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. Dr. Hiroaki Yokomori, of the Department of Internal Medicine, Kitasato Medical Center Hospital, Saitama, Japan, is the winner of the Japanese Society for Clinical Molecular Morphology Award for Promoting Young Researchers in 2007. Dr. Yokomori was recognized for his great contribution in elucidating the role of sinusoidal endothelial fenestrae in the physiology and pathology of the liver.     

Heparanase accelerates the proliferation of both hepatocytes and endothelial cells early after partial hepatectomy

Background and aims

Heparanase (HPSE) is an endo-β-D-glucuronidase, which cleaves heparan sulfate in the extracellular matrix (ECM) and has pro-angiogenic and pro-proliferative properties. The aim of this investigation was to study the effect of HPSE on hepatocytes and endothelial cells (EC) during liver regeneration.

Methods

Following 70% hepatectomy (PHP), rats were injected daily with 1–50 μg HPSE/rat. Liver samples were stained with H&;E and anti-bromodeoxyuridine (BrdU) antibody. mRNAs of hepatocyte growth factor (HGF), stem cell factor, tumor necrosis factor (TNF)-α, interleukin(IL)-6, and cyclinD1 were tested by real-time qPCR. Matrix metalloproteinases (MMPs) were tested by gel zymography.

Results

Compared to the saline control, HPSE increased hepatocyte proliferation 24 h, 48 h and 72 h after PHP, with the maximal effect found at 24 h with 50 μg HPSE (40.9 ± 2.5% vs. 8.6 ± 4.3%, p < 0.01 for BrdU staining; 5.5 ± 0.9% vs. 0.8 ± 0.5%, p < 0.05 for mitosis). Proliferation of the sinusoidal and the portal vein radical ECs was also increased (p < 0.05). HPSE caused a twofold increase in cyclinD1 mRNA (p < 0.05) and in pro-MMP-9 levels (p < 0.05). HPSE at all doses also caused significant reductions of TNF-α mRNA (p < 0.05) and IL-6 mRNA, and no change in HGF mRNA.

Conclusions

HPSE enhances liver regeneration by inducing proliferation of hepatocytes and both sinusoidal and vascular ECs. Since the effect of HPSE on hepatocytes occurred earlier than that observed in ECs, this effect is not related to HPSE's effect on ECs. The mechanism of HPSE action is probably indirect and is mediated by HPSE-dependent ECM cleavage and the release of pre-existing enzymes.     

Cell biology and pathology of liver sinusoidal endothelial cells

Growing evidence revealed that liver sinusoidal endothelial cells (SEC) play several important roles in physiology and pathology of the liver. It has been well understood that their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhancement of hepatic metabolic activity. In addition, SEC is now regarded as a member of the scavenger endothelial cells, which have potential to eliminate a variety of macromolecules from the blood circulation by receptor-mediated endocytosis. It is reported that molecules preferentially eliminated by SEC are denatured or modified proteins such as advanced glycation end products, extracellular matrix components including hyaluronic acid, and some lipoproteins. The nature of the scavenger receptors corresponding to these molecules remains to be clarified. Recently, it was noted that SEC has an antigen-presenting function similar to dendritic cells. Taken together, it is suggested that SEC, cooperating with Kupffer cells and hepatic dendritic cells, may partake of immunoregulatory functions in the liver. SEC also plays a pivotal role in the pathological process of ischemia-reperfusion injury following liver surgery and liver transplantation. Thus, it is of importance to elucidate the mechanisms of apoptosis and proliferation of SEC. Recent results on the regulation of growth and apoptotic signaling of SEC are discussed.     

