An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl₄) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and α‐smooth muscle actin (α‐SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl₄ and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl₄ mice, CV were enlarged, with marked sinusoidal‐free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and α‐SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl₄ and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.     

Beneficial Effect of Caffeic Acid Phenethyl Ester (CAPE) on Hepatocyte Damage Induced by Bile Duct Ligation: An Electron Microscopic Examination

Recently the authors have reported the potent beneficial effect of caffeic acid phenethyl ester (CAPE) on cholestatic oxidative liver injury induced by acute bile ligation in Swiss albino rats. Herein, they report the ultrastructural hepatocellular alterations induced by acute bile duct ligation and the effect of CAPE administration on these alterations. Bile duct ligation resulted in many degenerative changes, such as vacuolization, mitochondrial degeneration, endoplasmic reticulum dilatation, and lysosome accumulation within the cytoplasm of hepatocytes. Mitochondrial degeneration was also observed within the cytoplasm of the cells of biliary ductular epithelium. CAPE potentially protected the hepatocytes from the cholestasis-induced cellular injury.     

Alteration of adhesion molecule expression and cellular polarity in hepatocellular carcinoma

AIMS: To study the expression of adhesion molecules in human liver and their possible roles during hepatocarcinogenesis. METHODS AND RESULTS: The expression of adhesion molecules in normal liver tissues, benign including probable premalignant lesions and malignant lesions was systematically investigated by immunohistochemistry and Western blotting. In normal liver, both hepatocytes and bile duct cells expressed symplekin, desmoglein 1/2, desmocollin 2, desmoplakin and plakophilin 2, but did not express desmocollin 1/3 or plakophilin 1. In benign hepatocyte lesions, expression of the adherens junctions and desmosomes was uniform and slightly increased, but symplekin appeared to show reduced expression in dysplastic lesions. In hepatocellular carcinoma (HCC), the expression of adhesion molecules was often heterogeneous and of abnormal location. Tumour cells with an abnormal distribution or loss of adhesion molecules showed an apolar arrangement of tissue architecture. The expression levels of the adhesion molecules correlated with the differentiation grades of HCC cells. CONCLUSIONS: The decreased expression of symplekin may be an early step in the transformation of hepatocytes, whereas alteration of the expression of adherens junctions and desmosomes may indicate more serious changes.     

A cytokeratin immunohistochemical study of alcoholic liver disease: evidence that hepatocytes can express ''bile duct-type'' cytokeratins

A cytokeratin immunohistochemical study was performed on 40 liver biopsies diagnosed as alcoholic liver disease to further investigate the cytoskeletal changes occurring in this disease. On paraffin sections of 29 cases, a variable number of hepatocytes were reactive with a polyclonal antiserum that normally stains only bile ducts. Using monoclonal antibodies specific for a single cytokeratin polypeptide on cryostat sections, a variable number of hepatocytes were immunoreactive for cytokeratin no. 7 in 23 cases and also for cytokeratin no. 19 in seven cases. Both these polypeptides are restricted to bile duct cells in the normal liver. The number of hepatocytes positive for bile duct-type cytokeratins increased and their location changed with the severity of the disease. Mallory bodies were reactive with monoclonal antibodies CAM 5.2 and anti-cytokeratin no. 18 but unreactive with anti-cytokeratin no. 8. except in one case. In two cases, Mallory bodies reactive with both monoclonal antibodies anti-cytokeratin no. 7 and anti-cytokeratin no. 19 were found. These results clearly indicate that hepatocytes in alcoholic liver disease can express immunoreactivity for bile duct-type cytokeratins. Our data also demonstrate heterogeneity in the composition of Mallory bodies. Whether hepatocytes expressing bile duct-type cytokeratins are the precursors of Mallory body-containing cells is not clear at present.     

Expression of sialylated Lewis(x) antigen in chronic and neoplastic liver diseases.

