bromochloroacetic-acid and Cholestasis

The keratin intermediate filament (IF) cytoskeleton of hepatocytes has continuously gained medical relevance over the last two decades. Originally it was mainly recognized as a differentiation marker for diagnostic purposes in pathology. However, keratin IFs were soon identified as major cellular structures to be affected in a variety of chronic liver diseases, such as alcoholic and non-alcoholic steatohepatitis (ASH, NASH), copper toxicosis, and cholestasis. Based on observations in keratin gene knock-out mice, the insight into the functional role of keratins was extended from a mere structural role providing mechanical stability to hepatocytes, to an additional role as target and modulator of toxic stress and apoptosis. The functional relevance of keratins in human diseases has recently been underlined by the identification of mutations in keratin genes in patients with liver cirrhosis.

Topics: Biliary Tract; Cholestasis; Chronic Disease; Cytoskeleton; Epithelial Cells; Hepatitis; Humans; Keratins; Liver; Liver Diseases; Liver Neoplasms; Mutation

Proliferation of preexisting bile ducts, ductular metaplasia of hepatocytes and proliferation and differentiation of liver stem cells are discussed in the pathogenesis of neoductular structures in the liver. Under the condition of experimental bile duct obstruction and in extrahepatic bile duct stenosis neoductular structures are first the result of proliferation and sprouting of preexisting ducts and cholangioles. Especially in later stages of cholestasis but also in other chronic progredient liver diseases such as chronic alcoholic liver disease and chronic active hepatitis periportal hepatocytes may show a phenotypic shift towards ductular epithelia. In postnatal liver diseases hepatocytes first express keratin 7 and later keratin 19 during ductular transdifferentiation. This is in contrast to embryonal cholangiogenesis. In alpha-1-antitrypsin-deficiency, hemochromatosis, Wilson's disease, and chronic active hepatitis B cellular deposites typically located in hepatocytes such as alpha-1-AT, siderin, copper, HBs-Ag, and HBc-Ag can also be found in neoductular cells close to hepatocytes. These deposites seem to be retained during the ductular transdifferentiation of hepatocytes. Expression of bile duct-type integrin subtypes and TGF beta 1 in neoductular cells are involved in the changing parenchymal/mesenchymal interplay during neoductogenesis, resulting in periductular basal membrane and periductular fibrosis. In FNH the ductular transdifferentiation of hepatocytes is integrated in the histogenesis of micronodules and portal tract equivalents of these tumor-like lesions. Ductular structures in hepatoblastomas and especially in combined hepatocellular and cholangiocarcinomas (CHCC) may reflect the common embryologic derivation of hepatocytes and biliary epithelia. Non-neoplastic liver tissue in resection specimens of our CHCC showed a lower rate of cirrhosis, and a significantly higher Ki 67-LI of neoductular cells compared to liver tissue in resection specimens of HCC and liver metastases. 3 of 10 CHCC had developed in alpha-1-AT-deficiency, in which this protease-inhibitor was predominantly retained in periportal hepatocytes. These findings in non-neoplastic tumor-bearing liver tissue suggest that CHCC include a special histogenic type of primary liver carcinoma which in analogy to some experimental liver tumors might develop from periportal parenchymal cells.

Topics: alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; Animals; Bile Ducts; Bile Ducts, Extrahepatic; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Cholangiocarcinoma; Cholestasis; Copper; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Hepatoblastoma; Humans; Hyperplasia; Keratins; Liver; Liver Diseases; Liver Neoplasms; Metaplasia

Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis.. We generated liver-specific plectin knockout (Ple. Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in Ple. Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis.. Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.

