Contribution of Hepatic Parenchymal and Nonparenchymal Cells to Hepatic Fibrogenesis in Biliary Atresia
1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65595-2
ISSN1525-2191
AutoresGrant A. Ramm, Visalini Nair‐Shalliker, Kim R. Bridle, Ross W. Shepherd, Dorothy H. Crawford,
Tópico(s)Liver physiology and pathology
ResumoExtrahepatic biliary atresia is a severe neonatal liver disease resulting from a sclerosing cholangiopathy of unknown etiology. Although biliary obstruction may be surgically corrected by a "Kasai" hepatoportoenterostomy, most patients still develop progressive hepatic fibrosis, although the source of increased collagen deposition is unclear. This study examined the role of hepatic stellate cells (HSCs) and assessed the source of transforming growth factor-β (TGF-β) production in hepatic fibrogenesis in patients with biliary atresia. Liver biopsies from 18 biliary atresia patients (including 5 pre- and post-Kasai) were subjected to immunohistochemistry for α-smooth muscle actin and in situ hybridization for either procollagen α1 (I) mRNA or TGF-β1 mRNA. Sections were also subjected to immunohistochemistry for active TGF-β1 protein. The role of Kupffer cells in TGF-β1 production was assessed by immunohistochemistry for CD68. Procollagen α1 (I) mRNA was colocalized to α-smooth muscle actin-positive HSCs within the region of increased collagen protein deposition in fibrotic septa and surrounding hyperplastic bile ducts. The number of activated HSCs was decreased in only one post-Kasai biopsy. TGF-β1 mRNA expression was demonstrated in bile duct epithelial cells and activated HSCs and in hepatocytes in close proximity to fibrotic septa. Active TGF-β1 protein was demonstrated in bile duct epithelial cells and activated HSCs. This study provides evidence that activated HSCs are responsible for increased collagen production in patients with biliary atresia and therefore play a definitive role in the fibrogenic process. We have also shown that bile duct epithelial cells, HSCs, and hepatocytes are all involved in the production of the profibrogenic cytokine, TGF-β1. Extrahepatic biliary atresia is a severe neonatal liver disease resulting from a sclerosing cholangiopathy of unknown etiology. Although biliary obstruction may be surgically corrected by a "Kasai" hepatoportoenterostomy, most patients still develop progressive hepatic fibrosis, although the source of increased collagen deposition is unclear. This study examined the role of hepatic stellate cells (HSCs) and assessed the source of transforming growth factor-β (TGF-β) production in hepatic fibrogenesis in patients with biliary atresia. Liver biopsies from 18 biliary atresia patients (including 5 pre- and post-Kasai) were subjected to immunohistochemistry for α-smooth muscle actin and in situ hybridization for either procollagen α1 (I) mRNA or TGF-β1 mRNA. Sections were also subjected to immunohistochemistry for active TGF-β1 protein. The role of Kupffer cells in TGF-β1 production was assessed by immunohistochemistry for CD68. Procollagen α1 (I) mRNA was colocalized to α-smooth muscle actin-positive HSCs within the region of increased collagen protein deposition in fibrotic septa and surrounding hyperplastic bile ducts. The number of activated HSCs was decreased in only one post-Kasai biopsy. TGF-β1 mRNA expression was demonstrated in bile duct epithelial cells and activated HSCs and in hepatocytes in close proximity to fibrotic septa. Active TGF-β1 protein was demonstrated in bile duct epithelial cells and activated HSCs. This study provides evidence that activated HSCs are responsible for increased collagen production in patients with biliary atresia and therefore play a definitive role in the fibrogenic process. We have also shown that bile duct epithelial cells, HSCs, and hepatocytes are all involved in the production of the profibrogenic cytokine, TGF-β1. Extrahepatic biliary atresia is a progressive, sclerosing, inflammatory process in neonates, causing atresia of all or part of the extrahepatic biliary system and rapidly extending to involve the major intrahepatic biliary ducts.1Balistreri WF Neonatal cholestasis: medical progress.J Pediatr. 