Analysis of pulmonary heme oxygenase-1 and nitric oxide synthase alterations in experimental hepatopulmonary syndrome

BACKGROUND & AIMS: Cirrhosis and portal hypertension due to chronic common bile duct ligation reproduce the features of human hepatopulmonary syndrome, whereas portal hypertension alone due to partial portal vein ligation does not. Nitric oxide contributes to experimental hepatopulmonary syndrome, but the nitric oxide synthase forms involved remain controversial. Recently, increased pulmonary heme oxygenase-1 expression and carbon monoxide production have also been found after common bile duct ligation. Our aim was to explore the role of the heme oxygenase-1/carbon monoxide pathway in the pathogenesis of experimental hepatopulmonary syndrome. METHODS: Pulmonary heme oxygenase-1 expression and distribution were assessed in sham; 3-week partial portal vein ligation; and 1-, 2-, 3-, 4-, and 5-week common bile duct ligation animals by Northern, Western and immunohistochemical analysis relative to endothelial and inducible nitric oxide synthase levels and to hepatopulmonary syndrome development. In vivo heme oxygenase enzyme inhibition with tin protoporphyrin IX in common bile duct ligation animals was used to define effects on intrapulmonary vasodilatation and arterial blood gases. RESULTS: Heme oxygenase-1 expression in pulmonary intravascular monocytes/macrophages and arterial carboxyhemoglobin levels increased progressively from 3 to 5 weeks after common bile duct ligation relative to controls (5-week protein levels were 15.94 +/- 1.75-fold those of sham animals; P < 0.001). Inducible nitric oxide synthase increased transiently in pulmonary intravascular monocytes/macrophages in 3-week common bile duct ligation animals, whereas pulmonary microvascular endothelial nitric oxide synthase increases began at 2 weeks and correlated with the onset of hepatopulmonary syndrome. Tin protoporphyrin treatment normalized carboxyhemoglobin and improved arterial blood gases and intrapulmonary vasodilatation, reflecting partial reversal of hepatopulmonary syndrome. CONCLUSIONS: The heme oxygenase-1/carbon monoxide system is an important contributor to the progression of experimental hepatopulmonary syndrome in addition to alterations in the endothelial nitric oxide synthase/nitric oxide pathway.     

Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism in experimental hepatopulmonary syndrome

BACKGROUND/AIMS: In experimental hepatopulmonary syndrome (HPS), hepatic endothelin-1 (ET-1) release during common bile duct ligation (CBDL) and ET-1 infusion in pre-hepatic portal hypertension after portal vein ligation (PVL) initiate vasodilatation through an endothelin B receptor mediated increase in pulmonary endothelial nitric oxide synthase (eNOS). We evaluated if pulmonary ET receptor expression changes in experimental cirrhosis and portal hypertension and confers susceptibility to HPS.METHODS: In normal, PVL and CBDL animals, lung ET receptor expression and localization were assessed and ET receptor levels and functional analysis of ET-1 effects on eNOS levels were evaluated in intralobar pulmonary artery (PA) and aortic (AO) segments. Normal rats underwent evaluation for HPS after ET-1 infusion.RESULTS: There was a selective increase in ET(B) receptor expression in the pulmonary vasculature from PVL and CBDL animals. ET-1 stimulated NO production and an ET(B) receptor mediated increase in eNOS levels in PA segments from PVL and CBDL animals, but not normal animals. ET-1 did not alter lung eNOS levels or cause HPS in normal rats.CONCLUSIONS: ET(B) receptor expression and ET-1 mediated eNOS and NO production are enhanced in the lung vasculature in cirrhotic and portal hypertensive animals and correlate with in vivo susceptibility to ET-1 mediated HPS.     

