Increases in endothelial caveolin-1 and cavins correlate with cirrhosis progression.

BACKGROUND AND AIMS: Caveolin-1 is associated with flat caveolar domains, invaginated smooth plasmalemmal vesicles, and caveolae. Polymerase 1 and transcript release factor (PTRF) (cavin 1) and serum deprivation protein response (SDPR) (cavin 2) are required for the invagination of caveolae, and PRKCDBP (protein kinase C, delta-binding protein; cavin 3) is required for caveolae budding to form caveolar vesicles. To investigate whether cavins are involved in hepatic sinusoidal angiogenesis and remodeling during progression to cirrhosis, normal control liver specimens and early and late cirrhotic liver specimens were studied. MATERIALS AND METHODS: Cavin-1, cavin-2, and cavin-3 proteins and their gene expression were examined using immunohistochemistry (IHC), Western blotting, and laser capture microdissection (LCM)-polymerase chain reaction (PCR) during progression of cirrhosis caused by hepatitis C. According to the perfusion, fixation methods were designed to reevaluate the precise ultrastructural localizations and changes of cavin-1 and cavin-2 expression on liver sinusoidal endothelial cells (LSECs) facing the sinusoidal blood flow. RESULTS: For IHC, cavin-1 and cavin-2 expressions were found to be upregulated in small angiogenic LSECs with collagen deposition in the perisinusoidal space as well as in the vascular endothelial cells of the remarkably proliferated portal venules, hepatic arterioles, and arterial capillaries within the fibrotic septa of late-stage cirrhotic liver. Cavin-3 was mainly localized in large vessels, and it was detected only scantly on the central vein and hepatic sinusoids in the control liver. In late-stage cirrhotic liver, the intensity of cavin-3 was enhanced mainly on proliferative large vessels in regenerated nodules and in the peripheral regions of nodules and fibrous septa. On conducting immunoelectron microscopy, in the control liver tissue, cavin-1 was found to be localized on the caveolae of hepatic arterial and portal venous endothelial cells, but it was scantly localized on hepatic sinusoidal lining cells, and cavin-2 was found mainly on vesicles in LSECs. In the cirrhotic liver tissue, aberrant cavin-1 and cavin-2 expressions were observed on caveolae-like structures in LSECs. Significant overexpressions of cavin-1 at the protein and messenger RNA (mRNA) levels in a cirrhotic liver were demonstrated by Western blotting and LCM-PCR. CONCLUSIONS: Cavin-1 and cavin-2 are strongly expressed within caveolae-like structures and associated vesicles within LSECs of the hepatitis C-related cirrhotic liver. Cavin-1 would play a critical role in regulating aspects of caveolin-1 in LSECs. Moreover, these findings suggest a direct association of cavin-1 and cavin-2 with the process of differentiation and transformation of LSECs inducing hepatic sinusoidal capillarization related to the progression of cirrhosis.

Aquaporin-1 is associated with arterial capillary proliferation and hepatic sinusoidal transformation contributing to portal hypertension in primary biliary cirrhosis.

Although aquaporins (AQPs) in normal hepatobiliary system have been studied, little is known about AQP localization and changes in the hepatic microvascular system including sinusoids in cholestatic liver. The present study aimed to clarify the localization of AQP-1 in the microvessels in normal human liver and in primary biliary cirrhosis (PBC). Human normal liver (control) and PBC liver specimens were obtained. Immunohistochemistry, Western blotting, in situ hybridization (ISH) and electron microscopic examination for AQP-1 were conducted. In control liver and stages I-II PBC liver, AQP-1 immunoreactivity was mainly localized in portal venules, hepatic arterioles and bile ducts in the portal tract, but was hardly detected in the sinusoids. However, AQP-1 expression was enhanced in the proliferated bile ductules in PBC. In stages III-IV PBC liver tissues, AQP-1 was aberrantly expressed in proliferated arterial capillaries opening into the sinusoids at the peripheral edge of regenerating hepatic nodules and in the fibrotic septa. Overexpression of AQP-1 at protein and mRNA levels was demonstrated by Western blot and ISH, respectively. Angiogenetic and fibrotic responses are probably induced by AQP-1, leading to enhanced pouring of arterial blood into the sinusoids; thus, contributing to progression of portal hypertension in PBC.

Relation between ultrastructural localization, changes in caveolin-1, and capillarization of liver sinusoidal endothelial cells in human hepatitis C-related cirrhotic liver.

