Sinusoidal endothelial cell injury by superoxide anion and iron in the Propionibacterium acnes-pretreated and lipopolysaccharide-stimulated rat liver

AIMS/BACKGROUND: We attempted to measure the generation of superoxide anion, examine its site of release and determine its pathological role in Propionibacterium acnes-lipopolysaccharide-induced liver injury in the rat. METHODS: The P. acnes-pretreated (16 mg/kg i.v.) rat liver was perfused with buffer containing lipopolysaccharide (2.5 microg/ml). Chemiluminescence enhanced with Cypridina luciferin analog, MCLA, and reduction of nitro blue tetrazolium were used for detecting superoxide anion. Leakage of enzymes and release of cytokines into the perfusate, and histological specimens were also examined. RESULTS: Superoxide dismutase-inhibitable chemiluminescence peaked at 30 min of lipopolysaccharide infusion and blue formazan precipitate was histochemically deposited mainly on hepatic macrophages. Purine nucleoside phosphorylase (PNP) activity in the perfusate, as a marker of sinusoidal endothelial cell injury, reached its maximum at 50 min and aspartate aminotransferase (AST) activity, as a marker of hepatocyte injury, reached a plateau at 90 min. Simultaneous treatment with superoxide dismutase and deferoxamine mesylate significantly suppressed the leakage of PNP and AST. Release of tumor necrosis factor-alpha and growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 lagged behind PNP leakage. Light microscopy showed destruction of the sinusoids followed by hepatocyte necrosis. Electron microscopy revealed adherence of hepatic macrophages to sinusoidal endothelial cells. CONCLUSION: These results indicate that superoxide anion released from hepatic macrophages may induce sinusoidal endothelial cell injury via interaction with iron in the P. acnes-lipopolysaccharide-treated liver.     

Effect of FK506 on the activation state of hepatic macrophages in Propionibacterium acnes-treated rats

Activated hepatic macrophages can provoke massive liver necrosis following endotoxin stimulation through microcirculatory disturbances due to sinusoidal fibrin deposition in rats pretreated with heat-killed Propionibacterium acnes. In these rats, FK506 (tachlorinus) administered 24 h before and at the time of endotoxin injection, significantly attenuated liver injury compared with the rats given no FK506. The effect of FK506 on hepatic macrophage activation and its action sites were studied in Propionibacterium acnes-treated rats. When rats received Propionibacterium acnes intravenously, hepatic-mRNA expression of interferon-γ-inducing factor and interleukin-2 and splenic-mRNA expression of interferon-γ were significantly increased compared with normal rats. Hepatic-mRNA expression of CD14, a receptor for lipopolysaccharide and its binding protein complex, was also increased preceding the expressions of the three cytokines in the liver and spleen. FK506 administration attenuated hepatic-mRNA expression of interleukin-2 and both superoxide anions as well as tumour necrosis factor-α production by hepatic macrophages, but did not change CD14-mRNA expression in Propionibacterium acnes-treated rats. It is suggested that a cytokine network through interferon-γ-inducing factor, interferon-γ and interleukin-2 may operate during activation of hepatic macrophages in rats treated with heat-killed Propionibacterium acnes, while CD14 expression on the cells may increase independently of this network. FK506 seems to attenuate such activation by suppressing hepatic interleukin-2 expression, without affecting CD14 expression on the cells.     

Superoxide Anion Release Into the Hepatic Sinusoid After an Acute Ethanol Challenge and Its Attenuation by Kupffer Cell Depletion

Superoxide anion release into the hepatic sinusoids and subsequent damage to the endothelial cells of the hepatic sinusoids after ethanol challenge was examined. A 250 mg/kg body weight/hr dose of ethanol was given to rats for 3 hr, and superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome c method. Ethanol treatment resulted in superoxide anion release into the hepatic sinusoids (0.20 ± 0.01 vs. 0.12 ± 0.02 o.d., p < 0.05) and an increase in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate, a marker of damage to the endothelial cells of the hepatic sinusoids (0.003 ± 0.002 vs. 0.008 ± 0.002; p < 0.05). Tumor necrosis factor-alpha was not detectable in either group, and there were no significant differences in the population of hepatic macrophages, leukocytes, or Kupffer cells between the two groups. To clarify the role of Kupffer cells in the mechanism, 10 mg/kg of body weight of gadolinium chloride was given to rats twice, 24 hr apart, resulting in depletion of ED2-positive cells from the hepatic lobules. The superoxide anion release after the ethanol challenge was significantly attenuated in the Kupffer cell-depleted rats, compared with the controls (0.14 ± 0.02;p <0.05, compared with ethanol alone). The change was associated with a significant decrease in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate (0.004 ± 0.002; p < 0.05, compared with ethanol alone). Ethanol causes superoxide anion release into the hepatic sinusoid and subsequent damage to the sinusoidal endothelial cells. These changes were reduced by Kupffer cell depletion. This supports the view that Kupffer cell depletion has a protective effect on ethanol-induced liver injury.     

