Bile Ductular Cells Research Articles

Partial hepatectomy and bile duct ligation in rainbow trout (Oncorhynchus mykiss): histologic, immunohistochemical and enzyme histochemical characterization of hepatic regeneration and biliary hyperplasia.

Hepatic regeneration following partial hepatectomy (PH) and biliary hyperplasia subsequent to bile duct ligation (BDL) were characterized in rainbow trout (Oncorhynchus mykiss) by light microscopy using routine and special (immunohistochemical and enzyme histochemical) stains. Both PH and BDL involved initial hypertrophy and hyperplasia of bile preductular epithelial cells (BPDECs). BPDECs are small oval cells that form junctional complexes with hepatocytes and bile ductular cells and are commonly found in hepatic tubules of teleost liver. Proliferating BPDECs transitioned through intermediate cell types before final differentiation into large basophilic hepatocytes (following PH) or biliary epithelial cells (after BDL). Normal BPDECs and hepatocytes were both negative for cytokeratin intermediate filaments in control fish when screened with the monoclonal antibody AE1/AE3. In contrast, hyperplastic BPDECs and their progeny (intermediate cells, immature hepatocytes, ductal epithelial cells) were all strongly cytokeratin positive. Cytokeratin expression was transient in newly differentiated hepatocytes (expression decreased as hepatocytes acquired characteristics consistent with full differentiation) but was permanent in biliary epithelial cells (expression was very strong in large mature ducts). BPDECs, intermediate cells, and immature ductal cells were also strongly positive for alkaline phosphatase following BDL. Chronology of histologic events and cytokeratin and enzyme expression all support the hypothesis that BPDECs possess the capacity to differentiate into either hepatocytes or biliary epithelial cells. Thus, BPDECs may be the teleost equivalent of a bipolar hepatic stem cell in mammals.

Correction of defective CFTR function in bile ductular cells from a patient with cystic fibrosis

Correction of defective CFTR function in bile ductular cells from a patient with cystic fibrosis

Establishment of a novel rat cholangiocarcinoma cell culture model.

Furan cholangiocarcinogenesis in rat liver is proving to be a unique and useful animal model for investigating important aspects of the cellular and molecular pathogenesis of cholangiocarcinoma potentially relevant to the human disease. We now describe the first culture model of rat cholangiocarcinoma cells derived from a transplantable cholangiocarcinoma originally induced in the liver of a furan-treated rat. An epithelial cell isolate highly enriched in viable cholangiocarcinoma cells was consistently obtained from transplantable cholangiocarcinoma tissue utilizing a similar procedure to that recently developed by us to establish a new rat hyperplastic bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro. Primary cholangiocarcinoma cell cultures could be readily established with these isolated cells and, in addition, we established from one such culture a novel rat cholangiocarcinoma cell line designated C611B. Cultured C611B cholangiocarcinoma cells retained a number of important characteristic features of the carcinoma cells of the parent tumor, including marked expression of the tyrosine kinase growth factor receptor proteins c-Met and c-Neu. Under basal culture conditions, the C611B cell line exhibited a cell doubling time of approximately 24 h and was aneuploid, with a predominant chromosomal count of 43. Moreover, C611B cells on collagen gels were 100% tumorigenic when transplanted into inguinal fat pads of syngeneic rats. All tumors formed at the transplantation site were cytokeratin 19-positive, mucin-producing tubular adenocarcinomas whose histological and phenotypic features closely resembled those of the furan-induced parent transplantable rat cholangiocarcinoma. Based on our findings, we believe that this novel rat cholangiocarcinoma cell culture model can serve as a valuable resource for investigating aberrant growth properties and tumor progression in biliary cancer.

Human natural tumor necrosis factor alpha induces multiple endocrine and hematologic disorders in rats.