1985; 106: 171-184Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 2Kasai M Yakovac WC Koop CE Liver in congenital biliary atresia and neonatal hepatitis: a histopathological study.Arch Pathol. 1962; 74: 152-162PubMed Google Scholar This bile duct obliteration may be relieved by hepatoportoenterostomy (HPE) or the "Kasai procedure,"3Kasai M Suzuki S A new operation for "non-correctable" biliary atresia: hepatic portoenterostomy.Shujutsu. 1959; 13: 733-739Google Scholar, 4Kasai M Treatment of biliary atresia with special reference to hepatic portoenterostomy and its modifications.Prog Pediatr Surg. 1974; 6: 5-52PubMed Google Scholar, 5Otte JB de Ville de Goyet J Reding R Hausleithner V Sokal E Chardot C Debande B Sequential treatment of biliary atresia with Kasai portoenterostomy, and liver transplantation: a review.Hepatology. 1994; 20: 41S-48SPubMed Google Scholar in which >80% of infants will develop some biliary flow, particularly if HPE is performed within 60 days of birth (reviewed in 6Balistreri WF Grand R Hoofnagle JH Suchy FJ Ryckman FC Perlmutter DH Sokol RJ Biliary atresia: current concepts and research directions.Hepatology. 1996; 23: 1682-1692Crossref PubMed Scopus (308) Google Scholar). However, the majority of patients still develop progressive hepatic fibrosis, with approximately one-third developing liver failure and requiring liver transplantation within 12 to 14 months and a further one-third by the teenage years, and the remainder will live with some form of liver disease, including mild transaminase elevations, recurrent cholangitis, or an inactive cirrhosis with portal hypertension.7Kasai M Mochizuki I Ohkohchi N Chiba T Ohi R Surgical limitations for biliary atresia: indications for liver transplantation.J Pediatr Surg. 1989; 24: 851-854Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 8Laurent J Gauthier F Bernard O Hadchouel M Odievre M Valayer J Alagille D Long-term outcome after surgery for biliary atresia: study of 40 patients surviving for more than 10 years.Gastroenterology. 1990; 99: 1793-1797PubMed Scopus (130) Google Scholar, 9Stein JE Vacanti JP Biliary atresia and other disorders of the extrahepatic biliary tree.in: Suchy FJ Liver Disease in Children. Mosby, St. Louis1994: 426-442Google Scholar Overall, biliary atresia accounts for up to 70% of all pediatric cases progressing to liver transplantation.10Shepherd RW Liver transplantation in children.Med J Aust. 1990; 153: 509-510PubMed Google Scholar, 11Lynch SV Akiyama T Ong TH Pillay SP Balderson GA Matsunami H Shepherd RW Cleghorn GJ Patrick MK Strong RW Transplantation in children with biliary atresia.Transplant Proc. 1992; 24: 186-188PubMed Google Scholar Therefore, despite surgical relief of the obstruction deposition of collagen, progressive hepatic fibrosis and portal hypertension usually occur. Indeed, the development of hepatic fibrosis in this disease is more rapid and aggressive than any other disorder in adults. The mechanisms responsible for increased collagen production and hepatic fibrosis in neonatal liver diseases such as biliary atresia are unknown. A population of nonparenchymal cells known as hepatic stellate cells (HSCs) have been shown to be "activated" and therefore responsible for the increased production of type I collagen leading to hepatic fibrosis in pathological conditions of the adult human liver,12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 13Minato Y Hasumura Y Takeuchi J The role of fat-storing cells in Disse space fibrogenesis in alcoholic liver disease.Hepatology. 1983; 3: 559-566Crossref PubMed Scopus (175) Google Scholar, 14Friedman SL Hepatic stellate cells.Prog Liver Dis. 1996; 14: 101-130PubMed Google Scholar and in a number of experimental models of adult liver injury,15Ramm GA Li SCY Li L Britton RS O'Neill R Kobayashi Y Bacon BR Chronic iron overload causes in vivo activation of rat lipocytes.Am J Physiol. 