Sinusoidal Endothelial Fenestrae Research Articles

Carthami flos extract against carbon tetrachloride-induced liver fibrosis via alleviating angiogenesis in mice

BackgroundAngiogenesis is a pathological phenomenon contribute to the development of chronic liver diseases, and anti-angiogenic therapy is an effective strategy to alleviate liver fibrosis. Carthami flos, a medicinal and edible herb, has the effects of improving blood circulation and regulating angiogenesis. However, the anti-angiogenic effect of Carthami flos in liver fibrosis remains unknown. MethodsWe investigated the protective effect and therapeutic mechanism of Carthami flos extract (CFE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The liver injury and collagen deposition were observed and evaluated by conducting HE, Masson, and Sirius red staining, testing the serum biochemical indexes (ALT, AST, ALP, γ-GT), and measuring the contents of HYP and four indexes of liver fiber (Col-IV, LN, HA, PC-III). Simultaneously, the expressions of α-SMA and Collagen-I were detected to determine the activation of hepatic stellate cells (HSCs). Subsequently, we measured the expressions of angiogenesis-related proteins such as PDGFRB, ERK1/2, p-ERK1/2, MEK, p-MEK, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, and the mRNA levels of PDGFRB and VEGFA. Additionally, immunofluorescence staining and RT-qPCR assays were carried out to ascertain the expressions of continuous endothelial markers CD31, CD34 and vWF, and scanning electron microscope analysis was performed to observe the number of sinusoidal endothelial fenestrations. ResultsHerein, we found that CFE could significantly reduce liver injury and collagen deposition, like the same effect of colchicine. CFE significantly alleviated CCl4-induced liver injury and fibrosis, mainly manifested by reducing the levels of ALT, AST, ALP and γ-GT and decreasing the contents of HYP, Col-IV, LN, HA and PC-III. Additionally, CCl4 promoted the activation of HSCs by increasing the expressions of α-SMA and Collagen-I, while CFE could rectify the condition. Moreover, CFE treatment prevented the CCl4-induced the up-regulation of PDGFRB, p-MEK, p-ERK1/2, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, suggesting that CFE might provide the protection against abnormal angiogenesis. In the meantime, the gradual disappearance of sinusoidal capillarization after CFE treatment was supported by the decreased the contents of CD31, CD34 and vWF, as well as the increased number of sinusoidal endothelial fenestrae. ConclusionIn this study, the reduction of collagen deposition, the obstruction of HSCs activation, the inactivation of angiogenic signaling pathways and the weakening of hepatic sinusoidal capillarization jointly confirmed that CFE might be promising to resist angiogenesis in liver fibrosis via the PDGFRB/ERK/HIF-1α and VEGFA/AKT/eNOS signaling pathways. Nevertheless, as a potential therapeutic drug, the deeper mechanism of Carthami flos still needs to be further elucidated.

Inhibiting Th1/2 cells influences hepatic capillarization by adjusting sinusoidal endothelial fenestrae through Rho-ROCK-myosin pathway.

CD4+ T cells are considered to be vital in chronic liver diseases, but their exact roles in hepatic capillarization, the typical characteristic of liver fibrosis, are poorly understood. This study aimed to assess the roles of typical subtype of CD4+ T cells, named T helper 1 (Th1) and Th2 cells in liver fibrosis. Taking advantage of well established fibrotic rat model, we conducted in vitro and in vivo experiments to explore the interactions between liver sinusoidal endothelial cells (LSECs) and Th1/2 cells; meanwhile we evaluated the degree of hepatic capillarization when inhibiting these interactions with inhibitory antibodies. Our results showed that prohibiting interactions between Th2 cells and LSECs caused the restoration of fenestrae, increased cytokine level of Th1 cells and reduction of hepatic capillarization; inhibiting the interaction between Th1 cells and LSECs produced the opposite effects. Moreover, increased Rho and myosin light chain phosphorylation were observed when Th1 cells were inhibited with the corresponding inhibitory antibody; Th2 cell inhibition yielded the opposite results. This study indicated that Th1/2 cells steer the capillarization process in different directions and this effect is probably mediated by the Rho-Rho kinase (ROCK)-myosin signaling pathway.

