Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat

Abstract: Aims/Background: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. Methods: Sinusoidal endothelial cells were isolated from rat liver. After 2–3 days of primary culture, the cells were exposed to hypoxia (N₂/CO₂ 95/5) for 120 min and reoxygenation (O₂/CO₂ 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O₂ alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondria dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. Results: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. Conclusions: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.     

Antimycin A-induced defenestration in rat hepatic sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) possess fenestrae arranged in sieve plates. Hepatic endothelial fenestrae are open pores approximately 100 to 200 nm in diameter. Alterations in their number or diameter by hormones, xenobiotics, and diseases have important implications for hepatic microcirculation and function. Numerous reports of hepatotoxin-induced defenestration suggest that the cytoskeleton and the energy status of hepatic endothelial cells play a key role in the regulation of fenestrae. Therefore, we investigated the effect of antimycin A, an inhibitor of mitochondrial energy production, on the number of fenestrae in cultured LSECs using high-resolution microscopy and immunocytochemistry. Prolonged incubation (greater than 30 min) with antimycin A resulted in defenestrated cells and coincided with the appearance of F-actin dots, whereas the distribution of G-actin remained unchanged. Adenosine triphosphate (ATP) was depleted dramatically to less than 5% within 30 minutes within the LSECs. After treatment with antimycin A, unusual elevated fenestrated complexes were apparent, organized as a meshwork of anastomosing fenestrae at the center of and above the sieve plates. The position and appearance of these novel structures and their association with defenestration suggest that they are implicated in the process of defenestration. In conclusion, the results of experiments with antimycin A suggest that ATP is needed to maintain fenestrae and the underlying fenestrae-associated cytoskeleton rings that maintain fenestrae patency. Antimycin A-induced defenestration of LSECs is associated with the development of a structure in the sieve plate that appears to be intrinsically involved in defenestration.     

Antithrombin III stimulates prostaglandin I2 production by cultured rat hepatic sinusoidal endothelial cells

We examined the production of prostanoids, specifically prostaglandin (PG) I₂ and thromboxane (TX) A₂, in cultured rat hepatic sinusoidal endothelial cells treated with antithrombin (AT) III. When cells were treated for 3 h with various concentration of AT III, production of 6-keto-PGF_1α (a stable metabolite of PG I₂) increased significantly and in a dose-dependent manner, compared with production by untreated cells. In terms of kinetics, significant increases were noted at 3 h (20.13 ± 1.38 pg/ml), 4 h (21.23 ± 0.63 pg/ml) and 24 h (36.58 ± 4.93 pg/ml) with AT III (300 μg/ml) stimulation, compared with production by the untreated cells (10.29 ± 1.21, 10.34 ± 1.66 and 22.64 ± 2.59 pg/ml, respectively). Moreover, this production was significantly reduced with increasing concentrations of heparin. On the other hand, TX B₂ (a stable metabolite of TX A₂) production was unaffected by AT III treatment. These data suggest that AT III may ameliorate the liver damage or disturbances in the sinusoidal microcirculation by stimulating the PG I₂ production of hepatic sinusoidal endothelial cells.     

肝窦内皮细胞与肝窦毛细血管化研究进展

肝窦内皮细胞具有开放的、没有横膈膜的窗孔,内皮下没有基底膜。这种结构有利于调控肝细胞与肝窦血液的物质交换。肝窦内皮细胞能分泌内皮素-1和一氧化氮,并通过窗孔的变化,对肝脏微循环进行调节,同时可分泌大量的形成基底膜的成分,在肝窦毛细血管化的过程中起主导作用。肝窦内皮细胞表型标志发生变化,Ⅷ因子相关抗原、CD44等表达增强,也是肝窦毛细血管化的重要标志。笔者对肝窦内皮细胞的结构及其在肝窦毛细血管化中的功能和表型变化进行综述。     

Silybin counteracts lipid excess and oxidative stress in cultured steatotic hepatic cells

AIM: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis.METHODS: Rat hepatoma Fa O cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations(25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays.RESULTS: Lipid-loading resulted in intracellular triglyceride(TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors(PPARs) and sterol regulatory element-binding protein-1c(SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species(ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B(NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1(CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation.CONCLUSION: We demonstrated the direct antisteatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.     

肝窦内皮细胞与免疫耐受

肝脏似乎是一个免疫耐受多于免疫原性的器官．众多的肝脏组成细胞中许多参与肝脏免疫调节,如肝血窦内皮细胞(LSEC)．LSEC是定居在肝脏肝血窦的细胞,与经过肝脏的淋巴细胞直接作用,有大量的清道夫受体,能有效摄取抗原．LSEC处理抗原后以MHC限制性呈递给CD4T或者CD8T细胞．与LSEC作用的CD4T, CD8T细胞产生耐受性,是LSEC在肝脏重要的免疫功能:对循环中的可溶性抗原或口服抗原控制了免疫应答的类型．     

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. 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‐1‐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 trans‐endothelial transport.     