1995; 268: G451-G458PubMed Google Scholar, 16Pietrangelo A Gualdi R Casalgrandi G Geerts A De Bleser P Montosi G Ventura E Enhanced hepatic collagen type I mRNA expression into fat-storing cells in a rodent model of hemochromatosis.Hepatology. 1994; 19: 714-721Crossref PubMed Scopus (87) Google Scholar, 17Rockey DC Housset CN Friedman SL Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo.J Clin Invest. 1993; 92: 1795-1804Crossref PubMed Scopus (252) Google Scholar, 18Shiratori Y Ichida T Geerts A Wisse E Modulation of collagen synthesis by fat storing cells, isolated from CCl4- or vitamin A-treated rats.Dig Dis Sci. 1987; 32: 1281-1289Crossref PubMed Scopus (79) Google Scholar, 19Takahara T Kojima T Miyabayashi C Inoue K Sasaki H Muragaki Y Ooshima A Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat: immunoelectron microscopic observation of type I, type III collagens, and prolyl hydroxylase.Lab Invest. 1988; 59: 509-521PubMed Google Scholar, 20Matsuoka M Zhang MY Tsukamoto H Sensitization of hepatic lipocytes by high-fat diet to stimulatory effects of Kupffer cell-derived factors: implication in alcoholic liver fibrogenesis.Hepatology. 1990; 11: 173-182Crossref PubMed Scopus (63) Google Scholar including cholestasis.21Maher JJ McGuire RF Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo.J Clin Invest. 1990; 86: 1641-1648Crossref PubMed Scopus (367) Google Scholar, 22Hines JE Johnson SJ Burt AD In vivo responses of macrophages, and perisinusoidal cells to cholestatic liver injury.Am J Pathol. 1993; 142: 511-518PubMed Google Scholar, 23Ramm GA Crawford DHG Bridle KR Britton RS Bacon BR Tracy Jr, TF Biliary decompression results in rapid reversal of procollagen α1(I) mRNA gene expression in experimental biliary fibrosis.Hepatology. 1996; 24: A463Crossref PubMed Google Scholar, 24Olynyk JK Yeoh GC Ramm GA Clarke SL Hall PM Britton RS Bacon BR Tracy TF Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.Am J Pathol. 1998; 152: 347-352PubMed Google Scholar In liver injury, HSCs are transformed into myofibroblasts (activated HSCs), which produce increased levels of fibrillar collagen and express an intracellular microfilament protein, α-smooth muscle actin (SMA), which is traditionally used as a marker protein of the activated HSC phenotype (reviewed in 25Friedman SL The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.N Engl J Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). Activated HSCs also express a number of different cytokine receptors, including the transforming growth factor (TGF-β1) receptor.26Friedman SL Yamasaki G Wong L Modulation of transforming growth factor-β receptors of rat lipocytes during the hepatic wound healing response: enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo.J Biol Chem. 1994; 269: 10551-10558Abstract Full Text PDF PubMed Google Scholar TGF-β1 is an important profibrogenic cytokine and has been shown to increase collagen gene expression at the transcriptional level via binding of the transcription factors AP-1 and Sp-1.27Armendariz-Borunda J Simkevich CP Roy N Raghow R Kang AH Seyer JM Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression.Biochem J. 1994; 304: 817-824Crossref PubMed Scopus (72) Google Scholar, 28Rippe RA Almounajed G Brenner DA Sp-1 binding activity increases in activated Ito cells.Hepatology. 1995; 251: 241-251Google Scholar This study was designed to evaluate whether activated HSCs are the cellular source of increased collagen production in infants with biliary atresia and to determine the role of hepatic parenchymal and nonparenchymal cells in the expression of the profibrogenic cytokine, TGF-β1, in this age group. We were particularly interested in bile duct epithelia in view of the unique bile ductule hyperplasia seen in this disorder. Eighteen patients (6 male and 12 female) with extrahepatic biliary atresia and failed HPE were studied. Diagnosis of extrahepatic biliary atresia was confirmed in all cases at the time of HPE by histopathological evaluation, which revealed characteristic observations of portal or perilobular fibrosis, ductular