Slc:Wistar male rats treated with human natural tumor necrosis factor alpha (hn TNF-alpha, 3 X 10(5) Japan reference units/kg intravenously) for 3 months showed histologic vacuolation of basophils in the anterior pituitary, hyperplasia of the thyroidal follicular epithelium, and hyperplasia of the testicular interstitial cells. The vacuolated basophils were immunohistochemically shown to be thyrotrophs. In addition, there were decreases in plasma levels of triiodothyronine (T3), thyroxin (T4), and testosterone, and an increase in thyroid-stimulating hormone (TSH). The number of lymphocytes in the marginal zones of lymphoid follicles in spleen and lymph nodes and B-lymphocytes in the peripheral blood decreased. Hyperplasia of hematopoietic cells in the bone marrow and decreases in both leukocytes and erythrocytes in the peripheral blood were prominent. Hyperplasia of bile ductular epithelial cells with periportal mononuclear cell infiltration in the liver and increased cellularity in alveolar walls in the lung were also characteristic. In in vitro studies, hn TNF-alpha inhibited both proliferation and peroxidase activity of thyroid follicular epithelial cells. These findings demonstrate that hn TNF-alpha may induce histologic vacuolation of thyrotrophs by causing a decrease in plasma levels of T3 and T4; hyperplasia of the thyroid follicular epithelium, which may be attributed to the increased plasma level of TSH; hyperplasia of testicular interstitial cells, by lowering the plasma level of testosterone; hyperplasia of bile ductular epithelial cells; hyperplasia of hematopoietic cells in bone marrow; and the increase in cellularity in pulmonary alveolar walls. In addition, hn TNF-alpha may suppress the differentiation of B-lymphocytes.

Transforming growth factor betas and their receptors in human liver cirrhosis.

Transforming growth factor betas (TGF-betas) are a group of homologous polypeptides that exert pleiotropic effects on various cell types and stimulate the formation of extracellular matrix and fibrosis. To evaluate whether TGF-beta isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) and their receptors (types I-III) are also of importance in the pathophysiology of liver cirrhosis, we analysed their concomitant expression and localization in human liver cirrhosis. Cirrhotic liver tissue samples were obtained from 17 patients (four women, 13 men) with a median age of 41 years (range 22-67). Normal liver tissues from ten patients (seven women, three men) with a median age of 55 years (range 45-75) served as controls. The tissues were fixed in Bouin's solution and paraffin-embedded for histological analysis. For RNA analysis, freshly obtained tissue samples were snap-frozen in liquid nitrogen and stored at -80 degrees C until analysed. Northern blot analysis was used to examine the expression of TGF-beta1, beta2 and beta3 and their receptors, type I (TbetaR-I), type II (TbetaR-II) and type III (TbetaR-III). Immunohistochemistry was performed to determine the localization of the corresponding proteins in the normal and the cirrhotic liver. Northern blot analysis revealed enhanced expression (P < 0.05) of TGF-beta1 (twofold increase), TGF-beta2 (threefold increase) and TGF-beta3 (8.5-fold increase) and of TbetaR-II (threefold increase) mRNA in liver cirrhosis in comparison with normal controls. In contrast, TbetaR-I (ALK-5) and TbetaR-III mRNA expression showed no significant changes. No TGF-beta isoform immunoreactivity was present in hepatocytes in either normal livers or in liver cirrhosis. However, in liver cirrhosis, intense TGF-beta1 immunoreactivity was present in bile duct and ductular epithelial cells (including ductular proliferations) and in inflammatory cells. In a few sinusoidal lining cells, faint TGF-beta1 and moderate TGF-beta2 immunoreactivity was present. TGF-beta3 immunostaining was present in bile duct and ductular epithelial cells, in inflammatory cells and in fibroblast-like spindle cells in liver cirrhosis. For TbetaR-I and TbetaR-II, the immunoreactivity was localized in hepatocytes and biliary cells in normal and cirrhotic liver tissues, with higher intensity for TbetaR-II in the cirrhotic liver. Enhanced expression of all three TGF-bea isoforms and of TbetaR-II in liver cirrhosis suggests their involvement in this fibrotic disorder. The higher immunoreactivity of the three TGF-beta isoforms in the bile duct epithelial cells in cirrhotic tissues suggests a possible role of these cells in the pathogenesis of liver cirrhosis.