Topics: Animals; Biliary Tract; Cholestasis; Epithelium; Hepatocytes; Keratins; Liver; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Plectin; Protein Stability

Notch signaling has been demonstrated to be involved in ductular reactions and fibrosis. Previous studies have shown that Huang Qi Decoction (HQD) can prevent the progression of cholestatic liver fibrosis (CLF). However, whether HQD affects the Notch signaling pathway is unclear. In this study, CLF was established by common bile duct ligation (BDL) in rats. At the end of the first week, the rats were randomly divided into a model group (i.e., BDL), an HQD group, and a sorafenib positive control group (SORA) and were treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, and -4, Jagged (JAG) 1, and Delta like (DLL)-1, -3, and -4. The results showed that HQD significantly reduced the deposition of collagen and the Hyp content of liver tissue and inhibited the activation of HSCs compared with the BDL group. In addition, HQD significantly decreased the protein and mRNA expressions of TGF-[Formula: see text]1 and [Formula: see text]-SMA. In contrast, HQD significantly enhanced expression of the Smad 7 protein. HQD also reduced biliary epithelial cell proliferation, and reduced the mRNA levels of CK7, CK8, CK18, SRY-related high mobility group-box gene (SOX) 9, epithelial cell adhesion molecule (EpCAM) and the positive areas of CK19 and OV6. In addition, the mRNA and protein expressions of Notch-3, -4, JAG1, and DLL-1, -3 were significantly reduced in the HQD compared to the BDL group. These results demonstrated that HQD may prevent biliary liver fibrosis through inhibition of the Notch signaling pathway, and it may be a potential treatment for cholestatic liver disease.

Topics: Actins; Animals; Astragalus propinquus; Biliary Tract; Cell Proliferation; Cholestasis; Collagen; Common Bile Duct; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial Cell Adhesion Molecule; Epithelial Cells; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Keratins; Ligation; Liver; Liver Cirrhosis; Membrane Proteins; Rats; Receptors, Notch; RNA, Messenger; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta1

The aim of this study was to evaluate the possible protective effects of Urtica dioica (UD) against liver damage in the common bile duct-ligated rats. A total of 24 male Sprague Dawley rats were divided into three groups, namely, control, bile duct ligation (BDL) and BDL + received UD groups, containing eight animals in each group. The rats in UD-treated groups were given UD oils (2 ml/kg) once a day intraperitoneally for 2 weeks starting 3 days prior to BDL operation. The change demonstrating the bile duct proliferation and fibrosis in expanded portal tracts includes the extension of proliferated bile ducts into the lobules; inflammatory cell infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with UD attenuated alterations in liver histology. The α-smooth muscle actin, cytokeratin-positive ductular proliferation and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the BDL were observed to be reduced with the UD treatment. The data indicate that UD attenuates BDL-induced cholestatic liver injury, bile duct proliferation and fibrosis.

Topics: Actins; Animals; Antioxidants; Cell Proliferation; Cholestasis; Common Bile Duct; DNA Nucleotidylexotransferase; Fibrosis; Immunohistochemistry; In Situ Nick-End Labeling; Keratins; Liver; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; Urtica dioica

Ductular reaction (DR) in bile duct ligated rats generally appears from 2nd day after biliary obstruction (BO). However, we show that increased amount of ductular profiles is evident already in 6 hours after BDL. The study aims to explain the origin of such an early DR in response to BO. Male Lewis rats were subjected to common bile duct ligation (CBDL) for 3, 6, 12 and 24 hours and sham operation. Liver samples were studied histologically, immunohistochemically (Ki67, pan-Cytokeratin /AE1 + AE3/ and OV-6) and by immunoblotting analyses. It appeared that number of ductular profiles increase in time-related manner after BO. These ductular profiles are formed by biliary epitheliocyte-like cells; No mitotic activity was revealed. Part of hepatocytes reveals pan-Cytokeratin positivity on 12 and 24 hours after BO. Total cytokeratins content at 24 hours after CBDL was 37% higher in comparison with control data. The significant increase was observed for the cytokeratins with molecular weights: 61, 56 and 40 KDa. Thus, early DR after BDL is mediated by widening of the existed finest biliary ramifications and is not associated with proliferation activities. This DR is accompanied by differentiation of hepatocytes toward bile duct-like cells.

