Expression of vimentin in proliferating and damaged bile ductules and interlobular bile ducts in nonneoplastic hepatobiliary diseases.

Biliary epithelial cells express characteristically cytokeratin in their cytoplasm in normal and diseased livers. The present study disclosed that vimentin was frequently expressed in the cytoplasm of proliferating and damaged bile ductules and interlobular bile ducts, while their normal counterparts were negative for vimentin. Although this expression itself seemed nonspecific to any of the hepatobiliary diseases examined, bile ductules and interlobular bile ducts were frequently positive in chronic cholestatic and necroinflammatory liver diseases. In biliary epithelial cells, vimentin was localized around the nucleus or in the subnuclear regions, when present. Immunoelectron microscopically, reaction products for vimentin and for cytokeratin were found on bundles of intermediate filaments in the cytoplasm of biliary epithelial cells. The former was found mostly in the paranuclear and subnuclear regions, while the latter detected around the desmosomes, in addition to the paranuclear cytoplasm. Vimentin and cytokeratin were also seen together under immunoelectron microscopy on the same intermediate filaments. It seems likely that aberrant expression of vimentin in bile ductules and interlobular bile ducts and heterogeneous antigenic expression of intermediate filaments in the same biliary epithelial cells may be related to proliferation of, reorganization of, or damage to the ductular and ductal biliary cells in a variety of hepatobiliary diseases.     

Morphometric and immunohistochemical characterization of human liver regeneration.

Regeneration in human liver is characterized in part by the formation of ductular structures, so-called ductular hepatocytes in massive hepatic necrosis and bile ductules in mechanical biliary obstruction. In an attempt to characterize the liver regenerative process, we performed image analysis and immunohistochemical staining of the ductular structures in these well defined human liver disorders, 13 cases of massive hepatic necrosis and 9 cases of mechanical biliary obstruction. The proliferation index was determined and the expression of several antigens was localized by immunohistochemical staining using antibodies to alpha-fetoprotein, alpha-1-antitrypsin, albumin, and cytokeratin 19. The ductular structures in adult human liver were compared with the developing ductal plates in 11 fetal livers, ranging in age from 9 to 36 weeks of gestation. Image analysis demonstrated that the mean total area, mean nuclear area, and mean cell size of ductular hepatocytes were significantly larger than those of bile ductules (p < 0.05). The proliferation index of ductular hepatocytes and bile ductules was significantly higher than that of hepatocytes of normal livers (p < 0.02). Bile ducts, bile ductules in mechanical biliary obstruction, ductular hepatocytes in massive hepatic necrosis, and the ductal plate cells in fetal liver showed strong staining for cytokeratin 19, which characterizes intermediate filaments associated with bile duct epithelial cells. Albumin, a liver-specific protein, and alpha-1-antitrypsin, a protease inhibitor, were strongly expressed in ductal plate cells of fetal liver, hepatocytes, and ductular hepatocytes, whereas bile duct cells and bile ductules were negative for albumin. In summary, ductular hepatocytes demonstrate morphometric and immunophenotypic features of both hepatocytes and biliary epithelial cells, whereas bile ductules share characteristics primarily with fetal ductal plates and mature bile ducts. These findings suggest that ductular hepatocytes in massive hepatic necrosis may serve as bipotential progenitor cells, and bile ductules in mechanical biliary obstruction are related to ductal plates of fetal liver.     

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.     

Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells.

The origin of bile duct-like cells (oval cells) proliferating during chemical hepatocarcinogenesis is highly controversial. To illuminate this issue, we induced oval cell proliferation by feeding rats a choline-devoid diet containing 0.1% ethionine (CDE), a hepatocarcinogenic diet, for up to 60 days. At various times we studied 1) oval cell morphology by light and electron microscopy, 2) the immunohistochemical expression of albumin and intermediate filament proteins by the various hepatic cells, 3) hepatic incorporation of [3H]thymidine by histoautoradiography, 4) the fractional area occupied by duct-like structures in liver cross sections, 5) the biliary tree volume in vivo to establish the possible continuity of the proliferated structures to the existing biliary lumina, and 6) spontaneous bile flow rate and the choleretic responsiveness to the hormone secretin, which stimulates ductular secretory activity. The results demonstrated the following: 1) oval cells resemble bile duct cells with respect to their histologic and ultrastructural appearance and their formation of duct-like structures; 2) as normal and hyperplastic bile duct cells induced by bile duct ligation, oval cells are positive for cytokeratins 7 and 19 (markers of glandular epithelia) and 8 and 18 (markers of simple epithelia) and are negative for vimentin and desmin, markers of mesenchymal and muscular differentiation, respectively; 3) in general, oval cells are negative for albumin, which is expressive of hepatocyte lineage, even though a few are positive for this protein, particularly those morphologically resembling small hepatocytes; 4) after initiation of the CDE diet, DNA synthesis begins in biliary epithelial cells; and 5) the degree of oval cell proliferation parallels the increase in biliary tree volume, spontaneous bile flow rate, and responsiveness to secretin choleresis, as in bile duct cell hyperplasia induced by biliary obstruction. Although the involvement of a periductular progenitor compartment cannot entirely be eliminated, these findings are construed to indicate that oval cells proliferating during CDE hepatocarcinogenesis are biliary epithelial cells. In our view, oval cells represent the two-dimensional expression of spatially expanded cholangioles and intrahepatic bile ductules and/or ducts.     