Most vascular endothelial cells are continuously exposed to shear stress in vivo. Caveolae are omega-shaped membrane invaginations in endothelial cells (ECs) and are enriched in cholesterol, caveolins, and signaling molecules. This study was designed to elucidate the ultrastructural localization and change in caveolin-1 expression within human liver sinusoidal endothelial cells (LSECs) during the progression of cirrhosis caused by hepatitis C, using tissue sections prepared via perfusion-fixation. Normal control liver specimens and hepatitis C-related Child-Pugh A and C cirrhotic liver specimens were studied. Caveolin-1 in the liver sinusoids was examined via immunohistochemistry, Western blotting, and immunoelectron microscopy. In control liver tissue, caveolin-1 was localized on caveolae mainly in arterial and portal endothelial cells of the portal tract and was also found on vesicles and some fenestrae in LSECs around the central vein. In cirrhotic liver tissue, aberrant caveolin-1 expression was observed on caveolae-like structures in LSECs. Caveolin-1 was especially overexpressed in late-stage cirrhosis. This study demonstrates that caveolin-1 is strongly expressed within caveolae-like structures and associated vesicles within LSECs of the hepatitis C-related cirrhotic liver. These findings suggest a direct association of caveolin-1 in the process of differentiation of LSECs in cirrhosis-mediated capillarization.

Enhanced expressions of apelin on proliferative hepatic arterial capillaries in human cirrhotic liver.

AIM: Apelin (APLN), the endogenous ligand of angiotensin-like receptor 1 (APJ), is a peptide necessary for embryonic and tumor angiogenesis. Little is known about the localization and changes of APLN expression including the sinusoids in human cirrhotic liver, which might contribute to portal hypertension. This study was designed to elucidate the localization and change of APLN expression in human liver during the progression of cirrhosis. METHODS: Twelve normal liver specimens, eight specimens of Child-Pugh grade A cirrhosis, and 10 specimens of Child-Pugh grade C cirrhosis were studied. APLN protein and gene expression was examined by immunohistochemistry, western blotting, immunoelectronic microscopy, and laser captured microdissection (LCM) followed by polymerase chain reaction (PCR) in sinusoid. RESULTS: In control liver tissue, APLN was localized mainly on arterial endothelial cells and hepatic arterioles in the portal tract. In cirrhotic liver tissue, aberrant APLN expression was observed in periportal capillary endothelial cells corresponding to capillarized sinusoids, and in proliferated arterial capillaries in the fibrotic septa. Significant overexpression of APLN at protein level in cirrhotic liver was demonstrated by western blotting (P < 0.01 Child-Pugh A and C versus control, P < 0.01 Child-Pugh A versus C). APLN mRNA expression in the sinusoid was confirmed by LCM-PCR. CONCLUSION: In humans, APLN protein and gene were overexpressed in cirrhotic liver compared with normal liver, and the magnitude increased as cirrhosis progressed. Especially in end-stage cirrhosis, APLN was strongly expressed in proliferated arterial capillaries directly connected with the sinusoids, suggesting a role of APLN in the proliferation of arterial capillaries in cirrhosis.

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of "preneoplastic antigen"-like molecules.

Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases.

Recent advances in liver sinusoidal endothelial ultrastructure and fine structure immunocytochemistry.

Ultrastructure reports have described that liver sinusoidal endothelial cell (LSEC)s contain a cytoskeletal framework of filamentous actin. Small G protein has emerged as an important regulator of the actin cytoskeleton, and consequently, of cell morphology and motility. We investigated actin filaments in relation to SEF in LSECs using a heavy meromyosin-decorated reaction and thereby elucidated the roles of small G protein and actin cytoskeleton in the morphological and functional alterations of SEF. Caveolin-1 expression has also been found in fenestrations with many characteristics of liver sinusoidal endothelial cells. Currently, fenestral studies and human disease are revealing ways to increase the liver sieve's porosity, which is reduced through pathological mechanisms. Hepatic sinusoidal endothelial dysfunction, which is known to impair endothelium-dependent relaxation in the liver microcirculation, contributes to increased intrahepatic vascular resistance.

A new monoclonal antibody, 4F2, specific for the oligodendroglial cell lineage, recognizes ATP-dependent RNA helicase Ddx54: possible association with myelin basic protein.

Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2(+) , Sox10(+) , A2B5(+) , NG2(+) , Olig2(+) , O4(+) , and myelin basic protein (MBP)(+) cells but did not label any GFAP(+) or NeuN(+) cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2-positive cells were located initially in the ventricular zone as Musashi1(+) Tuj1(-) populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5-kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination.

