Tezosentan, an endothelin receptor antagonist, limits liver injury in endotoxin challenged cirrhotic rats

BACKGROUND/AIMS: Lipopolysaccharide (LPS) induces liver injury which is associated with upregulated endothelin (ET)-1 production. The aim of this study was to investigate the effects of tezosentan, a non-selective ETA and ETB receptor antagonist, in LPS challenged rats with cirrhosis. METHODS: Rats with cirrhosis received LPS and then tezosentan or placebo one hour later. Four hours after LPS administration, rats were killed to measure serum transaminase activity and plasma tumour necrosis factor alpha (TNF-alpha) levels. Hepatic inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), a marker of neutrophil infiltration, and cyclooxygenase (COX)-2 expression were also measured. RESULTS: LPS administration significantly decreased arterial pressure and significantly increased plasma endothelin levels. Following LPS and tezosentan administration, serum aspartate aminotransferase and alanine aminotransferase activities were similar to those in the control group while they were increased by more than 700% with LPS alone. Plasma TNF-alpha levels were significantly lower in rats receiving LPS and tezosentan (182 (38) pg/ml) compared with those receiving LPS alone (821 (212) pg/ml). Tezosentan significantly decreased hepatic MPO activity and hepatic neutrophils but had no effect on LPS induced iNOS or COX-2. Survival rate was significantly higher in rats receiving LPS plus tezosentan (80%) than in rats receiving LPS alone (50%). CONCLUSION: In LPS challenged cirrhotic rats, tezosentan administration prevents LPS induced liver injury by decreasing intrahepatic neutrophil infiltration. In addition, tezosentan increases survival in these rats.     

Identification of endothelin receptor subtypes in sheep choroid plexus

Endothelin (ET) and its G-protein-coupled receptors are distributed in a wide variety of tissues, including the brain. In this study, we have identified and characterized the endothelin receptor subtypes in sheep choroid plexus. Competitive binding experiments using [¹²⁵I]ET-1 and the receptor subtype-selective ligands, ET-1, ET-3, BQ-123, Sarafotoxin 6c, and [Ala^1,3,11,15]ET-1 demonstrated the presence of both ET_A and ET_B receptor subtypes in the ratio of 30:70. In addition, a small fraction of the total binding sites exhibited affinities for ET-1 in the subpicomolar range. Chemical crosslinking of [¹²⁵I]ET-1 withbis(sulfosuccinimidyl)-suberate (BS³) to choroid plexus membranes revealed the presence of two bands, with apparent molecular masses of 89 and 45 kDa, corresponding to the ET_A receptor, and three bands, with apparent molecular masses of 75, 58, and 33 kDa, corresponding to the ET_B receptor. Of considerable interest was the finding that dimers of the [¹²⁵I]ET-1-occupied ET_A receptor could be identified by crosslinking, as could apparent dimers and tetramers of [¹²⁵I]ET-1, but only when bound to receptor. In addition to mapping the distribution of ET receptors in sheep choroid plexus, our results strongly suggest that ET-1 binding to the ET_A receptor leads to dimer formation.     

The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver

Background/Aims: To characterize the effects of endothelin-1 and of Bosentan, a mixed endothelin antagonist, on hepatic hemodynamics in cirrhotic animals in vivo and on hepatic microvascular exchange in the perfused rat liver.Methods: Biliary cirrhosis was induced by bile duct ligation, and micronodular cirrhosis by chronic exposure to phenobarbital/CCl₄ in male rats. Hepatic hemodynamics were studied under basal conditions and after administration of Bosentan (3–30 mg/kg) by the microsphere technique. Microvascular exchange was assessed in the in situ perfused rat liver by the multiple indicator dilution technique.Results: Bosentan lowered portal pressure in a dose-dependent fashion; at the highest dose tested, this decrease averaged −29±11 and −26±8% in biliary and micronodular cirrhosis, respectively (p<0.01). This was achieved mainly via a marked decrease in hepatic arterial flow. In the perfused liver, endothelin-1 induced a dose-dependent vasoconstriction; up to 10⁻⁹ mol/l; this was not associated with any effect on viability. At this dose, endothelin-1 markedly decreased extravascular albumin space in both controls and micronodular cirrhosis; this could be antagonized by Bosentan 10⁻⁵ mol/l.Conclusions: Endothelin-1 affects hepatic microvascular exchange, presumably by a direct effect on hepatic sinusoidal endothelial cells. A mixed endothelin antagonist lowers portal pressure in vivo, presumably by acting on hepatic stellate cells, and counteracts the microvascular effects of endothelin-1 in vitro. These properties could prove useful in treatment of portal hypertension.     

