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• W1980464176 abstract "Hepatic fibrosis is a common outcome of a variety of chronic liver diseases. Here we evaluated the therapeutic efficacy of hepatocyte growth factor (HGF) on liver fibrosis induced by bile duct ligation (BDL) and investigated potential mechanisms. Mice underwent BDL, followed by intravenous injections of naked HGF expression plasmid or control vector. HGF gene therapy markedly ameliorated hepatic fibrotic lesions, as demonstrated by reduced α-smooth muscle actin (αSMA) expression, attenuated deposition of type I and type III collagen, and normalized total hydroxyproline content. HGF also suppressed transforming growth factor-β1 (TGF-β1) expression. Interestingly, colocalization of αSMA and cytokeratin-19 in bile duct epithelium was observed, suggesting the possibility of biliary epithelial to myofibroblast transition after BDL. Cells that were still positive for cytokeratin-19 but actively producing type I collagen were found in the biliary epithelia and periductal region. Laminin staining revealed an impaired basement membrane of the bile duct epithelium in diseased liver. These lesions were largely prevented by HGF administration. In vitro, treatment of human biliary epithelial cells with TGF-β1 induced αSMA and fibronectin expression and suppressed cytokeratin-19. HGF abolished the phenotypic conversion of biliary epithelial cells induced by TGF-β1. These results suggest that HGF ameliorates hepatic biliary fibrosis in part by blocking bile duct epithelial to mesenchymal transition. Hepatic fibrosis is a common outcome of a variety of chronic liver diseases. Here we evaluated the therapeutic efficacy of hepatocyte growth factor (HGF) on liver fibrosis induced by bile duct ligation (BDL) and investigated potential mechanisms. Mice underwent BDL, followed by intravenous injections of naked HGF expression plasmid or control vector. HGF gene therapy markedly ameliorated hepatic fibrotic lesions, as demonstrated by reduced α-smooth muscle actin (αSMA) expression, attenuated deposition of type I and type III collagen, and normalized total hydroxyproline content. HGF also suppressed transforming growth factor-β1 (TGF-β1) expression. Interestingly, colocalization of αSMA and cytokeratin-19 in bile duct epithelium was observed, suggesting the possibility of biliary epithelial to myofibroblast transition after BDL. Cells that were still positive for cytokeratin-19 but actively producing type I collagen were found in the biliary epithelia and periductal region. Laminin staining revealed an impaired basement membrane of the bile duct epithelium in diseased liver. These lesions were largely prevented by HGF administration. In vitro, treatment of human biliary epithelial cells with TGF-β1 induced αSMA and fibronectin expression and suppressed cytokeratin-19. HGF abolished the phenotypic conversion of biliary epithelial cells induced by TGF-β1. These results suggest that HGF ameliorates hepatic biliary fibrosis in part by blocking bile duct epithelial to mesenchymal transition. Liver fibrosis/cirrhosis is a common end-result of a wide variety of chronic hepatic diseases following diverse types of injurious insult, such as viral infection and alcoholic, drug, or chemical toxicity. It is often associated with severe morbidity and significant mortality, eventually resulting in the necessity of liver transplantation.1Pinzani M Rombouts K Liver fibrosis: from the bench to clinical targets.Dig Liver Dis. 2004; 36: 231-242Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar The pathogenesis of liver cirrhosis is characterized by the excess production and deposition of extracellular matrix components that lead to tissue scarring and the destruction of normal hepatic parenchyma.2Albanis E Friedman SL Hepatic fibrosis. Pathogenesis and principles of therapy.Clin Liver Dis. 2001; 5 (vvi): 315-334Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 3Eng FJ Friedman SL Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex.Am J Physiol. 