Early effect of a single intravenous injection of ethanol on hepatic sinusoidal endothelial fenestrae in rabbits

BackgroundIt has been postulated that ethanol affects hepatic sinusoidal and perisinusoidal cells. In the current experimental study, we investigated the early effect of a single intravenous dose of ethanol on the diameter of liver sinusoidal endothelial fenestrae in New Zealand White rabbits. The diameter of fenestrae in these rabbits is similar to the diameter found in humans with healthy livers. The effect of ethanol on the size of fenestrae was studied using transmission electron microscopy, because plastic embedding provides true measures for the diameter of fenestrae.ResultsAfter intravenous administration of a single dose of 0.75 g/kg, ethanol concentration peaked at 1.1 ± 0.10 g/l at ten minutes after injection. Compared to control rabbits (103 ± 1.1 nm; n = 8), the average diameter of fenestrae in ethanol-injected rabbits determined at 10 minutes after injection was significantly (p < 0.01) smaller (96 ± 2.2 nm; n = 5). Detailed analysis of distribution histograms of the diameters of fenestrae showed that the effect of ethanol was highly homogeneous.ConclusionA decrease of the diameter of fenestrae 10 minutes after ethanol administration is likely the earliest morphological alteration induced by ethanol in the liver and underscores the potential role of liver sinusoidal endothelial cells in alcoholic liver injury.

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

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). 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. 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. 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.

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.

An endothelin A receptor antagonist induces dilatation of sinusoidal endothelial fenestrae: implications for endothelin-1 in hepatic microcirculation

Sinusoidal endothelial fenestrae (SEF) regulate the sinusoidal circulation by altering their diameter and number. This study documented the effects of endothelin (ET) receptor antagonists on SEF and hepatic microcirculation. The portal pressure and hepatic tissue blood flow were measured with a hydromanometer and a laser Doppler blood flow meter, respectively. BQ-123 (ET(A) receptor antagonist) or BQ-788 (ET(B) receptor antagonist) was continuously infused into normal rats at the rate of 10 nmol/min for 10 min. The sinusoids were observed at 60 min after the infusion by scanning electron microscopy. The localization of ET-1 and ET(A) and ET(B) receptors was examined by the indirect immunoperoxidase method. When BQ-123 was infused, the portal pressure gradually decreased with time, and it showed a significant reduction compared with the control groups. On the other hand, a decrease in portal pressure was not evident in the BQ-788-infused groups. Hepatic tissue blood flow was maintained at the value prior to the infusion in both groups. BQ-123 also caused a marked dilatation of the SEF. The diameters of the SEF after BQ-123 infusion were almost three times those of normal SEF. ET-1 was evenly present along the sinusoidal walls, and the reaction products of the ET(A) receptors were recognized along the portal vein and in the sinusoidal cells, that is, the hepatic stellate cells and endothelial cells. Action of ET-1 via the ET(A) receptors may regulate the size of SEF in addition to hepatic microcirculation.

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

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. 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. 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. 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.

Diaphragmed fenestrae in the glomerular endothelium versus nondiaphragmed fenestrae in the hepatic endothelium

To the Editor: In a recent issue of Kidney International, Ballermann1.Ballermann B. Glomerular endothelial cell differentiation.Kidney Int. 2005; 67: 1668-1671Google Scholar discussed the development of diaphragmed fenestrae in glomerular endothelial cells. Major similarities exist between the hepatic and the glomerular endothelium regarding the process of fenestrae formation; intriguingly, an analogous controversy about the origin of fenestrae exists in both the hepatology and nephrology fields2.Braet F. How molecular microscopy revealed new insights in the dynamics of hepatic endothelial fenestrae in the past decade.Liver Int. 2004; 24: 532-539Google Scholar. Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. These membrane-bound pores lack a diaphragm and a basal lamina is also absent (Fig. 1A and B, arrows), in contrast to fenestrae described in the kidney, pancreas, and brain. Their biological relevance in various diseases has been widely acknowledged3.Braet F. Wisse E. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: A review.Comparative Hepatology. 2002; 1: 1Google Scholar. Evidence that VEGF-induced diaphragmed fenestrae are derived from fused caveolae has recently been accumulating. It has been postulated that a similar mechanism is used in the formation of hepatic fenestrae. Conversely, actin disruption in hepatic endothelial cells produces an increase in the number of fenestrae by using a distinct structure that appears to serve as a fenestrae-forming center Fig. 1C4.Braet F. Spector I. De Zanger R. Wisse E. A novel structure involved in the formation of liver endothelial cell fenestrae revealed using the actin inhibitor misakinolide.Proc Natl Acad Sci USA. 1998; 95: 13635-13640Google Scholar. Furthermore, it is important to emphasize the contradictions in the data reported about the origin of fenestrae in kidney, pancreas, and in the liver. For example, some data either clearly or indirectly indicate a possible relationship between caveolae, vesiculo-vacuolar organelles, and (diaphragmed) fenestrae; others suggest the opposite (reviewed in2.Braet F. How molecular microscopy revealed new insights in the dynamics of hepatic endothelial fenestrae in the past decade.Liver Int. 2004; 24: 532-539Google Scholar). Therefore, until this controversy is settled, no firm conclusions can be drawn, and stating that fenestrae, whether diaphragmed or not, correspond to fused and interconnected caveolae should be viewed with caution and remains as an emerging research topic.

