Roles of Endothelin B Receptors and Endothelial Nitric Oxide Synthase in the Regulation of Pulmonary Hemodynamic in Cirrhotic Rats.

INTRODUCTION: Hepatopulmonary syndrome and portopulmonary hypertension are common complications of liver disorders. This study aimed to determine roles of ET-B receptors and endothelial-derived NO synthase in the regulation of pulmonary hemodynamic in cirrhotic rats. METHODS: Male Sprague-Dawley rats were divided into the Sham and common bile duct ligation (CBDL) groups. After 28 days, animals were anesthetized, and the right ventricle, femoral artery, and vein cannulated. Then, intravenous injection of BQ-788 (a selective ET-B receptor antagonist) and L-NAME (eNOS inhibitor) were performed sequentially. RESULTS: After the first injection of BQ-788, the right ventricular systolic pressure (RVSP) and mean arterial systemic pressure increased only in the Sham group. L-NAME increased RVSP in the Sham and CBDL groups, whereas mean arterial systemic pressure elevated only in the Sham group significantly. Reinjection of BQ-788 increased RVSP in the Sham group, whereas it decreased RVSP in the CBDL group. Both plasma NO metabolites and lung endothelin-1 increased in the CBDL group. CONCLUSION: ET-B receptors on the endothelial cells play roles in the regulation of pulmonary and systemic vascular tone in normal condition through the NO-mediated pathway, whereas ET-B receptors on the smooth muscle cells have a role in the pulmonary vascular tone in liver cirrhosis.

Diammonium glycyrrhizinate alleviates hepatopulmonary syndrome via restoring superoxide dismutase 3 activity in rats.

Hepatopulmonary syndrome (HPS) has a fatal hypoxemia from pulmonary shunts. Superoxide dismutase 3 (SOD3) deficiency involves in this pathogenesis. The purpose of this study was to investigate the underlying mechanisms of diammonium glycyrrhizinate (DG) on HPS via SOD3. Carbon tetrachloride induced HPS rats were treated with captopril or DG for 56 days. Blood gas, pulmonary artery pressures, and histological changes were measured. Molecule dynamics of inducible (iNOS), endothelial (eNOS), neuronal nitric oxide synthase (nNOS) and SOD3 were assessed by immunohistochemistry, quantitative RT-PCR and western blot. The results showed that DG significantly increased partial pressure of oxygen (P<0.01), decreased alveolar-arterial oxygen gradient (P<0.01), and improved hypoxemia. In HPS model rats, anatomical pulmonary shunts were demonstrated as both constricted arterioles and dilated metarterioles, while physiological shunts were demonstrated by lowered pulmonary artery pressure in vivo. DG significantly reversed the vascular pathological changes. Elevated iNOS or eNOS, and decreased SOD3 expression in model rats indicated imbalance of nitric oxide (NO) bioavailability. Partial SOD3 potencies correlated with circulative events and NOSs, indicating that restorable SOD3 regulated arteriole constriction and metarteriole dilatation. DG reduced iNOS or eNOS, increased SOD3 expression, especially significantly increased the partial SOD3 located in pulmonary arteries (P<0.05), arterioles (P<0.05) and alveolus (P<0.05). These results suggested that DG relieved HPS shunts and limited HPS pathogenesis may associate with restoring SOD3 activity.

The involvement of aquaporin 1 in the hepatopulmonary syndrome rat serum-induced migration of pulmonary arterial smooth muscle cells via the p38-MAPK pathway.

Hepatopulmonary syndrome (HPS) is characterized by arterial oxygenation defects induced by intrapulmonary vascular dilation (IPVD). Pulmonary vascular remodeling (PVR) is an important pathological feature of IPVD; however, the details regarding the underlying mechanisms of this process remain undefined. Recent studies have determined that the abnormal migration of pulmonary arterial smooth muscle cells (PASMCs) plays a role in the pathogenesis of the PVR associated with HPS. Additionally, aquaporin 1 (AQP1) not only functions as a water channel molecule but also promotes cell migration by facilitating water transport in the lamellipodia of migrating cells. Common bile duct ligation (CBDL) rat is a well-accepted HPS model; we determined that the immunoperoxidase labeling of AQP1 was enhanced in the media of the pulmonary vessels in CBDL rats. HPS rat serum mediated the overexpression of AQP1 in PASMCs, and also upregulated PASMC migration. Small interfering RNAs (siRNAs) that targeted rat AQP1 caused significant downregulation of AQP1, which resulted in decreased PASMC migration. Furthermore, the inhibition of the p38-MAPK pathway abolished AQP1-dependent PASMC migration. In conclusion, this study demonstrated that AQP1 enhanced PASMC migration via the p38-MAPK pathway in rat with HPS and may represent a potential therapeutic strategy in the setting of pulmonary vascular remodeling associated with HPS.