Expression of cytokeratin 20 in developing rat liver and in experimental models of ductular and oval cell proliferation

Background/Aims: Recently, a novel type of cytokeratin (CK) has been added to the classical catalog of CKs as CK20. The aim of the present study was to examine the immunoreactivity for CK20 in normal and developing rat liver and in experimental models of bile ductular and oval cell proliferation. Methods: Eighty-five Fischer rats, subdivided into five groups, were utilized in this study: fetal rats, ranging from day 10 to day 21 of gestation; newborn-neonatal rats, from 2 h to 10 days of age; bile duct ligated (BDL) rats; alpha-naphthyl-isothiocyanate (ANIT)-treated rats; and rats fed a choline-deficient diet containing N-2 Fluorenylacetamide (CD-AAF rats). Frozen sections from each liver were stained with the CK20 specific monoclonal antibody IT-K s20.10. Results: The present study shows that CK20 is a “bile duct type” CK. In the fetal rat, CK20 appears late during intrahepatic bile duct development, at day 20 of gestation. A marked increase in CK20 expression occurs after birth, suggesting that intrahepatic bile duct maturation continues after birth and that CK20 may be considered as a “maturation” marker of the biliary tree. In BDL rats and in ANIT-treated animals, immunoreactivity of bile ductules for CK20 was strikingly heterogeneous. A variable number of proliferating biliary cells were weakly positive or negative for CK20 and their number increased with the duration of the obstruction or ANIT treatment. In CD-AAF-treated rats, we found a uniform staining of proliferating oval cells for CK20. This finding is in contrast with the observation in BDL and in ANIT groups, and suggests the existence of different mechanisms regulating the proliferation and differentiation of biliary cells under those conditions. Conclusions: In rat liver, CK20 may be added to the list of “bile duct type” cytokeratins. During development, CK20 expression may be related to the maturation stage of the biliary tree. Typical ductular proliferation induced by BDL or ANIT feeding clearly differs from the oval cell proliferation in the CD-AAF model in terms of immunoreactivity for CK20.

Stem cells of the liver

Liver regeneration can be accomplished either by the entry of normally proliferatively quiescent, differentiated hepatocytes into the cell cycle or, if hepatocyte regeneration is impaired, by bile ductular cells that migrate centrifugally from the portal tracts before differentiating into hepatocytes. These ductal cells are called oval cells, and their emergence when hepatocyte regeneration is defective has led to the conclusion that they are the progeny of facultative stem cells. Oval cells are of great biological interest because they may be the progenitor cells of both hepatocellular carcinomas and cholangiocarcinomas; furthermore, they could be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism.

EFFECT OF BILE DUCT OBSTRUCTION ON HEPATIC UPTAKE OF 1-METHYL-4-PHENYLPYRIDINIUM IN THE RAT

Previous studies have demonstrated that the organic cation 1-methyl-4-phenylpyridinium (MPP+) is avidly taken up by rat freshly isolated hepatocytes through at least two distinct transport mechanisms: the type I hepatic transporter of organic cations and P-glycoprotein. In this study, the effects of extrahepatic cholestasis induced by bile duct ligation for 4 days on the uptake of [3H]MPP+by rat freshly isolated hepatocytes and liver slices were determined. Bile duct ligation produced no significant alterations in the characteristics of [3H]MPP+uptake by freshly isolated hepatocytes. The strong correlation found between the effect of various drugs on [3H]MPP+uptake by hepatocytes from control and treated rats (r=0.958;P<0.0001;n=15) suggests that neither the type I hepatic transporter of organic cations nor P-glycoprotein were affected by bile duct ligation. On the contrary, uptake of [3H]MPP+by liver slices was markedly changed after bile duct ligation: (1) there was a significant increase (≅40%) in the amount of [3H]MPP+taken up by liver slices from bile duct-ligated rats; (2) there was no correlation between the effect of various drugs on [3H]MPP+uptake by liver slices from control and treated rats (r=0.772;P=0.072;n=6). On the basis of (1) the lack of effect of bile duct ligation on [3H]MPP+uptake by isolated hepatocytes; and (2) the profound morphological alterations of liver tissue observed 4 days after bile duct ligation (increase in volume density of bile ductules, ductular cells and infiltration of inflammatory cells), we suggest that non-parenchymal liver cells have an important participation in the hepatic uptake of [3H]MPP+after bile duct ligation in the rat.

Increased MUC6 apomucin expression is a characteristic of reactive biliary epithelium in chronic viral hepatitis.