Origin and structural evolution of the early proliferating oval cells in rat liver

We have analyzed the histological changes in rat liver after 2-acetylaminofluorene (AAF) administration. The data demonstrate that AAF-induced oval cells were preferentially generated by proliferation of the terminal biliary ductules that we suggest constitute the primary hepatic stem cell niche. The oval cells formed ductular structures, representing an extension of the canals of Hering. This histological organization provides continuous bile drainage of the hepatocytes and uninterrupted blood flow in the sinusoids. The oval cell ductules are surrounded by a continuous basement membrane that is intermittently disrupted by processes of stellate cells that form direct cell-cell contact with the oval cells. Although both AAF treatment and bile duct ligation results in proliferation of biliary epithelial cells, the mechanism(s) responsible for the proliferation of the biliary epithelium seems to differ in the two models. In contrast to the biliary proliferation stimulated by bile ligation, AAF-induced oval cell proliferation as well as the capacity of these cells to differentiate into hepatocytes, bile epithelial cells and possibly other cell lineages can be blocked by administration of dexamethasone.     

Neuroendocrine features of reactive bile ductules in cholestatic liver disease.

Various cholestatic liver diseases are accompanied by a striking increase in the number of bile ductules. This so-called ductular reaction is thought to arise both from ductular metaplasia of hepatocytes and from proliferation of pre-existing bile ductules. Previous studies have shown that these reactive bile ductules differ from their normal counterpart in enzyme and immunohistochemical make-up. Using monoclonal antibodies directed to neuroendocrine markers and immunohistochemistry, we found that reactive bile ductules in cholestatic liver disease display neuroendocrine features. In all cases of primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), extrahepatic obstruction, and acute hepatitis A, reactive bile ductules expressed the neural cell adhesion molecule (N-CAM) and reacted with monoclonal antibody A2B5. Both N-CAM and the ganglioside, recognized by A2B5, are restricted to neuroendocrine cells and tissues. In all but four of these cases, the same bile ductules expressed chromogranin-A, present in the matrix of neuroendocrine granules. Furthermore, in three cases of longstanding cholestasis, scattered periportal hepatocytes expressed chromogranin-A but not N-CAM. Other neuroendocrine markers such as neuron-specific enolase, synaptophysin, or myelin-associated glycoprotein were lacking from both bile ductules and hepatocytes. The neuroendocrine phenotype of bile ductules and hepatocytes was confirmed on electronmicroscopy, demonstrating various numbers of dense-cored, neuroendocrine granules near the peripheral cell membrane in bile ductules as well as in cells intermediate between hepatocytes and bile ductular cells. In 10 cases of normal liver tissue without ductular reaction, bile ductules lacked neuroendocrine markers except in two cases in which very weak reactivity for chromogranin-A was observed. These findings illustrate the presence of a new, neuroendocrine cell type that emerges in the liver during cholestasis. Elucidation of the significance of the neuroendocrine substance(s) produced in the dense cored granules of reactive bile ductules awaits their isolation and characterization. We can speculate that this molecule plays an autocrine or paracrine regulatory role in the process of ductular metaplasia of hepatocytes or growth of bile ductules.     

