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• W2022308645 abstract "Liver cirrhosis is characterized by hepatic dysfunction with extensive accumulation of fibrous tissue in the liver. In response to chronic hepatic injury, hepatic portal myofibroblasts and activated hepatic stellate cells (HSCs) play a role in liver fibrosis. Although administration or gene expression of hepatocyte growth factor (HGF) leads to improvement in hepatic fibrosis/cirrhosis, the related mechanisms are not fully understood. We investigated mechanisms involved in resolution from liver cirrhosis by HGF, focusing on growth regulation and apoptosis in portal myofibroblasts. Cultured rat HSCs could not proliferate, were withdrawn after passage, and were replaced by proliferating portal myofibroblasts during the passages. In quiescent HSCs, c-Met receptor expression was undetected whereas c-Met receptor expression was detected in activated HSCs and liver myofibroblasts expressing α-smooth muscle actin (α-SMA), suggesting that activated HSCs and portal myofibroblasts are targets of HGF. For cultured rat portal myofibroblasts, HGF counteracted phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and mitogenic stimulus induced by platelet-derived growth factor, induced c-jun N-terminal kinase (JNK) 1 phosphorylation, and promoted apoptotic cell death. In the dimethylnitrosamine rat model of liver cirrhosis, administration of HGF suppressed proliferation while promoting apoptosis of α-SMA-positive cells in the liver, events that were associated with reduced hepatic expressions of α-SMA and histological resolution from liver cirrhosis. Growth inhibition and enhanced apoptosis in portal myofibroblasts by HGF are newly identified mechanisms aiding resolution from liver fibrosis/cirrhosis by HGF. Liver cirrhosis is characterized by hepatic dysfunction with extensive accumulation of fibrous tissue in the liver. In response to chronic hepatic injury, hepatic portal myofibroblasts and activated hepatic stellate cells (HSCs) play a role in liver fibrosis. Although administration or gene expression of hepatocyte growth factor (HGF) leads to improvement in hepatic fibrosis/cirrhosis, the related mechanisms are not fully understood. We investigated mechanisms involved in resolution from liver cirrhosis by HGF, focusing on growth regulation and apoptosis in portal myofibroblasts. Cultured rat HSCs could not proliferate, were withdrawn after passage, and were replaced by proliferating portal myofibroblasts during the passages. In quiescent HSCs, c-Met receptor expression was undetected whereas c-Met receptor expression was detected in activated HSCs and liver myofibroblasts expressing α-smooth muscle actin (α-SMA), suggesting that activated HSCs and portal myofibroblasts are targets of HGF. For cultured rat portal myofibroblasts, HGF counteracted phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and mitogenic stimulus induced by platelet-derived growth factor, induced c-jun N-terminal kinase (JNK) 1 phosphorylation, and promoted apoptotic cell death. In the dimethylnitrosamine rat model of liver cirrhosis, administration of HGF suppressed proliferation while promoting apoptosis of α-SMA-positive cells in the liver, events that were associated with reduced hepatic expressions of α-SMA and histological resolution from liver cirrhosis. Growth inhibition and enhanced apoptosis in portal myofibroblasts by HGF are newly identified mechanisms aiding resolution from liver fibrosis/cirrhosis by HGF. Liver cirrhosis, which usually is as a long-term consequence of chronic hepatic injury caused by alcohol abuse or hepatitis virus infection, is characterized by extensive fibrous scarring of the liver.1Friedman SL The cellular basis of hepatic fibrosis.N Engl J Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar Advanced cirrhosis is generally irreversible and is often associated with variceal hemorrhage or development of hepatocellular carcinoma. Hence, liver cirrhosis is a major cause of morbidity and mortality worldwide. Approaches to promote the remodeling of the excess extracellular matrix (ECM) associated with reorganization of the hepatic structure are critical to establish a therapeutic base.In the liver, two different cell populations play a key role in the pathogenesis of liver cirrhosis as major sources of hepatic ECM. Quiescent hepatic stellate cells (HSCs) (also known as lipocytes, fat-storing cells, or Ito cells) synthesize low levels of ECM proteins, whereas in response to chronic hepatic injury, HSCs undergo phenotypic change into myofibroblast-like cells, a process termed “activation.”2Alcolado R Arther MJP Iredale JP Pathogenesis of liver fibrosis.Clin Sci. 1997; 92: 103-112Crossref PubMed Scopus (173) Google Scholar, 3Wu J Zern MA Hepatic stellate cells: a target for the treatment of liver fibrosis.J Gastroenterol. 2000; 35: 665-672Crossref PubMed Scopus (239) Google Scholar In addition to activated HSCs, liver myofibroblasts, which are located in periportal and perivenous areas in the normal liver, migrate to the site of hepatic injury and are involved in fibrotic change of the liver. Distinct from HSCs, portal myofibroblasts maintain proliferative ability and can be expanded in vitro and in vivo,4Knittel T Kobold D Saile B Grundmann A Neubauer K Piscaglia F Ramadori G Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential.Gastroenterology. 1999; 117: 1205-1221Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Bhunchet E Wake K Role of mesenchymal cell populations in porcine serum-induced rat liver fibrosis.Hepatology. 1992; 16: 1452-1473Crossref PubMed Scopus (121) Google Scholar, 6Tuchweber B Desmouliere A Bochaton-Piallat ML Rubbia-Brandt L Gabiani G Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.Lab Invest. 1996; 74: 265-278PubMed Google Scholar, 7Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (97) Google Scholar while portal myofibroblasts also express ECM proteins involved in fibrotic change of the liver. Although phenotypic change and cell proliferation in HSCs and portal myofibroblasts are a pathogenic cause of hepatic fibrosis, recent approaches indicated that apoptotic cell death in activated HSCs and liver myofibroblasts may be involved in the resolution from hepatic fibrosis in rats.7Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (97) Google Scholar, 8Iredale JP Benyon RC Pickering J McCullen M Northrop M Pawley S Hovell C Arthur MJP Mechanisms of spontaneous resolution of rat liver fibrosis: hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (931) Google Scholar, 9Iredale JP Hepatic stellate cell behavior during resolution of liver injury.Semin Liver Dis. 2001; 21: 427-436Crossref PubMed Scopus (291) Google Scholar, 10Saile B Knittel T Matthes N Schott P Ramadori G CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.Am J Pathol. 1997; 151: 1265-1272PubMed Google Scholar, 11Saile B Matthes N Neubauer K Eisenbach C El-Armouche H Dudas J Ramadori G Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-α.Am J Physiol. 2002; 283: G435-G444Google Scholar Thus, changes in cell fate and behavior of activated HSCs and portal myofibroblasts affect the pathogenesis and recovery from hepatic fibrosis/cirrhosis.Hepatocyte growth factor (HGF), originally identified and cloned as a mitogenic protein for hepatocytes,12Nakamura T Nawa K Ichihara A Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats.Biochem Biophys Res Commun. 1984; 122: 1450-1459Crossref PubMed Scopus (1086) Google Scholar, 13Nakamura T Nishizawa T Hagiya M Seki T Shimonishi M Sugimura A Tashiro K Shimizu S Molecular cloning and expression of human hepatocyte growth factor.Nature. 1989; 342: 440-443Crossref PubMed Scopus (1967) Google Scholar exerts diverse cellular responses through c-Met receptor tyrosine kinase in a wide variety of cells.14Birchmeier C Gherardi E Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase.Trends Cell Biol. 1998; 8: 404-409Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 15Matsumoto K Nakamura T Emerging multipotent aspects of hepatocyte growth factor.J Biochem. 