2000; 279: G7-G11PubMed Google Scholar Several lines of evidence indicate that α-smooth muscle actin (αSMA)-positive myofibroblasts are the principal effector cells that are primarily responsible for the overproduction of matrix components in fibrotic liver.2Albanis E Friedman SL Hepatic fibrosis. Pathogenesis and principles of therapy.Clin Liver Dis. 2001; 5 (vvi): 315-334Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar Therefore, delineation of the originality and mechanism of activation of the matrix-producing myofibroblast cells may be indispensable for designing rational therapeutic strategies for effective treatment of liver cirrhosis.In an effort to define the origin and regulation of myofibroblast activation in hepatic fibrosis, we have sought to investigate the pathogenesis of liver fibrosis in experimental animals induced by bile duct ligation (BDL). Hepatic fibrosis induced by BDL is unique in that the primary pathological lesions occur surrounding the bile duct epithelium. The maneuver of BDL introduces biomechanical stress to biliary epithelium and initially triggers the compensatory proliferation and expansion of biliary epithelial cells (BECs).4Ezure T Sakamoto T Tsuji H Lunz third, JG Murase N Fung JJ Demetris AJ The development and compensation of biliary cirrhosis in interleukin-6-deficient mice.Am J Pathol. 2000; 156: 1627-1639Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 5Lunz III, JG Contrucci S Ruppert K Murase N Fung JJ Starzl TE Demetris AJ Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.Am J Pathol. 2001; 158: 1379-1390Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Following this epithelial mitogenic phase, chronic obstruction of bile duct causes massive activation of myofibroblasts in the periductal region and ultimately results in biliary fibrosis/cirrhosis. There is little doubt that myofibroblast activation is one of the key steps that initiate fibrotic lesions in this model. However, the exact origin of these periductal myofibroblasts remains largely unknown, although it is often presumed that they are from local activation of residential fibroblasts or hepatic stellate cells.Recent studies from other organs have suggested that matrix-producing myofibroblasts may also come from epithelial cells through a phenotypic conversion known as epithelial to mesenchymal transition (EMT).6Kalluri R Neilson EG Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2059) Google Scholar, 7Liu Y Epithelial to mesenchymal transition in renal fibrogenesis: pathological significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (950) Google Scholar For instance, in the obstructive nephropathy induced by unilateral ureteral obstruction, kidney tubular epithelial cells can undergo phenotypic transition via EMT to give rise to matrix-producing myofibroblasts.8Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 9Iwano M Plieth D Danoff TM Xue C Okada H Neilson EG Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1696) Google Scholar Furthermore, blockade of this transition by hepatocyte growth factor (HGF) prevents renal interstitial fibrosis.10Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar These findings highlight that mature epithelial cells in adult animals may possess an incredible plasticity, with the ability to transform into myofibroblasts in response to chronic injury. Such epithelial cell plasticity is also observable in the liver.11Thorgeirsson SS Grisham JW Overview of recent experimental studies on liver stem cells.Semin Liver Dis. 2003; 23: 303-312Crossref PubMed Scopus (56) Google Scholar, 12Hixson DC Chapman L McBride A Faris R Yang L Antigenic phenotypes common to rat oval cells, primary hepatocellular carcinomas and developing bile ducts.Carcinogenesis. 1997; 18: 1169-1175Crossref PubMed Scopus (56) Google Scholar Numerous studies have shown that, under certain conditions, BECs may give rise to hepatic oval cells, which in turn may become mature hepatocytes.13Alison M Golding M Lalani EN Nagy P Thorgeirsson S Sarraf C Wholesale hepatocytic differentiation in the rat from ductular oval cells, the progeny of biliary stem cells.J Hepatol. 1997; 26: 343-352Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 14Alison MR Vig P Russo F Bigger BW Amofah E Themis M Forbes S Hepatic stem cells: from inside and outside the liver?.Cell Prolif. 2004; 37: 1-21Crossref PubMed Scopus (140) Google Scholar, 15Haque S Haruna Y Saito K Nalesnik MA Atillasoy E Thung SN Gerber MA Identification of bipotential progenitor cells in human liver regeneration.Lab Invest. 1996; 75: 699-705PubMed Google Scholar, 16Sirica AE Gainey TW Mumaw VR Ductular hepatocytes. Evidence for a bile ductular cell origin in furan-treated rats.Am J Pathol. 1994; 145: 375-383PubMed Google Scholar Vice versa, in the conditions of the organoid cultures or in vivo, hepatocytes can undergo phenotypic transition into BECs.17Michalopoulos GK Bowen WC Mule K Lopez-Talavera JC Mars W Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures.Hepatology. 