Moreover, the percentage of lung surface occupied by metastasis was 10-fold smaller in PG than CG (p<0.016). Pravastatin decreased liver matrix metalloproteinase (MMP)-9 activity and mostly suppressed MMP-2 activation (p<0.004), likely because it decreased expression of MMP-14 and tissue inhibitor of matrix metalloproteinases-2 (p<0.01), required for MMP-2 activation. CONCLUSIONS: Pravastatin reduces progression and limits metastatic diffusion of established HCC. This could be linked to the decreased MMP activity. These results, obtained in a very aggressive HCC model, further suggest the potential benefit of statins in human HCC.

Animals↗

PubMed2006

Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices.

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 microM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-beta and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-beta and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.

Animals↗

PubMed2006

Halofuginone suppresses the lung metastasis of chemically induced hepatocellular carcinoma in rats through MMP inhibition.

Halofuginone, an inhibitor of collagen synthesis, appears to be a promising antitumoral drug in preclinical studies. We used a relevant rat model of autochthonous, chemically induced, spontaneously metastasizing hepatocellular carcinoma (HCC) to test the efficacy of halofuginone on tumor progression and matrix metalloproteinase (MMP) expression. Following sequential administration of diethylnitrosamine and N-nitrosomorpholine for 14 weeks, all animals developed HCC and then received halofuginone or its solvent for 10 weeks. The final number of liver tumors was lower in the halofuginone group than in the solvent group (57.2 +/- 4.6 vs 68 +/- 5.0; P < .01). The percentage of the lung surface infiltrated by metastasis was much smaller in the halofuginone group (0.3 +/- 0.2%) than in the solvent group (13.5 +/- 10.1%; P < .02). MMP-9 activity was decreased in the halofuginone group by 89% and 63% in non-neoplastic parts of the liver and tumor, respectively. The percentage of active MMP-2 was reduced by 90% in non-neoplastic parts of the liver and by 61% in tumors. This was likely subsequent to a decreased expression of both MMP-14 and tissue inhibitor of matrix metalloproteinase-2, which are required for pro-MMP-2 activation. These results, obtained from a clinically relevant model, further suggest the potential benefit of halofuginone in HCC.

Angiogenesis Inhibitors↗

PubMed2006

Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis.

Matrix metalloproteinase (MMP)-3 is a protease involved in cancer progression and tissue remodeling. Using immunofluorescence and immunoelectron microscopy, we identified nuclear localization of MMP-3 in several cultured cell types and in human liver tissue sections. Western blot analysis of nuclear extracts revealed two immunoreactive forms of MMP-3 at 35 and 45 kd, with the 35-kd form exhibiting caseinolytic activity. By transient transfection, we expressed active MMP-3 fused to the enhanced green fluorescent protein (EGFP/aMMP-3) in Chinese hamster ovary cells. We showed that EGFP/aMMP-3 translocates into the nucleus. A functional nuclear localization signal was demonstrated by the loss of nuclear translocation after site-directed mutagenesis of a putative nuclear localization signal and by the ability of the MMP-3 nuclear localization signal to drive a heterologous protein into the nucleus. Finally, expression by Chinese hamster ovary cells of EGFP/aMMP-3 induced a twofold increase of apoptosis rate, compared with EGFP/pro-MMP-3, which does not translocate to the nucleus. Increased apoptosis was abolished by site-directed mutagenesis of the catalytic site of MMP-3 or by using the MMP inhibitor GM6001. This study elucidates for the first time the mechanisms of nuclear localization of a MMP and shows that nuclear MMP-3 can induce apoptosis via its catalytic activity.

Amino Acid Sequence↗

PubMed2005

The grape-derived polyphenol resveratrol differentially affects epidermal and platelet-derived growth factor signaling in human liver myofibroblasts.

The grape-derived polyphenol resveratrol is anti-proliferative for human liver myofibroblasts, which may be beneficial for the treatment of liver fibrosis. However, its mechanism of action is ill understood. Here, we have studied how resveratrol interfered with signaling pathways used by epidermal or platelet-derived growth factors to induce the proliferation of these cells. We found that resveratrol inhibited epidermal growth factor or platelet-derived growth factor-induced DNA synthesis. Resveratrol did not, however, decrease epidermal growth factor receptor autophosphorylation or activation of extracellular regulated kinases, but strongly inhibited the phosphorylation of Akt and of its substrate forkhead related transcription factor. This suggested that resveratrol inhibited epidermal growth factor-induced mitogenic signaling through inhibition of the phosphatidylinositol 3-kinase /Akt pathway. The phosphatidylinositol 3-kinase inhibitor LY 294002, also, inhibited epidermal growth factor-dependent DNA synthesis and Akt phosphorylation but did not decrease extracellular regulated kinases phosphorylation. In contrast, resveratrol inhibited platelet-derived growth factor-stimulated receptor autophosphorylation and every subsequent signaling step. Resveratrol did not directly inhibit phosphatidylinositol 3-kinase activity measured on immunoprecipitates from epidermal growth factor-stimulated myofibroblasts, but it strongly reduced the autophosphorylation of the phosphatidylinositol 3-kinase downstream target phospho-inositide-dependent kinase-1 that phosphorylates Akt. We, thus, show that resveratrol has growth factor-specific effects: it inhibits platelet-derived growth factor signaling via reduced receptor activation, whereas it reduces epidermal growth factor-dependent DNA synthesis via inhibition of the phosphatidylinositol 3-kinase/Akt pathway, possibly through inhibition of phospho-inositide-dependent kinase-1 activity.