1996; 119: 591-600Crossref PubMed Scopus (609) Google Scholar Multiple biological events of HGF include mitogenic, motogenic, morphogenic, and anti-cell death activities. Likewise, HGF stimulates expression and activity of proteases involved in breakdown of ECM proteins, including urokinase-type plasminogen activator and matrix metalloproteinases.16Pepper MS Matsumoto K Nakamura T Montesano R Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and uPA receptor expression in Madin-Darby canine kidney epithelial cells.J Biol Chem. 1992; 267: 20493-20496Abstract Full Text PDF PubMed Google Scholar, 17Dunsmore SE Rubin JS Kovacs SO Chedid M Parks WC Welgus HG Mechanisms of hepatocyte growth factor-stimulation of keratinocyte metalloproteinase production.J Biol Chem. 1996; 271: 24567-24582Google Scholar, 18Dohi M Hasegawa T Yamamoto K Marshall BC Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary fibrosis.Am J Respir Crit Care Med. 2000; 162: 2302-2307Crossref PubMed Scopus (130) Google Scholar, 19Ozaki I Zhao G Mizuta T Ogawa Y Hara T Kajihara S Hisatomi A Sakai T Yamamoto K Hepatocyte growth factor induces collagenase (matrix metalloproteinase-1) via the transcription factor Ets-1 in human hepatic stellate cell line.J Hepatol. 2002; 36: 169-178Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Physiologically, HGF plays a role in regeneration and protection of organs, including the liver.15Matsumoto K Nakamura T Emerging multipotent aspects of hepatocyte growth factor.J Biochem. 1996; 119: 591-600Crossref PubMed Scopus (609) Google Scholar Based on the trophic roles of HGF in tissue regeneration and protection, approaches to address the therapeutic potential of HGF have been made using a variety of experimental models. Importantly, HGF has therapeutic effects on liver cirrhosis, decreasing both ECM deposition and mortality in distinct models of liver cirrhosis in rats.20Matsuda Y Matsumoto K Ichida T Nakamura T Hepatocyte growth factor suppresses the onset of liver cirrhosis and abrogates lethal hepatic dysfunction in rats.J Biochem. 1995; 118: 643-649Crossref PubMed Scopus (277) Google Scholar, 21Yasuda H Imai E Shiota A Fujise N Morinaga T Higashio K Anti-fibrogenic effect of a deletion variant of hepatocyte growth factor on liver fibrosis in rats.Hepatology. 1996; 24: 636-642Crossref PubMed Google Scholar, 22Matsuda Y Matsumoto K Yamada A Ichida T Asakura H Komoriya Y Nishiyama E Nakamura T Preventive and therapeutic effects in rats of hepatocyte growth factor infusion on liver fibrosis/cirrhosis.Hepatology. 1997; 26: 81-89Crossref PubMed Scopus (181) Google Scholar, 23Ueki T Kaneda Y Tsutsui H Nakanishi K Sawa Y Morishita R Matsumoto K Nakamura T Takahashi H Okamoto E Fujimoto J Hepatocyte growth factor gene therapy of liver cirrhosis in rats.Nat Med. 1999; 5: 226-230Crossref PubMed Scopus (568) Google Scholar, 24Sato M Kakubari M Kawamura M Sugimoto J Matsumoto K Ishii T The decrease in total collagen fibers in the liver by hepatocyte growth factor after formation of cirrhosis induced by thioacetamide.Biochem Pharmacol. 2000; 59: 681-690Crossref PubMed Scopus (53) Google Scholar However, the molecular and cellular mechanisms by which supplements of HGF leads to recovery from cirrhosis are not fully understood.We now report evidence that the c-Met receptor is expressed in hepatic portal myofibroblasts and that HGF suppressed DNA synthesis and stimulated apoptotic cell death in portal myofibroblasts, and these processes were associated with recovery from liver cirrhosis in rats. We propose that newly identified mechanisms may well lead the way to therapeutic approaches for patients with liver cirrhosis.Materials and MethodsGrowth FactorsHuman recombinant HGF was purified from conditioned medium of Chinese hamster ovary cells transfected with an expression vector containing cDNA for human HGF deleted with five amino acid residues, as described.20Matsuda Y Matsumoto K Ichida T Nakamura T Hepatocyte growth factor suppresses the onset of liver cirrhosis and abrogates lethal hepatic dysfunction in rats.J Biochem. 1995; 118: 643-649Crossref PubMed Scopus (277) Google Scholar, 25Seki T Ihara I Sugimura A Shimonishi M Nishizawa T Asami O Hagiya M Nakamura T Shimizu S Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocyte.Biochem Biophys Res Commun. 