Hiroaki Yokomori

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

Abstract: 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.

Enhanced Expression of Endothelin B Receptor at Protein and Gene Levels in Human Cirrhotic Liver

Endothelin (ET) has been implicated in the regulation of hepatic microcirculation and development of portal hypertension. This study examined the localization of ETA receptor (ETAR) and ETB receptor (ETBR) in cirrhotic liver tissues from patients with hepatocellular carcinoma with hepatitis C-related cirrhosis, and normal liver samples from patients with metastatic liver carcinoma. Anti-ETAR and ETBR antibodies were used for immunohistochemistry and Western blot. Immunoelectron microscopy was conducted using immunoglobulin-gold and silver staining. For in situ hybridization (ISH), human ETAR and ETBR peptide nucleic acid probes were used with the catalyzed signal amplification system. In normal liver tissue, immunohistochemistry revealed that ETBR was predominantly expressed on hepatic sinusoidal lining cells, particularly on sinusoidal endothelial (SECs) and hepatic stellate cells (HSCs), and ETAR was scantily expressed. These findings were confirmed by Western blot and ISH. In cirrhotic liver tissue, overexpression of ETBR was demonstrated by Western blot and ISH. Morphometric analysis showed significant increase of ETBR expression on HSCs and SECs in cirrhotic liver, particularly on HSCs. ETAR expression was increased but remained low. Enhanced ETBR expression in cirrhosis may intensify the effect of endothelin on HSCs and increase hepatic microvascular tone.

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver

 Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Rho modulates hepatic sinusoidal endothelial fenestrae via regulation of the actin cytoskeleton in rat endothelial cells

The presence of actin-like microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. Rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The present study aimed to examine how a Rho stimulator; lysophosphatidic acid (LPA), and a Rho inhibitor; bacterial toxin C3 transferase (C3-transferase), affect the morphology of SEF. Monolayers of SEC culture were established by infusing a rat liver with collagenase for 30 min and then culturing in RMPI medium for 24 h. The cells were separated into three groups; control, LPA-treated (15 μM), and C3-transferase-treated (15 μg/ml) groups. SEF morphology was observed by scanning electron microscopy. Formation of F-actin stress fibers was observed by confocal microscopy. Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. Active Rho was measured by Rens modification. Treatment of SECs with LPA contracted the SEF, concomitant with increases in F-actin stress fiber and actin microfilament, and high expression of phosphorylated myosin light-chain kinase. Following treatment with C3-transferase, SEF dilated and fused, concomitant with a loss of F-actin and microfilament, and low expression of phosphorylated myosin light chain. Rho A expression does not change by both treatments. In conclusion, these results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.

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).

Elevated expression of caveolin-1 at protein and gene levels in human cirrhotic liver—Relationship to 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-caveoin-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.

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 sieves 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.

New insights into the dynamics of sinusoidal endothelial fenestrae in liver sinusoidal endothelial cells

Ultrastructural studies have shown that liver sinusoidal endothelial cells (LSECs) contain a cytoskeletal framework of filamentous actin, and that the presence of actin in the form of a calmodulin—actomyosin complex is responsible for regulation of the diameter of sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently of cell morphology. We investigated actin filaments in relation to SEF in LSEC using heavy meromyosin decorated reaction and elucidated the roles of Rho and actin cytoskeleton in morphological and functional alterations of SEF. Second, according to intracytoplasmic Ca2+ concentration, plasma membrane Ca2+Mg2+-ATPase activities were clearly demonstrated on the outer surface of the labyrinth-like SEF in the isolated LSECs. Furthermore, by investigating intracytoplasmic Ca2+ concentration, we have demonstrated plasma membrane Ca2+-Mg2+-ATPase activities on the outer surface of the labyrinth-like SEF in the isolated LSECs. Currently, the majority of fenestral studies are focused on finding ways to increase the liver sieve’s porosity, which is reduced through pathological mechanisms.

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

BackgroundThe 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.MethodsIn 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.ResultsThe 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-β.ConclusionsEnhanced expression of APJ on HSCs in cirrhosis indicates markedly increased vascular remodeling.

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. Andos 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.