The significance of bile ductular proliferation in the progression of various hepatobiliary diseases remains unclear. Increased expression of MUC6 apomucin, a major gastric mucin, has previously been noticed in proliferating bile ductules in chronic viral hepatitis. The purpose of the present study was to characterize MUC6 apomucin and mRNA expression in 35 histologically 'normal livers', 47 livers with chronic viral hepatitis, 28 with primary biliary cirrhosis, and seven with extrahepatic biliary obstruction. MUC6 protein was expressed focally in cytoplasm and/or on the luminal surface of septal and interlobular bile ducts in normal and diseased livers. Bile ductules in normal livers rarely expressed MUC6 protein. The MUC6 expression intensified and spread in proliferating bile ductules and small bile ducts in chronic viral hepatitis and to a lesser degree in other diseases. In the former, the extent and degree of MUC6 expression paralleled the degree of active necroinflammation. MUC6 mRNA expression resembled MUC6 protein expression in proliferating bile ductules and intralobular small biliary cells, suggesting increased transcription and synthesis of MUC6. In conclusion, proliferating bile ductular cells express MUC6 apomucin in diseased liver, especially in chronic viral hepatitis with active necroinflammation. This secreted mucin may act as a cytoprotective agent and represent a phenotype of reactive biliary epithelium in chronic viral hepatitis.

Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.

Hepatic oval cells (HOC) are a small subpopulation of cells found in the liver when hepatocyte proliferation is inhibited and followed by some type of hepatic injury. HOC can be induced to proliferate using a 2-acetylaminofluorene (2-AAF)/hepatic injury (i.e., CCl4, partial hepatectomy [PHx]) protocol. These cells are believed to be bipotential, i.e., able to differentiate into hepatocytes or bile ductular cells. In the past, isolation of highly enriched populations of these cells has been difficult. Thy-1 is a cell surface marker used in conjunction with CD34 and lineage-specific markers to identify hematopoietic stem cells. Thy-1 antigen is not normally expressed in adult liver, but is expressed in fetal liver, presumably on the hematopoietic cells. We report herein that HOC express high levels of Thy-1. Immunohistochemistry revealed that the cells expressing Thy-1 were indeed oval cells, because they also expressed alpha-fetoprotein (AFP), gamma-glutamyl transpeptidase (GGT), cytokeratin 19 (CK-19), OC.2, and OV-6, all known markers for oval cell identification. In addition, the Thy-1+ cells were negative for desmin, a marker specific for Ito cells. Using Thy-1 antibody as a new marker for the identification of oval cells, a highly enriched population was obtained. Using flow cytometric methods, we isolated a 95% to 97% pure Thy-1+ oval cell population. Our results indicate that cell sorting using Thy-1 could be an attractive tool for future studies, which would facilitate both in vivo and in vitro studies of HOC.

Beta-carotene reduces the ductular (oval) cell reaction in the liver of Wistar rats submitted to the resistant hepatocyte model of carcinogenesis

The morphology of livers of Wistar rats treated with beta-carotene (BC), vitamin A (VA, retinol acetate) or corn oil (CO, controls) and submitted to the resistant hepatocyte model of carcinogenesis was studied. Preneoplastic lesions (PNL) were smaller and less numerous in the BC group. The latter group also presented fewer placental glutathione-S-transferase (GST-P) positive and hematoxylin and eosin (H&E) distinguishable PNL, with smaller mean areas and smaller mean areas of the liver occupied by PNL. Clear cell foci predominated in BC livers. In picrosirius-stained liver sections, fibrosis, whether or not accompanying the bile ductular cells, surrounded only 16.67% of PNL in the BC group, as compared to 35.71% in the VA group and 87.72% in the CO group. Moreover, the ductular cell reaction was smaller in the BC group. Smooth muscle actin-positive cells surrounded some PNL, mostly in CO rats, and less frequently in the VA and BC groups. Examination by transmission electron microscopy (TEM) showed that cells with nuclei similar to those of perisinusoidal cells, devoid of cytoplasmic fat globules, probably represented myofibroblasts derived from Ito cells and accompanied the ductular cell reaction. On the basis of these results, we suggest that BC reduced not only the PNL but also the ductular (oval) cell reaction in this experimental model.

A new rat bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro.

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.

A new rat bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo. (Hepatology 1997 Sep;26(3):537-49)

The oval-shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and immunohistochemical staining for albumin and pyruvate kinase isoenzyme expression.