Aquaporin-1 associated with hepatic arterial capillary proliferation on hepatic sinusoid in human cirrhotic liver.

BACKGROUND: Aquaporins (AQPs) are key regulators not only of water transport in the cytoplasm but also of angiogenesis. Although AQPs in the normal hepatobiliary system have been studied in mammals, little is known about the localization and changes of AQPs in the hepatic microvascular system including sinusoids in cirrhotic liver, which might contribute to portal hypertension. AIMS: We designed this study to examine the localization of AQP1 in human cirrhotic liver. METHODS: Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunostaining, Western blotting, in situ hybridization (ISH) and laser-captured microdissection (LCM) were conducted. RESULTS: In control liver tissue, AQP1 was localized mainly in the portal venules, hepatic arterioles and bile ducts in the portal tract, although AQP1 was detected only slightly in the sinusoids. In cirrhotic liver tissue, AQP1 expression was evident, aberrantly observed on periportal sinusoidal endothelial cells corresponding to the capillarized sinusoids, on the proliferated arterial capillaries opening into the sinusoid in the generating hepatic nodule and on proliferated bile ductules at the peripheral edge of nodules and fibrotic septa. In cirrhotic liver, overexpression of AQP1 at protein and mRNA levels was demonstrated, respectively, using Western blot and ISH. AQP-1 of mRNA level in sinusoid was confirmed using LCM. CONCLUSIONS: Aberrant expressions of AQP1 in periportal sinusoidal regions in human cirrhotic liver indicate the proliferation of arterial capillaries directly connected to the sinusoids, contributing to microvascular resistance in cirrhosis.

Histopathological study of spinal meningioma originating from the arachnoid villi.

Although the histogenesis of meningiomas remains unclear, it is believed that arachnoid cells are the most likely origin of this type of neoplasm. Further, little attention has been paid to the histopathology of spinal meningiomas arising from the arachnoid villi. We came across a case of spinal meningioma that was locally attached to the arachnoid membrane. The associated arachnoid villi were investigated by light microscopy and immunohistochemical analysis. We confirmed the presence of tumor cells under the fibrous capsule that forms the outer component of the arachnoid villi. Tumor cells grew out from the apical portion of the arachnoid villi. Furthermore, immunohistochemical study suggested that arachnoid cells made the transition to tumor cells on the arachnoid cell layer.

Overexpression of apelin receptor (APJ/AGTRL1) on hepatic stellate cells and sinusoidal angiogenesis in human cirrhotic liver.

BACKGROUND: The apelin receptor (APJ) is related to angiotensin-like-receptor 1 (AGTRL1). This study was designed to elucidate the in vivo localization and changes of APJ in cirrhotic liver, and the in vitro changes of APJ expression in cultured hepatic stellate cells (HSCs) and capillarized sinusoidal endothelial cells (SECs) activated by growth factors. METHODS: In vivo studies used control liver samples, cirrhotic liver samples from patients with Child's A cirrhosis undergoing surgical resection (Child-A-LC), and cirrhotic liver samples from autopsied cases of decompensated Child's C cirrhosis (Child-C-LC). Immunohistochemical (IHC), Western blot, laser-capture microdissection (LCM) coupled with reverse transcription -polymerase chain reaction (RT-PCR), and immunoelectron microscopic (IEM) studies for APJ expression were conducted. In vitro examinations used commercial human HSCs and SECs. APJ expression was examined in cultured HSCs activated by growth factors and in capillarized SECs activated by angiogenic factors. RESULTS: The IHC study of liver samples revealed only slight APJ expression in hepatic sinusoids in control liver tissue. In cirrhotic liver (Child-A-LC and Child-C-LC), APJ expression was evident mainly along the sinusoids and on portal fibroblasts in fibrotic septa. Western blot analysis of whole-liver homogenate and LCM-PCR of sinusoids revealed overexpression of APJ in Child-C-LC samples. The results of IEM studies showed that APJ expression was increased significantly on HSCs, but it was sparse on SECs in Child-C-LC tissue. In vitro examination revealed that APJ was overexpressed in cultured HSCs activated by platelet-derived growth factor-ß. CONCLUSIONS: Enhanced expression of APJ on HSCs in cirrhosis indicates markedly increased vascular remodeling.

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver--re-evaluations of microlymphatic abnormalities.