proliferation, and canalicular and cellular biliary stasis.29Koop CE Biliary obstruction in the newborn.Surg Clin North Am. 1976; 56: 373-377PubMed Google Scholar All patients were referred for liver transplantation assessment because of progressive liver disease, and orthotopic liver transplantation was performed at a mean age of 2.6 ± 0.63 years (range, 7 months to 11.75 years). Twenty-three percutaneous liver biopsies, fixed in formalin and embedded in paraffin, were studied in these 18 patients. In 5 patients, both pre- and post-HPE biopsies were collected at a mean age of 1.8 ± 0.4 (mean ± standard error) and 8.2 ± 0.4 months, respectively. In the remaining 13 patients, liver biopsies were obtained at a mean age of 2.5 ± 0.8 years. For detection of procollagen α1 (I) mRNA, a 1500-bp fragment of human procollagen α1 (I) cDNA was subcloned into pGEM 11Z vector. For detection of TGF-β1 mRNA, a 1000-bp fragment of human TGF-β1 cDNA was subcloned into pGEM-3zf(+) vector. Both fragments were then subjected to alkaline hydrolysis to produce a 300-bp fragment for use in in situhybridization. Digoxigenin-labeled riboprobes, for sense (control) and antisense, were produced for both procollagen α1 (I) and TGF-β1 by in vitro transcription with SP6 and T7 polymerases. In situ hybridization was performed on 5-μm human liver sections, deparaffinized by xylol, and rehydrated by gradient alcohol before exposure to hydrochloric acid (0.2 mol/L), as previously described.30Rex M Scotting PJ Simultaneous detection of RNA and protein in tissue sections by nonradioactive in situ hybridization followed by immunohistochemistry.Biochemica. 1994; 3: 24-26Google Scholar Sections were permeabilized with 5 μg/ml proteinase K at 37°C for 15 minutes, followed by fixation in 4% paraformaldehyde for 20 minutes at room temperature. Prehybridization (50% formamide, 1% sodium dodecyl sulfate, 5× standard saline citrate, 500 μg/ml tRNA, and 50 μg/ml heparin) was performed at 70°C for 3 hours followed by hybridization for 16 hours at 70°C in a solution containing 1 μg/ml of digoxigenin-labeled riboprobe. Sections were then washed to remove unbound probe and incubated with alkaline phosphatase-conjugated anti-digoxigenin polyclonal sera (1:200) at room temperature for 2 hours. Unbound antibody was removed by washes, followed by visualization with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate in the dark at room temperature for 16 hours. Unbound complex was removed by washing, and sections were subjected to immunohistochemistry for SMA as previously described31Ooi LLPJ Crawford DHG Gotley DC Clouston AD Strong RW Gobé GC Halliday JW Bridle KR Ramm GA Evidence that "myofibroblast-like" cells are the cellular source of capsular collagen in hepatocellular carcinoma.J Hepatol. 1997; 26: 798-807Abstract Full Text PDF PubMed Scopus (79) Google Scholar to colocalize procollagen α1 (I) mRNA to activated HSCs (see Immunohistochemistry, below). All liver sections were incubated with a mouse monoclonal anti-SMA primary antibody (1:400, clone 1A4; Sigma Chemical Co., St. Louis, MO), followed by a biotinylated rabbit anti-mouse immunoglobulin G as the secondary antibody, as previously described.12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar The detection system used was a DAKO (Glostrup, Denmark) streptavidin-biotin complex/horseradish peroxidase kit, with 3,3-diaminobenzidine tetrahydrochloride as the chromogenic substrate. Sections were counterstained with eosin. Biopsies were graded histologically for SMA expression as previously described24Olynyk JK Yeoh GC Ramm GA Clarke SL Hall PM Britton RS Bacon BR Tracy TF Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.Am J Pathol. 