Topics: Animals; Bile Ducts; Cholestasis; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Immunohistochemistry; Keratins; Male; Rats; Rats, Inbred Lew

The intermediate filament cytoskeleton of hepatocytes is composed of keratin (K) 8 and K18 and has important mechanical and nonmechanical functions. However, the potential role of the K8/K18 network for proper membrane targeting of hepatocellular adenosine triphosphate-binding cassette transporters and bile formation is unknown. We therefore designed a comparative study in K8 and K18 knockout mice and respective wild-type controls to test the hypothesis that intermediate filaments of hepatocytes play a role in normal bile formation. In addition, we challenged mice either with a 1% cholic acid-supplemented diet or a diet containing the porphyrinogenic xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine to determine the effect of K8/K18 loss on bile flow/composition and liver injury under different physiological and toxic stress stimuli. Protein expression levels and membrane localization of various transporters and anion exchangers were compared using western blotting and immunofluorescence microscopy, respectively, and bile flow and composition were determined under various experimental conditions. Our results demonstrate that loss of the intermediate filament network had no significant effect on bile formation and composition, as well as expression levels and membrane targeting of key hepatobiliary transporters under baseline and stress conditions. However, loss of K8 significantly increased liver injury in response to toxic stress.. The intermediate filament network of hepatocytes is not specifically required for proper bile formation in mice.

Topics: Animals; ATP-Binding Cassette Transporters; Bile; Bile Acids and Salts; Cholestasis; Cholic Acid; Hepatocytes; Intermediate Filaments; Keratin-18; Keratin-8; Keratins; Liver; Mice; Mice, Knockout

Ischemia-reperfusion (I/R) injury in liver transplantation units and the influence of I/R injury on bile flow dynamics is being intensely investigated in various animal models, but the expression of intracellular intermediate filaments of biliary type as an early sign of cholestasis has not been yet explored.. We studied the hepatic elimination kinetics of indocyanine green (ICG), an exclusively biliary excreted cholephilic dye, and the functional and morphological integrity of liver cells in a canine liver transplantation model following I/R. During reperfusion following cold ischemia, we evaluated the ICG excretion curves, biochemical signs of liver damage, the bile canaliculus of the hepatocytes by electron microscopy, and the expression of intermediate filaments of cytokeratin type by immunohistochemistry.. Impairment of the biliary ICG excretion was directly related to ischemia time, but hepatocellular ICG uptake and bile flow rate were not significantly reduced. Liver enzymes increased as early as 6 h of ischemia and hepatocytes showed an increase of the bile canaliculus area. This was correlated to a membranous to cytoplasmatic staining of the cytoskeleton of the hepatocytes.. To the best of our knowledge, this is the first evidence of cholestatic changes starting early following cold ischemia in a canine isolated perfused liver transplantation model despite prompt recovery of the bile flow.

Topics: Animals; Bile; Cholestasis; Coloring Agents; Disease Models, Animal; Dogs; Hepatocytes; Immunohistochemistry; Keratins; Liver; Liver Transplantation; Microscopy, Electron; Reperfusion Injury

Hepatocyte "ballooning" is an often used but ill defined term in liver pathology to designate a special form of liver cell degeneration associated with cell swelling and enlargement found particularly in steatohepatitis. Alterations of the intermediate filament cytoskeleton of the hepatocyte may contribute to the pathogenesis of this microscopic change. Ballooning degeneration is considered a hallmark of steatohepatitis, but enlarged hepatocytes may also be observed in a variety of other acute and chronic liver diseases.. The intermediate filament cytoskeleton was investigated using keratin 8 and 18 immunohistochemistry in liver diseases associated with enlarged or ballooned hepatocytes.. Keratin 8/18 immunostaining was drastically reduced or lost in the cytoplasm of ballooned hepatocytes in alcoholic and non-alcoholic steatohepatitis, chronic cholestatic conditions, ischemia/reperfusion injury and in ballooned hepatocytes in chronic hepatitis C cases with concurrent steatohepatitis. In contrast, substantial decrease or loss of keratin 8/18 immunostaining was not noted in cases of acute hepatitis, giant cell hepatitis, chronic hepatitis B, or autoimmune hepatitis.. Loss of keratin 8/18 immunostaining can serve as an objective marker of a specific type of ballooning degeneration of hepatocytes. Oxidative stress may be a common denominator in the pathogenesis of keratin filament alterations in these conditions.