Appearance of denuded hepatic stellate cells and their subsequent myofibroblast-like transformation during the early stage of biliary fibrosis in the rat

To investigate the early in vivo response of hepatic stellate cells in biliary fibrosis, we examined rat livers during the first 7 days after bile duct ligation using light microscopy, immunohistochemistry, electron microscopy, and immunoelectron microscopy. At day 1 after bile duct ligation, alpha-smooth muscle actin-positive fibroblasts appeared and then increased in number around the proliferating bile ductules. With time, the destruction of the external limiting plate became accentuated because of the invasion of the proliferating bile ductules and periductural fibrosis. At day 7, stromal cells containing fat droplets appeared in the fibrous tissue adjacent to the periportal parenchyma; these are termed denuded hepatic stellate cells. In the fibrous tissue disconnected from the liver parenchyma, the denuded hepatic stellate cells were replaced by myofibroblast-like cells. Meanwhile, the expression of transforming growth factor-beta1 on biliary epithelial cells increased. These results indicate the dual origin of myofibroblasts in experimental biliary fibrosis, the periductural and periductal fibroblasts in the initial stage, and the denuded hepatic stellate cells in the subsequent stage. These two types of stromal cells may undergo myofibroblastic transformation by the transforming growth factor-beta1 secreted by the proliferating biliary epithelial cells.     

Role of mast cells in hepatic remodeling during cholestasis and its resolution: relevance to regulation of apoptosis.

BACKGROUND/AIMS: Mast cells are thought to be related to fibrogenesis, but recent studies have shown that fibrosis of the liver can be induced even in mast cell-deficient rats. To clarify the significance of mast cell accumulation in cholestatic liver diseases, the relations between such accumulation, bile ductule proliferation and apoptosis of biliary epithelial cells were examined in the rats during cholestasis and its resolution. METHODS: Cholestasis and its resolution were induced in rats by common bile duct ligation and spontaneous recanalization, respectively. The extent of bile ductule proliferation and the numbers of mast cells and apoptotic biliary epithelial cells were estimated quantitatively in liver sections. RESULTS: Recanalization of the ligated common bile duct led to an abrupt and transient increase in the number of mast cells, although the number of proliferated bile ductules decreased rapidly. The number of apoptotic biliary epithelial cells of the proliferated bile ductules increased rapidly and transiently, and the change paralleled that of the mast cells. CONCLUSIONS: Mast cells accumulating in the portal triads during cholestasis and its resolution may relate to the reduction of proliferated bile ductules, i.e., in hepatic remodeling, through the induction of apoptosis of biliary epithelial cells.     

Idiopathic hepatic fibrosis with cholestasis in broiler chickens: Immunohistochemistry of hepatic stellate cells

Immunohistochemical examinations of hepatic stellate cells (HSCs) were performed on six enlarged livers from broiler chickens with malformation of the extrahepatic biliary tract (group 1) and on eight broiler livers affected with naturally occurring cholangiohepatitis without biliary malformation (group 2). The livers from both groups were grossly enlarged, firm and tan-coloured, and histologically revealed severe diffuse fibrosis with proliferation of bile ductules. HSCs positive for muscle actin and desmin actively proliferated in the perisinusoidal space and around newly formed bile ductules. There was no difference in the immunohistochemical reactivities and location of HSCs between the two groups. The findings suggest that the diffuse hepatic fibrosis found in group 2 as well as group 1 results from reactive proliferation of HSCs.     

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes of the fetal mouse in vitro

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes was analysed in vitro. When fetal mouse liver fragments containing immature hepatocytes but no bile ducts were cultured organotypically, the immature hepatocytes differentiated into large hepatocytes. Some of these expressed bile duct markers such as cytokeratin and Dolichos biflorus agglutinin-binding sites, though only to a small extent, and typical intrahepatic bile duct cells failed to differentiate. Dexamethasone stimulated immature hepatocytes to differentiate into both mature hepatocyte and biliary epithelial cell lineages. Especially in the liver fragments cultured on Matrigel, dexamethasone stimulated the expression of bile duct markers (such as cytokeratin and binding sites for two types of lectin) in the immature hepatocytes. These results support the idea that immature hepatocytes can differentiate into both mature hepatocytes and biliary epithelial cells during normal development of the mouse liver, and suggest that glucocorticoids stimulate both these differentiation pathways. It also seems that basal laminar components may play a role in bile duct differentiation.     

Biliary tract of the rat as observed by scanning electron microscopy of cast samples.

