Abstract: Background & Aims: We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect. Methods: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3). Results: In vitro, the growth of GL2 - Dox, GL2 + Dox, and R2 - Dox cells was undistinguishable whereas that of R2 + Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid p-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2 + Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2 - Dox, GL2 + Dox, and R2 - Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation. Conclusions: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Abstract: AIM: To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells. METHODS: Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS: Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a close- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin. CONCLUSION: These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases. (C) 2010 Baishideng. All rights reserved
Abstract: Hepatocellular carcinogenesis is usually the result of a muti-step process. It begins with an exposure to various risk factors; followed by the development of a chronic hepatitis and cirrhosis that is a pre-neoplastic step; and finally after the occurrence of an hepatocellular carcinoma (HCC), different molecular events control aggressiveness of the tumors. The aim of this work was to identify in the international context, forces and priorities of the fundamental and translational HCC research.
Abstract: Reptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (El). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin. Conclusion: Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry. (HEPATOLOGY 2009;50:1871-1883.)
Abstract: Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl4) toxicity was similar in wildtype (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl4 or its solvent for 6 wk (300 mu l/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl4 treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.
Abstract: Studies in model organisms or cultured human cells suggest. potential implications in carcinogenesis for the AAA+ ATPases Pontin and Reptin. Both proteins are associated with several chromatin-remodeling complexes and have many functions including transcriptional regulation, DNA damage repair, and telomerase activity. They also interact with major oncogenic actors such as beta-catenin and c-myc and regulate their oncogenic function. We only now begin to get. insight into the role of Pontin and Reptin in human cancers.
Abstract: AIM: To test the effect of a standardized red wine polyphenolic extract (RWPE) on the phenotype of human liver myofibroblasts in culture. METHODS: Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Signaling events were analyzed by western blot with phospho-specific antibodies. Matrix-metalloproteinase activity was measured with gel zymography. RESULTS: We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor 1313 (PDGF-BB). Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix-metalloproteinases-1. CONCLUSION: Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis. (C) 2008 WJG. All rights reserved.
Abstract: 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. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Abstract: Mutations in multidrug resistance 3 gene (MDR3 or ABCB4) underlie progressive familial intrahepatic cholestasis type 3 (PFIC3), a severe pediatric liver disease progressing to cirrhosis. Abcb4-/- mice exhibit slowly developing hepatic lesions that can be accelerated by feeding a cholic acid (CA)-supplemented diet. We investigated the beneficial effects of a soybean lecithin (L)supplemented diet in this model of liver disease. Abcb4-/- mice and wild-type (WT) controls were divided in four groups by the diet they were fed: control (C) diet, L-supplemented diet, CA-supplemented diet, and L- and CA-supplemented (L+CA) diet. After 2 wk on these regimens, liver enzymes and bilirubin were measured in serum with bile flow, total bile acids, and cholesterol (CHOL) and phospholipid (PL) concentrations in bile. Ductular hyperplasia, portal fibroblastic cell proliferation, myofibroblast activation, and hepatic fibrosis were quantified on liver sections. Abcb4-/- mice fed the C diet exhibited mild liver damage. CA produced very high elevations of serum liver enzymes and bilirubin with significant bile duct proliferation, peribiliary fibroblast activation, and fibrosis. The L-supplemented diet dramatically mitigated the hepatic damage in CA-supplemented diet animals. We conclude that L is protective against liver disease in Abcb4-/- mice and suggest that it could offer potential benefit in PFIC3.
Abstract: Aims - The aim of this study was to evaluate the serum pattern of cytokines evolution after surgical radiofrequency ablation (SRFA) of colorectal metastases. Methods - Metastases of ten non consecutive patients were destroyed by radiofrequency ablation without concomitant resection after a complete surgical procedure including a laparotomy, a peritoneal examination, liver mobilisation and liver ultrasound. Serum levels of IL-6, TNF alpha, HGF, VEGF, bFGF, TGF beta 1 and CRP were assessed by ELISA assays at different time points. Results - TNF alpha and bFGF remained undetectable. IL-6 peaked at 3 hours and remained elevated during the entire study period. HGF increased by three-fold by Day 1 then decreased until Day 7 where it was still twice its baseline level. VEGF level increased from Day 5 onward. TGF beta 1 did not show significant variations. CRP was increased throughout the study. Conclusions - In contrast with cryotherapy, SRFA does not lead to high serum TNF alpha suggesting a better tolerance. Nevertheless high IL-6, HGF and VEGF serum levels are characteristic of a general inflammatory stress which should be taken into account.
Abstract: Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E-2 and its EP2 receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.
Abstract: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with P-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gamma c mice (P = 0.016). Conclusion: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.
Abstract: 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. (C) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Abstract: 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.
Abstract: 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 mu M 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.
Abstract: 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.
Abstract: 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.
Abstract: 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. (c) 2005 Elsevier Ltd. All rights reserved.
Abstract: 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 PABR 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.
Abstract: 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.
Abstract: 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.
Abstract: 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-I 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. (C) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Abstract: 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 alpha(1)-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 gp 130 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.
Abstract: Transforming growth factor (TGF)-beta1 is a major mediator of liver fibrosis. Connective tissue growth factor (CTGF) mediates TGF-beta1 pro-fibrogenic effects in vitro, but its in vivo role is unknown. Both TGF-beta1 and CTGF are overexpressed in hepatic stellate cells during liver fibrosis. We have used antisense oligonucleotides to examine the role of CTGF in carbon tetrachloride-induced liver fibrosis in mice. Mice received carbon tetrachloride together with CTGF or TGF-beta1 antisense oligonucleotides for 2 weeks (preventive model), or carbon tetrachloride for 2 weeks followed by carbon tetrachloride and oligonucleotides for 2 more weeks (curative model). In both models, CTGF and TGF-beta1 oligonucleotides decreased by more than 50 percent the mRNA expression of their targets. Type I collagen mRNA was also decreased by about 40 percent in the preventive experiment. Tissue inhibitor of matrix metalloproteinase-1 mRNA expression and fibrotic deposition evaluated by Sirius red staining were not modified in any group. In summary, our results suggest that hepatic stellate cells can be targeted in vivo with oligonucleotides, and that reducing CTGF levels can lead to a decrease in fibrogenesis as shown by the reduction in type I collagen expression. The lack of effect on fibrosis may be due to the persistence of high tissue inhibitor of matrix metalloproteinase-1 expression.
Abstract: 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 mug 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 mug 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. (C) RSNA, 2004.
Abstract: 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. hepaticits Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticits CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB Mutation. These results suggest that the effect of H. hepaticits CDT probably involves the DNase activity of CdtB. (C) 2004 Elsevier Inc. All rights reserved.
Abstract: The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin,A., Bedin, C., Balabaud, C., Foster, D. C., Desmouliere, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.
Abstract: Background: Somatostatin analogues have been used with conflicting results to treat advanced hepatocellular carcinoma (HCC). The aim of this study was to investigate expression of somatostatin receptor ( SSTR) subtypes in human liver, and to examine the effect of selective SSTR agonists on proliferation, apoptosis, and migration of hepatoma cells (HepG2, HuH7) and hepatic stellate cells (HSCs). Methods: Expression of SSTRs in cell lines, normal and cirrhotic liver, and HCC was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. Effects of SSTR agonists on proliferation and apoptosis of tumour cells and HSCs were assessed by the 5-bromo-2' deoxyuridine and TUNEL methods, respectively. The influence of SSTR agonists on migration was investigated using Boyden chambers. Results: In normal liver, both hepatocytes and HSCs were negative for all five SSTRs. Cirrhotic liver and HCC as well as cultured hepatoma cells and HSCs expressed all five SSTRs, both at the protein and mRNA levels, except for HuH7 cells which did not immunoreact with SSTR3. None of the agonists influenced proliferation or apoptosis. However, compared with untreated cells, L-797,591, an SSTR1 agonist, reduced migration of HepG2, HuH7, and HSCs significantly to 88 (7)% ( p< 0.05), 83 (11)% (p< 0.05), and 67 (13)% ( p< 0.01), respectively. Conclusions: Cirrhotic liver and HCC express SSTRs. Although the somatostatin analogues used in this study did not affect proliferation and apoptosis, stimulation of SSTR1 may decrease invasiveness of HCC by reducing migration of hepatoma cells and/or HSCs. Clinical trials evaluating somatostatin analogues for the treatment of HCC should take these findings into account.