Advanced glycation end products impair the scavenger function of rat hepatic sinusoidal endothelial cells

AIMS/HYPOTHESIS: We have previously reported that advanced glycation end products are eliminated from the circulation mainly by scavenger receptor-mediated uptake in hepatic sinusoidal endothelial cells. Our experiments showed that the degradation of AGE-modified protein after endocytosis in hepatic sinusoidal endothelial cells occurs slowly compared with that of other scavenger receptor ligands. The aim of this study was to investigate further the mechanism whereby AGE-modified protein affects the important scavenger function of hepatic sinusoidal endothelial cells. METHODS: Primary cultures of hepatic sinusoidal endothelial cells were pre-incubated with unlabelled ligand, unbound ligand was washed off, and the endocytic capacity was measured by addition of radiolabelled ligand, and immune electron microscopy. RESULTS: Pre-incubation with unlabelled AGE-modified bovine serum albumin reduced subsequent endocytosis of radiolabelled scavenger receptor ligands AGE-modified bovine serum albumin, formaldehyde-treated serum albumin, oxidized low density lipoprotein and acetylated low density lipoprotein by 50, 56, 32 and 20%, respectively. Non-scavenger receptor-mediated endocytosis was not affected by pre-exposure to AGE-modified protein. Pre-incubation with a number of non-AGE-ligands did not affect subsequent endocytosis via any of the major endocytosis receptors in hepatic sinusoidal endothelial cells. Incubation in fresh medium for 6 h after pre-exposure to AGE-modified protein almost completely restored normal scavenger receptor-mediated endocytic activity. Quantitative immune electron microscopy showed that the amount of a newly described scavenger receptor for AGE-modified protein is reduced after pre-incubation with AGE-modified protein. Subcellular fractionation showed that pre-incubation with AGE-modified protein delays intracellular transport of scavenger receptor ligands. CONCLUSION/INTERPRETATION: Endocytosis of AGE-modified protein leads to loss of scavenger receptors and delayed intracellular transport in hepatic sinusoidal endothelial cells.     

肝窦内皮细胞损伤在大鼠肝纤维化形成中的作用

目的 研究肝窦内皮细胞损伤在二甲基亚硝胺大鼠肝纤维化形成中的作用。方法 采用二甲基亚硝胺(dimethylnitrosamine,DMN)4周12次腹腔注射制备大鼠肝纤维化模型,应用电镜技术、免疫组织化学及图像分析方法结合血清生化测定,24周动态观察肝纤维化形成过程中肝窦内皮细胞损伤及其表型的改变。结果 造模2d后肝结构未见明显改变,肝窦内皮细胞(sinusoidal endothelial cell,SEC)远侧胞浆窗孔数减少、造模1周SEC失窗孔更明显,肝组织内未见明显变性坏死及纤维间隔形成,造模4周时见肝组织内大片出血坏死,有大量假小叶形成,内皮下出现SEC窗孔减少。SEC失窗孔早于肝细胞发生较为严重的坏死、肝纤维化的形成以及肝窦内皮下基底膜的形成。造模4周HA(ng/ml)和肝羟脯氨酸(ug/g)平均含量分别为231.30±143.80和223.04±37.09,对照组分别为56.50±18.10和61.55±20.85,t值在3.14～8.28,P<0.05。结论 DMN引起大鼠肝窦内皮细胞损伤及其表型改变可能是其诱导肝纤维化重要的始动机制之一。     

缺氧—复氧对培养的大鼠心肌细胞损伤与细胞凋亡的影响

目的探讨培养的大鼠心肌细胞在缺氧（缺血）和缺氧－复氧（再灌注）状态下心肌细胞损伤和细胞凋亡情况。方法采用１～３天新生ＳＤ大鼠,常规方式的培养心肌细胞,给予模拟缺氧（缺血）液、模拟复氧（再灌注）液以及干预因素（终浓度为２００Ｕ／ｍＬ的ＳＯＤ）处理,分为缺氧－复氧损伤组（Ｈ／Ｒ组）、缺氧－复氧损伤组＋ＳＯＤ（Ｈ／Ｒ＋ＳＯＤ组）、缺氧预处理组（ＨＰ组）和正常对照组（Ｃｏｎｔｒｏｌ组）,进行以下指标观察：     