Effects of salviainolic acid A (SA‐A) on liver injury: SA‐A action on hepatic peroxidation

Abstract: Background/Aims: This study aimed to investigate the actions of salviainolic acid A (SA‐A), an antiperoxidative component of Salvia miltiorrhiza (Sm), on rat liver injury and fibrosis. Methods: Acute and chronic rat liver injury models were established using carbon tetrachloride (CCl₄). After 48 h (acute) or during 6 weeks of CCl₄ injection, rats were further divided and treated with biphenyl dimethyl‐dicarboxylate (BDD) or colchicine, as a control antifibrotic treatment, with Sm, a herbal compound, or SA‐A, a water‐soluble extract of Sm. Liver function was investigated by assessing alanine transaminase (ALT) and aspartate transaminase (AST) activities, histological analysis, hydroxyproline (Hyp) and malondiadehyde (MDA) content. In vitro, isolated cultured hepatocytes were injured with CCl₄ gas for 24 h, followed by treatment with either vitamin E or various concentrations of SA‐A. The extent of hepatocyte injury was monitored by analyzing various lipid peroxidative parameters such as superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), lactase dehydrogenase (LDH), and glutathione peroxidase (GSH‐PX) levels in hepatocyte supernatants. Results: SA‐A significantly decreased abnormal serum ALT activity both in acutely and chronically injured rat livers, decreased abnormal serum AST activity, Hyp and MDA content and attenuated hepatic collagen deposition. After CCl₄ incubation and injury, the activities of AST, ALT CAT, GSH‐PX and LDH and MDA content in hepatocyte supernatants increased significantly, but GSH levels decreased significantly. SA‐A markedly improved these pathological changes in a dose‐dependent manner. 10^?4 mol/l SA‐A had stronger inhibitory action than vitamin E. Conclusions: Our studies suggest that SA‐A has antiperoxidative effects on injured hepatocytes in liver injury and fibrosis induced by CCl₄.     

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

Abstract: 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.     

Splenectomy attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide challenge.

BACKGROUND/AIMS: The aim of this study was to determine whether the spleen contributes to superoxide anion release into the hepatic sinusoids and subsequent damage to endothelial cells of the hepatic sinusoids after lipopolysaccharide challenge. METHODS: Rats were given 2 mg/kg body weight lipopolysaccharide. Three hours after the treatment, superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome C method. Damage to endothelial cells of the hepatic sinusoids was assessed from the purine nucleoside phosphorylase/glutamic-pyruvic transaminase ratio in the liver perfusate. To further characterize the mechanisms behind these changes, these studies were done in rats given superoxide dismutase or an anti-TNFalpha antibody. To study whether the spleen plays a role in the mechanisms, experiments with splenectomized rats were performed. RESULTS: Lipopolysaccharide challenge resulted in superoxide anion release into the hepatic sinusoids and damage to endothelial cells of the hepatic sinusoids. These changes were significantly attenuated by the treatments with superoxide dismutase or an antibody against TNFalpha, as well as by splenectomy. The hepatic macrophage and Kupffer cell populations after lipopolysaccharide challenge were significantly smaller in the rats given splenectomy than in those given a sham operation. There were no significant differences in the neutrophil populations between the two groups. Levels of TNFalpha were significantly lower in the former than the latter, whereas there were no significant differences in levels of Interleukin-8 between the two groups. CONCLUSIONS: Splenectomy reduced the superoxide anion release into the hepatic sinusoids caused by the lipopolysaccharide challenge and subsequent damage to endothelial cells of the hepatic sinusoids. This supports the view that splenectomy has a protective effect in lipopolysaccharide-induced liver injury.     

