The role of liver sinusoidal endothelial cells in liver remodeling after injury

Liver transplantation is the optimal treatment for patients with end-stage liver disease, metabolic liver diseases, and hepatic malignancies that are not amenable to resection. Hepatic ischemia-reperfusion injury (IRI) is the main problem in liver transplantation and liver resection, leading to parenchymal cell injury and organ dysfunction. The damage of liver sinusoidal endothelial cells (LSECs) is a critical event in IRI. LSECs work as an important regulating factor of liver regeneration after partial hepatectomy. This review primarily describes the mechanisms of LSECs injury in IRI and explores the roles of LSECs in liver regeneration, and briefly introduces the protective strategies targeting LSECs damaged in IRI.     

4,5,7-三羟基异黄酮对肝纤维化大鼠肝窦内皮细胞窗孔、增殖及合成一氧化氮的影响

目的 探讨酪氨酸蛋白激酶抑制剂4、5、7-三羟基异黄酮(genistein)对肝窦内皮细胞(sinusoidalendothelial cell, SEC)窗孔、增殖及合成一氧化氮(nitric oxide, NO)的影响。方法 用胶原酶原位灌注,Percoll不连续密度梯度离心法分离正常及CCl_4实验性肝纤维化大鼠的SEC并进行体外培养。采用扫描电镜技术,MTT法及硝酸还原酶法,分别观察genistein对SEC细胞窗孔、增殖及合成NO的影响。结果 Genistein对肝纤维化各级 SEC的窗孔数目及大小均无明显的影响。经不同浓度的genistein作用24h后,肝纤维化各级大鼠SEC的生长均受抑制,以100 μmol/L浓度的genistein对肝纤维化Ⅰ级大鼠SEC的作用最为明显[细胞增殖率为-(15.38±6.26)% vs(4.91±2.16)%,t=13.7. P＜0.05]。100 μmol/L浓度的genistein作用24h,可明显促进肝纤维化Ⅰ级大鼠SEC NO的合成[NO合成为(25.4±3.8)μmol/L vs(16.6±3.3)μmol/L,t=6.79,P＜0.05];但对肝纤维化Ⅱ级、Ⅲ级大鼠SEC的NO合成的影响却不明显。结论 体外实验中genistein可以抑制肝纤维化Ⅰ级SEC增殖,促进SEC细胞NO的合成;对肝纤维化SEC细胞的功能有一定的调节作用。     

Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide

In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 mumol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, N(G)-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca(2+)-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury.     

Chronic ethanol ingestion increases aortic endothelial nitric oxide synthase expression and nitric oxide production in the rat

Background:  Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study.
Methods:  Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose–Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production.
Results:  Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion.
Conclusions:  These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.     

Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats.

BACKGROUND: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. This study investigated the activity of nitric oxide synthase (NOS) in cirrhosis induced by bile duct-ligation (BDL) with NOS inhibitors. METHOD: Three days after operation, rats were randomized to receive aminoguanidine (AG, 25 mg/kg/day) or L-N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) for 21 days. RESULTS: Vascular NO production, which was increased in BDL cirrhotic rats, was reduced by 75% with AG but not L-NAME chronic administration. AG treatment attenuated liver damage, while L-NAME aggravated it. AG significantly suppressed inducible NOS (iNOS) expression in aorta of BDL rats at both mRNA and protein level, but much less efficient in reducing it in liver. In contrast, endothelial NOS (eNOS) expression was not markedly affected. Calcium-independent NOS activity, which was dramatically increased in aorta of BDL rats, was abolished by AG treatment. In liver, however, both calcium-dependent and -independent NOS activity were increased by AG treatment. CONCLUSION: Chronic administration of AG could reduce systemic NO levels as well as suppress iNOS expression and activity in aorta of BDL rats. It also improved liver function, possibly because of its ability to increase hepatic NOS activity, and to correct the systemic hemodynamic disorders by decreasing vascular NO production.     

