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.

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.

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.

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.

Elevated expression of caveolin-1 at protein and mRNA level in human cirrhotic liver: relation with nitric oxide.

BACKGROUND: Caveolin, the principal structural protein of caveolae, binds with endothelial nitric oxide synthase (eNOS) leading to enzyme inhibition. This study examined the expression of caveolin and eNOS at the protein and mRNA levels in patients with hepatocellular carcinoma and hepatitis C-related cirrhosis, and in control noncirrhotic liver specimens obtained from patients with metastatic liver carcinoma. METHODS: Anti-eNOS, anti-caveolin-1, and anti-calmodulin antibodies were used for Western blotting. For in situ hybridization (ISH), human eNOS and caveolin-1 peptide nucleic acid probes were used with a catalyzed signal amplification system. RESULTS: Western blotting showed marked overexpression of caveolin-1 protein in cirrhotic liver, while caveolin-1 was almost undetectable in control liver tissue. Endothelial NOS was expressed at a slightly higher level in cirrhotic liver than in control liver tissue. Calmodulin was expressed abundantly in control liver tissue and at a low level in cirrhotic liver tissue. By ISH, eNOS mRNA was localized on portal vein and hepatic lining cells, and caveolin-1 mRNA was almost undetectable in normal liver tissue. In cirrhotic liver tissue, caveolin-1 mRNA was overexpressed on hepatic sinusoidal lining cells, while eNOS mRNA expression was similar to that in normal liver. CONCLUSIONS: Enhanced caveolin-1 expression may be associated with a significant reduction in NO catalytic activity in cirrhosis.

Endothelin-1 suppresses plasma membrane Ca++-ATPase, concomitant with contraction of hepatic sinusoidal endothelial fenestrae.

Intracytoplasmic free calcium ions (Ca++) are maintained at a very low concentration in mammalian tissue by extruding Ca++ from the cytoplasm against a steep extracellular Ca++ concentration gradient, mainly through the activity of plasma membrane Ca++ pump-ATPase. The present study aimed to elucidate how endothelin-1 (ET-1) affects the morphology of sinusoidal endothelial fenestrae and ultrastructural distribution of plasma membrane ATPases and intracytoplasmic free Ca++ in isolated rat hepatic sinusoidal endothelial cells. Sinusoidal endothelial fenestrae were observed by scanning electron microscope. Ando's electron cytochemical method was used for ultrastructural localization of Ca++-Mg++-ATPase activity, electron immunogold postembedding method for Ca++ pump-ATPase immunoactivity, and antimonate method for intracytoplasmic free Ca++. Addition of ET-1 to sinusoidal endothelial cells significantly decreased Ca++-Mg++-ATPase activity and Ca++ pump-ATPase expression and increased intracytoplasmic free Ca++ concentration, concomitant with a decrease in diameter of sinusoidal endothelial fenestrae. Co-treatment with Bosentan abolished the actions of ET-1. These results suggest that ET-1 suppresses Ca++-Mg++-ATPase activity and Ca++ pump-ATPase expression on the plasma membrane of sinusoidal endothelial fenestrae, thereby attenuating the extrusion of intracytoplasmic free Ca++ into the extracellular space, leading to an increased concentration of intracytoplasmic free calcium ions and contraction of sinusoidal endothelial fenestrae.

Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells.

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and vascular permeability. Hepatic sinusoidal endothelial cells (SECs) possess sieve-like pores that form an anastomosing labyrinth structure by the deeply invaginated plasma membrane. Caveolin is the principal structural protein in caveolae. In this study, we examined the role of VEGF on the fenestration and permeability of SECs and the relation with caveolin-1. SECs isolated from rat livers by collagenase infusion method were cultured for 24 h with (10 or 100 ng/ml) or without VEGF. The cells were then examined by transmission and scanning electron microscopy (EM). The expression of caveolin was investigated by confocal immunofluorescence, immunogold EM, and Western blot. Endocytosis and intracellular traffic was studied using horseradish peroxidase (HRP) reaction as a marker of fluid phase transport in SECs. Both transmission and scanning EM showed an increased number of sinusoidal endothelial fenestrae (SEF) in SECs cultured with VEGF. By confocal immunofluorescence, SECs cultured with VEGF displayed prominent caveolin-l-positive aggregates in the cytoplasm, especially surrounding the nucleus region. Immunogold EM depicted increased caveolin-1 reactivity on vesicles and vacuoles of VEGF-treated SECs compared with VEGF-nontreated cells. However, there was no change in the level of caveolin-1 protein expression on Western blot. After HRP injection, an increase of electron-dense tracer filled the SEF in cells treated with VEGF. Our results suggested that VEGF induced fenestration in SECs, accompanied by an increased number of caveolae-like vesicles. Increased caveolin-1 might be associated with vesicle formation but not with fenestration. Increased fenestration may augment hepatic sinusoidal permeability and transendothelial transport.