Topics: Adaptor Proteins, Signal Transducing; Biliary Atresia; Cholestasis; Fatty Liver; Hepatocytes; Humans; Immunohistochemistry; Keratins; Liver Cirrhosis, Biliary; Oxidative Stress; Sequestosome-1 Protein; Ubiquitin

Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear.. Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated.. Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7.. In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

Topics: 1-Naphthylisothiocyanate; Animals; Basic Helix-Loop-Helix Transcription Factors; Bile Ducts; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Cell Proliferation; Cholestasis; Disease Models, Animal; Female; Gene Expression Regulation; Green Fluorescent Proteins; Homeodomain Proteins; Hyperplasia; Keratin-7; Keratins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Protein-Tyrosine Kinases; Receptors, Notch; Regeneration; Transcription Factor HES-1

The diagnostic utility of aquaporin (AQP)-1 in liver tumors was tested and compared with other well-established markers. In 30 cholangiocarcinomas (CCs), 20 hepatocellular carcinomas (HCCs), and 10 metastatic colorectal carcinomas (MCCs) of the liver, expression of AQP-1, CD10, cytokeratin (CK) 7, CK20, and polyclonal carcinoembryonic antigen (pCEA) was tested. In addition, staining patterns of CD10 and pCEA were analyzed. To compare the selectivity of AQP-1 and CK7 as possible markers for differentiated cholangiocytes, liver biopsies of cholestatic disease were also analyzed. Aquaporin-1 expression was found in 93% of all CCs compared with 0% of HCC (P < .000001) and with 30% of MCC (P < .01). CD10 was positive in 16.7% of CC compared with 40% of HCC (P < .04) and to 20% of MCC (not significant). Cytokeratin 7 was positive in 90% of CC compared with 10% of HCC (P < .00001) and with 20% of MCC (P < .0001). Cytokeratin 20 was positive in 90% of MCC compared with 16.7% of CC (P < .0001) and with 20% of HCC (P < .00001). Canalicular staining patterns of CD10 and pCEA were observed in HCC (100% and 89.5%, respectively) but typically not in CC (0% and 6.7%, respectively) and never in MCC. In cholestatic disease, AQP-1 was expressed in differentiated epithelial cells of the bile ducts, whereas CK7-positive hepatocytes of Rappaport zone 1 did not show any AQP-1 reactivity. Therefore, AQP-1 seems to be a highly selective marker for differentiated cholangiocytes and can be very helpful in the differential diagnosis of liver tumors.

Topics: Adult; Aged; Aged, 80 and over; Aquaporin 1; Biomarkers, Tumor; Capillaries; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Cholangiocarcinoma; Cholestasis; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratin-7; Keratins; Liver Neoplasms; Male; Middle Aged; Neprilysin

Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids.

Topics: Animals; Bile Acids and Salts; Bile Ducts; Cholestasis; Cholic Acid; Dicarbethoxydihydrocollidine; Diet; Hepatocytes; Humans; Inclusion Bodies; Keratins; Ligation; Liver; Male; Mice; Phosphorylation; Ubiquitin