The three-dimensional distribution of the biliary tract in the rat was studied by scanning electron microscopy of biliary casts. The casts were prepared by a retrograde infusion of a low viscosity or monomeric methacrylate resin mixture into the common bile duct. No resin flow from the bile canaliculi to sinusoidal capillaries was ever noted. Bile canaliculi formed intricate meshworks and drained via the Hering's canals into the bile ductules. The bile canalicular meshworks of adjacent lobules intercommunicated with each other. The bile ductules formed a marked periportal plexus around the portal vein branch, and drained into the intrahepatic bile duct running along the portal vein branch. The junctional zone of the Hering's canal and bile ductule usually showed an ampullary dilation. When the Hering's canal directly drained into a thick bile ductule or into a periportal plexus of bile ductules, such an ampullary dilation at the origin of the bile ductule was never replicated. The extrahepatic bile duct protruded many crypt-like projections which presumably corresponded to parietal glands. It is suggested that the periportal plexus of bile ductules may store the bile as a substitute for the gallbladder.     

Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.

The term oval cell describes small cells with oval nuclei that arise in the periphery of the portal tracts in rat models of hepatocarcinogenesis and injury and can differentiate into either hepatocytes or bile duct cells, ie, are bipotential. The presence of such cells in human liver is controversial. Here, immunolocalization of OV-6 and two biliary markers, cytokeratin 19 (CK-19) and human epithelial antigen 125 (HEA-125) is compared in normal adult human livers and in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) liver sections. CK-19 and HEA-125 stained bile ducts and ductules in normal liver as well as proliferating ductular structures in diseased livers. OV-6 did not label ducts or ductules in normal liver, but in PBC and PSC stained numerous proliferating ductular and periductular cells and lobular hepatocytes. In PBC, discrete OV-6-positive cells with a mature biliary-cell-like morphology were seen integrated into some intact bile ducts as well as occasional small immature oval-like cells. In addition, in PSC, hepatocytes in regenerating lobules were also strongly stained with OV-6, and on close inspection, in both PBC and PSC, oval cells and small hepatocytes at the margins of the lobules were strongly labeled. In contrast to the rat liver, OV-6 and CK-19 staining did not always co-localize. It is proposed that the small OV-6-positive oval cells are analogous to those seen in rat models and may represent human liver progenitor cells that may differentiate into OV-6-positive ductal cells or lobular hepatocytes.     

A scanning electron microscopic study of the intercalated portion of the biliary system in the rat liver.

The intercalated portion of the rat liver was studied by scanning electron microscopy (SEM) after removal of interlobular connective tissue by acid or alkaline hydrolysis. Biliary intercalated portions have generally been regarded as short straight links lying between the bile capillary network and the interlobular duct. The biliary system as observed by SEM lacked such specialized segments for linking. Instead, it contained long intercalated ductules taking winding and branching courses. The ductular branches frequently anastomosed with each other to form an extensive plexus along the limiting plate. The ductules repeatedly connected with the plate on their courses as well as at their terminals. This disposition of the ductules probably potentiates their tolerance to luminal obstruction. At the junction between the ductule and the limiting plate, ductular cells and hepatocytes shared the biliary lumen. The lumen sometimes approached the base of the ductule, providing a possible route for bile leakage. The intercalated ductule was composed of low fusiform epithelial cells throughout its length, meeting its classical criteria by light microscopy. Its basal surface was furrowed with narrow grooves along cell boundaries. The ductular cells extended numerous microplicae in the basal grooves and on their lateral surfaces, suggesting their secretory function.     

Storage of alpha-1-antitrypsin in intrahepatic bile duct cells in alpha-1-antitrypsin deficiency (Pi Z phenotype)

Storage of alpha-1-antitrypsin (AAT) has been found in a small number of bile duct cells in liver tissue specimens from patients with Pi MZ, Pi SZ and Pi ZZ phenotypes. The storage appeared in the form of intracellular AAT immunoreactive inclusions. On EM investigation, AAT-like material was detected within cisternae of the RER and SER. Such AAT inclusions were found in proliferating bile ductules in conditions such as cirrhosis, focal nodular hyperplasia and extrahepatic obstruction. They were also observed in normal biliary structures at the level of the canals of Hering, bile ductules and interlobular ducts in 13 out of 47 cases. These findings are interpreted as indicating that the intrahepatic bile duct cells are a further source of AAT, and that in case of defective export of AAT from the cell, as is the case for the Z protein, the protein accumulates not only in hepatocytes but in biliary cells as well.     