Abstract: 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. (C) 2004 Wiley-Liss, Inc.
Abstract: Background/Aim: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. Methods: Fibrosis was induced in rats by administration of CCl4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin ( ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. Results: After three weeks of CCl4, treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p=0.03) and the expression of TIMP-1 mRNA by 52% (p=0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl4, treatment with SSR182289 resulted in a significant decrease in fibrosis (-30%, p=0.04) and ASMA positive areas (-35%, p=0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl4 as shown by measuring levels of serum aminotransferases and the area of necrosis. Conclusions: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.
Abstract: 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 (CCl4) 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 CCl4-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 mug/ml) and decreased at higher concentrations, while adhesion on the IN-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.
Abstract: Trans-resveratrol, a phenolic compound present in wine, has been reported to be a potential cancer chemopreventive agent. However, although it has numerous biological activities in vitro, there are few data about its bioavailability and tissue distribution in vivo. The objectives of this study were to investigate the absorption and tissue distribution of C-14-trans-resveratrol following oral administration to mice. Male Balb/c mice were given a single oral dose of C-14-trans-resveratrol and were sacrificed at 1.5, 3 or 6 h postdose. The distribution of radioactivity in tissues was evaluated using whole-body autoradiography, quantitative organ-level determination and microautoradiography. In addition, identification of radioactive compounds in kidney and liver was done with high-performance liquid chromatography. Autoradiographic survey of mice sections as well as radioactivity quantification in various organs revealed a preferential fixation of C-14-trans-resveratrol in the organs and biological liquids of absorption and elimination (stomach, liver, kidney, intestine, bile, urine). Moreover, we show that C-14-trans-resveratrol derived radioactivity is able to penetrate the tissues of liver and kidney, a finding supported by microautoradiography. The presence of intact C-14-trans-resveratrol together with glucurono- and/or sulfoconjugates in these tissues was also shown. This study demonstrates that trans-resveratrol is bioavailable following oral administration and remains mostly in intact form. The results also suggest a wide range of target organs for cancer chemoprevention by wine polyphenols in humans. (C) 2003 Elsevier Science Inc. All rights reserved.
Abstract: Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects, of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependant manner (50-500 muM). Cell viability was significantly decreased by TLC and TCDC at 500 muM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases. (C) 2002 Elsevier Science Inc. All rights reserved.
Abstract: The membrane type-1 matrix metalloproteinase (MT1-MMP), a protease originally identified in breast carcinoma, is characterized by its capacity to Activate other MMPs (MMP-2 and MMP-13) and to degrade extracellular matrix. Our study was undertaken to localize and identify the MT1-MMP expressing cells in human breast adenocarcinomas. A textural analysis of images obtained by immunohistochemistry and in situ hybridization showed precisely the co-expression of alpha smooth muscle actin (alphaSM actin) and MT1-MMP in myofibroblasts. MT1-MMP expression is confined to myofibroblasts in close contact with tumor cells. In sharp contrast, the expression of MMP-2 was more widely distributed in both aSM actin positive and negative cells close to and at distance from cancer cell clusters. Our in vitro observations are consistent with the higher level of MT1-MMP expression and of MMP-2 activation observed in alphaSM actin positive fibroblasts derived from breast tumors, at compared to normal breast fibroblasts. Collectively, these results implicate myofibroblasts as major producer of MT1-MMP in breast cancer and emphasize the importance of stromal-epithelial cell interactions in their progression. (C) 2003 Wiley-Liss, Inc.
Abstract: It has been known for a long time that portal fibrosis consecutive to experimental common bile duct ligation is reversible following obstacle removal, but the mechanisms involved remain unknown. We have studied the effect of bilioduodenal anastomosis and of simple biliary decompression on the remodeling of the lesion in bile duct-ligated rats. Rats were subjected to common bile duct ligation for 7 days or 14 days. Bilioduodenal anastomosis was performed after 14 days of bile duct ligation and animals sacrificed at intervals. In other animals, after 7 days or 14 days of ligation, the common bile duct was merely decompressed by bile aspiration and animals sacrificed 24 h later. Collagen deposition, alpha-smooth muscle actin expression and apoptosis were evaluated. Bile was collected and the bile acid profile assessed. After anastomosis, collagen deposition and alpha-smooth muscle actin expression decreased and were back to control values after 7 days. These parameters remained practically unchanged 24 h after biliary decompression. Bile duct ligation by itself induced apoptosis of some fibroblastic and bile ductular cells after 7 days; this was back to normal after 14 days. After anastomosis or decompression, apoptosis of both fibroblastic and bile ductular cells increased greatly and was accompanied by ultrastructural features of extracellular matrix degradation. Total bile acid content decreased after common bile duct ligation, the proportion of dihydroxylated bile acids decreasing and that of trihydroxylated bile acids increasing. Biliary decompression and anastomosis did not modify total concentration and composition of the biliary bile acid pool. In summary, we show that mere biliary decompression, by relieving the mechanical stress, is as effective as bilioduodenal anastomosis to induce apoptosis of portal cells that likely triggers portal fibrosis regression.
Abstract: Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts. the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core. a structural protein. and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.
Abstract: Fibrillin-1, one of the main constituents of microfibrils, is present in normal adult liver and overexpressed in fibrotic area around cirrhotic nodules and hepatocellular carcinoma. In this work fibrillin-l expression was studied by immunohistochemistry in liver samples from children with various cholestatic diseases corresponding to paucity of intrahepatic bile ducts, biliary atresia, congenital hepatic fibrosis, Byler's disease, mitochondrial cytopathy, sclerosing cholangitis, or choledochal cyst. As controls, histologically normal liver samples were used. In control liver, as in adult, fibrillin-1 was expressed in vessel walls, sinusoids, and portal connective tissue, particularly at the interface with the limiting hepatocytic plate and close to the basement membrane of bile ducts. In paucity of intrahepatic bile ducts without fibrosis, the fibrillin-1 distribution was similar to controls. In cholestatic diseases associated with severe fibrosis, such as biliary atresia, congenital hepatic fibrosis, Byler's disease, mitochondrial cytopathy, or sclerosing cholangitis, an enhanced deposition of fibrillin-1 was observed in portal connective tissue and fibrous septa. The strong fibrillin-1 expression close to the basement membrane of biliary structures was lost in cholestatic diseases, except biliary atresia. Finally, in normal and pathologic tissues, fibrillin-1 was co-localized with its putative receptor alphaVbeta3 in sinusoids but not around biliary structures.
Abstract: Matrix metalloproteinases (MMPs) are a family of proteases that degrade extracellular matrix components and are involved in tumor progression and metastasis. We studied the pattern of expression of MMP-2 by human hepatoma cell lines and human hepatic myofibroblasts and its regulation in co-culture between these cell types. MMP-2 expression was studied by zymography and semi-quantitative RT-PCR. The expression of MT1-MMP (involved in MMP-2 activation) was studied by Western blot analysis and RT-PCR and the secretion of TIMP-2 by ELISA and RT-PCR. Myofibroblasts expressed high levels of MMP-2, MT1-MMP and TIMP-2. The hepatoma cell lines HepG2, HuH7 and Hep3B did not express MMP-2 and weakly expressed TIMP-2 and MT1-MMP. In co-culture, no modulation of MT1-MMP or TIMP-2 expression was observed. A strong decrease in myofibroblast MMP-2 expression was seen in the presence of hepatoma cell lines. This was partly reproduced by their conditioned media. We conclude that hepatoma cell lines dowri-regulate the expression of MMP-2 by myofibroblasts.