Interferon accumulation in cirrhotic rat liver

SUMMARY. In patients with chronic hepatitis C, the therapeutic effect of interferon (IFN) is influenced by the progression of liver disease. In a previous study, we showed that 2',5'-oligoadenylate synthetase activity in the liver homogenate was significantly lower in cirrhotic rats than in controls after injection of murine IFN. To determine the reason for this decrease, we injected IFN into rats with thioacetamide-induced cirrhosis and used microautoradiography with human lymphoblastoid interferon ([¹²⁵I]LyIFN). Accumulation of [¹²⁵I]LyIFN in cirrhotic rat livers was approximately half of that in control rats (2880±900 vs 5770±600mm², P <0.01). In the cirrhotic rat livers there were few grains on the hepatocytes, but many on collagen fibres. These results suggest that binding of IFN to its hepatocyte receptors is hindered in the presence of cirrhosis. The decreased amount of IFN reaching hepatocytes may contribute to the poor responses to IFN seen in patients with cirrhosis.     

UDP glucuronosyltransferase in the cirrhotic rat liver

In patients with cirrhosis, the elimination of drugs metabolized by glucuronidation is relatively preserved, in comparison with the metabolism of drugs by oxidation. This study explores this phenomenon at a molecular level. In cirrhotic rat livers the content of UDP-glucuronosyltransferase (UGT) was examined by immunohistochemistry and immunoblotting using three antibodies: (i) a polyclonal antibody directed against a broad number of UGT isoforms from both family 1 and family 2; (ii) a family 2-specific antibody; and a (iii) family 1-specific antibody. The steady state mRNA level of UGT of a family 2 isoform was also detected by northern blot analysis. The results demonstrate normal or increased UGT protein by immunohistochemistry and immunoblot in cirrhotic livers compared with controls. This was accompanied by increased steady state mRNA encoding the UGT isoform UGT2B1. In contrast, an isoform of cytochrome P450 (CYP2C11) was reduced markedly in both immunohistochemical staining and immunoblot analysis. These results suggest that in cirrhosis there is a comparative increase or at least a maintenance of UGT enzyme content and that this most likely occurs at a pretranslational level.     

Plasma endothelin levels in cirrhotic subjects.

Endothelin-1, a potent vasoconstrictor peptide with 21 amino acid residues, is released by the vascular endothelium. Plasma immunoreactive endothelin levels were measured in 23 patients with cirrhosis and in 20 healthy subjects. Concentrations were significantly lower in patients with non-uraemic cirrhosis than in normal subjects (19.4 +/- 8.9 pmol/l vs. 48.8 +/- 24.8 pmol/l, p less than 0.002). Plasma renin, aldosterone, atrial natriuretic peptide, arginine-vasopressin and catecholamines did not show significant correlations with plasma endothelin-1 levels. Furthermore, there were no significant differences in plasma endothelin levels for etiology of cirrhosis, presence of ascites or varices. These data suggest that low circulating endothelin may be involved in the development or maintenance of systemic vasodilatation in cirrhosis.     