Cell Proliferation↗

PubMed2005

Expression of protease-activated receptors and tissue factor in human liver.

Thrombin, acting via protease-activated receptors (PARs), and tissue factor (TF) are involved in inflammation, tissue repair and tumorigenesis. Hepatocellular carcinomas (HCCs) usually complicate chronic liver diseases characterised by inflammation and fibrosis. The aim of this study was to describe the expression of PARs and TF in normal liver, cirrhosis and HCCs. We performed an immunohistochemical detection of PAR-1, PAR-3, PAR-4 and human TF in human tissue samples from 19 subnormal livers, 33 cirrhosis and 30 HCCs. PAR-1 was found on endothelial cells of sinusoids and larger vessels. In cirrhosis, spindle-shaped cells within septa and T lymphocytes were PAR-1 positive. A few PAR-1-positive tumour cells were found in 10% of HCCs. PAR-4 expression was restricted to macrophages, B lymphocytes and nerves. PAR-3 expression was rare. Unexpectedly, TF was expressed in 95% of normal livers and in 94% of cirrhosis but only in 50% of HCCs (p<0.001). Staining was mostly hepatocellular. No association existed between TF labelling and clinicopathological characteristics of HCCs. In conclusion, the pattern of expression of PARs is compatible with its role in chronic liver disease by promoting inflammation via immune cells and neurogenic stimulation. However, our data do not support a role for PARs or TF in HCC progression.

Carcinoma, Hepatocellular↗

PubMed2005

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice.

BACKGROUND/AIMS: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied. METHODS: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis. RESULTS: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice. CONCLUSIONS: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.

Animals↗

PubMed2005

Proteomic analysis of differentially expressed proteins in hepatocellular carcinoma developed in patients with chronic viral hepatitis C.

Hepatocellular carcinoma (HCC) is a major complication of chronic viral hepatitis C. Therapy for HCC is still disappointing. It is thus of great importance to identify novel HCC markers for early detection of the disease, and tumor-specific proteins as potential therapeutic targets. We have used a proteomic approach to identify new proteins involved in HCC development. Four cases of HCC developing from chronic viral hepatitis C were analyzed by two-dimensional electrophoresis (2-DE), and results were compared to those of paired adjacent non-tumorous liver tissues. For MS fingerprinting, protein spots with differential intensity between HCC and non-tumorous liver were directly cut out of gels and processed for MALDI-MS and nano-LC-ESI-MS/MS analysis. Approximately 850 spots were visualized in each gel. The comparative analysis of paired samples indicated that 345 protein spots showed significant differences in expression level between non-tumor and tumor tissue. Among the 345 protein spots analyzed, 238 spots corresponding to 155 different proteins were identified; 49 proteins were up-regulated, whereas 106 proteins were down-regulated. Among these 155 proteins, 91 proteins were regulated in at least three cases. Although 52 out of these 91 proteins have been already described by previous proteomic or transcriptomic studies, or are already known to be involved in hepatocarcinogenesis, this experiment revealed 39 new proteins differentially expressed in HCC developing from viral hepatitis C. Variations in protein accumulation were confirmed for two selected proteins (apolipoprotein E, chloride intracellular channel 1) by Western blotting in ten additional cases of HCC developing in patients with viral hepatitis C.

Aged↗

PubMed2005

[Platelet-associated CD154: a new interface in haemostasis and in the inflammatory reaction].