Phenotypic expression of sialylated Lewis(x) antigen by means of the monoclonal antiserum SNH3 was studied in 87 livers, which included normal and steatotic livers and livers with chronic persistent and chronic active hepatitis, alcoholic hepatitis, allograft rejection, focal nodular hyperplasia, hepatocellular carcinoma, cholangiocarcinoma, metastatic carcinoma, cirrhosis of various causes (autoimmune, alcoholic, viral, drug induced, Wilson's disease, and primary biliary cirrhosis). The biotin-streptavidin-peroxidase method was used on formaldehyde-fixed, paraffin-embedded sections. Sialylated Lewis(x) antigen was not demonstrated in normal livers. Hepatocellular expression in a diffuse or perinodular honeycomb pattern was seen in cirrhosis, irrespective of cause. Sialylated Lewis(x) antigen was also observed in hepatocytes around metastatic carcinoma in the absence of inflammation, cirrhosis, or regeneration. Some bile ductules, most likely ductular hepatocytes, but not bile ducts, expressed sialylated Lewis(x) antigen. Sialylated Lewis(x) antigen was seen diffusely in fibrolamellar hepatocellular carcinoma, focally in other hepatocellular carcinomas, and either focally or diffusely in cholangiocarcinomas.     

Experimental induction of hepatocellular hyalin (Mallory bodies) in mice by griseofulvin treatment. 1. Light microscopic observation.

Griseofulvin (GF) feeding of mice resulted in protoporphyria, liver cell damage, bile duct alterations, and finally hepatoma formation. In addition, hepatocellular hyalin developed, resembling in its morphology classic Mallory bodies (MB) as seen in alcoholic and nonalcoholic liver disorders in man. Liver cells containing MB often displayed features of severe cell damage and MB were finally released into the sinusoids and degraded by macrophages. The rapid disappearance of MB following GF discontinuation and the reappearance after resumption of GF feeding suggest an intimate relationship between metabolic alterations in the hepatocytes exerted by the drug and MB formation. This assumption is further supported by the fact that MB change their tinctoreal properties in chromotrope aniline blue-stained sections after GF discontinuation, possibly relfecting degeneration. Long term GF treatment apparently primed the liver for MB formation since the cells were able to respond almost instantly with MB to a GF challenge after a 1-month GF-free period.     

Functional units in rainbow trout (Salmo gairdneri,Richardson) liver: III. Morphometric analysis of parenchyma,stroma, and component cell types

Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6–7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.     

Ultrastructural study of rat liver after administration of an aminated steroid (author's transl)]

The administration of amino-3 beta hydroxy-20 beta pregnene-5, to the male Wistar rat, per os, at doses of 100 and 200 mg/kg/24 h, induce the development of a chronic active hepatitis. The ultrastructural observation shows slight changes only in perilobular hepatocytes at the beginning of treatment; then hepatocellular alterations progressively increase and may be observed in the whole lobule after 40 and 80 days of treatment; the progression of hepatocellular damage is associated with collagen increase and bile duct proliferation. The interest of this experimental hepatitis, as a model analogous to human chronic active hepatitis, is discussed.     

Evidence for microfilament involvement in norethandrolone-induced intrahepatic cholestasis.

An experimental study of norethandrolone (NED)-induced intrahepatic cholestasis was made. NED was infused via a portal vein catheter into rat liver in vivo, and measurements were made of bile flow. Liver specimens were taken at intervals for light microscopy and for transmission and scanning electron microscopy. Bile-canalicular-rich membrane fractions were prepared. The effects of NED were also examined in isolated hepatocytes in suspension culture. NED infusion induced total cholestasis by 3 hours. Canalicular alterations commonly associated with cholestasis were found in in vivo infused liver and in isolated hepatocytes. Pericanalicular microfilament changes were also noted in both, with loss of filament structure and replacement by a granular zone. In isolated canalicular membrane fractions prepared from NED-treated animals, the normal investment of pericanalicular filaments was no longer present. Loss of the bile canalicular ruthenium red surface coat was also noted. In view of the identical findings in isolated hepatocytes and in in vivo liver, obstruction and mechanical factors can be excluded as possible causes. The results raise the possibility that the mechanism of NED-induced cholestasis may be related to disaggregation and/or detachment of microfilaments from the canalicular membranes.     