Cholestasis is associated with retention of potentially toxic bile acids and profound cytoskeletal alterations of hepatocytes. Given the well-established cytoprotective role of hepatocyte keratins this study aimed to determine the effects of cholestasis on the cytokeratin (CK) intermediate filament network in mouse liver. Mice were subjected to common bile duct ligation or sham operation. Mice were also fed a cholic acid or ursodeoxycholic acid (UDCA)-supplemented diet (0.1%, 0.5%, and 1%) or control diet for 7 days. CK 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction, in situ hybridization, Western blot analysis, and immunofluorescence microscopy. Common bile duct ligation and cholic acid feeding significantly stimulated CK 8 and CK 18 mRNA and protein levels compared to controls, whereas UDCA had no effect. CK overexpression was accompanied by pronounced phosphorylation. Our results show that potentially toxic bile acids induce hepatocytic CK 8 and CK 18 expression and phosphorylation whereas nontoxic UDCA has no effect on CKs. Thus, increased hepatocellular CK expression and phosphorylation in cholestasis may be caused by retention of toxic bile acids and reflect a hepatocellular stress response with potential beneficial effects.

Topics: Animals; Bile Acids and Salts; Bile Ducts; Cholestasis; Cytoskeleton; Diet; Hepatocytes; Humans; Immunohistochemistry; In Situ Hybridization; Intermediate Filaments; Keratins; Liver; Male; Mice

The involvement of hepatocytes in proliferation of bile ductule-like structures during cholestasis remains controversial. The present study was an attempt to address the issue of whether hepatocytes transform into ductular epithelial cells in response to cholestasis and, if so, which mechanisms are involved. Cholestasis was induced by common bile duct-ligation (CBDL) in rat liver for 2, 7, and 14 days. Immunofluorescence microscopy of cytokeratin 19 (CK19) was performed to assess the proliferation of bile ductules. Hepatocellular filamentous actin (F-actin) was studied using fluorescence microscopy of 7-nitrobenzene-2-oxa-1,3-diazole phallacidin and electron microscopy. Double labeling of F-actin and laminin was performed to reveal the relationship between rearrangement of F-actin and deposition of the extracellular matrix protein. The results showed that the localization of F-actin in hepatocytes underwent considerable changes after CBDL, from an even distribution at the entire plasma membrane in control liver to a more concentrated localization at one domain of the plasma membrane. This was followed by formation of rosette-like structures in pericentral and midzonal areas of the parenchyma. CK19 was expressed, as in the control liver, in the epithelial cells of proliferated bile ductules in enlarged portal tracts but not in rosette-like structures of CBDL livers. Furthermore, CBDL induced increasing amounts of laminin in the basal lamina of bile ducts and in connective tissue of portal tracts. In parenchyma, the newly deposited laminin was in close association with the rosette-like structures. It is concluded that the rearrangement of hepatocellular F-actin after CBDL precedes the formation of rosette-like structures. It is speculated that the altered F-actin contracts at one side of hepatocytes leading to tubular structures. Laminin may play an important role in this transformation process.

Topics: Actins; Animals; Cholestasis; Keratins; Laminin; Liver; Male; Microscopy, Electron; Rats; Rats, Wistar

Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adult rat liver by Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization analysis. We show that the GGT activity in the 18-day-old fetus results from the transcription of the gene from the promoter P3 in the hepatocytes. In contrast, the GGT promoter P4 activity appears to be specific of biliary cells in normal as well in cholestatic liver. Thus, sequences unique to the GGT transcripts initiated on these two alternate promoters provide unique molecular probes to discriminate between the biliary and the hepatocytic phenotypes in liver differentiation and cell lineage studies.

Topics: Aging; Animals; Animals, Newborn; Blotting, Northern; Cholestasis; Female; Gallbladder; gamma-Glutamyltransferase; Immunohistochemistry; In Situ Hybridization; Keratins; Liver; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Rats, Wistar; Transcription, Genetic

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by approximately 40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha-subunit (GsalphaS). The expression of either the large subunit (GsalphaL) or the common beta-subunit remained unchanged. The expression of Gialpha2 and Gialpha3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsalphaS and Galphai are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

Topics: Angiotensin II; Animals; Cholestasis; Colforsin; Common Bile Duct; Cricetinae; Cyclic AMP; Glucagon; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Keratins; Ligation; Liver; Male; Mesocricetus; Receptors, Glucagon; Taurochenodeoxycholic Acid; Ursodeoxycholic Acid

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.. 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-Ks20.10.. 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.. 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.