Oval cells observed in some experimental models of hepatocarcinogenesis can function as stem cells capable of differentiating into hepatocytes and bile ductular cells. Using markers which characterise embryonic hepatocytes, we showed that oval cells display different patterns of gene expression, suggesting some are more mature than others. In this study we looked for oval cells in developing liver, predicting that they are abundant in embryonic liver and decline in number during development. Albumin (ALB) serves as a liver-specific marker, and the isoenzymes of pyruvate kinase, M2-PK and L-PK, are used to identify immature and mature hepatocytes, respectively. Small oval-shaped cells expressing ALB, M2-PK and L-PK are found near the vascular spaces and portal areas in 20-day gestation (E20), E21, newborn, 3-day and 1-week-old rat liver. Similar cells expressing ALB and M2-PK, but not L-PK are seen only periportally in adult liver. These are abundant in early embryonic liver and decrease in number during development until only a few, located periportally, persist in the adult. Oval cells, located periportally a few days after commencing a choline-deficient, ethionine-supplemented diet, co-express ALB and M2-PK. Their similarity with respect to markers, morphology and location suggests that oval-shaped cells may be the progenitors of oval cells.

The apoptosis protector, bcl-2 protein, is downregulated in bile duct epithelial cells of human liver allografts

Background/Aims: Apoptosis of bile duct cells occurs in hepatic allografts and is correlated with acute rejection. bcl-2 protein counteracts apoptosis and prolongs cell survival. We therefore tested the expression of bcl-2 protein in bile ducts of liver grafts in comparison with those of liver cirrhosis. Methods: 115 biopsies from 17 liver allografts and 47 biopsies of liver cirrhosis were analyzed and compared with 22 normal controls and with biopsies from patients with primary sclerosing cholangitis or primary biliary cirrhosis. bcl-2 protein and PCNA (proliferating cell nuclear antigen) expression was assessed using immunohistochemistry, and apoptosis was analyzed employing in situ DNA end-labeling. Results: A high apoptotic rate was detected in bile duct cells of allograft biopsies. In contrast to controls, bile duct cells of allografts and liver cirrhosis had high proliferative activity (mean PCNA labeling index: 1.0% vs. 40.7% and 18.6%, respectively). In liver grafts, bcl-2 protein positivity of bile duct and ductular cells was found in 3.6% and 4.4% of sections, respectively, and in cirrhosis in 44% and 79%, respectively (allografts vs. cirrhosis p<0.01). In controls, only one biopsy was bcl-2 positive. Conclusions: Whereas increased proliferative activity of small bile ducts and ductules in cirrhosis is associated with a high degree of bcl-2 expression, bile duct and ductular cells in liver grafts have a very low bcl-2 protein reactivity, even though their proliferative activity is high. These findings suggest that downregulation of bcl-2 expression in allograft bile duct cells might play a role in the increased apoptosis of these cells in acute rejection.

Bile ductular proliferation and altered leukotriene elimination in thioacetamide-induced fibrosis of rat liver

Background/Aims: Liver fibrosis is accompanied by both bile ductular proliferation and inflammation under various conditions. The functional consequences and the interrelationships between these changes are unknown. Altered biliary elimination and retention of cholephilic mediators may be a factor in fibrogenesis. Therefore, the relationship between fibrosis, ductular proliferation and functional changes in biliary elimination was studied. Methods: Micronodular liver fibrosis was induced by thioacetamide in rats. The relative amount of bile ductular epithelial cells was determined by microscopic morphometry. The functional changes in bile secretion and metabolism of leukotriene C4 were assessed in isolated perfused livers of treated rats. Results: Pretreatment with thioacetamide in vivo resulted in enhanced bile fluid formation in subsequently isolated and perfused livers. Infusion of isoproterenol into the portal vein stimulated bile flow. Both unstimulated and isoproterenol-stimulated bile flows were increased in fibrotic livers and were correlated with liver content of bile ductular epithelia. In contrast, biliary secretion of infused leukotriene C4 was lowered in correlation with that of taurocholate. Enhanced metabolism resulted in a shift of major fraction in bile from leukotriene C4 to leukotriene D4. Conclusions: Thioacetamide-induced liver fibrosis is associated with an increased number of functionally intact bile ductules that are responsive to isoproterenol stimulating bile fluid formation. In contrast, biliary secretion of cysteinyl-leukotrienes and taurocholate is inhibited and the relative amount of leukotriene D4 is increased. Bile ductular proliferation as well as retention and altered metabolism of leukotrienes are factors associated with the development of liver fibrosis.