Sequential analysis of hepatic carcinogenesis: a comparative study of the ultrastructure of preneoplastic, malignant, prenatal, postnatal, and regenerating liver.

The objective of this study was to compare the fine structure of presumptive preneoplastic hepatocytes at various times during liver carcinogenesis with that of normal, developing, and regenerating liver and of hepatocellular carcinomas, using transmission and scanning electron microscopy. A new model of liver carcinogenesis was used in which several of the early steps are quite well synchronized. A single initiating dose of diethylnitrosamine induced isolated islands of altered hepatocytes. The cells were characterized by persistence of glycogen despite starvation, increase in smooth endoplasmic reticulum, and hypertrophic nucleoli. Following intense selection of the altered hepatocytes by dietary 2-acetylaminofluorene plus partial hepatectomy, the affected hepatocytes proliferated rapidly to produce basophilic foci. These early hyperplastic lesions revealed stellate-shaped dilated bile canaliculi lined by blebs and abnormally thick elongated microvilli, a decreased number of microvilli on the sinusoidal surface, a marked increase in smooth endoplasmic reticulum, large nucleoli, and bundles of pericanalicular microfilaments. A majority of the proliferating lesions reacquired a normal organizational pattern within several weeks after partial hepatectomy and could not be distinguished from normal liver. A small number continued to grow and become typical persistent hyperplastic nodules. These showed significant widening of intercellular spaces between hepatocytes, elongated microvilli over large regions of the cell surface, many invaginations of the cell membrane, and irregularly shaped bile canaliculi. Sequential changes in focal hyperplastic hepatocytes during carcinogenesis could be distinguished from normal, developing, and regenerating liver. The major differences involved the cell surfaces and cytoplasmic organelles. The findings are compatible with the hypothesis that a carcinogen may act by inducing alterations in a small number of hepatocytes and that hepatocellular carcinomas arise through stepwise evolutional changes in these cells.     

Ultrastructure of early phase hepatic metastasis of human colon carcinoma cells with special reference to desmosomal junctions with hepatocytes

To demonstrate ultrastructural events in the early phase of hepatic metastasis of human colon carcinoma, we intrasplenically injected a highly metastasizable, human colon carcinoma cell line LM-H3 (1 x 10(6) cells) into nude mice, and electron microscopically investigated the hepatic metastasis. At 24 h, tumor cells adhered to the endothelial wall of terminal portal venules and periportal sinusoids. At 48-72 h, after extravasation, they deeply invaded the hepatic cell plate and the interstitial tissue of the portal tract, in which they underwent proliferation and made the metastatic foci. Tumor cells were linked with each other or with surrounding hepatocytes by desmosomes. Desmosomes were maintained during the mitosis. When invading tumor cells were exposed to the bile canaliculi, they generated microvilli on the surface. Microvilli were also formed at the luminal surface of intracytoplasmic inclusions. In the interstitial tissue of the portal tract, tumor cells were closely associated with fibroblasts. However, no junctional specializations were seen between them. The present study demonstrated that human colon carcinoma cell line LM-H3 formed desmosomes with hepatocytes soon after invasion of the hepatic cell plate, suggesting the regulatory role of an interaction with hepatocytes in the growth of metastatic foci within the liver parenchyma.     

Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes.

The present study succeeded in cultivating normal adult rat hepatocytes for at least 85 days without losing their replicative potential and differentiation capacity. Small pieces of hepatocyte aggregates (clusters) were prepared from the primary culture of hepatocytes and used as starting material for the growth experiment. Some of the hepatocytes started to proliferate at 3 days when the clusters were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 mmol/L nicotinamide, 0.2 mmol/L L-ascorbic acid 2-phosphate, and 1% dimethylsulfoxide. Clusters continued to grow and formed colonies. All the cells covering colonies expressed normal hepatocyte-specific proteins. The number of albumin-expressing cells in the most replicative colonies increased sixfold during 32 days. Most of the cells were mononucleate and small in size and some of them expressed immature hepatocyte markers such as alpha-fetoprotein. Electron microscopy of cells in colonies revealed the presence of peroxisomes in the cytoplasm and desmosomes, tight junctions, and bile canaliculus-like structures between the cells. Depletion of one of the additives inhibited the growth of hepatocytes. The culture medium used also supported the growth of stellate cells (Ito cells) that had contaminated the original preparation in small numbers and seems to cooperatively stimulate a proliferative population of hepatocytes.     