Topics: 1-Naphthylisothiocyanate; Age Factors; Animals; Bile Ducts, Intrahepatic; Cell Division; Cholestasis; Choline Deficiency; Female; Intermediate Filament Proteins; Keratin-20; Keratins; Liver; Male; Pregnancy; Rats; Rats, Inbred F344

The ability to induce proliferation by temporary duct ligation suggested an hypothesis that retrovirus-mediated gene transfer into cells of the biliary tract could be accomplished. The time course of histologic changes, incorporation of 3H-thymidine and immunofluorescent staining with a monoclonal antibody to cytokeratin-19 (a marker for differentiated bile ducts) was studied in male Fischer F344 rats. A recombinant Gibbon ape leukemia virus (GALV), containing a gene encoding Escherichia coli beta-galactosidase was next introduced into 24 hr obstructed bile ducts. Gene transfer was maximal when virus was exposed to the obstructed duct for 12 hr (approximately 0.1%). The majority of X-gal positive cells were in cytokeratin-19 negative peribiliary tissues, which had the appearance of newly forming bile ducts. The data suggest that cells targeted by retroviral infection of the obstructed rat bile duct may be a precursor of mature, fully differentiated biliary epithelium.

Topics: Animals; beta-Galactosidase; Bile Ducts; Cell Cycle; Cell Division; Cholestasis; Epithelial Cells; Escherichia coli; Gene Transfer Techniques; Keratins; Leukemia Virus, Gibbon Ape; Ligation; Liver; Male; Rats; Rats, Inbred F344

The aim of this study is: 1. to isolate intrahepatic biliary epithelial cells and, 2. to study the toxicity of cholestatic biliary acids on these cells. Cells were isolated from rats with proliferated bile duct-cells, that were induced by a 21 days bile duct ligation. They were characterized by glutamyltranspeptidase and cytokeratins 7 and 19 using histochemical or immuno cytochemical methods. Isolated cells were incubated with different concentrations (250, 500, 1000 and 2000 microM) of cholestatic biliary acids, lithocholate sulfate (LCS) and taurolithocholate sulfate (TLCS. Cells viability is estimated by three methods: Trypan blue, LDH release and MTT test. We obtained purified fractions of non parenchymal liver cells enriched in biliary epithelial cells (> 95%). On these cells, we showed toxicity of LCS and TLCS and determined CI 10 and CI 50 of these acids which were respectively 800 microM and 2 mM for LCS; 1.4 and 2 mM for TLCS. These results indicate that cholestatic biliary acids (LCS and TLCS) are toxic for biliary cells. This cytotoxicity can be probably a possible mechanism of cholestasis.

Topics: Animals; Bile Acids and Salts; Bile Ducts, Intrahepatic; Cell Death; Cholestasis; gamma-Glutamyltransferase; Keratins; Ligation; Lithocholic Acid; Rats; Rats, Sprague-Dawley; Taurolithocholic Acid

The patterns of cytokeratin as determined by murine monoclonal antikeratin antibody lu-5 (mAb lu-5) were quantitated in paraffin-embedded liver tissue from normal and diseased subjects. In tissue from healthy medical students, mAb lu-5 was found to decorate 2-4 periportal and 2-3 perivenular cell layers. Alcoholic liver disease was accompanied by a marked increase in intensity of mAb lu-5 antigen expression in zone I and III hepatocytes. Moreover, additional liver cells of both zones were progressively recruited, so that in advanced lesions all three lobular zones became positive. In mechanical as well as in drug-induced cholestasis, a similar increase of mAb lu-5 antigen expression was already observed in earlier stages of disease, including an earlier recruitment of zone II hepatocytes. In both alcoholic and cholestatic biopsies the intensity and extent of mAb lu-5 epitope expression increased with the duration and severity of disease. In primary biliary cirrhosis (PBC) and seemingly also in primary sclerosing cholangitis the increase and extent was more marked in zone I, the zone of assumed cholate accumulation. Changes in zone III, the territory of histologic cholestasis (bilirubinostasis), became evident only in late stages of PBC. Mallory bodies of alcoholic and cholestatic liver disease showed an identical mAB lu-5 antigen expression, thus giving rise to four different staining patterns. Changes of cytokeratin expression are similar in alcoholic and cholestatic liver diseases. In chronic viral hepatitis, however, cytokeratin alterations are discrete and restricted to precirrhotic/cirrhotic stages.