2002; 36: 278-283Crossref PubMed Scopus (70) Google Scholar, 18Michalopoulos GK Barua L Bowen WC Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury.Hepatology. 2005; 41: 535-544Crossref PubMed Scopus (235) Google Scholar Thus BECs and hepatocytes appear to be reciprocally exchangeable. Along this line, we hypothesized that, in pathological circumstances, BECs may transform into αSMA-positive, matrix-producing myofibroblasts as well.HGF, a multifunctional protein that was originally characterized as a potent mitogen for mature hepatocytes,19Michalopoulos GK DeFrances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2864) Google Scholar has emerged as a potent anti-fibrotic cytokine that prevents tissue fibrosis in various organs, including the liver.20Liu Y Hepatocyte growth factor in kidney fibrosis: therapeutic potential and mechanisms of action.Am J Physiol Renal Physiol. 2004; 287: F7-F16Crossref PubMed Scopus (216) Google Scholar, 21Matsumoto K Nakamura T Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases.Kidney Int. 2001; 59: 2023-2038PubMed 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 It has been shown that HGF specifically preserves renal tubular epithelial cell phenotype by inhibiting epithelial to myofibroblast transition both in vitro and in vivo.10Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar Therefore, in this study, we evaluated the therapeutic potential of HGF in blocking hepatic fibrosis induced by DBL and explored the possibility of the biliary epithelial to myofibroblast transition in vivo and in vitro.Materials and MethodsCell Culture and TreatmentHuman intrahepatic biliary epithelial cells (HIBEpiCs) and epithelial cell medium were purchased from ScienCell Research Laboratories (San Diego, CA). HIBEpiCs were nonimmortalized cells isolated from human liver tissue, and the purity of these cells was almost 100% based on their positive staining for cytokeratin-19. The HIBEpiCs were seeded on six-well culture plates to approximately 60% to 70% confluence in complete medium containing 2% fetal bovine serum for 16 hours and then changed to serum-free medium after washing twice with medium. Recombinant human transforming growth factor (TGF)-β1 (R & D Systems, Minneapolis, MN) was added to the culture at a final concentration of 2 ng/ml. Recombinant human HGF (R & D Systems) was also added at the same time at the concentration of 40 ng/ml, except when otherwise indicated. The cells were typically incubated for 72 hours after addition of cytokines, except when noted otherwise, before harvesting and subjecting to Western blot and immunofluorescence staining as described below.AnimalsMale CD-1 mice that weighed approximately between 18 and 21 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were housed in the animal facilities of the University of Pittsburgh Medical Center with free access to food and water. Mice were randomly assigned to three groups: 1) sham control; 2) BDL followed by injection with empty vector pcDNA3; or 3) BDL followed by injection with HGF expression plasmid. BDL was performed as described elsewhere.4Ezure T Sakamoto T Tsuji H Lunz third, JG Murase N Fung JJ Demetris AJ The development and compensation of biliary cirrhosis in interleukin-6-deficient mice.Am J Pathol. 2000; 156: 1627-1639Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Briefly, under methoxyflurane anesthesia, the common bile duct was double-ligated using 4-0 silk after a midline abdominal incision. Sham-operated mice had their common bile duct exposed and manipulated but not ligated. Starting on the day of surgery, mice were administered human HGF cDNA plasmid under the control of cytomegalovirus (CMV) promoter (pCMV-HGF) through tail-vein injections at a concentration of 1 mg/kg body weight every other week (biweekly), as described previously.24Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar, 25Dai C Yang J Liu Y Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice.J Am Soc Nephrol. 