BACKGROUND: From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs. METHODS: Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels. RESULTS: Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue. CONCLUSION: It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.

Aberrant expressions of aquaporin-1 in association with capillarized sinusoidal endothelial cells in cirrhotic rat liver.

Aquaporins (AQPs) are key regulators of water channels across the cell cytoplasm. Little is known about AQP localization and changes in the hepatic microvascular system. This study aimed to clarify the localization of AQP-1 in the microvessels in normal and cirrhotic rat liver. To establish a rat cirrhosis model, thioacetamide (TAA) was injected for 24 weeks. AQP-1 in liver specimens was examined by immunohistochemistry (IHC), Western blotting, and immunoelectron microscopy (IEM). IHC revealed that AQP-1 was localized in hepatic sinusoids, especially on the liver sinusoidal endothelial cells (LSECs), predominantly in zone 1 in control rats, whereas AQP-1 immunoreactivity was increased on LSECs in central portions of regenerative nodules in cirrhotic rats, and was expressed especially strongly on the outer side of the duplicated liver cell cords. IEM demonstrated that, in control livers, AQP-1 was mainly expressed on the plasma membrane of LSECs in zone 1. In cirrhotic livers, many immunogold particles showing the presence of AQP-1 were seen on the LSECs in central portions of regenerative nodules, and the number was significantly greater than that in zone 3 of control liver. Protein levels of AQP-1 examined by Western blot were almost the same in the cirrhotic liver and control liver. AQP-1 immunoreactivities were aberrantly expressed on LSECs in central portions of regenerative nodule (CPRN) of cirrhotic liver, which may be associated with capillarization of LSECs and remodeling in this region.

A possible connection between psychosomatic symptoms and daily rhythmicity in growth hormone secretion in healthy Japanese students.

BACKGROUND: Students suffering from psychosomatic symptoms, including drowsiness and feelings of melancholy, often have basic lifestyle problems. The aim of this study was to investigate whether psychosomatic complaints may be related to circadian dysfunction. METHODS: We examined 15 healthy students (4 men and 11 women) between 21 and 22 years old. To assess the presence of psychosomatic symptoms among the subjects, we developed a self-assessment psychosomatic complaints questionnaire consisting of five items pertaining to physical symptoms and five items concerning mental symptoms. The subjects rated their psychosomatic symptoms twice a day (08:00 and 20:00 h). We also assessed growth hormone secretion patterns by fluorescence enzyme immunoassay (FEIA). Salivary samples were collected from the subjects at home five times a day (20:00, 24:00, 04:00, 08:00, and 12:00 h) in Salivette tubes. RESULTS: The results indicated a relationship between the self-assessment scores and the salivary levels of growth hormone. Subjects with high self-assessment scores showed significant variability in growth hormone secretion over the day, whereas subjects with low self-assessment scores did not. CONCLUSION: Psychosomatic symptoms may be associated with circadian dysfunction, as inferred from blunted rhythmicity in growth hormone secretion.

A new monoclonal antibody, A3B10, specific for astrocyte-lineage cells recognizes calmodulin-regulated spectrin-associated protein 1 (Camsap1).

Recent studies of adult neurogenesis of the mammalian central nervous system have suggested unexpected plasticity and complexity of neural cell ontogenesis. Redefinition and reconstitution of cell classification and lineage relationships, especially between glial and neural precursors, are an urgent and crucial concern. In the present study, we describe a new monoclonal antibody, A3B10, which was produced by immunizing mice with the membrane fraction prepared from astrocyte-enriched primary neural cell cultures. Immunohistochemistry of brain sections, including brains from glial fibrillary acidic protein (GFAP)-deficient mice and primary mixed neural cell cultures, as well as immunoblot analysis and immunoelectron microscopy, have revealed that 1) A3B10 recognizes a majority of cells in ependyma in neonatal and adult rats, 2) A3B10 stains almost all GFAP(+) cells and some S100beta(+) cells in the corpus callosum, 3) A3B10 specifically stains astrocytes in vitro in primary cultures of rat embryonic cerebral hemispheres, 4) A3B10 equally stains ependymal cells of wild-type and GFAP-deficient mice, and 5) A3B10 antigen might construct intermediate filament bundles with GFAP and/or vimentin. These data suggested that the antibody labels a wide array of astorcytic-lineage cells including astrocytes, astrocyte precursors, and neural stem cells. Screening a cDNA library derived from rat embryonic brain has revealed that the antibody recognizes calmodulin-regulated spectrin-associated protein 1 (Camsap1). Thus this antibody may provide not only a new marker to identify astrocyte-lineage cells but also a new target molecule to elucidate the ontogeny, development, and pathophysiological functions of astrocyte-lineage cells.