Abstract: The effect of adrenergic innervation and/or circulating catecholamines on the function of liver fibrogenic cells is poorly understood. Our aim was to investigate the effects of noradrenergic antagonism on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Two weeks of CCl4 induced a similar to5-fold increase in the area of fibrosis as compared with controls. The addition of 6-hydroxydopamine (OHDA), a toxin that destroys noradrenergic fibers, decreased fibrosis by 60%. After 6 weeks Of CCl4, the area of fibrosis increased about 30-fold in CCl4-treated animals and was decreased by 36% with OHDA. At 2 weeks, OHDA abrogated the CCl4-induced increase in mRNA level of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), an inhibitor of extracellular matrix degradation, and it greatly reduced it at 6 weeks. Finally, when rats treated with CCl4 for 2 weeks also received prazosin, an antagonist of alpha(1)-adrenergic receptors, fibrosis was decreased by 83%. In conclusion, destruction of noradrenergic fibers or antagonism of noradrenergic signaling through alpha(1) receptors inhibited the development of liver fibrosis. Because adrenoreceptor antagonists have a very sound safety profile, they appear as attractive drugs to reduce liver fibrogenesis.
Abstract: Intra-hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF-induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose-dependently decreased HGF-induced invasion of HepG2 cells with a maximum of 82.7 +/- 13.3% at 20 muM. TIMP-2, a natural inhibitor, decreased invasion up to 51.2 +/- 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT-PCR. MMPs 1, 7-9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP-2 and MMP-13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP-3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF-stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose-dependently stimulated the secretion of pro-MMP-3 in the culture medium. The role of MMP-3 in HGF-induced invasion was directly confirmed by using an antibody to MMP-3, that blocked invasion. Finally, RT-PCR demonstrated MMP-3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP-3, mediate HGF-induced invasion of HCC cells. The in vivo expression of MMP-3 in HCC suggests a role for this protease in HCC progression. (C) 2002 Wiley-Liss, Inc.
Abstract: Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. lmmunohistochemical staining was performed for CRBP-1 and alpha-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and alpha-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic alpha-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and alpha-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when alpha-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and alpha-SM actin expression. In both cell populations, transforming growth factor-beta1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.
Abstract: During liver fibrogenesis or long term culture. hepatic stellate cells (HSCs) evolved from ''quiescent" to activated phenotype called "myofibroblast-like". a transition prevented by retinoic acid (RA). Little is known about RA generation by HSCs. Our study aimed to check the ability of these cells to produce RA from retinol (Rol) and the alterations of this metabolic step by ethanol. To study this metabolic pathway, primary Cultures of HSCs represent the most physiological model but technically suffer several drawbacks. To circumvent these problems, an immortalized rat HSC line (named PAV-1) has been established. We validated PAV-1 cell line as a convenient model to study retinoids metabolism by HSCs. Then, we showed that PAV-1 cells express Rol-binding proteins (RBPs), enzymes and nuclear receptors involved in RA signaling pathway. We also demonstrated in situ generation of functional all-trans-RA (ATRA), using transient transfections with a RA-sensitive reporter gene. in situ modulation of tissue transglutaminase (tTG) activity and HPLC experiments. This production was Rol dose-dependent 4-methylpyrazole. citral, and ethanol-inhibited which argues in favor of an enzymatic process. In conclusion. v, e first demonstrate in situ RA generation from Rol in a newly immortalized rat HSC line, named PAV-1. Inhibition of RA production by ethanol in PAV-1 and recent data. suggesting fundamental role of RA to prevent fibrosis development in the liver. allow us to hypothesize that Rol metabolism could be a primary target for ethanol during development of hepatic fibrosis. (C) 2002 Elsevier Science Ltd. All rights reserved.
Abstract: Hepatic fibrosis is a scaring process leading to cirrhosis, a major complication of numerous chronic liver diseases. Hepatic stellate cells play a central role in the fibrotic process, After parenchymal or biliary injury, cytokines and growth factors allow the recruitment, proliferation, and activation, of stellate cells toward myofibroblasts, which secrete the extracellular matrix, Fibrosis, resulting from the failure of the balance between synthesis and degradation of extracellular matrix, is an evolutive and potentially reversible process. Histological examination is the main investigation to quantify fibrosis. Serological tests are warranted to allow a non invasive follow up of patients. Development of antifibrotic therapies should soon permit to slow down the evolution toward cirrhosis, limiting the needs for hepatic transplantation. (C) 2002 Editions scientifiques et medicales Elsevier SAS.
Abstract: Background/Aims: We have shown that hepatocyte growth factor, secreted by human liver myofibroblasts, promoted in vitro invasion of human hepatocellular carcinoma cell lines. The aim of this work was to measure hepatocyte growth factor expression in 29 human hepatocellular carcinomas and the corresponding peri-tumoral livers. Methods: We used reverse transcription-polymerase chain reaction, in situ hybridization, ELISA and Western blot. Results: Sixty-two of tested hepatocellular carcinomas were positive by reverse transcription-polymerase chain reaction. With in situ hybridization, a signal was found in every sample. In many cases, the signal was localized in cells labeled with an anti-smooth muscle alpha -actin antibody, while hepatocytes were mostly non-labeled. ELISA, performed in 15 pairs of hepatocellular carcinomas and surrounding livers, detected hepatocyte growth factor in every sample with wide variations. Hepatocellular carcinomas that had developed in non-cirrhotic livers contained essentially the same amount of hepatocyte growth factor as the matching non-tumoral liver, In cirrhotic livers, the hepatocyte growth factor content of the tumors was significantly lower than that of the surrounding cirrhotic livers. Conclusions: These data indicate that hepatocyte growth factor is expressed at significant levels in every hepatocellular carcinoma tested and that its expression takes place in the stromal myofibroblasts. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
Abstract: Background: The expression and the distribution of fibrillin-1 and elastin were studied in normal and pathological human liver samples. Methods: As controls, histologically normal/subnormal liver samples (n = 24) were used. Pathological samples corresponded to seven cirrhosis and eight hepatocellular carcinomas (HCC) developed on cirrhotic (four) or non-cirrhotic (four) liver. Results: In normal liver, fibrillin-1 and elastin co-localized in vessel walls and portal tract connective tissue. Fibrillin-1 alone was detected along sinusoids and in portal spaces at the interface with the limiting hepatocytic plates and close to the basement membrane of bile ducts. By transmission electron microscopy, typical bundles of microfibrils were detected both in Disse space and in portal zones, Cirrhotic nodules were usually rich in fibrillin-1 along sinusoids; fibrillin-1 and elastin were co-localized in fibrotic septa surrounding nodules. In HCC, fibrillin-1 was present between tumoral hepatocytes; stromal reaction around the tumors contained both fibrillin-1 and elastin. Conclusions: Fibrillin-1 was associated with elastin in portal mesenchyme and vessel walls of normal liver, in fibrotic septa around cirrhotic nodules and stromal reaction around HCC, but was expressed alone in the perisinusoidal space. The functional roles for fibrillin-1 in non-elastic tissues, such as the liver, remain to be elucidated. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.