2002; 13: 411-422Crossref PubMed Google Scholar Control mice received biweekly injections of the same volume of empty vector (pcDNA3). Animals were sacrificed at 4 and 12 weeks after BDL (n = 4–6). At the time of sacrifice, liver tissues were collected for histology and immunohistochemical studies, as well as for mRNA and protein analyses.Biochemical Measurement of Hepatic Hydroxyproline ContentTotal hepatic hydroxyproline level at different time points after BDL was determined in the hydrolysates of liver samples.26Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (131) Google Scholar Briefly, precisely weighed liver tissue samples (30 to 40 mg) were homogenized in distilled H2O. The homogenates were hydrolyzed in 10 N HCl by incubation at 110°C for 18 hours. The hydrolysates were dried by speed vacuum centrifugation over 3 to 5 hours and redissolved in a buffer containing 0.2 mol/L citric acid, 0.2 mol/L glacial acetic acid, 0.4 mol/L sodium acetate, and 0.85 mol/L sodium hydroxide, pH 6.0. Hydroxyproline levels in the hydrolysates were biochemically measured according to the procedures previously described.26Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (131) Google Scholar, 27Kivirikko KI Laitinen O Prockop DJ Modifications of a specific assay for hydroxyproline in urine.Anal Biochem. 1967; 19: 249-255Crossref PubMed Scopus (968) Google ScholarHistology and ImmunostainingLiver sections from paraffin-embedded tissues were prepared at 3-μm thickness using a routine procedure. Sections were stained with hematoxylin/eosin for general histology. Another set of sections was stained using the Masson's trichrome staining method for identifying interstitial collagen by blue color. A computer-aided morphometric analysis was used for quantitatively determining the area of fibrosis, as reported previously.28Moal F Chappard D Wang J Vuillemin E Michalak-Provost S Rousselet MC Oberti F Cales P Fractal dimension can distinguish models and pharmacological changes in liver fibrosis in rats.Hepatology. 2002; 36: 840-849PubMed Google Scholar Briefly, a series of digital images was captured from the Masson's trichrome-stained sections. The areas of fibrosis were measured by using morphometric analysis software (MetaMorph; Universal Imaging Corp., Downingtown, PA).29Dai C Huh CG Thorgeirsson SS Liu Y β-Cell-specific ablation of the hepatocyte growth factor receptor results in reduced islet size, impaired insulin secretion, and glucose intolerance.Am J Pathol. 2005; 167: 429-436Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar The percentage of the fibrotic area per total liver area was calculated based on each individual animal.Immunofluorescence and immunohistochemical staining were performed on liver tissue cryosections and paraffin-embedded sections, respectively. All immuno-stainings were performed by using the Vector M.O.M. Immunodetection kit according to the procedures specified by the manufacturer (Vector Laboratories, Burlingame, CA). The primary antibodies used for staining were as follows: goat polyclonal antibodies against type I and type III collagen (Southern Biotechnology Associates, Birmingham, AL) (dilution 1:50); anti-TGF-β1 (sc-146), anti-TGF-β type I receptor (sc-398), and anti-heat shock protein 47 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) (dilution 1:100); mouse monoclonal anti-αSMA antibody (clone 1A4, dilution 1:50) and monoclonal antibodies for cytokeratin-19 (DakoCytomation, Carpentaria, CA, dilution 1:20); monoclonal anti-fibronectin (clone 10, dilution 1:1000; BD Transduction Laboratories, San Diego, CA); and anti-laminin (Chemicon Inc., Temecula, CA, dilution 1:100). Double immunofluorescence staining was performed on paraffin-embedded sections (3 μm) or cryosections (4 μm) by using different combinations of antibodies. For instance, for double staining for cytokeratin-19 and αSMA, liver sections were first incubated with monoclonal anti-cytokeratin-19 for 1 hour at room temperature. Biotin-conjugated anti-mouse IgG1 (A85–1; BD Pharmingen, San Diego, CA) was then added, and the mixture was incubated for 30 minutes. The staining re-action was developed with cyanine Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes. The sections were then incubated with anti-αSMA (isotype IgG2a; Dako, Carpentaria, CA) for 1 hour, followed by incubation with fluorescein isothiocyanate anti-mouse IgG2a (R19–15, BD Pharmingen, dilution 1:100) for 30 minutes. As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred.Immunofluorescence staining of HIBEpiCs was performed using an established procedure. Briefly, control or cytokine-treated HIBEpiCs cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 minutes on ice and blocked with 20% normal donkey serum in phosphate-buffered saline buffer for 30 minutes at room temperature and then incubated with the specific primary antibodies described above. To visualize the primary antibodies, cells were stained with fluorescein isothiocyanate- or