LIGHT AND ELECTRON MICROSCOPIC STUDIES ON THE DEVELOPMENT OF PERIPORTAL BILE DUCTS OF THE HUMAN EMBRYO

In order to clarify the development of periportal bile duct in the human embryo, the liver tissue of a 13 week-old human embryo was studied using the electron as well as light microscope.
The findings observed in this study lead to the following conclusions: The biliary epithelial cell originates from the periportal hepatoblast under certain influences of the connective tissue. The periportal bile duct is formed by the proliferation of the biliary epithelial cells surrounding the biliary space. The so-called intermediate cell between the biliary epithelial cell and the hepatoblast is not recognized in the human fetal liver of 13 weeks gestation.     

Somatostatin analogue (octreotide) inhibits bile duct epithelial cell proliferation and fibrosis after extrahepatic biliary obstruction.

Extrahepatic biliary obstruction leads to bile duct epithelial cell proliferation. Somatostatin and its analogue, octreotide, have been shown to inhibit DNA synthesis and proliferation in hepatocytes. We investigated the effect of octreotide on the biliary epithelial cell proliferative responses to biliary obstruction. Male Sprague-Dawley rats underwent common bile duct ligation and subcutaneous injection of either saline or octreotide (6 micrograms/kg) twice daily for 7 days. Morphometric analysis of hepatocytes, bile duct epithelial cells, and periportal connective tissue was performed by computerized point counting. Hepatocyte volume was preserved with octreotide treatment, which also significantly decreased bile duct proliferation and periportal extracellular matrix deposition in response to biliary obstruction compared with saline treated, duct-ligated animals. These results indicate that octreotide prevents the morphological changes that accompany extrahepatic biliary obstruction.     

Gadolinium chloride suppresses hepatic oval cell proliferation in rats with biliary obstruction.

Liver injury due to bile duct ligation (BDL) is histologically characterized by cholestasis, bile ductular proliferation, hepatocellular damage, portal fibrosis, and ultimately biliary cirrhosis. Stem cells within the liver may act as progenitor cells for small epithelial cells termed oval cells that can differentiate into bile duct cells or hepatocytes, whereas myofibroblasts are the principal source of collagen production in fibrosis. The aims of this study were to determine 1) whether BDL induces oval cell proliferation and 2) whether blockade of Kupffer cells affects oval cell proliferation, bile duct proliferation, and myofibroblast transformation in experimental biliary obstruction. Male Sprague-Dawley rats were divided into two groups to receive either a single dose of gadolinium chloride (a selective Kupffer cell blocking agent) or vehicle. One day later, the gadolinium- and vehicle-treated groups were further subdivided to receive either BDL or sham operation. The rats were sacrificed on day 7 postoperatively. Serum was collected for measurement of aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin levels. Liver tissue was taken for evaluation of fibrosis, bile ductular cells, oval cells, and myofibroblasts. BDL resulted in elevated aspartate aminotransferase, gamma-glutamyl transpeptidase, and bilirubin in serum, and gadolinium pretreatment did not modify these effects. BDL induced marked oval cell proliferation, which was completely prevented by gadolinium pretreatment. Gadolinium did not affect the induction of bile duct expansion or myofibroblasts after BDL. We conclude that experimental biliary obstruction induces oval cell proliferation, which can be prevented by gadolinium pretreatment. This suggests that bile ductular proliferation and myofibroblast transformation are not mediated by Kupffer cells and that ductular proliferation can proceed in the absence of oval cells. Alternatively, gadolinium may directly affect oval cell proliferation after BDL.     

A HISTOCHEMICAL STUDY ON THE ACTIVITY OF GAMMA-GLUTAMYL TRANSPEPTIDASE IN LIVER DISEASE

Comparative histochemical study for γ-GTP and alkaline phos-phatase was performed in several disorders of liver.
Areas of extensively proliferated fibrosis in chronic active hepatitis and areas of newly formed fibrosis in cirrhosis showed prominent activity of γ-GTP but poor activity for alkaline phosphatase. On the other hand, alkaline phosphatase activity was remarkably high in the connective tissue around proliferating bile ductules.
Activity of both enzymes was prominent in the bile canaliculi and sinusoidal walls in obstructive jaundice, but marked decrease or loss of activity of the enzymes was observed in the degenerated or necrotic areas. While fatty degeneration of hepatocytes showed no increase in activity of the enzymes, prominent activity of γ-GTP was noted in the connective tissue surrounding degenerated hepatocytes in alcoholic liver disease.
There was marked activity of γ-GTP in most tumor cells of hepatoma, while in metastatic tumor the activity of both enzymes was noted in only bile canaliculi and sinusoidal wall adjacent to the tumor.