Preventive effect of cyclosporin A on experimentally induced acute liver injury in rats

Abstract: We examined the effect of cyclosporin A (CsA) on the pathogenesis of acute experimental liver injury in rats induced by injection of heat-killed Propionibacterium acnes (P. acnes) and subsequent injection of lipopolysaccharide (LPS). Pretreatment with CsA significantly reduced serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α) production, without changing the TNF-α mRNA level in the liver, and plasma interferon-γ (IFN-γ), following LPS injection in this model. Twenty-four-hour mortality was also markedly improved, from 100% in the P. acnes plus LPS group to 0% in the CsA-pretreated group. Although direct addition of CsA to isolated hepatic macrophages from P. acnes-pretreated rats did not prevent the production of TNF-α and active oxygen species, isolated hepatic macrophages from P. acnes plus CsA-pretreated rats significantly reduced their production in response to the addition of LPS. These results suggest that CsA protects against P. acnes plus LPS-induced acute liver injury, not by direct inhibition of hepatic macrophage activation, but by indirect prevention of hepatic macrophage activation, presumably related to the reduction in plasma IFN-γ levels.     

All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134 ± 119 IU/L and 72.7%) compared with the control group (713 ± 411 IU/L and 18.2%; P < 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-α (TNF-α) and superoxide anions produced by activated macrophages. Serum levels of TNF-α 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5 ± 7.0 ng/mL) as compared with the control group (105.2 ± 39.3 ng/mL; P < 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

Glutathione Depletion Enhances the Formation of Superoxide Anion Released Into Hepatic Sinusoids After Lipopolysaccharide Challenge

Background: We examined the effects of glutathione depletion on the level of superoxide anion released into hepatic sinusoids after lipopolysaccharide challenge. Methods: Rats were given 1 mg/kg of maleic acid diethyl ester to deplete glutathione in vivo and then 0.5 mg/kg body weight of lipopolysaccharide. Results: This treatment significantly depleted serum reduced glutathione (32.7 ±1.7 vs. 23.0 ± 3.2 mM, p = 0.002). However, it did not affect the serum oxidized glutathione concentration (2.88 ± 0.56 vs. 3.10 ± 0.78 mM, not significant). The lipopolysaccharide challenge caused significant superoxide anion formation as compared with controls (0.12 ± 0.04 vs. 0.22 ± 0.05 o.d., p < 0.001), and it was enhanced significantly by glutathione depletion (0.28 ± 0.04 o.d., p < 0.05). There were no significant differences in levels of lipopolysaccharide (2142 ± 452 vs. 2503 ± 612 pg/ml) and tumor necrosis factor α (277 ± 186 vs. 252 ± 88 pg/ml) after the lipopolysaccharide challenge between the glutathione-depleted and nondepleted rats. Moreover, the purine nucleoside phosphorylase/glutamic-pyruvic transaminase ratio in liver perfusates, a marker of damage to endothelial cells in hepatic sinusoids, was significantly higher in the glutathione-depleted rats than in the nondepleted rats. Conclusions: The reduced form of glutathione can decrease levels of the superoxide anion released into hepatic sinusoids and can decrease subsequent damage to endothelial cells in these sinusoids caused by lipopolysaccharide; that is, it can reduce lipopolysaccharide-induced liver injury.     

Protection by Glycyrrhizin against Warm Ischemia‐Reperfusion–Induced Cellular Injury and Derangement of the Microcirculatory Blood Flow in the Rat Liver

Background/Aim: The mechanism by which ischemia‐reperfusion (I/R)‐induced derangement of the hepatic microcirculation leads to tissue injury is not fully understood. We postulated that alterations to the hepatic microcirculation, including hemodynamic derangement and increased leukocyte‐endothelium interaction, play a role, and that glycyrrhizin exerts its hepatoprotective effects, in part, by reducing these microcirculatory changes. Materials and Methods: Wistar rats were subjected to 30–60 minutes segmental hepatic ischemia, followed by 120 minutes of reperfusion. Glycyrrhizin was administered prior to ischemia. Using intravital fluorescence microscopy, the administration of fluorescein isothiocyanate–conjugated erythrocytes allowed the measurement of erythrocyte‐velocity (RBC_vel), lobular, and sinusoidal perfusion. Bleb formation was observed by electron microscopy. Blood and tissue were taken for the assessment of liver injury. Results: Glycyrrhizin reduced I/R‐induced liver injury (histology, liver enzymes) and reduced hepatocyte apoptosis (TUNEL, caspase‐3 activity). Glycyrrhizin inhibited hepatocyte bleb formation and reversed the I/R‐induced reductions in lobular perfusion and RBC_vel. Leukocyte rolling and adherence in postsinusoidal venules and neutrophil infiltration were reduced by glycyrrhizin. I/R‐induced elevation in HMGB1 was prevented by glycyrrhizin. Conclusions: Early bleb formation with deranged microcirculatory flow and leukocyte‐endothelium interaction would appear to contribute to I/R‐induced hepatocellular injury. Glycyrrhizin exerts its hepatoprotective effect by preventing these changes, in addition to a direct cellular effect.     