1998; 152: 347-352PubMed Google Scholar using the following classification: 0, normal staining pattern for SMA with expression in smooth muscle cells within portal blood vessels only; 1+, mild perisinusoidal staining for SMA within activated HSCs; 2+, periportal staining for SMA, proliferation of SMA-expressing HSCs, and moderate SMA expression in perisinusoidal HSCs; 3+, septal and bridging SMA expression between portal tracts; and 4+, SMA expression within cirrhotic bands linking portal tracts. All liver sections were subjected to antigen retrieval by heating in a microwave oven on high power for 8 minutes in 0.01 mol/L citrate buffer (ph 6.0) and then incubated with a mouse monoclonal anti-TGF-β1 -β2, and -β3primary antibody to active TGF-β (150 μg/ml; Genzyme Diagnostics, Cambridge, MA) for the cellular localization of TGF-β protein. The sections were then subjected to the identical detection methodology as for SMA.12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar All liver sections were subjected to antigen retrieval by autoclaving in 0.01 mol/L citrate buffer (pH 6.0) at 121°C for 10 minutes. Immunohistochemistry for CD68, a specific marker for Kupffer cells, was performed by incubating sections with a mouse monoclonal anti-CD68 primary antibody (1:50, clone PG-M1; DAKO), followed by identical detection methodology as described for SMA.12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar This technique allowed assessment of Kupffer cells as a potential source of TGF-β1 mRNA in the livers of patients with biliary atresia. The negative controls used for each immunohistochemical assessment used nonimmune normal mouse immunoglobulin G antisera (Santa Cruz, San Diego, CA) in place of the primary antibody for either SMA, TGF-β, or CD68 (results not shown). All sections were subjected to hematoxylin/Van Gieson stain for the detection of collagen protein deposition. There was evidence of canalicular and cellular biliary stasis, variable inflammatory changes, bile duct hyperplasia with expanded portal tracts, periportal and bridging fibrosis, and mild to severe cirrhosis in all biopsies examined, including pre- and post-Kasai HPE livers. Liver biopsies were subjected to immunohistochemistry for the intracellular microfilament protein, SMA, which has been shown to be an excellent marker for the activated HSC phenotype. Activated HSCs were demonstrated morphologically both by their stellate shape and by the expression of SMA (Figure 1A) in the extracellular matrix surrounding hyperplastic bile ducts and within fibrous septa bridging between portal tracts (Figure 1B). Furthermore, procollagen α1 (I) mRNA expression was shown to colocalize to SMA-positive HSCs (Figure 1, A and B), demonstrating that activated HSCs are the cellular source of increased collagen leading to hepatic fibrosis in biliary atresia. Procollagen α1 (I) mRNA expression was not seen in hepatocytes, bile duct epithelial cells, or smooth muscle cells of the portal tract vasculature. Procollagen α1 (I) mRNA signal specificity for the antisense probe was demonstrated by the absence of signal over SMA-expressing stellate cells using the sense probe (results not shown). Five patients were examined both pre- and post-Kasai HPE for evidence of HSC activation. Only one of five patients showed a decrease in the expression of SMA (grade 4+ to 2+) and hence in the number of activated HSCs surrounding hyperplastic bile ducts and within fibrous bridging septa after HPE (Figure 1, C and D). All five of these patients subsequently progressed to liver transplantation. Liver biopsies were examined histologically for collagen protein deposition using hematoxylin/Van Gieson stain. Figure 2A demonstrates grossly enlarged bile ducts surrounded by excessive collagen protein deposition. Figure 2B demonstrates increased numbers of activated HSCs showing colocalization of SMA and procollagen α1 (I) mRNA in the identical region of increased collagen protein deposition. Elevated numbers of procollagen α1 (I) mRNA-expressing, activated HSCs (Figure 2D) were also demonstrated in the identical region of collagen protein deposition within fibrous tissue between portal tracts (Figure 2C). Immunohistochemistry for TGF-β protein revealed that TGF-β was predominantly expressed by bile duct epithelial cells within hyperplastic bile ducts and also by activated HSCs in the extracellular matrix of scar tissue (Figure 3A). TGF-β was also expressed to a lesser extent in hepatocytes in close proximity to areas of fibrosis at the interface of the regenerative nodule (Figure 3B). TGF-β expression was not evident in hepatocytes at a distance from scar tissue (results not shown). In situ hybridization for TGF-β1 mRNA demonstrated that TGF-β1 mRNA was expressed in bile duct epithelial cells within hyperplastic bile ducts (Figure 3, C and E) and was also observed colocalized to SMA-positive HSCs (Figure 3C). Increased expression of TGF-β1 mRNA was also demonstrated in hepatocytes along the interface of the regenerative nodules and fibrotic scar tissue (Figure 3D). TGF-β1 mRNA was not detected in hepatocytes within the acinus distal from scar tissue (results not shown). Figure 3E demonstrates the localization of increased numbers of Kupffer cells as assessed by CD68 immunohistochemistry, in sinusoidal and perisinusoidal regions of the regenerative hepatocyte nodule, and within scar tissue. TGF-β1 mRNA expression was not detected in CD68-positive Kupffer cells in close proximity to the interface of the fibrotic scar tissue, indicating that Kupffer cells may not contribute to the TGF-β1 mRNA expression seen in Figure 3D. In addition, CD68-positive cells within the scar tissue did not demonstrate colocalization of TGF-β1 mRNA, and therefore, these cells do not appear to play a role in collagen production by HSCs surrounding bile ducts. TGF-β1 mRNA signal specificity for the antisense probe was demonstrated by the absence of signal over bile duct epithelial cells, hepatocytes, and HSCs using the sense probe (Figure 3F). This study has demonstrated that activated HSCs, identified by the colocalization of procollagen α1 (I) mRNA expression to cells expressing the HSC activation marker, SMA, are responsible for the production of increased levels of type I collagen leading to hepatic fibrosis in young patients with biliary atresia. In addition, this study has shown that the hyperplastic bile duct epithelium is the predominant source of the profibrogenic cytokine TGF-β1within the portal tract and that hepatocytes produce increased levels of TGF-β1 along fibrous septa bridging portal tracts, which forms the fibrotic scar leading to cirrhosis. TGF-β1 was also produced by activated HSCs within the fibrous matrix but to a lesser degree than other cells. Finally, this study has demonstrated that the number of activated HSCs was decreased in only one of five patients after Kasai HPE. Many different theories have been proposed to explain the pathogenesis of biliary atresia, including infectious, genetic, and immune-mediated etiologies, although convincing evidence to support these hypotheses is lacking (reviewed in 6Balistreri WF Grand R Hoofnagle JH Suchy FJ Ryckman FC Perlmutter DH Sokol RJ Biliary atresia: current concepts and research directions.Hepatology. 1996; 23: 1682-1692Crossref PubMed Scopus (308) Google Scholar, 32Middlesworth W Altman RP Biliary atresia.Curr Opin Pediatr. 1997; 9: 265-269Crossref PubMed Scopus (24) Google Scholar). Furthermore, there is a paucity of knowledge concerning the mechanisms involved in the fibrogenesis associated with this condition. In a recent study, Malizia and colleagues33Malizia G Brunt EM Peters MG Rizzo A Broekelmann TJ McDonald JA Growth factor and procollagen type I gene expression in human liver disease.Gastroenterology. 1995; 108: 145-156Abstract Full Text PDF PubMed Scopus (82) Google Scholar examined five patients with advanced biliary atresia and cirrhosis and showed increased expression of procollagen α1 (I) mRNA associated with "spindle-shaped fibroblast-like cells" in the fibrous tissue surrounding regenerative hepatocyte nodules and some proliferating bile ductules. The identification of the responsible cell type was not established in this study, although the cells were described as "vimentin-positive mesenchymal cells,"33Malizia G Brunt EM Peters MG Rizzo A Broekelmann TJ McDonald JA Growth factor and procollagen type I gene expression in human liver disease.Gastroenterology. 