Over expression of transforming growth factor-alpha and epidermal growth factor receptor in human hepatic cirrhosis tissues

BACKGROUND/AIMS: Transforming growth factor-alpha has 30% amino acid homology to epidermal growth factor and binds with the same membrane-bound receptor, epidermal growth factor receptor. The purpose of this study was to investigate the expression of transforming growth factor-alpha and epidermal growth factor receptor in human hepatic cirrhosis tissues. METHODOLOGY: Expression of transforming growth factor-alpha and epidermal growth factor receptor was evaluated by immunohistochemistry stain in sixty-three hepatic cirrhosis specimens and five normal liver specimens. RESULTS: The transforming growth factor-alpha and epidermal growth factor receptor expression rates were 84.1% (53/63) and 52.4% (33/63), respectively. These positive granules were brown and most common in cytoplasm or cell membrane of hepatocytes. There was prominently positive correlation between transforming growth factor-alpha and epidermal growth factor receptor (P<0.05, gamma=0.32). In five normal liver tissues, transforming growth factor-alpha and epidermal growth factor receptor were not detectable in hepatocytes and bile ducts. CONCLUSIONS: Hepatic cirrhosis might be under the autocrine regulation of transforming growth factor-alpha and its receptor, epidermal growth factor receptor. Increasing expression of transforming growth factor-alpha and epidermal growth factor receptor might be one of the important events in hepatic cirrhosis pathogenesis. Furthermore, transforming growth factor-alpha might play a role in morphogenesis and regeneration of intrahepatic bile ducts.     

Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats.

BACKGROUND/AIMS: Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor beta subunit in a rat model of chronic cholestasis. METHODS: Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor beta subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver. RESULTS: In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells. CONCLUSIONS: These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.     

Common bile duct ligation as a model of hepatopulmonary syndrome and oxidative stress

BACKGROUND: The hepatopulmonary syndrome is characterized by hepatic dysfunction and presence of dilated pulmonary vessels, with alterations in air diffusion that can be demonstrated in the experimental model of common bile duct ligation. AIM: To evaluate the oxidative stress in pulmonary tissue of cirrhotic rats with common bile duct ligation. MATERIAL/METHODS: We used 12 male Wistar rats weighing between 200-300 g divided in two groups: control (Co = 6) and cirrhotic (Ci = 6). We evaluated aminotransferases, arterial gasometry, lipoperoxidation and chemoluminescence), and antioxidant enzymatic activity with superoxide dismutase. The tissues analyzed for hepatopulmonary syndrome were cirrhotic liver and lung. RESULTS: The animals with common bile duct ligation showed alterations in the following aminotransferases: aspartate aminotransferase, Co = 105.3 +/- 43/Ci = 500.5 +/- 90.3, alanine aminotransferase, Co = 78.75 +/- 37.7/Ci = 162.75 +/- 35.4, and alkaline phosphatase, Co = 160 +/- 20.45/Ci = 373 +/- 45.44. The lipoperoxidation and the antioxidant response had significant differences between the groups when evaluated in lung (lipoperoxidation) Co = 0.87 +/- 0.3/Ci = 2.01 +/- 0.9, chemoluminescence Co = 16008.41 +/- 1171.45/Ci = 20250.36 +/- 827.82 superoxide dismutase Co = 6.66 +/- 1.34/Ci = 16.06 +/- 2.67. CONCLUSIONS: Our results suggest that in this experimental model of cirrhosis using common bile duct ligation, there is an increase in lipoperoxidation in pulmonary tissue as well as an increase in superoxide dismutase's antioxidant activity, suggesting a pulmonary injury caused by secondary biliary cirrhosis.     

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis.

BACKGROUND AND AIMS: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin-converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. METHODS: Rats were treated with captopril (100 mg. kg(-1). day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals served as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. RESULTS: Captopril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. CONCLUSIONS: Captopril significantly attenuates the progression of hepatic fibrosis in the rat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.     

Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat

BACKGROUND & AIMS: During hepatic fibrogenesis, the hepatic extracellular matrix changes to fibrillar collagens types I and III, and cirrhosis is believed to produce an irreversible scar. In this study, we investigated whether gene delivery of human matrix metalloproteinase-1, which degrades collagens types I and type III, would attenuate established hepatic fibrosis in the rat, induced by either thioacetamide or bile duct ligation. METHODS: Hepatic fibrosis induced by thioacetamide for 7 weeks was persistent for at least 2 months, even after discontinuation of the treatment. The rats were infected once with a recombinant adenovirus, Ad5MMP-1, into which human pro-human matrix metalloproteinase-1 complementary DNA was packaged, or with a control adenovirus, Ad5LacZ. RESULTS: In Ad5MMP-1-infected, but not in Ad5LacZ-infected, rats, the fibrosis was dramatically attenuated at 2 weeks after the infection. It is interesting to note that the number of activated hepatic stellate cells was also decreased in Ad5MMP-1-infected rats. Moreover, disorganization of the hepatic trabecula, heterogeneity in the size of hepatocytes, and increased dried liver weight were observed only in Ad5MMP-1-treated rats, suggesting that human matrix metalloproteinase-1 stimulated hepatocyte proliferation, which was confirmed by bromodeoxyuridine staining. After 4 weeks, the proliferative effect of human matrix metalloproteinase-1 almost disappeared, but the hepatic fibrosis remained attenuated, whereas the fibrosis in Ad5LacZ-treated rats persisted. Furthermore, the administration of Ad5MMP-1, but not Ad5LacZ, decreased type I collagen and generated a small collagen fragment in hepatic fibrosis induced by bile duct ligation. CONCLUSIONS: Our findings show that transient human matrix metalloproteinase-1 overexpression in the liver effectively attenuates established fibrosis and induces hepatocyte proliferation.     

The role of endothelin-1 and the endothelin B receptor in the pathogenesis of hepatopulmonary syndrome in the rat

Endothelin-1 (ET-1) stimulation of endothelial nitric oxide synthase (eNOS) via pulmonary endothelial endothelin B (ET(B)) receptors and pulmonary intravascular macrophage accumulation with expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are implicated in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Our aim was to evaluate the role of ET-1 in the development of experimental HPS. The time course of molecular and physiological changes of HPS and the effects of selective endothelin receptor antagonists in vivo were assessed after CBDL. Effects of ET-1 on intralobar pulmonary vascular segment reactivity and on eNOS expression and activity in rat pulmonary microvascular endothelial cells (RPMVECs) were also evaluated. Hepatic and plasma ET-1 levels increased 1 week after CBDL in association with a subsequent increase in pulmonary microvascular eNOS and ET(B) receptor levels and the onset of HPS. Selective ET(B) receptor inhibition in vivo significantly decreased pulmonary eNOS and ET(B) receptor levels and ameliorated HPS. CBDL pulmonary artery segments had markedly increased ET(B) receptor mediated, nitric oxide dependent vasodilatory responses to ET-1 compared with controls and ET-1 triggered an ET(B) receptor dependent stimulation of eNOS in RPMVECs. Pulmonary intravascular macrophages also accumulated after CBDL and expressed HO-1 and iNOS at 3 weeks. Selective ET(B) receptor blockade also decreased macrophage accumulation and iNOS production. In conclusion, ET-1 plays a central role in modulating pulmonary micovascular tone in experimental HPS.     

Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFbeta1) in biliary atresia and paucity of intrahepatic bile ducts during infancy.

BACKGROUND/AIMS: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts. METHODS: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization. RESULTS: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. CONCLUSIONS: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.     

Hepatitis C virus-replicating hepatocytes induce fibrogenic activation of hepatic stellate cells