Blood platelets play a crucial part in the blood clotting process by forming the platelet plug. Recent evidence indicates that they are likely to play a key role in the inflammatory reaction via CD154/CD40 interactions. CD40 was known to be widely expressed, for instance on cells of the vasculature including endothelial cells, smooth muscle cells and macrophages. It was also known that the triggering of CD40 on these cells led to the acquisition of an activated pro-inflammatory and pro-coagulant phenotype. It was subsequently shown that platelets express CD154 which is cryptic in unstimulated platelets but is expressed at the platelet surface upon platelet activation. When expressed at the platelet surface and exposed to CD40-expressing vascular cells, the platelet-associated CD154 triggers a variety of pro-inflammatory and pro-coagulant responses including induction of adhesion receptors, release of cytokines and chemokines, induction of tissue factor and of metalloproteinases. Platelet-associated CD154 is also involved in platelet/platelet interactions during platelet aggregation. Furthermore, in vivo models have emphasized the critical role of the platelet-associated CD154 in the progression of atherosclerotic disease and in the stabilization of arterial thrombi. Recent data show that CD40-bearing cells involved in fibrosis such as hepatic stellate cells and glomerular mesangial cells also respond to platelet-associated CD154, thus suggesting a new mechanism by which platelets may be instrumental in the inflammatory diseases of the liver or the kidney. Finally, platelet-associated CD154 has been shown to have immune competence both in vitro and in vivo, observations that open new fields of research on the potential implications of platelets in the immune response and auto-immune diseases.

B-Lymphocytes↗

PubMed2004

Expression of leukemia inhibitory factor (LIF) and its receptor gp190 in human liver and in cultured human liver myofibroblasts. Cloning of new isoforms of LIF mRNA.

BACKGROUND: The cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver. RESULTS: LIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4. CONCLUSIONS: We show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation.

Journal Article↗

PubMed2004

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver.

PURPOSE: To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs). MATERIALS AND METHODS: This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically. RESULTS: SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48 hours with 50 microg of iron per milliliter was 50 sec(-1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability (P > .27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100 microg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs. CONCLUSION: MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver.

Adipocytes↗

PubMed2004

Expression and activity of the cytolethal distending toxin of Helicobacter hepaticus.

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.

Amino Acid Sequence↗

PubMed2004

Expression of fibrillin-1 in focal nodular hyperplasia of the liver: a role in microcirculation adaptability.

INTRODUCTION: It has been suggested that the elastic network plays an important role in the tissue response to mechanical stress. The components of the elastic network have been poorly studied in liver diseases. Therefore, in this work, the expression and distribution of fibrillin-1 and elastin were studied in hepatic focal nodular hyperplasia and compared with surrounding liver and hepatocellular adenoma. METHODS: Immunohistochemical studies for fibrillin-1 and elastin were performed on unfixed cryostat sections of focal nodular hyperplasia (22 cases), hepatocellular adenoma (15 cases) and surrounding liver (34 cases). RESULTS: Surrounding normal liver showed only a continuous, thin and regular immunostaining of fibrillin-1 in the space of Disse, whereas elastin was nearly absent. In focal nodular hyperplasia, fibrillin-1 was more strongly expressed in the perisinusoidal space, compared with surrounding liver; in contrast, in adenomas fibrillin-1 immunostaining was irregular and very low in perisinusoidal space, more intense in peliotic areas. CONCLUSIONS: In focal nodular hyperplasia, the increased microfibrillar network containing fibrillin-1 in the space of Disse could reflect an adaptation of the sinusoidal wall to an increased arterial blood flow in sinusoids. In hepatocellular adenoma, the different patterns of fibrillin-1 could be related to the heterogeneity of the arterial vascularization and to the frequent necrotico-hemorrhagic changes. This study comparing the elastic network in two types of lesions with vascularization abnormalities and in the surrounding liver provides interesting new data for understanding the structural role of fibrillin-1 in the space of Disse.

Journal Article↗

PubMed2004

Innervation of the sinusoidal wall: regulation of the sinusoidal diameter.

In the livers of humans, cats, guinea pigs, and tupaia, nerve endings are distributed all over the hepatic lobules. Nerve endings in the intralobular spaces are localized mainly in the Disse spaces and are oriented toward the hepatic stellate cells (HSCs), sinusoidal endothelial cells, and hepatocytes. They are especially closely related to HSCs. Various neurotransmitters such as substance P exist in the nerve endings. In addition, HSCs possess endothelin (ET) and adrenergic receptors and contract in response to the corresponding agonists. In contrast, nitric oxide (NO) inhibits the contraction of HSCs. HSCs thus appear to be involved in the regulation of hepatic sinusoidal microcirculation by contraction and relaxation. In the cirrhotic liver, intralobular innervation is decreased, but ET, ET receptors, and NO are overexpressed in the HSCs. These findings indicate that HSCs in cirrhotic liver may play an important role in the sinusoidal microcirculation through agents such as ET or NO rather than through intralobular innervation.

Animals↗

PubMed2004

Cellular retinol-binding protein-1 expression in normal and fibrotic/cirrhotic human liver: different patterns of expression in hepatic stellate cells and (myo)fibroblast subpopulations.