Caveolin-1 and Rac regulate endothelial capillary-like tubular formation and fenestral contraction in sinusoidal endothelial cells.

BACKGROUND/AIMS: Rho guanidine triphosphatases (GTPases) are major regulators of cell migration. We investigated the cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cells (LSECs) cultured on Matrigel while stimulated by the vascular endothelial growth factor (VEGF). METHODS: To obtain primary monolayers, LSECs were cultured on Matrigel for 5-17 h with or without VEGF. Sinusoidal endothelial fenestrae (SEF) morphology was observed using scanning electron microscopy and transmission electron microscopy. RhoA, Rac1 and phosphorylated myosin light-chain kinase, Rho-binding domain of Rhotekin and the p21-binding domain of p21-activated protein kinase were analysed using Western blotting. RESULTS: The LSECs showed cellular protrusions and or cords of aligned cells resembling primitive capillary-like structures, with SEF contraction. Time course analyses of Rac1 activation matched specific morphological changes. Rac1 activity increased progressively to 17 h in cells cultured without VEGF, but markedly increased at 7 h in the presence of VEGF. RhoA activity was slightly elevated at 5 h. The levels of endogenous caveolin-1 (CAV-1) expression increased in a time-dependent manner, reaching a peak at 7 h. CAV-1 expression occurred immediately before the formation of the capillary-like tube. Moreover, treatment with VEGF regulated CAV-1 expression in LSECs. CONCLUSIONS: Spatial activation of Rac1 is involved in the formation of a capillary-like tubular network accompanying SEF contraction in LSECs, implying that endothelial migration and adhesion are necessary for LSECs tubular formation in the liver. CAV-1 might play an important positive role in the regulation of LSEC tubular formation.

The endothelin-1 receptor-mediated pathway is not involved in the endothelin-1-induced defenestration of liver sinusoidal endothelial cells.

BACKGROUND/AIMS: We previously reported that endothelin (ET)-1 may be involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently cell morphology. To clarify the role of ET receptors [endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] in ET-1-induced defenestration, we studied the size of hepatic SEF under various experimental conditions. METHODS: Liver sinusoidal endothelial cells (LSECs) isolated from rat livers by collagenase perfusion were cultured and divided into four groups: control, ET-1 (10(-6) -10(-10) M)-treated, ET-1+selective ETAR antagonist (BQ610)-treated and ET-1+ETBR antagonist (BQ788)-treated groups. SEF morphology was observed by scanning electron microscopy. Protein expressions of ETAR and ETBR, Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. F-actin stress fiber formation was observed by confocal microscopy. Active Rho was measured by Ren's modification. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluorescence digital imaging using fura-2 AM by Aqua cosmos. RESULTS: ET-1 induced a reduction in the number and size of SEF. ETAR antagonist pretreatment inhibited defenestration induced by low ET-1 concentrations (10(-8) -10(-10) M), whereas ETBR antagonist pretreatment did not block defenestration at low to high ET-1 concentrations (10(-6) -10(-10) M). F-actin stress fibers, Rho A levels and phosphorylated myosin light-chain kinase levels remained the same in various treatments. Active Rho was not detected in control and various treatments. ET-1 did not increase [Ca2+]i. Western blot showed prominent ETBR but scarce ETAR protein expression in LSECs. CONCLUSIONS: The present findings demonstrated that ETBR- and ETAR-induced contractile mechanisms are not involved in ET-1-induced defenestration, and that Rho is also not activated. Therefore, ET-1 induces hepatic defenestration by mechanisms other than receptor-mediated contraction.

Suppression of proinflammatory cytokine production in macrophages by lansoprazole.