BACKGROUND & AIMS: The mechanism by which hepatitis C virus induces liver fibrosis remains largely obscure. To characterize the profibrogenic potential of hepatitis C virus, we used the hepatitis C virus replicon cell line Huh-7 5-15, which stably expresses the nonstructural hepatitis C virus genes NS3 through NS5B, and hepatic stellate cells as fibrogenic effector cells. METHODS: Rat and human hepatic stellate cells were incubated with conditioned media from replicon cells, and expression of fibrosis-related genes was quantified by using real-time polymerase chain reaction, protein, and functional assays. Transforming growth factor beta1 activity was determined by bioassay. RESULTS: Hepatitis C virus replicon cells release factors that differentially modulate hepatic stellate cell expression of key genes involved in liver fibrosis in a clearly profibrogenic way, up-regulating procollagen alpha1(I) and procollagen alpha1(III) and down-regulating fibrolytic matrix metalloproteinases. Transforming growth factor beta1 expression and bioactivity were increased severalfold in hepatitis C virus-replicating vs mock-transfected hepatoma cells. However, transforming growth factor beta1 activity was responsible for only 50% of the profibrogenic activity. CONCLUSIONS: Hepatitis C virus nonstructural genes induce an increased expression of transforming growth factor beta1 and other profibrogenic factors in infected hepatocytes. The direct induction of profibrogenic mediators by hepatitis C virus in infected hepatocytes explains the frequent observation of progressive liver fibrosis despite a low level of inflammation and suggests novel targets for antifibrotic therapies in chronic hepatitis C.     

大鼠胆汁淤积性肝硬化的动态病理变化及其意义

目的：观察胆汁淤积性大鼠肝硬化形成过程中模型动态变化特点。方法：结扎大鼠胆总管制备大鼠胆汁淤积性肝硬化模型；结扎后3天、1周、2周、3用、4周、5周后杀鼠取材，观测大鼠一般状况，肝功能、肝组织羟脯氨酸（Hyp）含量测定，肝组织病理学及CK-19免疫组织化学观察。结果：与假手术组大鼠比较。模型组大鼠肝重、脾重、肝／体比、脾／体比均呈先增加后降低的动态变化；血清Alb含量持续下降，而TP含量呈先高后低的变化；血清ALT活性在结扎3天后最高，2周时最低；GGT活性及TBil含量自造模后呈持续增高；而ALP活性在2周时最高；肝组织Hyp含量从结扎后呈持续增加；结扎3天后汇管区已见胆管增生，至3周时增生逐渐加重。CK-19免疫组织化学染色观察，胆管上皮细胞第3天起开始增生，1周、2周时增生明显，3周后增生渐减。4周、5周时肝组织呈花环样改变，肝实质细胞显著减少。可见肝细胞去分化现象。结论：①胆汁淤积性大鼠肝硬化模型主要病理改变有胆汁淤积、胆管上皮细胞增生、肝细胞减少、胶原增生和沉积与新生的胆管上皮细胞关系密切。②血清ALT水平由高到低，5周末仅比假手术组稍高。AST活性一直维持在较高水平。③肝细胞的减少方式除了凋亡、坏死外，可见去分化现象。     

Differential effects of jaundice and cirrhosis on beta-adrenoceptor signaling in three rat models of cirrhotic cardiomyopathy

BACKGROUND/AIMS: Attenuated cardiac function has been reported in cirrhosis as well as in jaundice, but the mechanisms remain unclear. This study aimed to explore the differential effects of jaundice and cirrhosis on the heart. METHODS: Three rat models of cirrhosis were studied: chronic bile duct ligation, bile duct ligation followed by choledochojejunostomy to relieve jaundice, and a less jaundiced model induced by thioacetamide administration. Controls underwent a sham operation. Cardiac function was assessed by measuring isolated ventricular papillary muscle contractility. Cardiac beta-adrenergic receptor signaling was studied by measuring cAMP production stimulated at the receptor, G-protein, and adenylyl cyclase levels in the signaling pathway, using isoproterenol, aluminum fluoride and forskolin, respectively. RESULTS: Serum bilirubin and bile salt levels were markedly elevated in the bile duct-ligated group, moderately increased in the thioacetamide rats, and normal in the choledochojejunostomy and sham-operated controls. Papillary muscle contractile force after maximal beta-adrenergic receptor stimulation was decreased to a similar extent in all three cirrhotic models. In the bile duct-ligated and thioacetamide-induced cirrhotic rats, production of cAMP by all three drugs was significantly attenuated. However, the cAMP production in the choledochojejunostomy group was blunted only with isoproterenol and fluoride, and remained intact with forskolin stimulation. CONCLUSIONS: These results demonstrate that cirrhosis per se impairs cardiac function by attenuating the portion of the beta-adrenergic receptor signaling pathway upstream of adenylyl cyclase. Furthermore, significant jaundice and/or cholemia can inhibit adenylyl cyclase, which may contribute to blunted cardiac contractility in jaundiced patients.     