Triiodothyronine and interleukin‐6 (IL‐6) induce expression of HGF in an immortalized rat hepatic stellate cell line

Abstract: Background/Aims: Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens. Methods: In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index. In vitro studies: a rat hepatic stellate cell line (HSC‐6T) was cultured with T3, IL‐6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL‐6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT‐PCR. Results: In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor α1, but not β1. Proliferation of stellate cells is not affected by T3, with or without IL‐6. T3 has no effect on secreted levels of IL‐6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL‐6 show significantly increased amounts of secreted HGF after 48 h in culture. Conclusion: T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL‐6.     

Red blood cells attenuate sinusoidal endothelial cell injury by scavenging xanthine oxidase‐dependent hydrogen peroxide in hyperoxic perfused rat liver

Abstract: Aims/Background: Rat liver perfused with an oxygenated buffered solution alone results in degenerative changes even when the perfusion flow is accelerated to give a sufficient oxygen supply. On the other hand, perfusion media supplemented with red blood cells (RBCs) preserve the viability of the liver. The present study was conducted to clarify how RBCs protect the isolated perfused liver. Methods: The liver was perfused with and without RBCs in a perfusate equilibrated with supra‐physiological oxygen tension at regulated inflow pressures, and controlled hepatic oxygen consumption. We examined alanine aminotransferase and purine nucleoside phosphorylase activity in the perfusate as specific markers of liver cells injury. Hydrogen peroxide (H₂O₂) production and morphological changes were determined using cerium electron microscopy. Apoptosis was detected by measuring CPP 32 protease activity and using TdT‐mediated dUTP‐digoxigenin nick end‐labeling. Results: When the liver was perfused with RBC‐free buffer, H₂O₂ production and consequent injury progressing to apoptosis were initiated in the sinusoidal endothelial cells (SECs). After SECs were injured, H₂O₂ appeared in the hepatocytes. H₂O₂ production and associated degenerative changes were attenuated both morphologically and enzymatically by the addition of RBCs, a specific xanthine oxidase (XOD) inhibitor and the H₂O₂ radical scavenger, catalase. Conclusions: In the liver perfused with RBC‐free buffer, H₂O₂ production and consequent injury were initiated in SECs. RBCs attenuate liver injury by scavenging XOD‐dependent H₂O₂.     

Hyaluronic acid and endothelial damage due to paracetamol‐induced hepatotoxicity

Abstract: Background: Damage to endothelial cells may be an important factor in the complications of acute liver failure, resulting in multi‐organ failure. The aim of this study was to assess endothelial cell function in patients with severe hepatotoxicity due to paracetamol ingestion. Patients and methods: Fifty‐eight patients with paracetamol‐induced hepatotoxicity were studied for up to 7 days. Serum hyaluronic acid (HA), as a marker of hepatic sinusoidal endothelial cell function, was determined using an enzyme‐linked binding assay. Plasma von Willebrand Factor, thrombomodulin and interleukin‐8 were also determined using ELISA. Results: Serum HA on admission was significantly increased (median 6777 ng/ml, range 24–50 967 ng/ml) as compared to normal controls (n = 10, median 21 ng/ml, range 0–50 ng/ml; P < 0.001). In non‐survivors (n = 21) HA levels peaked on day 2 after admission (P = 0.044), and then decreased. In the survivors (n = 37) the levels of HA did not increase further. Plasma von Willebrand Factor, plasma thrombomodulin and serum interleukin‐8 were significantly increased in the patients as compared to the normal controls (P < 0.001). Serum interleukin‐8 was significantly higher in non‐survivors in the first 2 days. Conclusions: Endothelial function is abnormal in paracetamol‐induced hepatotoxicity. Damage to hepatic sinusoidal endothelial cells assessed by serum HA was greater in non‐survivors than survivors.     