Caspase-3 inhibition prevents the development of hepatopulmonary syndrome in common bile duct ligation rats by alleviating pulmonary injury.

BACKGROUND & AIMS: Common bile duct ligation (CBDL) rats is an accepted experimental model of hepatopulmonary syndrome (HPS), defined as liver disease and intrapulmonary vascular dilatation and hypoxaemia. Pulmonary Akt and ERK activation followed by angiogenesis in the later stages of CBDL, contribute to the pathogenesis of HPS. However, the mechanisms behind Akt and ERK activation remain undefined. Pulmonary injury induced by increased bilirubin, endotoxin and inflammatory mediators occurs in the early stages of CBDL. We assessed the effects of relieving pulmonary injury on Akt and ERK activation and on the development of HPS following CBDL. METHODS: Pulmonary injury, angiogenesis, arterial oxygenation, cell proliferation and, phospho-Akt and ERK1 were evaluated in CBDL animals with or without caspase-3 inhibition (Z-DEVD-FMK). Pulmonary injury was assessed by histology and quantifying apoptosis and aquaporin-1 (AQP1) levels. Lung angiogenesis was assessed by quantifying AQP1 level, vWF-positive cells and microvessel count. RESULTS: Pulmonary apoptosis and caspase-3 activation were markedly increased in the early stages of CBDL. Caspase-3 inhibition alleviated apoptosis, the reduction in AQP1, phospho-Akt and ERK1 levels and pulmonary injury 1 week after CBDL. Caspase-3 inhibition also reduced AQP1, phospho-Akt and ERK1 levels, vWF-positive cells, cell proliferation, microvessel count, and microvascular dilatation and improved arterial oxygenation 3 weeks following CBDL. CONCLUSIONS: Caspase-3 inhibition alleviates pulmonary injury, thereby preventing angiogenesis as well as the development of HPS in CBDL rats. These effects are related to the regulation of the Akt and ERK1 pathways.

Atividade da mieloperoxidase está aumentada na síndrome hepatopulmonar em ratos / Myeloperoxidase activity is increased in hepatopulmonary syndrome in rats

RACIONAL: A síndrome hepatopulmonar é formada por tríade clínica composta de doença hepática, dilatação vascular intrapulmonar e alterações de gases sanguíneos. Sua patogênese não é bem definida, mas especula-se que uma combinação de fatores, tais como o desequilíbrio das respostas dos receptores de endotelina, remodelação microvascular pulmonar e predisposição genética, leva à translocação bacteriana e dilatação vascular intrapulmonar. OBJETIVO: Avaliar a atividade da mieloperoxidase em modelo experimental de síndrome hepatopulmonar em ratos. MÉTODO: Foram estudados 29 animais divididos em grupos controle, sham e experimental de síndrome hepatopulmonar. O modelo experimental utilizado para induzir a síndrome foi a ligadura de ducto biliar comum. RESULTADOS: Os níveis de mieloperoxidase foram significativamente maiores no grupo ligadura de ducto biliar comum em comparação com os outros grupos. A atividade da mieloperoxidase foi maior no grupo ligadura de ducto biliar comum que o grupo controle (p<0,05) e do grupo sham (p<0,05). CONCLUSÃO: A atividade da mieloperoxidase estava aumentada na síndrome hepatopulmonar experimentais em ratos.