Biliary cyst fluid from common bile duct-ligated rats stimulates endothelial nitric oxide synthase in pulmonary artery endothelial cells: a potential role in hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from pulmonary microvascular dilatation in cirrhosis and is associated with increased pulmonary endothelial nitric oxide synthase (eNOS) levels. In the common bile duct ligation (CBDL) model, endothelin-1 (ET-1) released from the liver contributes to the rise in pulmonary eNOS and intrapulmonary vasodilatation. Whether substances, including ET-1, are found in the biliary tree and selectively enter the circulation after CBDL to influence the pulmonary vasculature is unknown. We assessed if control bile and fluid obtained from the obstructed biliary tree in CBDL animals contains ET-1 and alters eNOS expression and activity in bovine pulmonary artery endothelial cells (BPAECs). Control bile and biliary cyst fluid contained concentrations of ET-1 25- to 42-fold normal plasma levels, and hepatic venous concentrations of ET-1 were selectively increased after CBDL. Biliary cyst fluid caused a dose-dependent induction of eNOS messenger RNA (mRNA) (1.9-fold control), protein (2.5-fold control), and enzyme activity (2.2-fold control) maximal at a 1:10 dilution. The increases were associated with enhanced nitric oxide (NO) production (3.1-fold control) and were inhibitable with an ET(B) receptor antagonist. Bile from sham and portal vein-ligated animals did not increase eNOS expression and at dilutions of 1:100 and 1:10 caused cell toxicity. These results show that bile and biliary cyst fluid contain high concentrations of ET-1 that are specifically increased in hepatic venous blood after CBDL. Biliary cyst fluid increases eNOS expression and activity in an ET(B) receptor-dependent manner in BPAECs. The findings suggest a novel mechanism for the susceptibility of CBDL animals to the HPS.     

Adiponectin and its receptors in rodent models of fatty liver disease and liver cirrhosis

INTRODUCTION Obesity and especially visceral fat accumulation cause insulin resistance, a common risk factor for hepatic steatosis. Fatty liver is thought to represent the first step towards the subsequent development of liver fibrosis. Impaired mitochond…     

Effect of sensory denervation with capsaicin on liver fibrosis induced by common bile duct ligation in rat

Hepatic fibrosis represents an important stage in the progression of chronic liver disease to cirrhosis. In the present paper we have investigated whether capsaicin-sensitive neuropeptide-containing sensory neurons may participate in the development of liver fibrosis. The expression of hepatic fibrosis induced by common bile duct obstruction has been studied both in capsaicin- and vehicle-treated rats. Common bile duct-induced liver fibrosis was less marked in capsaicin-treated rats than in vehicle-treated rats. Diffuse alterations of liver parenchyma structure with marked collagen deposition and nodular regeneration occurred 8 weeks after common bile duct ligation in vehicle-treated animals, while none of the capsaicin-treated rats exhibited the formation of complete connective septa altering the parenchyma architecture. Both vehicle- and capsaicin-treated rats showed an increasing number of desmin-positive cells in the perivenular zone, but the density of these cells was lower in treated animals than in untreated rats. The hydroxyproline content of the liver increased after common bile duct ligation in a time-dependent manner. Eight weeks after bile duct obstruction vehicle-treated rats showed a 7-fold increase of liver collagen content in comparison to normal animals. This enhancement was about 3.5-fold in capsaicin-treated rats. These findings raise the possibility that the peripheral release of neuropeptides stored in sensory nerves might participate in the development of liver fibrosis following common bile duct obstruction.