大鼠肝窦内皮细胞的分离、培养及鉴定

肝脏是由实质细胞和非实质细胞组成的具有复杂功能的器官，其中肝窦内皮细胞(sinusoidal endothelial cells，SEC)占肝非实质细胞的40％，在肝窦内形成一连续的带窗孔结构的衬垫细胞层，是肝内物质交换的屏障。与普通血管内皮细胞相比，SEC具有特殊的表面标志、结构和功能，在肝脏的生理、病理及器官移植过程中具有重要作用。但由于SEC的原代分离培养十分困难，因此阻碍了对其功能的深入研究。本研究旨在建立大鼠SEC的分离、培养及鉴定方法，为其生物学特性及功能的研究奠定基础。     

Globular adiponectin upregulates nitric oxide production in vascular endothelial cells

Aims/hypothesis Adiponectin, also called ACRP30, is a novel adipose tissue-specific protein that has been shown to improve insulin sensitivity and to exert anti-atherogenic effects. It is known that knockout mice lacking endothelial NO synthase (eNOS) develop hypertension, insulin resistance, hyperlipidaemia, and show augmented ischaemia-reperfusion damage. Thus, we examined whether globular adiponectin activates eNOS to produce NO.Methods To analyze NO production in bovine aortic endothelial cells (BAE), NOx (nitrite and nitrate) was measured in the medium with an automated NO detector/high-performance liquid chromatography system. eNOS activation was assessed by phosphorylation of the enzyme and its activity was evaluated by citrulline synthesis in human umbilical vein endothelial cells (HUVEC). eNOS mRNA and protein expressions in HUVEC were evaluated by Realtime PCR and Western blot analysis.Results Gobular adiponectin increased NO production in BAE. It also caused eNOS phosphorylation and potentiated eNOS activity in HUVEC. In addition, globular adiponectin up-regulated the eNOS gene to increase protein expression in HUVEC.Conclusion/interpretation Globular adiponectin increases NO production through two mechanisms, namely, by activation of eNOS enzyme activity and via an increase in eNOS expression. Activation and up-regulation of eNOS could explain some of the observed vasoprotective properties of globular adiponectin, as well as its beneficial effects on the cardiovascular system.Abbreviations NO nitric oxide - eNOS endothelial NO synthase - BAE bovine aortic endothelial cells - HUVEC human umbilical vein endothelial cells - ACRP30 adipocyte complement-related protein of 30 kDa - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Low-dose ethanol attenuates gut ischemia/reperfusion-induced liver injury in rats via nitric oxide production

BACKGROUND AND AIMS: The acute administration of low-dose ethanol was demonstrated to attenuate liver injury elicited by gut ischemia/reperfusion (I/R). Nitric oxide (NO) has been found to be a modulator of adhesive interactions between leukocytes, platelets, and endothelial cells, but there has been much controversy about the effects of ethanol on NO regulation. The objective of this study was to investigate the role of NO in ethanol-reduced hepatic microvascular dysfunction elicited by gut I/R. METHODS: Male Wistar rats were exposed to 30 min of gut ischemia followed by 60 min of reperfusion. Intravital microscopy was used to monitor leukocyte recruitment and non-perfused sinusoids (NPS). Plasma alanine aminotransferase (ALT) activities were measured 6 h after the onset of reperfusion. In another set of experiments, ethanol (10%, 1 g/kg) was administered before ischemia. RESULTS: Gut I/R elicited increases in the number of stationary leukocytes, NPS, and plasma ALT activities; all of which were attenuated by pretreatment with ethanol or an NO donor. Gut I/R caused the apoptosis of hepatocytes, which was prevented by pretreatment with ethanol. Pretreatment with an NO synthase inhibitor diminished the protective effects of ethanol. The administration of ethanol increased plasma nitrite/nitrate levels. CONCLUSION: These results suggest that low-dose ethanol attenuates the gut I/R-induced hepatic microvascular dysfunction and sequential liver injury by increasing sinusoidal NO levels.     