Abstract: We have shown that liver myofibroblasts stimulate in vitro invasion of hepatocellular carcinoma cell lines through a hepatocyte growth facror/urokinase-dependent mechanism. Resveratrol, a grapevine-derived polyphenol, has been shown to inhibit cellular events associated with tumor initiation, promotion and progression. The aim of this study was to evaluate the effects of trans-resveratrol on invasion of the human hepatoma cell line HepG2. Cell invasion was assessed using a Boyden chamber assay. Activation of the HGF signal transduction pathways was evaluated by Western blot with phospho-specific antibodies. Urokinase expression was measured by RT-PCR and zymography. Trans-resveratrol decreased hepatocyte growth factor-induced cell scattering and invasion. It also decreased cell proliferation without evidence for cytotoxicity or apoptosis. Trans-resveratrol did not decrease the level of the hepatocyte growth factor receptor c-met and did not impede the hepatocyte growth factor-induced increase in c-met precursor synthesis. Moreover, trans-resveratrol did not decrease hepatocyte growth factor-induced c-met autophosphorylation, or Akt-1 or extracellular-regulated kinases-l and -2 activation. Finally, it did not decrease urokinase expression and did not block the catalytic activity of urokinase. In conclusion, our results demonstrate that trans-resveratrol decreases hepatocyte growth factor-induced HepG2 cell invasion by an as yet unidentified post-receptor mechanism.
Abstract: Hepatitis C virus is a major risk factor for hepatocarcinogenesis in humans. In situ detection of the virus in early sequential lesions of hepatocarcinogenesis could provide information about the role of the virus in the transformation and promotion process. Parallel in situ detection of HCV proteins and RNA in human tissues were performed in 55 posthepatitis C cirrhosis, 17 dysplastic nodules (DN), and 25 hepatocellular carcinomas (HCC), using inmnunohistochemistry and tissue quantitative RT-PCR A consistent cytoplasmic hepatocellular staining was obtained in 73% of cirrhosis cases (with or without HCC) and in 55% DN cases. A few tumoral hepatocytes were unambiguously stained in 28% HCC. The percentage of positive cells and the intensity of immunostaining significantly decreased from cirrhosis to HCC through DN, whereas there was no difference in the prevalence of positivity or the number of viral copies between cirrhosis and HCC using tissue-quantitative RT-PCR, Finally, RT-PCR levels were found parallel with the immunostaining in cirrhosis but not in HCC. These results suggest that HCV protein synthesis may persist but be down-regulated during sequential hepatocarcinogenesis. A putative role of HCV proteins on cell proliferation and differentiation during the early steps of carcinogenesis cannot therefore be excluded.
Abstract: Liver myofibroblasts are major actors in the development of liver fibrosis and cancer progression. There is a large interest in drags that might deactivate these cells. Many studies have shown that the grapevine-derived polyphenol, trans-resveratrol, and other stilbenes have therapeutic potential in some diseases. In this work, we have studied the effect of grapevine polyphenols on cultured human liver myofibroblasts. We have shown that trans-resveratrol profoundly affects myofibroblast: phenotype. Trans-resveratrol induced morphological modifications. It markedly reduced proliferation of myofibroblasts in a dose-dependent manner. Trans-resveratrol also decreased the expression of a smooth muscle actin (alpha-SMA) without affecting vimentin or beta-cytoplasmic actin expression. It decreased myofibroblast migration in a monolayer wounding assay. We also showed chat trans-resveratrol inhibited the messenger RNA (mRNA) expression of type I collagen. Finally it decreased the secretion of matrix metalloproteinase 2 (MMP-2). We conclude that trans-resveratrol can deactivate human liver myofibroblasts. In the second part of this study, we have shown that neither trans-piceid (a glycosylated analog) nor trans-piceatannol (a hydroxylated analog) reproduces trans-resveratrol effects on liver myofibroblasts. We finally show that, although trans-resveratrol decreases the proliferation of skin fibroblast and vascular smooth muscle cells, it does not affect their expression of alpha-SMA, which indicates some cell specificity.
Abstract: The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in tumour invasion, Previous studies have shown by RT-PCR that uPA and mRNAs are expressed in human hepatocellular carcinoma (WCC). Here, in situ hybridization, immunohistochemistry, and double immunofluorescence were used to identify the cells expressing uPA and uPAR in 26 HCCs, The results indicate that uPA and uPAR were expressed in every case, almost exclusively in stromal cells, mostly myofibroblasts and macrophages, except for rare tumoural hepatocytes expressing cytokeratin 7, These results show the important role of stromal cells of HCC in the pericellular proteolysis which facilitates cancer cell invasion. Copyright (C) 2000 John Wiley & Sons, Ltd.
Abstract: Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is characterized by a high rate of intra-hepatic invasion. The stroma of HCC is infiltrated by myofibroblasts. We have previously shown that hepatocyte growth factor (HGF) secreted by human liver myofibroblasts greatly increased the in vitro invasiveness of 3 human HCC cell lines. In this study we show that the conditioned medium (CM) from the same HCC cell lines dose-dependently stimulates HGF secretion by myofibroblasts. This effect was post-transcriptional as no increase in HGF mRNA was observed. We show that the effect of CM is not due to IL-1, lL-6, IGF-1, bFGF or PDGF, previously shown to stimulate HGF synthesis in other models. Our data demonstrate that HCC cells increase HGF secretion by liver myofibroblasts in a paracrine way that could act to enhance invasion.
Abstract: We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-8). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII, Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.
Abstract: Hepatocellular carcinoma (HCC) is the main type or primary liver cancer, and it develops from hepatocytes. The stroma of HCC is infiltrated by myofibroblasts. In other settings, such as liver fibrosis, myofibroblasts are derived mainly from the activation of hepatic stellate cells (HSC). In this study, we investigated whether tumoral hepatocytes were able to activate HSC. HSC were isolated from normal rats and were plated in dishes coated with Matrigel, to prevent their spontaneous activation. HSC were exposed to conditioned medium (CM) from the rat HCC lines Fao and H5. Tumor cell CM elicited major morphologic changes, such as spreading and generation of cytoplasmic processes. Fao and H5 CM increased HSC proliferation to 1.60 and 1.76 times control values, respectively. The expression of alpha-smooth muscle actin was low or undetectable in control cells and was markedly increased by both tumor cell CM but not by normal rat hepatocyte CM. Desmin expression was also enhanced. Gelatinase A secretion was significantly increased 1.20-fold by Fao CM and 1.55-fold by H5 CM. Expression of beta-type platelet-derived growth factor receptor mRNA was increased 5.8-fold by H5 GM but was decreased to 13% of control levels by Fao CM. HSC activation by tumor cell CM was not prevented by urokinase or matrix metalloproteinase inhibitors, suggesting that Matrigel degradation was not central to the activation process. Finally, a blocking antibody to transforming growth factor-beta 1 did not impede Fao CM-induced activation but significantly blocked the increase in matrix metalloproteinase-2 expression induced by H5 CM. Our results show that tumoral rat hepatocyte CM is able to induce the activation of rat HSC in culture. The lack of induction of beta-type platelet-derived growth factor receptor mRNA by Fao CM indicates that, in some cases, tumor-induced activation differs from classic fibrosis-type activation. Our data thus suggest that HSC recruitment and activation in HCC could be under the control of tumor cells.