Reply from the Authors

Braet and Soon question the conclusions we have drawn in a recent review1.Ballermann B.J. Glomerular endothelial cell differentiation.Kidney Int. 2005; 67: 1668-1671Google Scholar about the relationship between caveolae and fenestrae in glomerular endothelium. They suggest that we were too hasty in stating that fenestrae do not represent fused caveolae. There is little doubt about the dynamic nature of fenestrae. Work with liver sinusoidal endothelial cells is particularly interesting because these cells retain very nice sieve plate structures in culture which can change rapidly by alterations in actin microfilament dynamics2.Braet F. Wisse E. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: A review.Comp Hepatol. 2002; 1: 1Google Scholar. Also, VEGF-A can induce endothelial fenestration. Conversely, interference with VEGF-A function in renal glomeruli, for instance, in preeclampsia3.Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Google Scholar or with podocyte VEGF-A haploinsufficiency4.Eremina V. Sood M. Haigh J. et al.Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.J Clin Invest. 2003; 111: 707-716Google Scholar leads to an absence of glomerular fenestrae. Finally, VEGF-A seems to induce caveolin-1-containing vesicles to fuse and to form transendothelial cell channels5.Vasile E. Qu H. Dvorak H.F. et al.Caveolae and vesiculo-vacuolar organelles in bovine capillary endothelial cells cultured with VPF/VEGF on floating Matrigel-collagen gels.J Histochem Cytochem. 1999; 47: 159-167Google Scholar , 6.Yokomori H. Oda M. Yoshimura K. et al.Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells.Liver Int. 2003; 23: 467-475Google Scholar. Such circumstantial evidence has suggested that fenestrae represent fused caveolae. However, in caveolin-1-deficient mice known to lack caveolae altogether7.Razani B. Engelman J.A. Wang X.B. et al.Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Google Scholar, we found glomerular endothelium with fenestrae that were normal in size and density, even though no caveolin-1 and no caveolae were observed at all8.Sorensson J. Fierlbeck W. Heider T. et al.Glomerular endothelial fenestrae in vivo are not formed from caveolae.J Am Soc Nephrol. 2002; 13: 2639-2647Google Scholar. In normal rat glomeruli examined for caveolin-1 localization by immunogold electron microscopy, fenestrated portions of glomerular endothelium were completely devoid of caveolin-1, similar to the report by Esser et al9.Esser S. Wolburg K. Wolburg H. et al.Vascular endothelial growth factor induces endothelial fenestrations in vitro.J Cell Biol. 1998; 140: 947-959Google Scholar for the choroid plexus endothelium. Now, our experiments leave open the possibility that fenestrae in other vascular beds could represent fused caveolae, and that caveolar precursor vesicles lacking caveolin-1 could fuse to form fenestrae. But if caveolae are defined as specialized plasma membrane invaginations stabilized by caveolins, then our findings in caveolin-deficient mice seem to prove that fenestrae in glomerular endothelium cannot represent fused caveolae.

Endothelial nitric oxide synthase and caveolin-1 are localized in the sinusoidal endothelial fenestrae in hepatic sinusoidal endothelial cell: Hiroaki Yokomori, Kitasato Institute Medical Center, Saitama Japan; Masaya Oda, Internatinal University of Health and Welfare, Tokyo Japan; Hirosama Ishii, Keio University, Tokyo Japan

Endothelial nitric oxide synthase and caveolin-1 are localized in the sinusoidal endothelial fenestrae in hepatic sinusoidal endothelial cell: Hiroaki Yokomori, Kitasato Institute Medical Center, Saitama Japan; Masaya Oda, Internatinal University of Health and Welfare, Tokyo Japan; Hirosama Ishii, Keio University, Tokyo Japan

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.

Sinusoidal endothelial fenestrae organization regulated by myosin light chain kinase and Rho-kinase in cultured rat sinusoidal endothelial cells

The presence of actin filaments in the neighborhood of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. We examined the roles of Rho-kinase and myosin light chain kinase (MLCK) in the organization of SEF. Cultured SEC were treated with MLCK inhibitor (ML-7) and Rho-kinase inhibitor (Y-27632). SEF morphology was observed by scanning electron microscopy. F-actin stress fibers were observed by confocal microscopy and heavy meromyosin-decorated reaction under transmission electron microscopy. Y-27632 caused disassembly of stress fibers in the center of the cell, while SEF clustered and dilated. However, stress fibers located in the periphery of the cell were not severely affected by Y-27632. ML-7 caused disruption and/or shortening of peripheral stress fibers, leaving the central stress fibers relatively intact. ML-7, but not Y-27632, caused cells to lose the spreading morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. Thus, there are at least two different stress fiber systems in SEC. The central stress fiber system and SEF microfilaments depend more on the activity of Rho-kinase, while the peripheral stress fiber system depends on MLCK. These results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.

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 Ren's 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.

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.

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.

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.