1995; 108: 145-156Abstract Full Text PDF PubMed Scopus (82) Google Scholar which could describe either Kupffer cells, endothelial cells,34Tsutsumi M Takada A Takase S Characterization of desmin-positive rat liver sinusoidal cells.Hepatology. 1987; 7: 277-284Crossref PubMed Scopus (108) Google Scholar or ductal plate or biliary epithelial cells.35Haruna Y Saito K Spaulding S Nalesnik MA Gerber MA Identification of bipotential progenitor cells in human liver development.Hepatology. 1996; 23: 476-481Crossref PubMed Scopus (175) Google Scholar These authors also described the collagen-producing cells as desmin negative, suggesting that HSCs may not be the major cell type involved in fibrogenesis, based on a previous report that identified human HSCs as desmin-positive cells.36Friedman SL Rockey DC McGuire RF Maher JJ Boyles JK Yamasaki G Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.Hepatology. 1992; 15: 234-243Crossref PubMed Scopus (247) Google Scholar However, the literature on desmin reactivity of human HSCs is conflicting. Other studies have shown that the detection of desmin in human HSCs, either in vitro or in vivo, is quite variable and often unsuccessful.12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 37Schmitt-Graff A Kruger A Bochard F Gabbiani G Denk H Modulation of α smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human liver.Am J Pathol. 1991; 138: 1233-1242PubMed Google Scholar, 38Geerts A De Bleser P Hautekeete ML Niki T Wisse E Fat-storing (Ito) cell biology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter D Shafritz DA The Liver: Biology and Pathobiology. Raven, New York1994: 819-838Google Scholar Our study, however, clearly documents the identification of activated HSCs, as evidenced by both SMA expression and cell morphology, as the cellular source of increased procollagen α1 (I) mRNA in extrahepatic biliary atresia. The hepatic histopathological presentation of biliary atresia is classically characterized by ductular proliferation, canalicular and cellular biliary stasis, swelling and vacuolization of biliary epithelial cells, portal tract edema and fibrosis, and monocytic inflammatory cell infiltration of portal tracts.39McEvoy CF Suchy FJ Biliary tract disease in children.Pediatr Clin North Am. 1996; 43: 75-98Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Although the mechanisms responsible for many of these phenomena are not known, portal fibrosis and cirrhosis are arguably the most damaging and have the greatest prognostic significance. It is now clear that activated HSCs are responsible for the increased production of type I collagen leading to hepatic fibrosis in biliary atresia similar to that of pathological conditions of the adult liver12Ramm GA Crawford DHG Powell LW Walker NI Fletcher LM Halliday JW Hepatic stellate cell activation in genetic hemochromatosis: lobular distribution, effect of increasing hepatic iron and response to phlebotomy.J Hepatol. 1997; 26: 584-592Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 13Minato Y Hasumura Y Takeuchi J The role of fat-storing cells in Disse space fibrogenesis in alcoholic liver disease.Hepatology. 1983; 3: 559-566Crossref PubMed Scopus (175) Google Scholar, 14Friedman SL Hepatic stellate cells.Prog Liver Dis. 1996; 14: 101-130PubMed Google Scholar and in experimental models of cholestatic liver injury.21Maher JJ McGuire RF Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo.J Clin Invest. 1990; 86: 1641-1648Crossref PubMed Scopus (367) Google Scholar, 22Hines JE Johnson SJ Burt AD In vivo responses of macrophages, and perisinusoidal cells to cholestatic liver injury.Am J Pathol. 1993; 142: 511-518PubMed Google Scholar, 23Ramm GA Crawford DHG Bridle KR Britton RS Bacon BR Tracy Jr, TF Biliary decompression results in rapid reversal of procollagen α1(I) mRNA gene expression in experimental biliary fibrosis.Hepatology. 1996; 24: A463Crossref PubMed Google Scholar, 24Olynyk JK Yeoh GC Ramm GA Clarke SL Hall PM Britton RS Bacon BR T
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