Kayo Fujimaki

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).

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.

Actin filaments around endothelial fenestrae in rat hepatic sinusoidal endothelial cells.

The presence of microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) suggests that the cytoskeleton of liver sinusoidal endothelial cells (LSEC) plays an important role in the modulation of SEF. In this study, we investigated actin filaments around SEF in LSECs. Monolayers of LSEC culture were established by infusing a rat liver with collagenase for 30 min and then culturing in RMPI medium for 24 h. Cells were reacted with 0.1% Triton X for 5 s and 15% glycerinated PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl, pH 6.9) containing heavy meromyosin for 10 min and observed under a transmission electron microscope. By electron microscopy with the modified heavy meromyosin decorated reaction, actin filaments were clearly demonstrated around SEF in LSEC.

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

Abstract: 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.

Antigens of monoclonal antibody NB3C4 are novel markers for oligodendrocytes.

We produced NB3C4, a novel monoclonal antibody specific for oligodendrocytes, using human neuroblastoma IMR-32 cells. NB3C4 specifically recognized oligodendrocytes in the CNS, although it bound to neuroblastoma IMR-32 cells and oligodendrocytes in vitro. Double immunofluorescence staining of rat brain using NB3C4 and anti-GST-π, anti-glial fibrillary acidic protein (GFAP), or anti-neurofilament 200 (NF) antibody revealed that anti-GST-π antibody identified an oligodendro- cyte marker recognizing NB3C4-positive cells, while both anti-GFAP and anti-NF antibody did not. Western blotting of rat brain homogenates showed that NB3C4 bound three proteins of 22–28 kDa, while the anti-GST-π recognized a 27 kDa protein. Therefore, antigens recognized by NB3C4 could be novel markers for oligodendrocytes.