Absence of growth-hormone receptor in hepatocellular carcinoma and cirrhotic liver

Hepatocellular carcinoma is a hormone-sensitive tumor. It has been reported that thyroxine and prolactin significantly stimulated hepatoma growth, whereas growth hormone failed to do so. To learn whether the growth hormone receptor is present in human hepatocellular carcinoma, we used radioreceptor assays in samples of human hepatocellular carcinoma. The liver tissues adjacent to hepatocellular carcinoma (mostly cirrhotic) and control liver tissues (taken during various surgical procedures) were also studied. The study results showed that the affinity constant and capacity of high-affinity growth hormone receptor in normal liver tissues were 6.6 +/- 2.0 x 10(10) mol/L-1 (mean +/- SE, n = 7) and 20.7 +/- 11.5 fmol/mg protein, respectively. The affinity constant and capacity of low-affinity growth hormone receptor in normal liver tissues were 8.9 +/- 3.3 x 10(9) mol/L-1 and 64.7 +/- 32.1 fmol/mg protein, respectively. The absence of growth hormone receptor in human hepatocellular carcinoma and cirrhotic liver samples may explain the absence of growth hormone in the stimulation of hepatoma growth and the decrease of somatomedin levels in cirrhosis.     

Hypoxia potentiates transforming growth factor-beta expression of hepatocyte during the cirrhotic condition in rat liver.

BACKGROUND/AIMS: Many studies have reported that hypoxia might be associated with angiogenesis and fibrogenesis, and the level of transforming growth factor-beta1 (TGF-beta1) was increased in fibrotic liver and maximal at cirrhosis. Therefore, we examined the expression of TGF-beta1, phosphorylated-Smad2/3 (p-Smad2/3) of the TGF-beta immediate down stream signaling system and hypoxic status during hepatic fibrogenesis. METHODS: Fibrosis of rats was induced by carbon tetrachloride. Collagens were detected with Azan stain. Immunohistochemistry and immunoblotting was used. RESULTS: TGF-beta1 was mainly produced by hypoxic hepatocytes at cirrhosis although myofibroblasts (MFBs) and macrophages producing TGF-beta1 were decreased. Moreover, distribution of p-Smad2/3 in hepatocytes was consistent with those of hypoxic hepatocytes regardless of MFBs. Furthermore, in recovery, most MFBs disappeared, whereas positive reactions of p-Smad2/3 still existed in the hepatocytes of hypoxic areas. Therefore, TGF-beta1 expression in hepatocytes might have been associated with hypoxia. CONCLUSIONS: We put forward the hypothesis that TGF-beta1 is mainly produced by MFBs and macrophages at early and middle stages of fibrotic processes, but it is predominantly released by hypoxic hepatocytes in the last fibrotic stage or cirrhosis.     

Enhanced vasoconstrictor response of the isolated perfused cirrhotic rat liver to humoral vasoconstrictor substances found in portal venous blood

Humoral vasoconstrictor factors in portal venous blood have an important influence on hepatic vascular tone. The aim of this study was to determine whether there is altered reactivity of the intrahepatic portal vascular bed of cirrhotic livers to such factors. Isolated perfused rat liver preparations (IPRLP) obtained from rats with carbon tetrachloride-induced cirrhosis and from normal controls were treated with small aliquots of fresh, heparinized venous blood (4% vol/vol) added to a synthetic perfusate composed of 2.5% bovine serum albumin in Krebs-Henseleit buffer. Compared with blood from the inferior vena cava, portal venous blood produced a greater increase in perfusion resistance of normal IPRLP (2.8 +/- 0.7 vs 15 +/- 3%, P less than 0.05). There was no significant difference in the response of normal IPRLP to portal venous blood obtained from cirrhotic animals compared with portal blood from normal controls (10 +/- 4 vs 15 +/- 3%). However, cirrhotic IPRLP were significantly (P less than 0.05) more responsive to portal venous blood than were control livers, regardless of whether the blood was obtained from control (28 +/- 6%) or cirrhotic (24 +/- 6%) rats. The response of both control and cirrhotic IPRLP to portal blood could be partially inhibited by the alpha-adrenoceptor antagonist phentolamine (5 x 10(-6) mol/L) and cirrhotic IPRLP were more responsive than controls to exogenous noradrenaline (518 +/- 27 vs 363 +/- 21%, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced expressions of apelin on proliferative hepatic arterial capillaries in human cirrhotic liver