Heparan Sulfate Proteoglycan Expression in Chronic Cholestatic Human Liver Diseases

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.

Heparan sulfate proteoglycan expression in chronic cholestatic human liver diseases

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. (Hepatology 1996 Sep;24(3):524-32)

Electron microscopic study of the liver with biliary atresia and neonatal hepatitis

Eleven cases of biliary atresia (BA) and eight of neonatal hepatitis (NH) were studied, using transmission electron microscopy, to document their different ultrastructural characteristics and to elucidate the possible pathogenesis of biliary atresia. Among 30 consecutive liver biopsies obtained from 19 infants with BA or NH, 21 specimens composed (13 BA, 8 NH) were examined ultrastructurally. The electron microscopic features of NH (patients' age range, 35 to 60 days) were (1) giant hepatocytic transformation with scattered areas of dilated endoplasmic reticulum, indicative of intracytoplasmic degeneration, (2) frequent cytoplasmic biliary necrosis, and (3) relatively intact microvilli in most bile canaliculi, which contained some hepatocytic cytoplasmic fragments. These features strongly suggest that the main pathological process in NH is hepatocellular injury rather than bile duct damage. In contrast, all cases with BA (age range, 27 to 130 days) demonstrated (1) marked hepatocellular cholestasis associated with many lysosomes and myelin figures, (2) marked loss of bile canalicular microvilli, (3) degenerated bile ductular cells containing bile pigments, and (4) periductal inflammatory fibrosis. These features suggest that the main pathological process in BA involves the biliary system. A few viral inclusions were observed in two cases with BA, whic suggests that viral infection is a potential cause. In two BA cases (aged 40 and 43 days at the time of first biopsy), the ultrastructural findings essentially were the same as those of NH, and follow-up biopsy specimens (at 48 and 94 days) showed findings consistent with BA. Such results support Landing's hypothesis that BA and NH are different manifestations of a single pathological process.

Dual phenotypic expression of hepatocytes and bile ductular markers in developing and preneoplastic rat liver.

This study supports the existence of a pluripotent liver stem cell population which has the potential to differentiate into hepatocytes and bile ductular cells. We compared the expression of hepatocyte-specific and bile ductular-specific markers in fetal and preneoplastic rat liver. L-pyruvate kinase (L-PK) and alpha glutathione S-transferase (GST) were used as adult hepatocyte-specific markers, while cytokeratin 19 (CK19) was used as a bile ductular-specific marker. pi GST and M2-pyruvate kinase (M2-PK), which are fetal hepatocyte-specific and expressed at high levels in the oval and duct-like cells, were also used. We characterized fetal liver derived from 13-21 days of gestation (E13-E21). pi GST was detected in the E18 hepatoblasts, which form the intrahepatic bile ducts, while CK19 was detected at E19. Some of these cells express alpha GST and L-PK from E19 to E21. Oval, duct-like and bile ductular cells in rats treated with a choline-deficient diet containing 0.07% ethionine (CDE diet) for up to 8 weeks were characterized by double immunocytochemistry. L-PK and alpha GST are absent from bile ductular cells in the normal adult liver and up to 3 weeks of CDE treatment. After 4-5 weeks on CDE treatment, the majority of bile ductular cells express L-PK, while at 6 weeks some co-express L-PK and alpha GST. There are two populations of oval cells, a major population expressing only the fetal hepatocyte markers, while a minor population expresses the fetal hepatocyte, adult hepatocyte and bile ductular markers. There are at least three different duct-like cell populations which co-express different markers and have characteristics of fetal hepatocytes at sequential stages of differentiation. One population co-expresses pi GST and M2-PK and is similar to fetal hepatocytes derived from E13-E14 fetuses. The second expresses the two fetal markers and L-PK, and this reflects characteristics of E15 hepatocytes. The third expresses pi GST, M2-PK, L-PK and alpha GST which is characteristic of E16-E19 hepatocytes. Upon withdrawal of the CDE diet, autoradiography using tritiated thymidine shows that oval and duct-like cells differentiate into hepatocytes. This study demonstrates that oval and duct-like cells express both hepatocytic and bile ductular markers, and have the capacity to differentiate into hepatocytes, characteristics similar to hepatoblasts in the developing rat liver.