1990; 172: 321-327Crossref PubMed Scopus (172) Google Scholar Recombinant human transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB were obtained from R&D Systems (Minneapolis, MN).Animal TreatmentFive-week-old male Sprague-Dawley rats were used. All animal experiments were done in accordance with National Institutes of Health guidelines, as dictated by the animal care facility at Osaka University Graduate School of Medicine. For development of experimental hepatic cirrhosis, rats were intraperitoneally administered dimethylnitrosamine (DMN) dissolved in saline at 10 mg per kg of body weight, on 3 consecutive days a week for 5 continuous weeks, then these rats were intraperitoneally given saline alone or HGF daily in a dose of 300 μg per kg of body weight for 1 week. Seven or eight rats were included in each experimental group.Histological ProceduresTissues were fixed in 10% neutralized formaldehyde or 70% ethanol and embedded in paraffin. For immunohistochemical detection of desmin and α-smooth muscle actin (α-SMA), the tissue sections were, respectively, incubated with monoclonal anti-mouse desmin antibody (EPOS system; DAKO, Carpinteria, CA) and monoclonal anti-human α-SMA antibody (EPOS system) for 1 hour. The reaction was visualized with use of 0.025% diaminobenzidine and 0.003% hydrogen peroxide. For detection of the c-Met receptor in cultured cells, cells were washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol for 10 minutes at room temperature. After washing three times with PBS, these cells were incubated for 1 hour with rabbit anti-mouse c-Met antibody (SP260; Santa Cruz Biotechnology, Santa Cruz, CA) and Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 hour. Nuclei were stained with TO-PRO-3 iodide (642/661) (Molecular Probes). In the control specimen, the c-Met primary antibody was preabsorbed with an excess of c-Met blocking peptide (SP260P, Santa Cruz Biotechnology). For double immunohistochemistry of desmin and c-Met, tissue sections were incubated overnight with anti-desmin antibody and polyclonal anti-mouse c-Met antibody (SP260; Santa Cruz Biotechnology). Tissue sections were sequentially incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG and rhodamine-conjugated anti-rabbit IgG (ICN Pharmaceuticals Inc., Aurora, OH). Fibrous tissue area was quantitated in hepatic tissue sections stained for α-SMA, using a computerized morphometric analysis using the NIH image software (Wayne Rasband Analytics, NIH, Bethesda, MD).For detection of cells undergoing DNA synthesis, 5′-bromo-2′-deoxyuridine (BrdU, 100 mg per kg) was intraperitoneally injected into rats, 1 hour before killing the animals. Tissues were fixed in 70% ethanol and sections were denatured in 2 mol/L HCl for 30 minutes at room temperature, followed by neutralization with 0.1 mol/L borate buffer for 30 minutes. After washing in phosphate-buffered saline (PBS), tissue sections were incubated with monoclonal anti-BrdU antibodies (1:500 dilution, DAKO) for 1 hour, followed by sequential incubation with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) and peroxidase-labeled avidin-biotin complex, using the Vectastain Elite ABC kit (Vector Laboratories). The reaction was detected in substrate solution containing diaminobenzidine. For detection of cells undergoing apoptosis, tissue sections were subjected to terminal dUTP nick-end labeling (TUNEL), using an In Situ Apoptosis Detection kit (MK-500; Takara Shuzo Co., Tokyo, Japan). After BrdU or TUNEL staining, sections were successively incubated with monoclonal anti-human α-SMA antibody and alkaline phosphatase-conjugated anti-mouse IgG, and the reaction was detected using a commercial kit (Vectastain ALP ABC, Vector Laboratories). The number of cells double positive for BrdU and α-SMA or TUNEL and α-SMA was counted in the stained sections at 400-fold field magnification under a microscope, using at least 10 randomly selected microscopic fields per each section.Western Blots for Desmin and α-SMA in Hepatic TissuesHepatic tissues were homogenized