Abstract: Background/Aims: Marked changes in extracellular matrix occur in the stroma of hepatocellular carcinoma, as compared to normal or cirrhotic liver. The cell types responsible for extracellular matrix synthesis within hepatocellular carcinoma have not been clearly identified. Methods: In vivo collagen synthesis was studied by in situ hybridization and immunohistochemistry for types I, IV, V and VI collagen, together with immunolabeling of alpha-smooth muscle actin, a myofibroblast marker, and CD34, an endothelial cell marker. In vitro, extracellular matrix deposition by cultured myofibroblasts was studied by reticulin staining, immunocytochemistry and RNase protection. Results: All collagens studied were expressed in the stroma of the tumor, with a higher level of type VI and IV collagens than of type I and V. The majority of the cells expressing collagen transcripts in human hepatocellular carcinoma stroma were alpha-actin positive and CD 34 negative. In vitro experiments demonstrated that the hepatocellular carcinoma cell lines HepG2, HuH7 and Hep3B markedly increased extracellular matrix deposition by human liver myofibroblasts, This increase was mediated by a soluble mediator present in tumor cell conditioned medium. It was not explained by an increase in mRNA levels of extracellular matrix components, nor by a decrease in the secretion of matrix-degrading proteinases by myofibroblasts. Conclusions: Myofibroblasts are the main source of collagens in the stroma of hepatocellular carcinoma. Our data also indicate that tumoral hepatocytes increase extracellular matrix deposition by cultured myofibroblasts, probably by post-transcriptional mechanisms. The generation of hepatocellular carcinoma stroma by myofibroblasts could thus be under control of tumoral cells.
Abstract: Background/Aims: We have shown that hepatocyte growth factor secreted by human hepatic myofibroblasts increased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. Methods: Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay, Cell invasion was assayed on Matrigel-coated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-beta chain-specific antibody. Results: HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. Conclusion: Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.
Abstract: Background/Aims: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy, Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta 1 in biliary atresia and paucity of bile ducts. Methods: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta 1 mRNAs were detected by in situ hybridization. Results: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta 1 was expressed around biliary ductules, probably in inflammatory cells, land also in biliary cells. Osteonectin expression was also increased in the lobules, In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta 1, except in the only child with marked fibrosis, However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. Conclusions: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta 1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta 1 could explain the slow progression of fibrosis in paucity of bile ducts.
Abstract: Osteonectin (ON)/SPARC is a glycoprotein involved in extracellular matrix remodelling. ON expression by myofibroblasts has been reported in fibrotic human liver. As ON also plays a role in cell adhesion, differentiation, and proliferation, this study was designed to document its expression in human hepatocellular carcinoma (HCC), Tissues from 26 HCCs of various histological grades and architecture and from surrounding non-tumour liver (23 cirrhotic or fibrotic, three non-fibrotic) were tested by in situ hybridization and immunohistochemistry. Immunohistochemical detection of alpha-smooth muscle actin (alpha-SMA) was performed on serial sections or in combination with hybridization. Large amounts of ON mRNA and protein were detected in the tumour capsule, in the fibrous bands, and along capillaries within HCCs. The signal was located in cells suggestive of myofibroblasts, as confirmed by positive staining for alpha-SMA, In HCC, ON protein was always detectable, with strong staining in high-grade tumours, whereas it was mostly undetectable in non-tumour tissues. A clear difference was also shown for ON transcripts, except in a few cases,vith chronic active hepatitis, where ON transcripts were also expressed at a high level. Overexpression of ON transcripts in HCC vs. non-tumour liver was confirmed by RNA blot in 20/22 patients tested. In conclusion, ON is strongly expressed by the stromal myofibroblasts of human HCC, especially of high grade This expression could play a role in tumour progression. Copyright (C) 1999 John Whey & Sons, Ltd.
Abstract: Background/Aims: The aim of this study was to assess the effect of the early and chronic administration of interferon alpha in the prevention of hepatic fibrosis and portal hypertension. Methods: Rats with liver fibrosis due to bile duct ligation or CCl4 were divided into three groups: sham, placebo and interferon alpha(2a) 100 000 UI/day. Liver fibrosis was assessed by the area of fibrosis (image analysis), liver hydroxyproline and mRNA (fibronectin, procollagen alpha 2(I)) contents, and serum hyaluronate. Systemic and splanchnic hemodynamics were also evaluated. Results: Interferon alpha significantly decreased fibrosis in the CCl4 model only: area of fibrosis: 13.9+/-3.7 vs 10.5+/-3.3% (p<0.05), hydroxyproline: 1.8+/-0.6 vs 1.2+/-0.2 mg/g wet liver (p<0,001), respectively placebo vs interferon ex. There was a significant correlation between the area of fibrosis and hydroxyproline liver content (r=0.77 in the biliary model and r=0.87 in the CCl4 model, p<0.0001). Interferon decreased spleno-renal shunt blood flow (2.0+/-1.8 vs 0.9+/-0.7 ml/min; p<0.05) but not portal pressure in the CCl4 model, No significant effects were observed in rats with biliary fibrosis, Conclusions: The early and chronic administration of interferon alpha prevents the development of liver fibrosis and porto-collateral circulation in the CCl4 model but not in the biliary model. However, the antifibrotic effects of interferon need to be confirmed in further studies.
Abstract: The aim of this study was to assess the effect of the early and chronic administration of octreotide in the prevention of hepatic fibrosis and portal hypertension (PHT). Two experimental models of liver fibrosis caused by bile duct ligation (BDL) or CCl4 were divided into 4 rat groups: sham, placebo, and octreotide (10 and 100 mu g/kg twice daily, subcutaneously). Liver fibrosis was assessed by the area of fibrosis (image analysis), liver hydroxyproline and fibronectin mRNA contents, and serum hyaluronate. Systemic and splanchnic hemodynamic changes were also evaluated, including the splenorenal shunt blood flow by the transit-time ultrasound (TTU) technique. Zn both models, splenorenal shunt blood flow was significantly lower in the octreotide groups than in the placebo group (P < .05), while portal pressure was not significantly decreased. There was a significant decrease in fibrosis by octreotide in the CCl4 model only: area of fibrosis: 13.9% +/- 3.7% vs. 9.8% +/- 2.5% (P < .01), hydroxyproline: 1.8 +/- 0.6 vs. 1.3 +/- 0.4 mg/g wet liver (P < .05), respectively, placebo vs, octreotide 10 mu g/kg. There was a significant correlation between the area of fibrosis and hydroxyproline liver content (r = .87 in the biliary model and r = .91 in the CCl4 model; P < .0001). The early and chronic administration of octreotide prevents the development of portocollateral blood flow without reducing portal pressure in two models of liver fibrosis and the development of Liver fibrosis in the CCl4 model.
Abstract: Myofibroblasts (MF) differentiate from precursor cells during wound healing, and persist when tissue repair is abnormal, i.e., during tissue fibrosis or the stromal reaction to tumors. MF precursors in the liver are hepatic stellate cells (HSC) and portal fibroblasts. We have established a culture model for human liver MIS that allowed us to characterize their growth regulation and extracellular matrix synthesis. We found that MF strikingly increased the invasive ability of hepatocellular carcinoma (HCC cells) via the secretion of hepatocyte growth factor. Our data also show that HCC cells can induce the differentiation of HSC into MF: Drugs such as interferons, simvastatin, or pentoxifylline can prevent HSC activation and modulate the MF phenotype. Preliminary data indicate that trans-resveratrol, a polyphenol from grapes, is also able to modulate these parameters. It also interferes with the MF-induced stimulation of HCC cell invasion. Work is in progress to define structure-function relationships within polyphenols and to specify the targets of trans-resveratrol.