Hyperglycemia and oxidative stress in cultured endothelial cells - a comparison of primary endothelial cells with an immortalized endothelial cell line

Diabetes mellitus is a major risk factor for the development of cardiovascular disease and oxidative stress plays an important role in this process. Therefore, we investigated the effects of hyperglycemia on the formation of reactive oxygen species (ROS) and nitric oxide/cGMP signaling in two different endothelial cell cultures. Human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells showed increased oxidative stress and impaired NO-cGMP signaling in response to hyperglycemia. The major difference between the two different cell types was the dramatic decrease in viability in HUVEC whereas EA.hy cells showed rather increased growth under hyperglycemic conditions. Starvation led to an additional substantial decrease in viability and increased superoxide formation in HUVEC. Both endothelial cell types, HUVEC and EA.hy 926, may be used as models for vascular hyperglycemia. However, high growth medium should be used to avoid starvation-induced oxidative stress and cell death.     

Occurrence of antibody against rat hepatic sinusoidal endothelial cells in sera of patients with autoimmune hepatitis

To determine whether an antibody against hepatic sinusoidal endothelial cells was present in sera of patients with autoimmune hepatitis (AIH) type 1, we measured the serum IgG bound to the glutaraldehyde-fixed cultured rat sinusoidal endothelial cells by enzyme-linked immunosorbent assay. IgG bound to the cells was detected significantly more in patients with autoimmune hepatitis type 1 (97.1%) than in those with primary biliary cirrhosis (13.0%), chronic hepatitis C (5.9%) or B (7.9%), or healthy controls (0%). IgG-F(ab')₂ fragments from autoimmune hepatitis patients also bound to the cells, and this binding was observed after absorption of the fragments with rat hepatoma cells, but not after absorption with bovine carotid endothelial cells. Culture of sinusoidal endothelial cells in the presence of IgG from AIH patients significantly reduced the number of viable attached cells. In conclusion, anti-sinusoidal endothelial cell antibody occurred in the sera from patients with autoimmune hepatitis type 1.This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, Mitsui Life Social Welfare Foundation and Asahi Life Insurance Foundation.     

Prostanoid secretion by rat hepatic sinusoidal endothelial cells and its regulation by exogenous adenosine triphosphate

We investigated the secretory profiles of prostanoids in two types of nonparenchymal cell from the rat liver, sinusoidal endothelial cells and Kupffer cells, in primary culture both under basal conditions and after stimulation with adenine nucleotides. Prostaglandin (PG) E₂ was the main prostanoid secreted by both types of hepatic nonparenchymal cell in the basal and adenosine triphosphate (ATP)-stimulated states. Time- and concentration-dependent effects of ATP-mediated PGE₂ secretion were noted in sinusoidal endothelial cells, whereas the profile of the relative potencies of individual nucleotides was consistent with the presence of P_2y and P₁ purinergic receptors. In Kupffer cells, the regulation of prostanoid secretion by adenine nucleotides was essentially the same as that in sinusoidal endothelial cells except that adenosine did not stimulate prostanoid secretion and that prostanoid secretion differed somewhat; Kupffer cells secreted relatively more PGF_2α and less 6-keto-PGF_1α than sinusoidal endothelial cells in the presence of ATP, suggesting the presence of only P_2y receptors. In summary, PGE₂ is the main prostanoid secreted by hepatic nonparenchymal cells and its secretion may be stimulated by adenine nucleotides and adenosine.     

氧化应激在大鼠创伤性脑损伤后应激性肝损害中的作用

目的:探讨氧化应激(OS)在大鼠创伤性脑损伤(TBI)后应激性肝损害(HSI)中的作用.方法:用改良Allen法建立TBI模型.40只健康Wister大鼠随机分为5组,正常对照组、颅脑致伤后6、12、24、48 h时相组.酶学法检测血清ATL和AST水平,ABC-ELISA法测定血清TNF-α水平.硫代巴比妥酸法测定MDA水平变化,化学发光法测定SOD水平变化.光镜及电镜下观察肝脏组织学改变.结果:颅脑致伤后12 h,各组血ALT和AST、TNF-α水平及肝组织MDA明显增加,肝组织SOD显著减少,与对照组比较差异均显著(252.92±56.29 vs 41.17±7.88;283.12±45.28vs 45.22±6.57;1138.27±212.02 vs 210.56±28.22;15.21±0.36 vs 6.14±0.25;78.13±3.12vs 135.58±5.58,P<0.01或0.05);TBI各组光镜和电镜可观察到肝组织不同程度受损.结论:TBI后早期可出现HSI,OS可能参与了其发病过程.