Treatment for postoperative liver failure after major hepatectomy under hepatic total vascular exclusion

 Hepatic total vascular exclusion (HTVE) with clamping of the portal triad and the inferior vena cava below and above the liver is a useful technique in the resection of major hepatic lesions situated close to the hepatic veins and inferior vena cava. From 1996 to 2000, five patients underwent major hepatectomy under HTVE; among these, liver failure occurred in two patients because of liver cirrhosis or hepatic artery interruption. In the former case, apheresis therapy (plasma exchange: 9 times), continuous prostaglandin E₁ (PGE₁) infusion via the hepatic artery (0.01 μg/kg/min) for 7 days, and hyperbaric oxygen therapy (3 times: 2 ATA, 60 min) were applied. In the latter case, apheresis therapy (plasma exchange: 9 times, continuous hemodiafiltration: 12 days) and continuous PGE₁ infusion via the superior mesenteric artery for 7 days were applied. With these treatment modalities, both cases were cured of postoperative liver failure. Received: November 5, 2002 / Accepted: March 6, 2003     

四氯化碳诱导肝纤维化早期大鼠肝窦内皮细胞及基底膜形态改变的动态研究

 目的：探讨四氯化碳诱导肝纤维化早期大鼠肝窦毛细血管化的形成过程。方法：清洁级雄性SD大鼠采用随机数字表法随机分为2组：正常对照组（N组,6只）和肝纤维化模型组（M组,32只）。M组大鼠腹腔注射50%四氯化碳蓖麻油混合液, N组大鼠腹腔注射生理盐水,剂量为2 mL/kg,每周2次,共4周。分别于造模第3天、1周、2周和4周处死大鼠,HE染色和Masson染色观察肝脏组织炎症及纤维化的改变,透射电镜观察肝窦内皮细胞（LSECs）窗孔与基底膜（BM）的改变,免疫组织化学检测LSECs表面标志物CD31及基底膜成分IV型胶原（Col IV）和层黏连蛋白（LN）的改变。结果：HE及Masson染色显示四氯化碳造模4周早期肝纤维化已形成。肝组织透射电镜显示四氯化碳造模第3天后开始出现LSECs窗孔直径变小及数目减少,随着造模时间的延长,LSECs失窗孔现象逐步严重,至第4周时局部内皮下可见连续的基底膜。免疫组化染色显示LSECs表面标志物CD31表达随着LSECs窗孔数目的减少而逐渐增强;基底膜成分Col IV于造模第2周时表达开始显著增强并随着造模时间延长表达逐渐增强,LN于造模第4周时表达开始显著增强。结论：肝纤维化早期大鼠局部肝组织可见典型的肝窦毛细血管化形成;肝窦壁内LN沉积是肝窦毛细血管化时形成连续基底膜的关键因素。     

Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.     

Thrombin potentiates volume-activated chloride currents in pulmonary artery endothelial cells

 In bovine pulmonary artery endothelial cells, ionic currents and the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) were measured with a combined patch clamp and Ca²⁺-fluorimetric method (Fura-2). Volume-activated Cl^––currents (I_Cl,vol) were activated by a 13 or 28% decrease in tonicity. Thrombin, 1 U/ml, strongly potentiated I_Cl,vol preactivated by low hypotonicity (13% HTS) but had no effect on I_Cl,vol preactivated by stronger hypotonic challenges (28% HTS). The thrombin-induced potentiation was not affected by buffering [Ca²⁺]_i at 50–100 nmol/l and omitting extracellular Ca²⁺. A peptide agonist of the thrombin receptor, SFLLRN, also potentiated I_Cl,vol, while an enzymatically inactive thrombin analogue, DIP-thrombin, was without effect. These results suggest that proteolytic activation of the thrombin receptor sensitises the activation of I_Cl,vol in endothelial cells in a Ca²⁺-independent mechanism. Received: 27 September 1996 / Received after revision: 16 January 1997 / Accepted: 30 January 1997     

Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats

Rats received a dose of dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4). In the liver of rats given DMN, apoptosis of fat-storing cells occurred at 7.5 h, and sinusoidal endothelial cell degeneration followed, with parenchymal cell necrosis after 9 h. Fibrin thrombi appeared in the sinusoids as well as in these necrotic areas after 12 h. In contrast, in the liver of rats given CCl4, parenchymal cell degeneration was seen after 6 h and necrosis with fibrin thrombi developed after 9 h. Fat-storing cells and endothelial cells were almost intact, and fibrin thrombi were not present in the sinusoids. SGPT values increased with decreased plasma levels of fibrinogen and antithrombin III and prolonged prothrombin time after 3 and 6 h, in the CCl4 and DMN models, respectively. An extensive reduction in plasma factor VIIIC levels and peripheral platelets was seen after 18 and 24 h, respectively, only in the DMN model. These results suggest that endothelial cells destruction can cause fibrin formation in the hepatic sinusoids in acute liver injury. Fat-storing cell injury may contribute to the destruction.     