Aim: Apelin (APLN), the endogenous ligand of angiotensin-like receptor 1 (APJ), is a peptide necessary for embryonic and tumor angiogenesis. Little is known about the localization and changes of APLN expression including the sinusoids in human cirrhotic liver, which might contribute to portal hypertension. This study was designed to elucidate the localization and change of APLN expression in human liver during the progression of cirrhosis. Methods: Twelve normal liver specimens, eight specimens of Child–Pugh grade A cirrhosis, and 10 specimens of Child–Pugh grade C cirrhosis were studied. APLN protein and gene expression was examined by immunohistochemistry, western blotting, immunoelectronic microscopy, and laser captured microdissection (LCM) followed by polymerase chain reaction (PCR) in sinusoid. Results: In control liver tissue, APLN was localized mainly on arterial endothelial cells and hepatic arterioles in the portal tract. In cirrhotic liver tissue, aberrant APLN expression was observed in periportal capillary endothelial cells corresponding to capillarized sinusoids, and in proliferated arterial capillaries in the fibrotic septa. Significant overexpression of APLN at protein level in cirrhotic liver was demonstrated by western blotting (P < 0.01 Child–Pugh A and C versus control, P < 0.01 Child–Pugh A versus C). APLN mRNA expression in the sinusoid was confirmed by LCM-PCR. Conclusion: In humans, APLN protein and gene were overexpressed in cirrhotic liver compared with normal liver, and the magnitude increased as cirrhosis progressed. Especially in end-stage cirrhosis, APLN was strongly expressed in proliferated arterial capillaries directly connected with the sinusoids, suggesting a role of APLN in the proliferation of arterial capillaries in cirrhosis.     

Differential expression of somatostatin receptor subtypes in hepatocellular carcinomas

BACKGROUND/AIMS: Somatostatin analogues inhibit cell proliferation by stimulation of distinct somatostatin receptor (SSTR) subtypes. In recent years, these compounds have been introduced into the therapy of advanced hepatocellular carcinoma (HCC). The efficacy of this treatment is under debate due to the controversial results of clinical trials. Despite the widespread clinical use of somatostatin analogues in HCC, little is known about the expression of each of the five SSTRs in these tumors. METHODS: We analyzed the expression of SSTR subtypes in 56 HCCs by immunohistochemistry using subtype-specific antibodies. Six of the samples were also investigated by RT-PCR using subtype-specific oligonucleotide primers. RESULTS: HCCs display differential, individual expression patterns as well as variable expression levels for SSTRs. The overall expression rate of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 is 46, 41, 64, 0, and 75%, respectively. No significant correlation was observed between SSTR expression and tumor stage, differentiation, histological tumor type, or underlying liver disease. CONCLUSIONS: Individual patterns and levels of SSTR expression might determine the response to treatment with somatostatin analogues in HCC. Selective treatment of these tumors based on the analysis of SSTR subtype expression might lead to an increase in response rates.     

肝癌组织生长抑素受体亚型的表达及其意义

目的研究原发性肝癌患者肝癌组织中生长抑素受体亚型(SSTR2、3和5)的表达情况及其意义。方法应用免疫组化法检测50例原发性肝癌患者癌组织、癌旁组织以及24例非癌肝组织中SSTR2、3和5的表达;采用RT-PCR法检测10例肝癌组织及非癌肝组织SSTR2、3和5mRNA的表达。结果肝癌组织中SSTR2、3和5蛋白表达阳性率分别为72%、34%、14%,SSTR2的表达率明显高于SSTR3和5,差异有统计学意义(P0.05);SSTR2在肝癌组织中表达率较非癌肝组织高(x2=7.372,P0.01),差异有统计学意义;肝癌组织SSTR2、3和5mR-NA表达率分别为100%、80%和30%,均高于非癌肝组织。结论肝癌组织SSTR亚型表达增高,SSTR2为肝癌组织中主要表达的生长抑素受体亚型。     

Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands.

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.