Kupffer cell depletion attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide treatment

BACKGROUND AND AIM: The mechanisms involved in the beneficial effect of gadolinium chloride against endotoxin-induced liver damage were studied. METHODS: Superoxide anions released into the hepatic sinusoids were examined in a liver perfusion model using the cytochrome C method. RESULTS: Gadolinium chloride treatment fully depleted ED2-positive cells from the liver and significantly attenuated superoxide anion release after a lipopolysaccharide or tumor necrosis factor-alpha (TNF-alpha) challenge. Moreover, gadolinium chloride treatment resulted in a significant decline in endothelial cell damage in the hepatic sinusoids as assessed by the purine nucleoside phosphorylase/glutamic-pyruvic transaminase ratio in the liver perfusate. Although gadolinium chloride treatment did not affect the level of serum TNF-alpha, it significantly reduced that of interleukin (IL)-8 and neutrophil migration in the hepatic sinusoids after the lipopolysaccharide challenge. CONCLUSION: These data suggest that a reduction of the superoxide anion level in the hepatic sinusoids in acute endotoxemia and subsequent reduction of neutrophil migration into the liver may indicate that gadolinium chloride treatment suppresses the progression of liver damage in acute endotoxemia.     

Overexpression of granulocyte‐macrophage colony‐stimulating factor in mouse liver enhances the susceptibility of lipopolysaccharide leading to massive apoptosis of hepatocytes

Abstract: Background/Aims: We examined whether antigen‐nonspecific accumulation of dendritic cells (DCs) and macrophages in the liver by the overexpression of granulocyte macrophage‐colony stimulating factor (GM‐CSF) could prime severe liver injury after LPS injection. Methods: We injected a recombinant adenovirus encoding GM‐CSF intravenously (AdGM), and LPS was administered 7 days later. Liver histology, serum alanine aminotransferase (ALT) levels and apoptosis of hepatocytes were examined. Results: Liver histology of the AdGM‐primed mice showed marked infiltrates of mononuclear cells (DCs and macrophages) without granuloma formation on day 7. Expression of toll‐like receptor‐4 on intrahepatic mononuclear cells isolated from AdGM‐primed mice was up‐regulated. After LPS injection, serum ALT levels in AdGM‐primed mice reached about 6000 IU/l at 12 h, and all those mice died within 24 h. Hemorrhagic liver injury with massive apoptosis of hepatocytes was histologically recognized. When AdGM and LPS were injected in FasL‐deficient C57BL/6J‐gld/gld mice, serum ALT levels were not elevated by the pretreatment with a neutralizing anti‐TNF‐α antibody. Conclusions: Our present study provides a new model of severe liver injury, in which antigen‐nonspecific accumulation of DCs and macrophages in the liver by overexpressing GM‐CSF enhances the susceptibility to LPS, leading to hemorrhagic liver injury with massive hepatocyte apoptosis after LPS injection.     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

Effects of exogenous superoxide anion and nitric oxide on the scavenging function and electron microscopic appearance of the sinusoidal endothelium in the isolated, perfused rat liver

BACKGROUND/AIMS: Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. METHODS: Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. RESULTS: At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glutathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. CONCLUSIONS: The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.     

Salvage effect of E5564, Toll‐like receptor 4 antagonist on d‐galactosamine and lipopolysaccharide‐induced acute liver failure in rats

Background and Aims: The transmembrane protein Toll‐like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro‐inflammatory cytokine release. Activation of the pro‐inflammatory cytokine cascade, including tumor necrosis factor‐alpha (TNF‐α), has a pivotal role in the progression of severe liver injury. D‐galactosamine (GalN) and lipopolysaccharide (LPS)‐induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF‐α plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin‐stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS‐induced acute liver failure (ALF) in rats. Methods: ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 µg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS‐induced ALF rats were compared between those with and without E5564 treatment. Results: The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF‐α levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS‐induced ALF rats at 24 h (8% vs 43%). Conclusions: TLR4 antagonist E5564 reduced GalN+LPS‐induced acute liver injury in rats and improved the overall survival rate of GalN+LPS‐induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin‐induced TNF‐α overproduction of macrophages.