Lipoxygenase products regulate nitric oxide and inducible nitric oxide synthase production in interleukin-1beta stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMCs), interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. IL-1beta also activates phospholipase A2 (PLA2), and induces lipoxygenase (LOX) and cyclooxygenase-2 (COX-2). The present study investigated whether these metabolites are involved in the regulation of IL-1beta-induced NO production and iNOS expression. Pretreatment with ONO-RS-082, the secretory PLA2 (sPLA2) inhibitor, at 1 to 10 micromol/l reduced IL-1beta-stimulated nitrite production and iNOS expression. Nordihydroguaiaretic acid (NDGA, 1 to 10 micromol/l), the LOX inhibitor, also reduced IL-1beta (10 ng/ml)-stimulated nitrite production and iNOS expression in a dose-dependent manner. Exogenous 12(S)-hydroxyeicosatetraenoic acids (HETE) enhanced the IL-1beta-stimulated nitrite production and iNOS expression. On the other hand, the COX inhibitors, indomethacin and NS-398, had little effect on nitrite production or iNOS expression. These results suggest that LOX products play important roles in the regulation of stimulus-induced NO production in VSMCs.     

肝窦内皮细胞在肝损伤中的功能研究进展

肝窦内皮细胞（liver sinusoidal endothelial cells, LSECs）位于肝血窦表面，是肝脏与血液接触的第一道防线，也是肝脏中含量最多的非实质细胞。在生理情况下，LSECs通过参与物质运输、代谢废物清除而诱导肝脏免疫耐受，从而维持肝脏稳态；在病理情况下，LSECs通过抗原递呈促进肝脏炎症反应。LSECs在维持肝再生和肝纤维化平衡中发挥了重要的调节作用。本文对LSECs功能、LSECs在肝损伤中的变化、调节LSECs功能相关的信号通路以及LSECs与肝内其他细胞的相互作用等四方面研究进展进行综述，从而进一步明确LSECs的功能及在肝损伤中的作用。     

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.

Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.     

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

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Endothelial NOS and caveolin are co-localized on sinusoidal endothelial fenestrae, suggesting that interaction of the two may modulate cellular regulation of NO synthesis.     

Role of oxidative stress in hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells

To characterize the role of oxidative stress in cultured rat sinusoidal endothelial cells, we studied the production of superoxide after reoxygenation, the relationship of reduced glutathione (GSH) levels to cell injury, and the protective efficacy of antioxidants. Hypoxia (pO(2) 1-2 mm Hg) was achieved by culturing cells under 95% N(2)5% CO(2) for 4 hours. Reoxygenation was then reestablished, and viability was determined at 24 hours by trypan blue exclusion; putative protective agents were added at the time of reoxygenation (4 hours). As previously reported, reoxygenation after 4 hours hypoxia accentuated sinusoidal cell death fourfold compared with hypoxic or normoxic controls (P <.0001). Superoxide was not produced on reoxygenation, and superoxide dismutase provided no protection against reoxygenation injury. Cellular levels of GSH fell to 37 +/- 4% of normoxic controls (P <.0001) following reoxygenation. These changes were essentially abrogated by Trolox (Aldrich Chemical Co., Milwaukee, WI) and dimethyl sulfoxide, both of which also completely protected against reoxygenation injury. When cellular GSH levels were lowered by addition of diethylmaleate (which conjugates GSH), this reduced the viability of endothelial cells cultured under normoxic conditions and greatly augmented reoxygenation injury. Conversely, addition of exogenous GSH partially protected endothelial cells against hypoxia-reoxygenation injury. Desferrioxamine also protected against reoxygenation injury, but catalase was only partly protective. It is concluded that sinusoidal endothelial cells undergo significant intracellular oxidative stress following reoxygenation, and their viability is critically dependent on GSH levels. Reactive oxygen species are likely mediators of oxidative stress in hepatic sinusoidal endothelial cells.