BACKGROUND: Hepatopulmonary syndrome is formed by a triad of liver disease, intrapulmonary vascular dilatation and changes in blood gases. Its pathogenesis is not well defined, but it is speculated that a combination of factors, such as the imbalance of endothelin receptor responses, pulmonary microvascular remodeling, and genetic predisposition, leads to bacterial translocation and intrapulmonary vascular dilatation. AIM: To evaluate the myeloperoxidase activity in hepatopulmonary syndrome in rat model. METHOD: Twenty-nine rats were divided into control, sham and experimental hepatopulmonary syndrome groups. Was evaluated the myeloperoxidase activity and the experimental model used to induce hepatopulmonary syndrome was common bile duct ligation. RESULTS: The myeloperoxidase activity levels were significantly increased in the common bile duct ligation group as compared with the other groups. Myeloperoxidase activity was higher in the common bile duct ligation group than control group (p<0.05) and than sham group (p<0.05). CONCLUSION: The myeloperoxidase activity is increased in experimental hepatopulmonary syndrome in rats.

Myeloperoxidase activity is increased in hepatopulmonary syndrome in rats.

BACKGROUND: Hepatopulmonary syndrome is formed by a triad of liver disease, intrapulmonary vascular dilatation and changes in blood gases. Its pathogenesis is not well defined, but it is speculated that a combination of factors, such as the imbalance of endothelin receptor responses, pulmonary microvascular remodeling, and genetic predisposition, leads to bacterial translocation and intrapulmonary vascular dilatation. AIM: To evaluate the myeloperoxidase activity in hepatopulmonary syndrome in rat model. METHOD: Twenty-nine rats were divided into control, sham and experimental hepatopulmonary syndrome groups. Was evaluated the myeloperoxidase activity and the experimental model used to induce hepatopulmonary syndrome was common bile duct ligation. RESULTS: The myeloperoxidase activity levels were significantly increased in the common bile duct ligation group as compared with the other groups. Myeloperoxidase activity was higher in the common bile duct ligation group than control group (p<0.05) and than sham group (p<0.05). CONCLUSION: The myeloperoxidase activity is increased in experimental hepatopulmonary syndrome in rats.

Predominant vascular dilatation with NOS expression in lung lower lobe of thioacetamide induced-cirrhotic rat.

BACKGROUND: About eighteen percent of cirrhotic patients come along with decreased systemic arterial oxygenation and expansion of pulmonary venous plexus which triggered by nitric oxide. The level of nitrate and iNOS significantly increase in the cirrhotic patients. However the localization of nNOS and iNOS in the lung tissue has not yet been clarified. OBJECTIVE: The present study, therefore, aimed to demonstrate the sites of expansion of pulmonary blood vessels and to localize nNOS and iNOS in the lung tissue of cirrhotic rat models induced by thioacetamide (TAA). MATERIAL AND METHOD: The rats were divided into 5 groups. The first group was the control. The other four groups were treated with 200 mg/kg body weight of TAA 3 times per week for 1, 2, 3, or 4 month(s), respectively. At the end of each month rats in each treated group were sacrificed. Lung histology and pulmonary NOS expression was studied by light microscope and immunohistochemical technique, respectively. RESULTS: It was found that diameter of blood vessels were highest increased in the right lower lobe of the 4-months TAA-treated group. In addition, iNOS and nNOS expression was localized at epithelium of respiratory tract, endothelium of pulmonary vessel and macrophage at this age. CONCLUSION: The present study demonstrated that the pulmonary blood vessels at the right lower lobe with cirrhotic background got enormous dilatation. iNOS and nNOS were immunostained at epithelium of respiratory tract, pulmonary endothelium and macrophages. Our observations suggested that enhanced NOS expression is important in the development of systemic hyperdynamic circulatory abnormalities in cirrhosis. As appearance of vasodilatation at right lower lobe of lung, it could, therefore, be evidence confirming that there was a real connection between inferior pulmonary vein and azygos vein at the embryonic period but obliterated later.

Clinical significance of a myeloperoxidase gene polymorphism and inducible nitric oxide synthase expression in cirrhotic patients with hepatopulmonary syndrome.

The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO -463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95+/-1.58 kPa and 6.81+/-0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62+/-0.20 kPa and 5.92+/-0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36+/-11.62 and 13.23+/-4.81 microg/L; 10.27+/- 3.20 and 4.95+/-1.12 microg/L) than in blood (16.66+/-5.24 and 4.87+/-1.73 microg/L; 5.79+/-2.31 and 2.35+/-0.84 microg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome.