in buffer composed of 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L phenylmethyl sulfonyl fluoride, and 5 μg/ml leupeptin. The homogenates were centrifuged and the supernatants were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and probed with monoclonal anti-mouse desmin (DAKO) or α-SMA antibodies (DAKO). Proteins reacting with these antibodies were detected using an enhanced chemiluminescence reagent (Amersham Life Science Inc., Arlington, IL).Cultivation of HSCs and Liver MyofibroblastsHSCs were isolated according to Knook and colleagues.26Knook DL Seffelaar AM de Leeuw AM Fat-storing cells of the rat liver. Their isolation and purification.Exp Cell Res. 1982; 139: 468-471Crossref PubMed Scopus (221) Google Scholar Briefly, the liver was digested in situ with solutions containing collagenase type I. The digested liver was filtrated and parenchymal hepatocytes were removed after centrifugation at 50 × g for 1 minute. Nonparenchymal cells obtained after centrifugation at 1400 × g were resuspended and subjected to a Nycodenz density gradient centrifugation. Cells in the top layer were recovered. The cells were cultured in proline-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Portal myofibroblasts were obtained after serial passages (three to five passages) of the culture. Populations of different cell types in cultures were analyzed by vitamin A autofluorescence and immunocytochemistry using antibodies against fibulin-2 (for portal myofibroblasts), Mac-1 (for macrophages), and von Willebrand factor (for endothelial cells).Western Blot Analysis of c-Met Receptor and α-SMA in Cultured CellsTo detect c-Met receptors using Western blots, cells were lysed in the sample buffer for SDS-PAGE. The cell lysate was subjected to SDS-PAGE and proteins were electroblotted onto polyvinylidene difluoride membranes. After blocking with PBS containing 5% skim milk, the membrane was incubated with rabbit anti-mouse c-Met antibody (SP260, Santa Cruz Biotechnology). For analysis of α-SMA expression, the membrane was successively incubated with mouse monoclonal anti-human α-SMA antibody, biotinylated anti-mouse IgG (Vector Laboratories), and peroxidase-labeled avidin-biotin complex (Vectastain, Vector Laboratories). Immunoreactive proteins were visualized using an enhanced chemiluminescence reagent.Measurement of DNA Synthesis, Apoptosis, and Lactate Dehydrogenase (LDH) ActivityCells were seeded at a density of 2 × 104 cells per well in 24-well plates and cultured for 24 hours. After being serum-starved in DMEM supplemented with 0.2% fetal bovine serum for 24 hours, the cells were incubated in the absence or presence of varying concentrations of HGF with or without 10 ng/ml of PDGF-BB for 20 hours, then pulse-labeled with 1.0 μCi per ml [methyl-3H] thymidine (Amersham Life Science Inc.) for 6 hours. The number of viable cells was determined using trypan blue dye extrusion assays. To detect apoptotic cells, cells were fixed in 70% ethanol and apoptotic cells were stained using the TUNEL reaction, and In Situ Apoptosis Detection kits (MK-500; Takara Shuzo Co). LDH activity in the culture media was determined using a kit (Wako Pure Chemicals, Co., Osaka, Japan).Analysis of Extracellular Signal-Regulated Kinase (ERK)-1/2 and c-jun N-Terminal Kinase (JNK)For analysis of ERK-1/2 phosphorylation, cells were serum-starved in DMEM supplemented with 0.2% fetal bovine serum for 24 hours and treated with 10 ng/ml of PDGF in the absence or presence of HGF for 24 hours. For analysis of JNK, serum-starved cells were treated with 10 ng/ml of HGF then were lysed with sample buffer for SDS-PAGE and the lysate was subjected to SDS-PAGE, followed by electroblotting on polyvinylidene difluoride membrane. After blocking, the membrane was sequentially incubated with anti-phospho-p44/p42 mitogen-activated protein kinase (ERK-1/2) antibody (E10; New England BioLabs Inc., Beverly, MA) or an anti-phosphorylated JNK antibody (Phosphoplus SAPK/JNK antibody kit; Cell Signaling Technology, Beverly, MA), biotinylated anti-mouse IgG (Vector Laboratories), and horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech UK, Little Chalfont, UK). Immunoreactive proteins were detected using an enhanced chemiluminescence reagent. To detect total ERK1/2 or JNK1 proteins, the cells lysate were subjected to Western blots as described above except for use of an anti-ERK-1 antibody (K-23; Santa Cruz Biotechnology) or an anti-JNK antibody (Phosphoplus SAPK/JNK antibody kit).Data AnalysisData represent the mean ± SD. Statistical analyses were made using unpaired Student's t-test (two-tailed) or analysis of variance. Differences were considered to be statistically significant at the P < 0.05 levels.ResultsResolution of Liver Cirrhosis by HGFDMN was given to rats intraperitoneally for 3 consecutive days each week for 5 weeks (Figure 1a), and liver specimens were subjected to histological analysis (Figure 1b). Formation of fibrotic septa joining the central area and the nodular pattern of parenchymal cells separated by fibrous septa were evident and reticulin fibers had spread radially throughout the liver. These pathological changes are characteristic of liver cirrhosis. To evaluate expansion of activated HSCs and liver myofibroblasts, hepatic sections were subjected to immunostaining for α-SMA and desmin (Figure 1b). Desmin and α-SMA are expressed in activated HSCs and liver myofibroblasts.7Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (97) Google Scholar Cells positive for α-SMA and desmin localized in the fibrous area spread throughout the liver, indicating that expansion of activated HSCs and portal myofibroblasts is associated with fibrotic changes in the liver. Associated with these changes, TGF-β1 levels in the cirrhotic liver tissues increased to 5.1-times higher level than in the normal liver (not shown).To determine whether HGF affects resolution from liver cirrhosis, rats subjected to DMN treatment for 5 weeks were daily given HGF or saline for 1 week. In control rats given saline alone, there was little change in the histopathology of the liver: fibrous areas still spread radially throughout the liver and cells positive for α-SMA and desmin were entirely localized in fibrous areas (Figure 1b). In contrast, in rats treated with HGF, fibrotic septa joining the central area and reticulin fibers either disappeared or were markedly decreased. Likewise, cells positive for α-SMA and desmin were remarkably decreased by HGF treatment (Figure 1b). When expansion of fibrous tissue areas was quantitated by image analysis in hepatic tissue sections stained for α-SMA, fibrous area markedly increased by DMN treatment (Figure 1c). The fibrous area in DMN-treated rats given HGF for 1 week decreased to 41% of DMN-treated rats given saline alone.The remarkable decrease in fibrous tissue expansion and hepatic expression of α-SMA by HGF suggested that HGF directly or indirectly affects the fate of activated HSCs and/or portal myofibroblasts. One potential mechanism for reduction in α-SMA expression might be that HGF facilitates a phenotypic reversal of activated HSCs to a quiescent phenotype. Therefore, the number of cells that expressed α-SMA or desmin was quantitated in hepatic sections subjected to immunohistochemistry (Figure 1d). The number of α-SMA- and desmin-positive cells, respectively, reached 98.4 ± 12.3 and 87.6 ± 10.3 cells per microscopic field at 6 weeks in control rats not given HGF treatment. In rats subjected to a 1-week treatment with HGF, the number of α-SMA-positive cells decreased to 51.4 ± 7.6 cells per microscopic field at 6 weeks. If HGF facilitates a reversal of activated HSCs (positive for both α-SMA and desmin) to a quiescent phenotype (desmin-positive but α-SMA-negative), decrease in the number of α-SMA-positive cells would be associated with an increase in the number of desmin-positive cells. However, the number of desmin-positive cells was also decreased by HGF to 51.2 ± 13.5 cells, a value being the same as that of α-SMA-positive cells at 6 weeks in HGF-treated rats. Consistently, when hepatic tissue extracts were subjected to Western blots, hepatic expressions of both α-SMA and desmin were decreased by HGF treatment (Figure 1e). These findings strongly suggested that HGF did not facilitate a reversal of activated HSCs to the quiescent phenotype.c-Met Receptor Expression in Activated Liver MyofibroblastsTo determine how HGF directly or indirectly affects the fate of α-SMA-positive cells, c-Met receptor expression was analyzed in HSCs and portal myofibroblasts isolated from the rat liver. In agreement with previous findings, HSCs had vitamin A-containing vacuoles characterized by autofluorescence and had a star-like appearance within a few days after plating (not shown). Three days after plating, cells with a fibroblast-like appearance appeared. After passage of the culture, most of cells had a fibroblast-like appearance. Previous studies indicated that HSCs in culture had little potential to proliferate, were withdrawn because of cell death, and could be maintained in culture at maximum until passage 2 only.7Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (97) Google Scholar Instead, portal myofibroblasts showed rapid proliferative ability and could be expanded up to the 10th passage. Taken together, we speculated that portal myofibroblasts present in primary culture of HSCs proliferated and HSCs were replaced by portal myofibroblasts. Because fibulin-2 is specifically expressed in portal myofibroblasts but not in HSCs,7Ramadori G Saile B Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (97) Google Scholar we analyzed fibulin-2 expression in cultures of HSCs (Figure 2a). In primary culture of HSCs (3 days after plating), there were contaminating portal myofibroblasts positive for fiblulin-2, whereas 75 to 85% of cells were positive for fibulin-2 after serial passages of the culture (at passage 3). The result indicated that HSCs were replaced by proliferating portal myofibroblasts during serial passages and cultivation. Knittel and colleagues4Knittel T Kobold D Saile B Grundmann A Neubauer K Piscaglia F Ramadori G Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential.Gastroenterology. 1999; 117: 1205-1221Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar reported that 50 to 100% of rat portal myofibroblasts were positive for fibulin-2.Figure 2c-Met receptor expression in cultures of HSCs and portal myofibroblasts, and in normal and cirrhotic livers. a: Expression of fibulin-2 in cultures of HSCs and portal myofibroblasts. Fiblin-2 expression was analyzed by immunocytochemistry in primary cultures of HSCs on day 3 after plating and the cells after three passages. b: Changes in expression of c-Met receptor and α-SMA in cultured HSCs and portal myofibroblasts. Expressions of c-Met receptor and α-SMA were analyzed using Western blots. c: c-Met receptor expression in HSCs and activated liver myofibroblasts as detected immunocytochemically. C-Met expression was analyzed in primary culture of HSCs on day 9 after plating and the culture of cells after three passages. d: Localization of c-Met receptor expression in normal and cirrhotic livers. Hepatic sections were subjected to immunohistochemical staining for desmin (green) and c-Met receptor (red). Nuclei were detected by staining with Hoechst 33342. Note the merged image of desmin and c-Met receptor staining. Original magnifications: ×200; ×400 (insets).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In quiescent HSCs in primary culture (day 0), α-SMA was undetectable in Western blots, but was detectable in cells cultured for 3 days (Figure 2b). Expression of α-SMA remarkably increased in cells expanded after serial passages and cultured in the presence of 10 ng/ml of TGF-β1 for 3 days. When c-Met receptor expression was analyzed in these cells using Western blots, the c-Met receptor was undetec" @default.
• W2022308645 created "2016-06-24" @default.
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• W2022308645 date "2005-04-01" @default.
• W2022308645 modified "2023-10-10" @default.
• W2022308645 title "Growth Inhibition and Apoptosis in Liver Myofibroblasts Promoted by Hepatocyte Growth Factor Leads to Resolution from Liver Cirrhosis" @default.
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