Abstract: Myofibroblasts (MF) are a major effector cell type in liver fibrogenesis, where they are thought to derive from the activation of hepatic stellate cells. Cultured human MF, grown from liver explants, retain most of the in vivo characteristics of liver MF but are in limited supply. A continuous MF cell line would therefore be valuable in studying human liver fibrogenesis. For this purpose, we sought to immortalize human liver MF with polyoma virus large T antigen. MF were obtained from explants of human liver and transfected with a plasmid containing the coding sequence of polyoma virus large T antigen. This procedure yielded an actively growing cell line, designated GREF-X, which did not express large T antigen. Nevertheless, this cell line has been passaged repeatedly for almost 1 year and is thus likely immortalized. The morphology of GREF-X resembles that of primary liver MF. These cells have a doubling time of approximately 72 hours and are density-inhibited, and their growth is serum-dependent. Moreover, GREF-X cells do not grow in soft agar or induce tumors in nude mice, suggesting that they are not transformed. They stain positively for MF markers, such as smooth muscle a-actin and vimentin; express collagens type I, IV, V, and VI, fibronectin, and laminin; and secrete matrix-metalloproteinase-2. In addition, GREF-X cells are able to take up and esterify [H-3]retinol, suggesting that they actually derive from hepatic stellate cells. Finally, these cells respond to transforming growth factor-beta 1, a major mediator of liver fibrogenesis, by increasing secretion of fibronectin and plasminogen activator-inhibitor type 1. Transient transfection experiments showed that plasminogen activator-inhibitor type 1 regulation, by transforming growth factor-beta 1, was transcriptional. We believe, therefore, that GREF-X would be a useful tool for studying the pathophysiology and pharmacology of liver fibrogenesis.
Abstract: Osteonectin/SPARC is a glycoprotein involved in the regulation of cell shape, adhesion, migration, and proliferation. It also has complex effects on extracellular matrix synthesis and turnover. We found that osteonectin mRNA was very abundant in a human Liver myofibroblast library. Using Northern and Western blot, immunoprecipitation, and radioimmunoassay, we found that cultured liver myofibroblasts actively secreted osteonectin. Myofibroblasts are very rare in normal liver but proliferate during liver fibrosis where they synthesize extracellular matrix components. Thus, we studied the distribution of osteonectin in normal and fibrotic human liver using in situ hybridization. Osteonectin mRNA expression was weak in normal liver but very high in fibrotic liver within fibrous septae and scattered sinusoidal cells. Serial sectioning and double staining experiments with an antibody to smooth muscle alpha-actin showed that osteonectin transcripts were mostly co-localized with myofibroblasts. In conclusion, osteonectin is highly expressed in human liver myofibroblasts in culture as well as in human liver fibrosis in vivo. The many biological properties of osteonectin make it a candidate effector of human liver fibrogenesis.
Abstract: Extracellular matrix (ECM) plays a major role in cell differentiation, proliferation, and gene expression, both in physiological and in pathological conditions. Immunohistochemistry has been used to investigate modifications of ECM and related receptors, the integrins, in 26 small nodular lesions developed in human cirrhotic livers, on the basis that these lesions could represent sequential steps of hepatocarcinogenesis: the lesions mere 16 macroregenerative nodules (MRNs), either of ordinary (n=5) or atypical (n=11) type, and ten small (<15 mm) hepatocellular carcinomas (HCCs), Data mere compared with those obtained in the surrounding cirrhotic tissue, in large HCCs, and in normal liver, The results indicate similarities between ordinary MRNs and cirrhosis, on the one hand, and between atypical MRNs and small HCCs, on the other, Strong and homogenous deposition of collagen type IV and laminin in sinusoids and overexpression of alpha 6 integrin by sinusoidal cells and hepatocytes were especially noticeable in dysplastic areas characteristic of atypical MRN's, as in small HCCs. In addition, the staining of alpha 2 and alpha 6 integrins in MRNs revealed the presence of widespread atypical ductular proliferation expanding from periportal and perinodular areas, containing epithelial cells with transitional (hepato-biliary) phenotype. These findings suggest a transition from atypical MRNs to small HCCs and a possible role for liver epithelial precursor cells ('stem cells') in the development and evolution of MRNs. (C) 1997 by John Wiley and Sons, Ltd.
Abstract: The stroma of hepatocellular carcinomas (HCC) is infiltrated with myofibroblasts (MFs). Preliminary in vivo data have suggested that liver MF express hepatocyte growth factor (HGF), a cytokine that has been implicated in several tumor models. Our aim was to investigate the role of MF and HGF in HCC. Cultured liver MF expressed HGF messenger RNA (mRNA) and secreted HGF in their medium, as shown by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Addition of MF-conditioned medium to the HepG2 HCC cell line induced cell scattering. This was associated with a decrease in cell proliferation. MF also increased about 100-fold the ability of HepG2 to invade Matrigel. Increased invasiveness was also shown for HuH7 cells, but no scattering was observed and cell proliferation was stimulated. All the effects of MF on both tumor cell types were blocked by addition of an antibody to HGF and they all could be reproduced by adding recombinant HGF to the tumor cells. RT-PCR and Western blot analysis confirmed that both tumor cell lines expressed c-met, the receptor for HGF. The effects of MF-conditioned medium were not reproduced by acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), or platelet-derived growth factor (PDGF-BB). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that HGF was expressed in human HCC. Our data show that human liver MF act on HCC cells to increase their invasiveness and suggest that MF-derived HGF could be involved in the pathogenesis of HCC.
Abstract: Hepatic stellate cells were studied by immuno-cytochemistry with anti smooth muscle a-actin antibody (an activation marker for these cells) and electron microscopy, in eleven patients transplanted for fulminant or subfulminant hepatitis. Numerous smooth muscle a-actin positive cells were found in necrotic areas. In both fulminant and subfulminant hepatitis, hepatic stellate cells appeared enlarged, often irregular, with spikes. There were numerous signs of activation and many contained numerous small lipid droplets. In the cases of fulminant hepatitis, hepatic stellate cells presented, at times, some subcellular damage. Hepatic stellate cells processes, often in several layers, displayed numerous cytoplasmic microfilaments with conspicuous dense plaques below the plasma membrane. Hepatic stellate cells were never surrounded by a basement membrane. The extracellular matrix was loose and granulofibrillar. In areas of multiacinar nodules (in cases of map-like pattern), hepatic stellate cells were grossly normal. These results are in agreement with in vitro data showing that acutely damaged hepatocytes activate hepatic stellate cells but do not fully transform them into myofibroblasts.
Abstract: The mechanism of bile ductular reaction and accompanying fibrogenesis depends on interactions of ductular cells with the matrix and growth factors, Heparan sulfate proteoglycans (HSPGs) are essential cofactors in cell-matrix adhesion processes, in cell-cell recognition systems, and in receptor-growth factor interactions, We used monoclonal antibodies specific for the cell surface HSPGs (syndecans, glypican), for matrix HSPG (perlecan), and for heparan sulfate carbohydrate (HS) to investigate their immunohistochemical expression in 20 specimens with chronic cholestatic liver disease and in five normal human liver specimens, Because activated hepatic stellate cells (HSC) are a major source of fibrosis in the liver, we also examined HSPG expression in primary cultures of human activated HSC using immunocytochemistry and Western blotting and for syndecan-1 also Northern blotting, In comparison with bile ductular cells of normal liver, reactive ductules in chronic cholestasis were marked by an elevated expression of syndecan-1, surrounded by an increased perlecan expression, In acinar zone 1, large stimulated macrophages and HSC, present in increased numbers, were strongly positive for syndecan-3, Cultured HSC showed a membranous staining pattern for syndecan-1, syndecan-3, and heparan sulfate, and in addition intracellular staining for syndecan-2, -3, and -4, Perlecan immunoreactivity was detected as intercellular strings, Western blotting revealed positive bands with all antibodies and Northern blotting for syndecan-1 was also positive, These results show that cultured human HSC can synthesize all four syndecans, glypican, and perlecan, These data reveal changes in the expression of syndecan-1, syndecan-3, and perlecan in human chronic cholestatic liver disease, that may be important in the deposition of matrix components and activation of growth factors that support ductular reaction and accompanying fibrogenesis.