BACKGROUND/AIMS: Cellular retinol-binding protein-1 (CRBP-1) which is involved in vitamin A metabolism is highly expressed in liver cells, particularly in hepatic stellate cells (HSCs). In this work, the CRBP-1 expression was studied by immunohistochemistry in the different liver cell populations, including HSCs and portal fibroblasts, of normal liver and of fibrotic and cirrhotic liver. METHODS: Normal liver, fibrotic liver in different stages and cirrhotic liver sections were studied. Immunohistochemistry was performed using antibodies against CRBP-1, alpha-smooth muscle actin (SMA), CD 68 and CD 34. RESULTS: In normal liver, quiescent HSCs expressed CRBP-1, while portal fibroblasts did not. In fibrotic or cirrhotic liver, activated HSCs co-expressed CRBP-1 and alpha-SMA; a variable proportion of portal and septal (myo)fibroblasts, more important in cirrhosis, neo-expressed both CRBP-1 and alpha-SMA. Biliary epithelial cells both in normal and pathological situations expressed CRBP-1. Neither Kupffer cells, nor endothelial cells showed CRBP-1 expression. CONCLUSIONS: Our study demonstrates that CRBP-1 is a good marker to identify HSC in normal human liver. Furthermore, in fibrotic or cirrhotic liver, the different patterns of expression for CRBP-1 and alpha-SMA allow the distinction of different subsets of fibroblastic cells involved in fibrogenesis and septa formation.

Actins↗

PubMed2004

Fibrillin-1 expression in normal and fibrotic rat liver and in cultured hepatic fibroblastic cells: modulation by mechanical stress and role in cell adhesion.

Fibrillin-1, together with elastin, is the main component of elastic fibers found throughout the extracellular space and responsible for the biomechanical properties of most tissues and organs. In this work, fibrillin-1 expression and modulation were explored in experimental rat liver fibrosis and in vitro; furthermore, the role of fibrillin-1 fragments on cell adhesion was analyzed. Fibrosis was induced by subjecting rats to common bile duct ligation for 72 h and 7 days or carbon tetrachloride (CCl(4)) treatment for 2 and 6 weeks. Immunohistochemistry showed that, after bile duct ligation, fibrillin-1, elastin, and alpha-smooth muscle actin colocalized in the developing portal connective tissue. In CCl(4)-treated animals, a similar colocalization was observed in septa; however, elastin deposition was not observed around activated alpha-smooth muscle actin-positive stellate cells of the parenchyma. Treatment with the profibrogenic mediator transforming growth factor-beta1 (TGF-beta1) greatly increased the fibrillin-1 expression of cultured liver fibroblasts. The level of fibrillin-1 expression was significantly higher in cells grown in restrained (stressed) collagen lattices compared with those grown in unrestrained collagen lattices. Cell adhesion on the C-terminal fragment of fibrillin-1 containing the RGD sequence (rF6H) slightly increased (between 0.3 and 2.5 microg/ml) and decreased at higher concentrations, while adhesion on the N-terminal fragment of fibrillin-1 (rF16) was dose-dependently decreased. In addition, the rF16 fragment decreased cell adhesion to fibronectin. In conclusion, our study illustrates the important deposition of fibrillin-1 that occurs in two mechanistically distinct settings of liver fibrogenesis. Furthermore, the induction of fibrillin-1 expression by TGF-beta1 and mechanical stress, and the antiadhesive properties of fibrillin-1 fragments suggest important implications for physiological and pathological fibrillin-1 catabolism during tissue remodeling.

Actins↗

PubMed2004

Expression of tissue factor pathway inhibitor-2 in murine and human liver regulation during inflammation.

Tissue factor pathway inhibitor-2 (TFPI-2) is a recently described serine proteinase inhibitor. Human and murine TFPI-2 share about 50% homology. The aim of this study was to investigate the cellular localization of human and murine TFPI-2 in the liver and the regulation of their expression during acute inflammation. Northern blot, in situ hybridization and studies on isolated hepatocytes demonstrated a high-level expression of TFPI-2 in murine hepatocytes. On the other hand, very little TFPI-2 mRNA expression could be detected in human liver. Studies with isolated human liver cells suggested that TFPI-2 expression in human liver was mainly observed in liver sinusoidal endothelial cells rather than hepatocytes. Liver murine TFPI-2 expression was greatly increased after lipopolysaccharide administration with a delayed kinetics as compared to alpha1-acid glycoprotein, a classical acute-phase reactant. Accordingly, studies with isolated cells showed that the increase in TFPI-2 transcripts occurred in non-hepatocytic cells. Moreover, the LPS response was abolished in mice with a hepatocyte-specific KO for the gp130 receptor, thus indicating that a mediator from hepatocytes is involved in the up-regulation of TFPI-2 in non-parenchymal cells. In conclusion, murine TFPI-2 is highly expressed in hepatocytes in the normal murine liver and is upregulated in non-parenchymal cells in the context of inflammation. The large difference in the level of liver expression of human and murine TFPI-2 suggests that despite significant sequence similarities, these proteins presumably have different functions in the two species.

Acute-Phase Reaction↗