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.

Ligadura de ducto biliar como modelo de estudo da síndrome hepatopulmonar e estresse oxidativo / Common bile duct ligation as a model of hepatopulmonary syndrome and oxidative stress

RACIONAL: A síndrome hepatopulmonar é caracterizada por uma disfunção hepática e pela existência de dilatações dos vasos pulmonares, levando a alterações nas trocas gasosas, tendo algumas das suas características observadas de forma experimental no modelo de ligadura de ducto biliar. OBJETIVOS: Avaliar o estresse oxidativo no tecido pulmonar de ratos cirróticos por ligadura de ducto biliar comum. MATERIAIS E MÉTODOS: Foram utilizados 12 ratos machos Wistar, pesando entre 200 e 300 g, divididos em dois grupos: controles (Co = 6) e cirróticos (Ci = 6). Foram realizadas avaliações de transaminases, gasometria arterial, avaliação da lipoperoxidação (substâncias reativas ao ácido tiobarbitúrico e quimiluminescência) e quantificação da atividade enzimática antioxidante através das concentrações da enzima superóxido dismutase. Os tecidos analisados para avaliação da síndrome hepatopulmonar foram o fígado cirrótico e o pulmão. RESULTADOS: Os animais com ligadura de ducto biliar apresentaram alteração nas transaminases: aspartato aminotransferase, Co = 105,3 ± 43/Ci = 500,5 ± 90,3 alanina aminotransferase, Co = 78,75 ± 37,7/Ci = 162,75 ± 35,4 e fosfatase alcalina, Co = 160 ± 20,45/Ci = 373,25 ± 45,44. Em relação à lipoperoxidação e à resposta antioxidante, estas também apresentaram diferenças estatisticamente significativas quando avaliadas no pulmão (substâncias reativas ao ácido tiobarbitúrico) Co = 0,87 ± 0,3/Ci = 2,01 ± 0,9; quimiluminescência Co = 16008,41 ± 1171,45/Ci = 20250,36 ± 827,82; superóxido dismutase Co = 6,66 ± 1,34/Ci = 16,06 ± 2,67. CONCLUSÕES: Os dados obtidos sugerem que no modelo experimental de cirrose por ligadura de ducto biliar há aumento significativo da lipoperoxidação no tecido pulmonar, bem como aumento na atividade da enzima antioxidante superóxido dismutase, sugerindo a presença de dano pulmonar decorrente da cirrose biliar secundária.

[Common bile duct ligation as a model of hepatopulmonary syndrome and oxidative stress]. / Ligadura de ducto biliar como modelo de estudo da síndrome hepatopulmonar e estresse oxidativo.

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.

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.

Endothelin-1 stimulation of endothelial nitric oxide synthase in the pathogenesis of hepatopulmonary syndrome.

Biliary cirrhosis in the rat triggers intrapulmonary vasodilatation and gas exchange abnormalities that characterize the hepatopulmonary syndrome. This vasodilatation correlates with increased levels of pulmonary microcirculatory endothelial nitric oxide synthase (eNOS) and hepatic and plasma endothelin-1 (ET-1). Prehepatic portal hypertension induced by portal vein ligation (PVL) does not cause similar changes, suggesting that ET-1 in cirrhosis may modulate pulmonary eNOS and vascular tone. We assessed whether ET-1 altered eNOS expression and nitric oxide production in bovine pulmonary artery endothelial cells (BPAECs) and if a 2-wk low-level intravenous ET-1 infusion in PVL animals modulated pulmonary eNOS levels, microcirculatory tone, and gas exchange. ET-1 caused a 2.5-fold increase in eNOS protein in BPAECs, inhibitable with an endothelin B receptor antagonist, and an increase in eNOS mRNA and nitrite production. ET-1 infusion in PVL animals caused increased pulmonary eNOS levels, intrapulmonary vasodilatation, and gas exchange abnormalities without increasing pulmonary arterial pressure. ET-1 produced during hepatic injury may contribute to the hepatopulmonary syndrome by modulating eNOS and inducing pulmonary microcicrulatory vasodilatation.