Abstract: Ito cells play a pivotal role in the development of liver fibrosis associated with chronic liver diseases. During this process, Ito cells acquire myofibroblastic features, proliferate, and synthesize fibrosis components, Considering the reported mitogenic properties of endothelin-1 (ET-1), we investigated its effects on the proliferation of human Ito cells in their myofibroblastic phenotype. Both ET receptor A (ETA: 20%) and ET receptor B (ETB: 80%) binding sites were identified, using a selective ETA antagonist, BQ 123, and a selective ETB agonist, sarafotoxin S6C (SRTX-C). ET-1 did not stimulate proliferation of myofibroblastic Ito cells. In contrast, ET-1 inhibited by 60% DNA synthesis and proliferation of cells stimulated with either human serum or platelet-derived growth factor -BB (PDGF-BB), PD 142893, a nonselective ETA/ETB antagonist totally blunted this effect. SRTX-C was as potent as ET-1, while BQ 123 did not affect ET-1-induced growth inhibition. Analysis of the intermediate steps leading to growth-inhibition by ET-1 revealed that activation of mitogen-activated protein kinase by serum or PDGF-BB was decreased by 50% in the presence of SRTX-C. In serum-stimulated cells, SRTX-C reduced c-jun mRNA expression by 50% whereas c-fos or krox 24 mRNA expression were not affected. We conclude that ET-1 binding to ETB receptors causes a potent growth inhibition of human myofibroblastic Ito cells, which suggests that this peptide could play a key role in the negative control of liver fibrogenesis. Our results also point out that, in addition to its well known promitogenic effects, ET-1 may also exert negative control of growth on specific cells.
Abstract: During the course of ongoing liver fibrogenesis, Ito cells acquire myofibroblastic features, proliferate, and synthesize increased amounts of extracellular matrix components. Interferon (IFN) alfa and IFN gamma have been shown to elicit antiproliferative and/or antifibrogenic effects in various cell cultures of mesenchymal origin. The aim of this study was to investigate the effects of IFN-alpha and IFN-gamma on cultured human myofibroblastic Ito cells (MFBIC) proliferation and collagen synthesis and secretion. Serum-stimulated incorporation of [H-3]thymidine into DNA of MFBIC was dose-dependently decreased by both cytokines. IFN-alpha (10(4) U/mL) and IFN-gamma (10(3) U/mL) decreased DNA synthesis by 69% and 66%, respectively. Inhibition of cell proliferation was confirmed by cell counting. Similar results were observed when cell growth was stimulated with platelet-derived growth factor (PDGF-BB, PDGF-AA) or transforming growth factor (TGF)-beta 1. Collagen secretion per cell was inhibited by both cytokines, as assessed by [H-3]hydroxyproline incorporation. After a 6-day treatment, IFN-gamma showed a greater potency than IFN-alpha in inhibiting secretion of newly synthetized collagen (41% and 48% of control in the presence of 10(2) U/mL of IFN-gamma and 10(4) U/mL of IFN-alpha, respectively). Both IFN-alpha and IFN-gamma concurrently decreased steady-state expression of type I and type III procollagen messenger RNAs (mRNAs) in quiescent MFBIC. Viability assays ruled out cytotoxic effects of the two molecules. Finally, both IFNs decreased smooth muscle alpha-actin (SM alpha-actin) expression, whether assayed by immunobloting or by Northern blot analysis. We conclude that IFN-alpha and IFN-gamma inhibit proliferation as well as collagen synthesis in human MFBIC.
Abstract: During human fibrogenesis, myofibroblastlike cells proliferate and are the main source of fibrosis components. We have used cultured myofibroblastlike cells obtained by outgrowth from explants of human liver to study the expression of extracellular matrix (ECM) components and matrix-metalloproteinase-2 (MMP-2). These cells contained types I, III, IV, and V procollagen messenger RNAs (mRNAs). They also expressed mRNAs for laminin B1 chain and for cellular and plasma fibronectin. The corresponding proteins were detected by immuno-cytochemistry. MMP-2 expression was shown by Northern blot and gelatin zymography. Because transforming growth factor beta 1 (TGF beta 1) is considered an important mediator in liver fibrogenesis, we examined its effect on expression of ECM components by cultured human myofibroblastlike cells. TGF beta 1 increased collagen mRNAs steady-state levels and total collagen secretion in the culture medium. It also increased fibronectin mRNA levels but had no effect on laminin mRNA or (MMP-2 expression. In summary, cultured human myofibroblastlike cells express those ECM components that accumulate during hepatic fibrogenesis, indicating the usefulness of this model to study mechanisms of human liver fibrogenesis, In addition to the mitogenic effect of TGF beta 1 on human myofibroblastike cells, we now demonstrate its stimulation of ECM accumulation in these cells, thus emphasizing the central role of TGF beta 1 and myofibroblastlike cells in the pathophysiology of human hepatic fibrosis.
Abstract: Background & Aims: During liver fibrogenesis, myofibroblastic liver cells proliferate and synthesize components of fibrosis, Fibroblast growth factor 2 (FGF-2) is expressed in vivo in myofibroblastic liver cells (MFLCs) during fibrogenesis, and exogenous FGF-2 is mitogenic for MFLCs. The aim of this study was to study the expression and role of endogenous FGF-2 in cultured human MFLCs. Methods: FGF-2 and FGF-2 receptors were studied using immunoblotting. All RNA studies used ribonuclease protection, Growth of MFLCs was studied using [H-3]thymidine incorporation and direct cell counting. Results: MFLCs expressed FGF-2 and its receptors FGF receptor 1 and FGF receptor 2. An antibody to FGF-2 blocked the mitogenic effect of transforming growth factor beta 1 (TGF-beta 1) for MFLCs but not TGF-beta 1-induced increase in cellular fibronectin messenger RNA (mRNA). TGF-beta 1 increased levels of FGF-2 and FGF receptor mRNAs in MFLCs. We have previously shown that TGF-beta 1 also increased platelet-derived growth factor (PDGF) A chain mRNA in these cells and that anti-PDGF antibody blunted the mitogenic effect of TGF-beta 1. The present results show that anti-FGF-2 and anti-PDGF-AA are not additive and that FGF-2 and PDGF-AA are not sequentially induced by TGF-beta 1. Conclusions: FGF-2 mediates the mitogenic but not the profibrogenic effect of TGF-beta 1 for human MFLCs, and autocrine FGF-2 and PDGF-A interact in the mediation of the mitogenic effect of TGF-beta 1.
Abstract: During hepatic fibrogenesis, Ito cells proliferate, acquire a myofibroblastlike phenotype and synthesize increased amounts of extracellular matrix components. In this study, we have assessed the effects of simvastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, on the growth of human myofibroblastlike Ito cells. Cells were grown from explants of normal human liver and characterized by a positive staining for desmin and smooth muscle alpha-actin. Simvastatin (0.1 to 10 mu mol/L) induced a marked dose-dependent decrease of [H-3]thymidine incorporation in human Ito cells, whether stimulated by human serum or by purified growth factors. Simvastatin-induced inhibition of DNA synthesis was cofirmed by nuclear autoradiography and was not explained by a cytotoxic effect. The growth inhibitory effect of simvastatin was specifically due to inhibition of hydroxy-methylglutaryl-coenzyme A reductase because it was overcome by addition of mevalonic acid, the product of the enzymatic reaction. The reduction in [H-3]thymidine incorporation was not affected by supplementation of culture medium with purified cholesterol-low density lipoprotein or isopentenyl adenine. It was partially reversed by addition of farnesol. These results show that simvastatin decreases the growth of human Ito cells, independently of its effect on cholesterol synthesis. This decrease may be due in part either to reduced farnesylation of proteins involved in growth factor signaling pathway or to inhibition of N-linked protein glycosylation. Whether this effect exists in vivo and could thus lead to a parallel decrease of fibrosis deposition within the liver requires further study.