Circulating endothelial cells in patients with heart failure and left ventricular dysfunction

Introduction and Aims: Acute and chronic heart failure may manifest different degrees of endothelial damage and angiogenesis. Circulating endothelial cells (CEC) have been identified as marker of vascular damage. The aim of our study was to evaluate the evolution of the CEC at different stages of patients with heart failure. We also investigated a potential correlation between CEC and markers of vascular damage and angiogenesis. Methods: We studied 32 heart failure patients at hospital admission (acute phase) and at revision after 3 months (stable phase) and 32 controls. Circulating markers of endothelial damage (CEC; von Willebrand factor, vWF and soluble E-selectin, sEsel) and angiogenesis (vascular endothelial growth factor, VEGF and thrombospondin-1) were quantified. Results: Levels of CEC, vWF, sEsel and VEGF are significantly higher in heart failure patients than in controls. Levels of CEC (36.9 ± 15.3 vs. 21.5 ± 10.0 cells/ml; p< 0.001), vWF (325 ± 101 vs. 231 ± 82%; p< 0.001) and VEGF (26.3 ± 15.2 vs. 21.9 ± 11.9 ng/ml; p< 0.001) are significantly higher in the acute phase than in the stable phase of heart failure. CEC levels correlate with vWF and VEGF. Results show than 100% of patients in acute phase and 37.5% in stable phase have levels of CEC higher than the 99th percentile of the distribution of controls (16 cells/ml). Therefore, increases in CEC represent a relative risk of 9.5 for heart failure patients suffering from acute phase. Conclusions: CEC, in addition to being elevated in heart failure, correlate with vWF levels, providing further support for CEC as markers of endothelial damage. Levels of CEC are associated with the acute phase of heart failure and could be used as a marker of the worsening in heart failure.     

Morphological and functional analysis of PTA granules in human NK cells

 Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3⁻, CD16⁺, CD56⁺) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNα, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation. Accepted: 24 March 1997     

Cell-surface arylsulfatase A and B on sinusoidal endothelial cells,hepatocytes, and Kupffer cells in mammalian livers

Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.     

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.     

Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2-/- knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2K(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.     

Expression of cell adhesion molecules and their beta1 and beta2 integrin ligands in human liver peliosis

The expression of the following cell adhesion molecules and their beta1 and beta2 integrin ligands was investigated in the liver tissue from 3 patients with non-bacillar peliosis using light and electron microscope immunohistochemistry: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), leukocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1), and very late antigen-4 (VLA-4). We found a parallel enhancement of the adhesion molecules expression in the dilated sinusoids and cavities in all 3 cases with peliosis. Mononuclear blood cells were detected in the sinusoids and sometimes perisinusoidally. These cells were mainly ICAM-1-, LFA-1-, and VLA-4-positive. At the ultrastructural level, ICAM-1-positive immune deposits were observed on the membrane of sinusoidal endothelial cells, Kupffer cells, and hepatocytes. The expression of cell adhesion molecules on liver sinusoids in peliosis is probably triggered by factors released from damaged endothelial cells and hepatocytes. The prevalence of the ICAM-1/LFA-1 and VCAM-1/VLA-4 patterns of mononuclear blood cell/sinusoidal cell interactions could support the macrophage-induced or lymphocyte-induced type of liver injury. PECAM-1 was also included in the non-specific immune response in peliosis. The presence of erythrostasis or thrombosis in liver sinusoids could participate in the induction of adhesion molecule expression in peliosis.     