cyanine Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Cells were counterstained with 4′,6-diamidino-2-phenylindole, HCl to visualize the nuclei. Stained cells were viewed under a Nikon Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Melville, NY).Western Blot AnalysisLiver samples were homogenized by using a Polytron homogenizer in radioimmune precipitation assay lysis buffer (1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 100 μg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mmol/L sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml antipain, and 2 μg/ml leupeptin in phosphate-buffered saline) on ice. The supernatants were collected after centrifugation at 13,000 × g at 4°C for 20 minutes. Protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma), and whole tissue lysates were mixed with an equal amount 2× SDS loading buffer (125 mmol/L Tris-HCl, 4% SDS, 20% glycerol, 100 mmol/L dithiothreitol, and 0.2% bromphenol blue), as described previously.10Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar, 24Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar HIBEpiCs and cytokine-treated cells were lysed with SDS sample buffer. Samples were heated at 100°C for 5 to 10 minutes before loading and were separated on 10% SDS-polyacrylamide gels. The proteins were electrotransferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) in transfer buffer containing 48 mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at 4°C for 1 hour. Nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% nonfat milk in TBS buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The membranes were then incubated for 16 hours at 4°C with various primary antibodies in blocking buffer containing 5% milk at the dilutions specified by the manufacturers, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA) for 1 hour in 5% nonfat milk dissolved in Tris-buffered saline. Membranes were then washed with Tris-buffered saline buffer, and the signals were visualized using the enhanced chemiluminescence system (ECL, Amersham Biosciences).Determination of Tissue TGF-β1 Levels by Reverse Transcription-Polymerase Chain ReactionTotal RNA was isolated from liver samples by using Ultraspec RNA isolation kit according to the instructions specified by the manufacturer (Biotecx Laboratories, Houston, TX). The first strand cDNA was synthesized by using a Reverse Transcription System (Promega, Madison, WI) with random primers at 42°C for 30 minutes. Polymerase chain reaction (PCR) was performed using a standard PCR kit on 1-μl aliquots of cDNA and HotStarTaq polymerase (Qiagen Inc., Valencia, CA) with specific primer pairs for rat TGF-β1 and β-actin. The sequences of the primers were as follows: TGF-β1, 5′-GCAACATGTGGAACTCTACCAGAA-3′ (sense) and 5′-GACGTCAA-AAGACAGCCACTCA-3′ (antisense); β-actin, 5′-AGGC-ATCCTCACCCTGAAGTA-3′ (sense) and 5′-CACACG-CAGCTCATTGTAGA-3′ (antisense). The PCR protocol consisted of 35 (for TGF-β1) or 30 (for actin) cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1.5 minutes, followed by a final extension step at 72°C for 7 minutes. PCR products were size-fractionated on agarose gels and detected by ethidium bromide staining.Statistical AnalysesAnimals were randomly assigned to control and treatment groups. All data were expressed as mean ± SEM, For Western blot analysis, quantitation was performed by scanning and determination of the intensities of the hybridization signals. Statistical analyses of the data were performed by Student's t-test using SigmaStat software (Jandel Scientific, San Rafael, CA). Values of P < 0.05 were considered significant.ResultsHGF Suppresses Hepatic Fibrosis Induced by Bile Duct LigationTo assess the potential effect of exogenous HGF on liver fibrosis, mice were injected through the tail vein with human HGF expression plasmid (pCMV-HGF) or control vector pcDNA3 every other week after common BDL. Our earlier studies have shown that substantial HGF transgene expression was detected in the liver by using this simple gene transfer approach.24Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar Figure 1 shows representative micrographs of the Masson's trichrome staining of liver tissue sections at 12 weeks after BDL. In mice treated with control pcDNA3 vector, extensive peribiliary (Figure 1A) and interstitial (Figure 1C) collagen deposition was evident, as shown by positive Masson's trichrome staining. However, delivery of HGF gene largely inhibited hepatic collagen accumulation after BDL (Figure 1). Collagen staining was much weaker in the enlarged periductal area (Figure 1B), and essentially no collagen accumulation was found in liver interstitium after HGF treatment (Figure 1D). A computer-aided morphometric analysis revealed that the area of fibrosis in the liver sections at 12 weeks after BDL was reduced by 74% after pCMV-HGF administration, when compared with the pcDNA3 controls.To quantitatively evaluate the therapeutic efficacy of HGF on hepatic fibrosis induced by BDL, we determined the hydroxyproline content in the hydrolysates extracted from liver tissue by using biochemical methods. This assay is based on the observation that essentially all of the hydroxyproline in animal tissues is exclusively found in collagen.27Kivirikko KI Laitinen O Prockop DJ Modifications of a specific assay for hydroxyproline in urine.Anal Biochem. 1967; 19: 249-255Crossref PubMed Scopus (968) Google Scholar As shown in Figure 2A, the hydroxyproline content was progressively increased in the liver extracts at 4 and 12 weeks after BDL, suggesting increased hepatic fibrosis. However, exogenous HGF substantially reduced the hydroxyproline levels in the liver tissue extracts at different time points, suggesting that HGF is capable of protecting the liver from the development of fibrotic lesions after BDL.Figure 2HGF reduces hepatic hydroxyproline content and suppresses type I and type III collagen deposition. A:Total tissue hydroxyproline content was determined by biochemical assay. Data were expressed as micrograms of hydroxyproline per milligram of tissue and presented as mean ± SEM (n = 4–6). *, P < 0.05 versus sham; **, P < 0.01 versus sham; †, P < 0.05 versus pcDNA3 control. B–E: Representative micrographs showed the deposition of type I (B and C) and type III (D and E) collagen in the portal tract region of liver sections. B and D: pcDNA3; C and E: pCMV-HGF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We further examined the accumulation and deposition of the specific extracellular matrix components by immunofluorescence staining. As demonstrated in Figure 2 (B and D), liver sections at 12 weeks after BDL displayed stronger staining for type I and type III collagen, respectively. Consistent with the hydroxyproline assay (Figure 2A), delivery of HGF gene largely blocked hepatic type I and type III collagen deposition in the peribiliary region (Figure 2, C and E).HGF Inhibits TGF-β1 Expression Induced by BDLBecause TGF-β1 is a fibrogenic cytokine that is believed to play a central role in regulating tissue fibrosis,30Bissell DM Roulot D George J Transforming growth factor β and the liver.Hepatology. 2001; 34: 859-867Crossref PubMed Scopus (335) Google Scholar, 31George J Roulot D Koteliansky VE Bissell DM In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: a potential new therapy for hepatic fibrosis.Proc Natl Acad Sci USA. 1999; 96: 12719-12724Crossref PubMed Scopus (321) Google Scholar we next investigated the effect of exogenous HGF on hepatic TGF-β1 expression after BDL. As presented in Figure 3A, BDL induced a dramatic increase in hepatic TGF-β1 gene expression, as determined by reverse transcription-PCR analyses. The steady-state levels of TGF-β1 mRNA in the liver at 12 weeks after BDL were increased by more than 10-fold, compared to the sham controls (Figure 3B). However, exogenous HGF markedly suppressed hepatic TGF-β1 mRNA abundance. Figure 3B shows the quantitative determination of the relative TGF-β1 mRNA abundance in different groups. We also examined TGF-β1 protein expression in the liver by using immunohistochemical staining. As shown in Figure 3D, TGF-β1 was predominantly localized in the biliary epithelium at 12 weeks after BDL. Strong staining for TGF-β1 was also observed in the periductal region (Figure 3D). Consistent with mRNA expression, TGF-β1 protein level was extremely low in normal liver (Figure 3C), and exogenous HGF largely suppressed its induction after BDL (Figure 3E). These results are consistent with the notion that biliary epithelial cells are the major source of hepatic TGF-β1 after BDL.Figure 3HGF inhibits hepatic TGF-β1 expression. A: Reverse tran" @default.
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• W1980464176 title "Hepatocyte Growth Factor Attenuates Liver Fibrosis Induced by Bile Duct Ligation" @default.
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• W1980464176 doi "https://doi.org/10.2353/ajpath.2006.050747" @default.
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