Abstract: Hepatic fibrosis complicates most chronic liver diseases. It carries a high morbidity and mortality rate, due to its endpoint, cirrhosis of the liver. The cell type responsible for the major part of fibrosis deposition is the Ito cell. This perisinusoidal cell, involved in retinol metabolism in the normal liver, switches to a myofibroblastic phenotype, proliferates and synthetizes extracellular matrix components during fibrognesis. The mechanisms responsible for this ''activation'' have been studied in detail. A 3-step model of activation has been proposed. In the first step, necrotic hepatocytes initiate Ito cell proliferation by releasing a mitogenic mediator. In the second step, mononuclear cells, activated by the products of hepatocyte lysis, release several cytokines, including transforming growth factor beta 1, that affect the phenotypic modulation of Ito cells into myofibroblasts. Finally, myofibroblastic Ito cells are then able to sustain the activation by secreting multiple cytokines acting through an autocrine/paracrine pathway by themselves. The activation is also influenced by the disruption of the normal extracellular matrix by metalloproteinases secreted by Ito and Kupffer cells. In parallel, the breakdown of the newly synthetized matrix is impaired due to decreased expression of interstitial collagenase together with increased expression of the tissue inhibitor of metalloproteinases-1 in the diseased liver. An adequate treatment for hepatic fibrosis is still lacking. Future research is aiming at several targets : decreasing the activation of Ito cells, stimulating fibrosis degradation and developing profibrogenic cytokines antagonists.
Abstract: Ito cells play a major role in liver fibrosis but the mechanisms controlling their activation in vivo are poorly understood. Heparin-binding growth factors (HBGF) types 1 and 2 are mitogenic for cultured Ito cells. They have been found in liver extracts but their cellular localization is unknown. We have studied by immunohistochemistry HBGF-1 and -2 expression in normal rat liver and in carbon tetrachloride (CCl4)-induced fibrosis. In normal liver, HBGF-1 was present only in sinusoidal cells whereas HBGF-2 was also detected in endothelial cells lining major vessels. At the acute stage of CCl4 intoxication, HBGF-2 was expressed in centrilobular clusters of mononuclear phagocytes that were surrounded by many HBGF-2-negative Ito cells. In the later stages, HBGF-2 was expressed by Ito cells within the fibrous bands. No modulation of HBGF-1 expression was noted at any stage. These results suggest that (1) at the acute stage of CCl4 intoxication, HBGF-2 produced by mononuclear phagocytes could participate in the recruitment of Ito cells; and (2) during the CCl4-induced fibrotic process, HBGF-2 could contribute to Ito cell proliferation and the synthesis of fibrosis components. In this in vivo model of hepatic fibrosis, the hyperexpression of HBGF-2 is a relatively specific event since the expression of a structurally related molecule, HBGF-1 was not modulated.
Abstract: We assessed the effect of transforming growth factor-beta1 on the proliferation of human Ito cells. Ito cells in their myofibroblastlike phenotype were grown from explants of human liver and were characterized with electron microscopy and positive immunostaining for desmin and smooth muscle alpha-actin. Transforming growth factor-beta1 was mitogenic for human Ito cells whatever the culture conditions, although it was, as previously described, inhibitory of growth for rat Ito cells. The mitogenic effect of transforming growth factor-beta1 was likely due to induction of autocrine platelet-derived growth factor chain secretion by Ito cells themselves because (a) the mitogenic effect of transforming growth factor-beta1 was blocked by specific platelet-derived growth factor antibodies, (b) transforming growth factor-beta1 increased platelet-derived growth factor-A chain messenger RNA expression and platelet-derived growth factor-AA secretion by human Ito cells and (c) human Ito cells expressed the alpha-type platelet-derived growth factor-A receptor messenger RNA. Exogenous platelet-derived growth factor-AA was also mitogenic for human Ito cells, mimicking the effect of transforming growth factor-beta1. Our data suggest that results obtained with rat Ito cells must be extrapolated with caution to human ones. The mitogenic effect of transforming growth factor-beta1 on human Ito cells probably has pathophysiological relevance because transforming growth factor-P 1 has been demonstrated in vivo at sites of active liver fibrogenesis.
Abstract: The liver is composed of six main cellular types: hepatocytes, endothelial cells, Kupffer cells, Ito cells, biliary epithelial cells and pit cells. This article describes some of the interactions which occur between these cells. Vitamin A, coming from the portal vein, enters the hepatocytes; it is then secreted as a complex with retinol-binding protein which binds to Ito cells where vitamin A is finally stored. Kupffer cells are involved in the glycogenolytic effect of endotoxin and platelet-activating factor via secretion of prostaglandin D2 which activates phosphorylase b in hepatocytes. Native ferrotransferrin cannot bind to hepatocytes but must transit through endothelial cells where it is partly desialylated, becoming then a ligand for the hepatocyte asialoglycoprotein receptor. Ito cells are exposed to mitogenic and profibrogenic signals originating mainly from Kupffer cells; these effects could be counter-balanced by molecules secreted by endothelial cells and hepatocytes. Following 2/3 hepatectomy, hepatocyte proliferation is stimulated by growth factors synthetized by non-parenchymal cells (and hepatocytes themselves); this proliferation could be put to a stop by endothelial cell-derived transforming growth factor beta-1. Cultured hepatocytes rapidly lose their differentiated functions; when co-cultured with liver epithelial cells, they can maintain these functions for several weeks, probably due to interactions involving membrane proteins.
Abstract: We have recently demonstrated that polymorphonuclear neutrophils were toxic to hepatocytes through a protease-mediated mechanism. Since synthesis of antiproteases is markedly increased during acute inflammatory reaction, the aim of this work was to investigate the toxicity of neutrophils against normal vs. inflammatory rat hepatocytes. Acute inflammatory reaction was induced by subcutaneous injection of turpentine 24 hr before the experiments. Hepatocytes from normal and turpentine-treated rats were isolated by collagenase digestion. They were incubated with human neutrophils stimulated by 1 mg/ml opsonized zymosan. Cytotoxicity was quantified by the percentage of alanine aminotransferase activity released by hepatocytes in culture medium after an 18-hr incubation period. By comparison to normal hepatocytes, inflammatory hepatocytes were more resistant to the toxicity of neutrophils. At a neutrophil/hepatocyte ratio of 20:1, the alanine aminotransferase activity releases were 53.7% +/- 5.4% (mean +/- 1 S.E.) and 27.4% +/- 4.8% for normal and inflammatory hepatocytes, respectively. Similarly, inflammatory hepatocytes were found to be less sensitive than normal hepatocytes to the toxic effect of purified neutrophil cathepsin G. In contrast, both types of hepatocytes exhibited the same sensitivity to H2O2 generated by a system consisting of glucose and glucose oxidase. Two arguments suggested that the resistance of inflammatory hepatocytes to protease toxicity was explained by an increased production of antiproteases by these cells: (a) when tested against cathepsin G and procine pancreatic elastase activities, the protease inhibitory capacity of conditioned medium from inflammatory hepatocytes was higher than that of conditioned medium from normal hepatocytes; (b) conditioned medium from inflammatory hepatocytes markedly reduced the toxicity of stimulated neutrophils as that of cathepsin G. These results show that, during an acute inflammatory reaction, the increased synthesis of antiproteases by hepatocytes may inhibit the protease-mediated toxicity of neutrophils. In the clinical disorders in which a pathogenetic role of neutrophils has been suggested, the accompanying inflammatory reaction might thus have a beneficial effect by reducing neutrophil-mediated tissue injury.