Localization of vascular endothelial growth factor in synovial membrane mast cells: examination with ”multi-labelling subtraction immunostaining”

 Mast cells are believed to play a novel part in the development of destructive synovial pannus in rheumatoid arthritis (RA). This study was undertaken to investigate the localization of vascular endothelial growth factor (VEGF) in the synovial membrane using a unique immunostaining technique. Synovial specimens of RA patients were examined immunohistochemically and were compared with specimens from non-RA controls. Multi-labelling subtraction immunostaining, a modification of double- and triple-labelling immunostaining, revealed that the VEGF-positive cells were identical to tryptase-positive cells (mast cells). No other cell types were found to be positive for VEGF. The synovium of RA patients showed a larger number of VEGF-positive mast cells than that of non-RA controls (P<0.001). The study suggests that mast cell-derived VEGF may contribute to the development of synovial pannus in RA. Received: 1 April 1998 / Accepted: 13 July 1998     

Light and electron microscopic examination of endothelial cells from bovine retinal vessels in long-term cultures

The aim of the present work was to examine and compare the ultrastructure of bovine retinal endothelial cells (BRECs) in vitro during several passages in a medium selective for endothelial cells. The identity of the endothelial cells was confirmed immunohistochemically, up to the tenth passage. Changes in their ultrastructure in comparison to endothelial cells in vivo occurred at the onset of culturing and not progressively with repeated passages. The cultured BRECs show high metabolic activity in all passages. While retaining their identity as endothelial cells, they modify their lipid metabolism, so that lipids are stored. This change in lipid metabolism was induced by the medium. Received: 19 May 1999 / Accepted: 4 August 1999     

小鼠肝脏窦状内皮细胞分离和培养的一种新方法

目的：建立小鼠肝窦状内皮细胞(liver sinusoidal endothelial cells, LSEC)的分离培养方法, 研究其生物学特性。方法：中性蛋白酶消化小鼠肝脏组织,Percoll密度梯度离心分离消化后的细胞悬液,用内皮细胞筛选培养基及酶差异消化法纯化LSEC;免疫细胞化学染色检测LSEC的表面标志,分析LSEC的超微结构,观察DiI荧光标记的乙酰低密度脂蛋白（acetylated low density lipoprotein,DiI Ac LDL）摄取能力,以体外成血管能力分析LSEC的功能。结果：分离纯化后的细胞呈典型鹅卵石样内皮细胞形态,这些细胞表达VIII因子,不表达CD31,CD90和CK19,具有窦状内皮细胞特征性的窗孔结构;能摄取DiI Ac LDL,并有一定的成血管能力。结论：建立了一种分离、培养LSEC的方法,为研究LSEC的功能奠定了基础。     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

 The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells. Received: 23 May 1996 / Received after revision: 3 September 1996 / Accepted 16 September 1996     

肝窦内皮细胞损伤和表型改变在大鼠肝硬化门脉高压发生中的作用

目的：探讨肝窦内皮细胞（SECs）损伤和表型改变与大鼠肝硬化门脉高压的关系。 方法： 采用二甲基亚硝胺（DMN）4周12次腹腔注射复制大鼠肝硬化模型，分别于造模后1 d、2 d、3 d、1周、2周、4周、6周、8周作动态观察；肠系膜前静脉分支插管法测门脉压力（Ppv）；透射电镜观察肝组织超微结构；免疫组化观察肝窦壁CD44和Ⅷ因子相关抗原（vWF）表达；Northern blot检测肝组织内皮素-1（ET-1） mRNA和内皮型一氧化氮合酶（eNOS）mRNA 表达；Western blot检测肝组织eNOS表达；放射免疫法测定血清透明质酸（HA）和肝组织ET-1含量。 结果： DMN造模1 d后CD44染色明显弱于正常对照组（P<0.05），SECs窗孔减少，血清HA含量显著高于正常对照组(P<0.05)；DMN造模2 d后vWF阳性染色明显强于正常对照组(P<0.05)；DMN大鼠的Ppv与肝窦壁vWF表达量和血清HA含量呈显著正相关(P<0.05)；造模2 d和3 d时ET-1 mRNA表达强于正常对照组，ET-1含量轻度高于正常对照组；造模1 d、2 d和3 d时eNOS mRNA表达强于正常对照组，而eNOS一直呈低水平状态。 结论：SECs损伤和表型改变是DMN大鼠肝硬化门脉高压形成的病理基础之一；ET-1和NO产生的平衡失调，使肝内血流阻力增加，在门脉高压形成过程中起重要作用。