Topics: Antibodies, Monoclonal; Biopsy; Cholestasis; Chronic Disease; Epithelium; Hepatitis, Viral, Human; Humans; Keratins; Liver Diseases, Alcoholic; Paraffin Embedding; Reproducibility of Results; Retrospective Studies

Proliferation of bile duct-like structures is a hepatic cellular reaction observed in most forms of human liver disease and in a variety of experimental conditions associated with liver injury. Yet the origin, means of initiation, and significance of this hyperplasia are unknown. To clarify these issues we induced bile duct proliferation in rats by ligating the common bile duct and studied (a) hepatic incorporation of [3H]thymidine by histoautoradiography, (b) hepatic morphometry, (c) biliary tree volume using [3H]taurocholate as a marker of biliary transit time, (d) immunohistochemical expression of cytokeratin no. 19, (e) the effect of indomethacin, and (f) the role of increased biliary pressure, in the absence of physiological and biochemical evidence of cholestasis, on [3H]thymidine incorporation by the bile-duct cells. The results have demonstrated that (a) the proliferating bile duct-like cells are products of the extant biliary epithelium and retain its characteristics; (b) bile duct cells divide irrespective of the size of the duct in which they are located and form a system with a lumen continuous with the preexisting one; (c) bile duct proliferation results mainly in elongation, not in circumferential enlargement or sprouting of side branches; (d) portal macrophage infiltration does not play a role in the hyperplastic reaction, and (e) increased biliary pressure is the initiating factor in bile duct cell division. Our results provide evidence that under the present conditions, ductular metaplasia of hepatocytes does not occur and there is no functioning stem cell for biliary epithelial growth segregated in any particular duct size or within the portal connective tissue.

Topics: Animals; Autoradiography; Bile Ducts, Intrahepatic; Cell Division; Cholestasis; Common Bile Duct; Hyperplasia; Indomethacin; Keratins; Ligation; Male; Rats; Rats, Inbred Strains

The novel extracellular matrix glycoprotein tenascin was studied immunohistochemically in normal and fibrotic human liver. Its localization was compared to that of laminin, fibronectin and collagen type IV. In the normal liver, a weak staining for tenascin was detected along sinusoids, while portal tracts were negative. In both alcoholic and cholestatic liver disease and acute and chronic hepatitis, sinusoidal immunoreactivity for tenascin was variably increased as compared to the normal liver. Most striking, however, was the preferential accumulation of tenascin at connective tissue-parenchymal interfaces between proliferating ductules and in areas of piecemeal necrosis. As compared to laminin, fibronectin and collagen type IV, tenascin has the most restricted distribution. Our findings indicate that tenascin is a component of the extracellular matrix of the human liver. Its preferential expression at connective tissue-parenchymal interfaces in fibrosing areas in contrast to its absence from mature fibrous septa suggest a transient role in early matrix organization.