Macrophages (MPs) produce increased levels of proinflammatory cytokines in Crohn's disease; these cytokines are thought to play a central role in the occurrence of the disease. Biologics are currently available for anti-cytokine therapy, but treating intestinal inflammation through direct suppression of proinflammatory cytokine production could be more effective. P-ATPase inhibitors have been reported to be anti-inflammatory, and these inhibitors might suppress the production of MP proinflammatory cytokines. In this study, we examined the effect of two types of ATPase inhibitors on the expression patterns of typical proinflammatory cytokines. Peritoneal MPs from 6- to 8-week-old mice were cultured for 48 h in the presence of lansoprazole (P-ATPase inhibitor), bafilomycin A(1) (V-ATPase inhibitor), or the control solvent dimethylsulfoxide. The MPs were then examined for cytokine expression by quantitative real-time polymerase chain reaction (PCR), and culture supernatants were examined for cytokine production with a multiplex assay in a suspension array system. The possible existence of P-ATPase mRNA in MPs was explored using reverse-transcriptase PCR. P-ATPase mRNA was not detected in MP cells. However, all examined proinflammatory cytokines decreased significantly in their mRNA and protein expression in the lansoprazole-treated group. Conversely, bafilomycin A(1) increased the levels of these cytokines. Lansoprazole might be useful for the treatment of inflammatory bowel diseases (IBDs), including Crohn's disease, as it suppresses the production of relevant MP proinflammatory cytokines. However, because P-ATPase was not detected in MPs, the mechanism is unclear and remains to be studied further in an IBD animal model.

Expression of adhesion molecules on mature cholangiocytes in canal of Hering and bile ductules in wedge biopsy samples of primary biliary cirrhosis.

AIM: To examine the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) expression on canals of Hering (CoH) and bile ductules associated with the autoimmune process of bile duct destruction in primary biliary cirrhosis (PBC). METHODS: Ten wedged liver biopsies of PBC (five cases each of stages 2 and 3) were studied. The liver specimens were processed for transmission electron microscopy. Immunohistochemistry was performed using anti-ICAM-1 and anti-LFA-1 mouse mAbs. In situ hybridization was done to examine the messenger RNA expression of ICAM-1 in formalin-fixed, paraffin-embedded sections using peptide nucleic acid probes and the catalyzed signal amplification (CSA) technique. Immunogold-silver staining for electron microscopy was performed using anti-ICAM and anti-LFA-1 mouse mAbs. The immunogold particles on epithelial cells of bile ductules and cholangiocytes of CoH cells were counted and analyzed semi-quantitatively. Western blotting was performed to confirm ICAM-1 protein expression. RESULTS: In liver tissues of PBC patients, immunohistochemistry showed aberrant ICAM-1 expression on the plasma membrane of epithelial cells lining bile ductules, and also on mature cholangiocytes but not on hepatocytes in CoH. LFA-1-positive lymphocytes were closely associated with epithelial cells in bile ductules. ICAM-1 expression at protein level was confirmed by Western blot. In situ hybridization demonstrated ICAM-1 mRNA expression in bile ductules and LFA-1 mRNA in lymphocytes infiltrating the bile ductules. By immunoelectron microscopy, ICAM-1 was demonstrated on the basal surface of epithelial cells in bile ductules and on the luminal surfaces of cholangiocytes in damaged CoH. Cells with intermediate morphology resembling progenitor cells in CoH were not labeled with ICAM-1 and LFA-1. CONCLUSION: De novo expression of ICAM-1 both on mature cholangiocytes in CoH and epithelial cells in bile ductules in PBC implies that lymphocyte-induced destruction through adhesion by ICAM-1 and binding of LFA-1-expressing activated lymphocytes takes place not only in bile ductules but also in the CoH.

High expressions of caveolins on the proliferating bile ductules in primary biliary cirrhosis.

AIM: An increase in bile ductular structures is observed in diverse human liver diseases, especially in primary biliary cirrhosis (PBC). These structures harbor the progenitor cell component of the liver. Caveolins are cholesterol-binding proteins involved in the regulation of several intracellular processes including cholesterol transport. This study aims to examine the role of caveolin in PBC. METHODS: Immunohistochemical and Western blotting studies were performed on human liver specimens obtained from patients with PBC and normal liver samples. The expression of caveolin (CAV)-1 and -2 was determined using specific antibodies. RESULTS: In normal liver, scanty immunostaining for CAV-1 and -2 was observed in bile ductules. In PBC liver samples, the expression levels of CAV-1 and -2 were increased on proliferating bile ductules especially in stage 3 cases, but was sparse on interlobular bile duct in stage 1 specimens. Especially, the regenerating bile ductules at the interface of portal tracts and necrotic areas were immunostained intensely for CAV-1 and -2. These phenomena were confirmed by Western blot. CONCLUSION: The present results demonstrate increased expression of caveolins in proliferating bile ductules in PBC, which may be related to the homeostasis of cholesterol transport in regenerating bile ductules in PBC liver.