Ultrastructure of hepatocellular neoplasms in aflatoxin B1 (AFB1)-initiated rainbow trout (Oncorhynchus mykiss).

The fine structure of hepatocellular neoplasms from aflatoxin B1 (AFB1)-initiated rainbow trout was studied by transmission electron microscopy. Large, usually uniform hepatic nuclei, large nucleoli, abundant, dilated rough-surfaced endoplasmic reticulum, and reduced glycogen storage were common findings in both hepatocellular adenomas and hepatocellular carcinomas. In addition, the presence of poorly developed microvilli in the space of Disse and in bile canaliculi, the occurrence of few or no bile preductule cells and a striking increase in the size and number of intercellular spaces characterized hepatocellular carcinomas. The three latter characteristics of hepatocellular carcinomas suggest loss of inter-relationships between hepatocytes and the microvascular system (sinusoids), between hepatocytes and the biliary system, and between individual hepatocytes, respectively. With respect to these parameters, adenomas were more similar to normal liver than to carcinomas.     

Detection of Ki-67 in liver biopsy specimens using monoclonal antibody to Mib-1

Ki-67 antigen was visualized in formalin-fixed, paraffin-embedded liver biopsy specimens using monoclonal antibody to Mib-1 to identify the proliferating hepatocytes. Thirty liver specimens obtained from 10 patients with chronic hepatitis (CH) or liver cirrhosis (LC) and 10 patients with hepatocellular carcinoma were studied. Liver specimens were treated with a pepsin solution or heated with autoclave or treated with microwave as a part of antigen retrieval system; then stained with an immunoperoxidase method using a monoclonal antibody to Ki-67 (Mib-1). Stable stainings were obtained in the sections treated with autoclave. Ki-67 was detected in the nuclei of hepatocytes, bile duct epithelium, fibroblast and infiltrating mononuclear cells. In patients with CH and LC, the numbers of hepatocytes positive for Ki-67 has a good co-relation with serum GPT level (p < 0.01), while has no relationship with the degree of fibrosis. The number of hepatocytes positive for Ki-67 has a good co-relation with the degree of the differentiation of hepatocellular carcinoma. Detection of proliferating hepatocytes using Mib-1 is useful to understand the degree of proliferation.     

Hepatocarcinoma-Intestine-Pancreas/Pancreatic Associated Protein (HIP/PAP) Is Expressed and Secreted by Proliferating Ductules as well as by Hepatocarcinoma and Cholangiocarcinoma Cells

Hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP) gene was identified because of its increased expression in 25% of human hepatocellular carcinoma. HIP/PAP protein, a C-type lectin, binds laminin, acts as an adhesion molecule for hepatocytes, and has also been described as an acute phase secretory protein during acute pancreatitis in humans and rats. We investigated HIP/PAP protein expression in patients with various liver diseases associated with ductular reaction. At the same time, we analyzed patients with hepatocellular carcinoma and cholangiocarcinoma, and tested HIP/PAP protein levels in sera to establish the pattern of secretion. Our data show that HIP/PAP expression was not restricted to hepatocellular carcinoma, but was also detected in cholangiocarcinoma cells as well as in reactive non-malignant bile ductules. In contrast, HIP/PAP protein expression was undetectable in normal mature hepatocytes, but some ductular cells localized at the interface of portal tracts with parenchyma were HIP/PAP immunoreactive in normal liver. Finally, we present evidence that HIP/PAP serum levels were increased in 21/28 (75%) patients with hepatocellular carcinoma, and in 25/51 (49%) patients with nonmalignant cirrhosis. Altogether, these results suggest that HIP/PAP protein may be implicated in hepatocytic and cholangiolar differentiation and proliferation.