Topics: Alcoholism; Cell Adhesion Molecules, Neuronal; Cholestasis; Chronic Disease; Collagen; Extracellular Matrix; Fibronectins; Hepatitis; Humans; Immunohistochemistry; Keratins; Laminin; Liver; Liver Cirrhosis; Liver Diseases; Tenascin

Cholestatic and hepatitic liver cell rosettes, gland-like formations found respectively in chronic cholestasis and in chronic active hepatitis, represent structural modifications of liver cell plates in response to injury. Differences in cytokeratin expression, ultrastructure and three-dimensional (3-D) configuration have been investigated. Cholestatic rosettes are considered to be a form of biliary metaplasia of hepatocytes, linking with newly-formed bile ductules in adjacent septa and probably providing some protection from injury caused by abnormal bile constituents. Hepatitis rosettes, by contrast, are a form of liver cell regeneration developing in isolated surviving hepatocytes or small groups of hepatocytes within areas of collapse.

Topics: Bile Canaliculi; Biopsy; Cholestasis; Hepatitis C; Hepatitis, Chronic; Humans; Immunoenzyme Techniques; Keratins; Liver; Liver Cirrhosis, Biliary; Liver Regeneration; Microscopy, Electron

In the present study we have isolated and purified fractions of nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% alpha-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36-40 ml/min. In this fraction 1.8-3.8 x 10(6) cells per liver were recovered and up to 55% of them were positive for gamma-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 x 10(6) per liver, and as many as 60%-85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or alpha- naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.

Topics: Animals; Bile Ducts, Intrahepatic; Cell Separation; Cholestasis; Epithelial Cells; gamma-Glutamyltransferase; Hyperplasia; Immunohistochemistry; Keratins; Liver; Male; Microscopy, Electron; Phenotype; Rats; Rats, Inbred Strains

The architectural framework of the pericanalicular sheath composed of cytokeratin intermediate filaments (IFs) was examined after phalloidin treatment, bile duct ligation, and alcoholic fatty liver in rats to assess the role of IFs in experimental cholestasis. Electron microscopy examination of whole mount unembedded extracted liver slices was employed to visualize the cytoskeleton. Immunofluorescence staining and immunoelectron microscopy of the sheath were also performed using monoclonal antibodies to rat hepatocyte cytokeratins CK49 and CK55. The thickness of the wall and the diameter of the lumens were measured. In the phalloidin-treated rats, the pericanalicular sheath was markedly dilated and thickened. Immunofluorescence staining showed that the CK49 and CK55 IFs were localized in the pericanalicular region, particularly in the pericentral area. Immunoelectron microscopy documented that the IFs at the thickened pericanalicular sheath consisted of both CK49 and CK55, which means that the thickening of the bile canaliculus was in part due to an increase of IFs and not just due to an increase in actin filaments. In the livers where the bile duct was ligated, the pericanalicular sheath was irregularly dilated and some parts of the sheath appeared thinned out or missing. The belt desmosome also appeared absent focally in the pericanalicular sheath. Immunofluorescence studies showed that the staining for CK49 and CK55 was reduced focally in the pericanalicular region. The CK55 antibody stained the cytoplasm of hepatocytes in the periportal area more intensely when compared with the controls. These results indicated that the pericanalicular sheath and the belt desmosome were focally disrupted in response to extrahepatic bile duct obstruction. In the ethanol-fed rats, the pericanalicular sheath was dilated, thickened and tortuous, and appeared focally flattened by large fat droplets. IFs in the cytoplasm were pushed to the cell periphery and were compressed against each other by the fat droplets. CK55 and CK49 appeared increased as indicated by the observed immunofluorescence at the pericanalicular region. Immunoelectron microscopy showed that IFs of the thickened pericanalicular sheath were composed of CK55 and CK49. It is suggested that the pericanalicular sheath functions to mechanically provide a scaffolding for the bile canaliculus which is vulnerable to the different forces involved in cholestasis of different pathogenesis such as focal compression a

Topics: Actins; Animals; Bile Canaliculi; Bile Ducts; Bile Ducts, Intrahepatic; Cholestasis; Cytoskeleton; Fatty Liver, Alcoholic; Fluorescent Antibody Technique; Intermediate Filaments; Keratins; Ligation; Liver; Male; Microscopy, Electron; Phalloidine; Rats; Rats, Inbred Strains