Role of endothelin receptors in endothelin-1-induced morphological changes of hepatic sinusoidal endothelial fenestrae: morphometric evaluation with scanning electron microscopy

Endothelin (ET)-1, a potent vasoconstrictor, is involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF) through ET-1 receptors. To clarify the role of each receptor (R) in ET-1 induced contraction of SEF, we studied the size of hepatic SEF under various experimental conditions. Scanning electron microscopy was used for morphometric analysis of the fenestrae of sinusoidal endothelial cells isolated from male Wistar rats under the following conditions: (1) control, (2) ET-1, (3) Bosentan (ET A-R+ET B-R antagonist)→ET-1, (4) BQ485 (ET A-R antagonist)→ET-1, (5) BQ788 (ET B-R antagonist)→ET-1. Each experiment was based on the observations of 200–205 fenestrae (15–20 fenestrae per cell, two cells per dish and six dishes). The diameter of the endothelial pores of the isolated sinusoidal endothelial cells was 123±35 nm in group (1), 46±21 nm in group (2), 130±40 nm in group (3), 72±28 nm in group (4), and 130±27 nm in group (5). The differences between groups (2) and (4), and between groups (2) and (5), were statistically significant ( P<0.05, P<0.01, respectively). Endothelin B receptor (ET B-R) antagonist pretreatment abolished the ET-1-induced contraction of SEF, whereas endothelin A receptor (ET A-R) antagonist pretreatment appeared to partially block this contraction. The present findings indicate that ET B-R plays a primary role in endothelin-1 induced SEF morphological changes, while ET A-R plays a subsidiary role.

Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review

This review provides a detailed overview of the current state of knowledge about the ultrastructure and dynamics of liver sinusoidal endothelial fenestrae. Various aspects of liver sinusoidal endothelial fenestrae regarding their structure, origin, species specificity, dynamics and formation will be explored. In addition, the role of liver sinusoidal endothelial fenestrae in relation to lipoprotein metabolism, fibrosis and cancer will be approached.

Endothelial nitric oxide synthase and caveolin-1 are co-localized in sinusoidal endothelial fenestrae.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS leading to enzyme inhibition in a reversible process modulated by Ca++-calmodulin. The aim of the present study was to clarify the ultrastructural localization of eNOS and caveolin-1 in hepatic sinusoidal endothelium by an electron immunogold method. Male Wistar rats were used. Liver tissues and hepatic sinusoidal endothelial cells isolated from rat livers by collagenase infusion were studied. For immunohistochemistry, liver specimens were reacted with anti-eNOS or anti-caveolin-1 antibody. The ultrastructural localization of eNOS or caveolin-1 was identified by electron microscopy using an immunogold post-embedding method. Immunohistochemical studies using liver tissues localized endothelial NOS in hepatic sinusoidal lining cells, portal veins and hepatic arteries; and caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein and hepatic arteries. Immunogold particles indicating the presence of eNOS and caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae in liver tissue and also in isolated sinusoidal endothelial cells. Endothelial NOS and caveolin are co-localized on sinusoidal endothelial fenestrae, suggesting that interaction of the two may modulate cellular regulation of NO synthesis.

Transmission Electron and Immunoelectron Microscopic Studies on Microcystin-LR-Induced Hepatic Injuries in Mice.

A cyanobacterial toxin, microcystin-LR (MCLR), is a potent inhibitor of protein phosphatase that disrupts cytoskeleton network in hepatocytes. Conventional transmission electron and immunoelectron microscopic studies were conducted in the liver from mice that received a single dose of MCLR and were sacrificed at 24 hours after dosing. The two types of death of hepatocytes, necrosis and apoptosis, were observed concomitantly, and the hepatic injuries, therefore, could be classified as a class of mixed apoptotic and oncotic necrosis according to the recommended criterion by the Society of Toxicologic Pathologists. Apoptotic hepatocytes were characterized by ballooning with numerous intracytoplasmic vacuoles in addition to the common features of apoptotic cells referred in the literatures. In non-parenchymal cells, significant changes indicating microcirculatory alterations and inflammatory reactions included activation of endothelial cells and Kupffer cells, widening of sinusoidal endothelial fenestrae, apoptosis of endothelial cells, accumulation of platelets and polymorphonuclear neutrophils, and fibrin deposition. Pre-embedding immunoelectron microscopy with biotinylated anti-microcystin antibody demonstrated that MCLR was heterogeneously apparent in the cytoplasm of hepatocytes and strong immunoreactivity was evident in apoptotic cell/bodies. In non-parenchymal cells, there were no positive reactions except for phagocytic apoptotic bodies in Kupffer cells. These results suggest that liver damages induced by MCLR are ascribed to primary cytotoxic effects of the toxin localized in hepatocytes and secondary microcirculatory alterations and inflammatory reactions involved non-parenchymal cells.