RESUMO
BACKGROUND & AIMS: Sepsis is associated with microvascular dysfunction, which contributes to organ failure. Intrahepatic endothelial dysfunction occurs after exposure to lipopolysaccharide (LPS). The upregulation of inducible nitric oxide synthase (iNOS) has been shown to contribute to systemic vascular dysfunction after LPS administration. However, little is known about the effects of iNOS induction on the liver microcirculation. This study aimed at exploring, in the isolated rat liver perfusion model, the role of iNOS induction in liver microvascular dysfunction associated with endotoxemia. METHODS: All experiments were conducted in male Wistar rats, after 24 h of LPS (5 mg/kg i.p.) or saline administration in the presence or absence of the iNOS inhibitor 1400 W (3 mg/kg i.p.), administered 3 and 23 h after LPS/saline injection. Liver microvascular function was assessed by isolated liver perfusion, followed by molecular studies and liver function tests. RESULTS: At 24 h, LPS induced liver endothelial dysfunction, as shown by a decreased vasodilatory response to acetylcholine and decreased eNOS phosphorylation at Ser(1176). This was associated with liver injury, assessed by an increase in liver transaminases and decreased indocyanin green clearance, and increased nitrooxidative stress. iNOS inhibition prevented liver endothelial dysfunction, blunted the development of liver injury and attenuated LPS-induced nitrooxidative stress. CONCLUSIONS: iNOS upregulation contributes to liver microvascular dysfunction in endotoxemia. This suggests that this mechanism deserves further exploration in studies addressing liver protection in the context of severe acute bacterial infection.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Células Endoteliais/fisiologia , Endotoxemia/fisiopatologia , Lipopolissacarídeos/efeitos adversos , Fígado/fisiopatologia , Óxido Nítrico Sintase Tipo II/fisiologia , Acetilcolina/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotoxemia/induzido quimicamente , Fígado/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Regulação para Cima , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
Increased hepatic vascular resistance mainly due to elevated vascular tone and to fibrosis is the primary factor in the development of portal hypertension in cirrhosis. Leptin, a hormone associated with reduction in nitric oxide bioavailability, vascular dysfunction, and liver fibrosis, is increased in patients with cirrhosis. We aimed at evaluating whether leptin influences the increased hepatic resistance in portal hypertension. CCl4-cirrhotic rats received the leptin receptor-blocker ObR antibody, or its vehicle, every other day for 1 wk. Hepatic and systemic hemodynamics were measured in both groups. Hepatic nitric oxide production and bioavailability, together with oxidative stress, nitrotyrosinated proteins, and liver fibrosis, were evaluated. In cirrhotic rats, leptin-receptor blockade significantly reduced portal pressure without modifying portal blood flow, suggesting a reduction in the intrahepatic resistance. Portal pressure reduction was associated with increased nitric oxide bioavailability and with decreased O2(-) levels and nitrotyrosinated proteins. No changes in systemic hemodynamics and liver fibrosis were observed. In conclusion, the present study shows that blockade of the leptin signaling pathway in cirrhosis significantly reduces portal pressure. This effect is probably due to a nitric oxide-mediated reduction in the hepatic vascular tone.
Assuntos
Cirrose Hepática/patologia , Pressão na Veia Porta/efeitos dos fármacos , Receptores para Leptina/antagonistas & inibidores , Resistência Vascular/efeitos dos fármacos , Animais , Anticorpos , Fígado/metabolismo , Fígado/patologia , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos WistarRESUMO
UNLABELLED: Increased production of vasoconstrictive prostanoids, such as thromboxane A2 (TXA2 ), contributes to endothelial dysfunction and increased hepatic vascular tone in cirrhosis. TXA2 induces vasoconstriction by way of activation of the thromboxane-A2 /prostaglandin-endoperoxide (TP) receptor. This study investigated whether terutroban, a specific TP receptor blocker, decreases hepatic vascular tone and portal pressure in rats with cirrhosis due to carbon tetrachloride (CCl4 ) or bile duct ligation (BDL). Hepatic and systemic hemodynamics, endothelial dysfunction, liver fibrosis, hepatic Rho-kinase activity (a marker of hepatic stellate cell contraction), and the endothelial nitric oxide synthase (eNOS) signaling pathway were measured in CCl4 and BDL cirrhotic rats treated with terutroban (30 mg/kg/day) or its vehicle for 2 weeks. Terutroban reduced portal pressure in both models without producing significant changes in portal blood flow, suggesting a reduction in hepatic vascular resistance. Terutroban did not significantly change arterial pressure in CCl4 -cirrhotic rats but decreased it significantly in BDL-cirrhotic rats. In livers from CCl4 and BDL-cirrhotic terutroban-treated rats, endothelial dysfunction was improved and Rho-kinase activity was significantly reduced. In CCl4 -cirrhotic rats, terutroban reduced liver fibrosis and decreased alpha smooth muscle actin (α-SMA), collagen-I, and transforming growth factor beta messenger RNA (mRNA) expression without significant changes in the eNOS pathway. In contrast, no change in liver fibrosis was observed in BDL-cirrhotic rats but an increase in the eNOS pathway. CONCLUSION: Our data indicate that TP-receptor blockade with terutroban decreases portal pressure in cirrhosis. This effect is due to decreased hepatic resistance, which in CCl4 -cirrhotic rats was linked to decreased hepatic fibrosis, but not in BDL rats, in which the main mediator appeared to be an enhanced eNOS-dependent vasodilatation, which was not liver-selective, as it was associated with decreased arterial pressure. The potential use of terutroban for portal hypertension requires further investigation.
Assuntos
Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/fisiopatologia , Naftalenos/farmacologia , Naftalenos/uso terapêutico , Pressão na Veia Porta/efeitos dos fármacos , Propionatos/farmacologia , Propionatos/uso terapêutico , Receptores de Tromboxanos/antagonistas & inibidores , Animais , Ductos Biliares/fisiopatologia , Tetracloreto de Carbono/efeitos adversos , Modelos Animais de Doenças , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Ligadura , Cirrose Hepática/induzido quimicamente , Masculino , Óxido Nítrico Sintase Tipo III/fisiologia , Pressão na Veia Porta/fisiologia , Ratos , Ratos Sprague-Dawley , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , Vasodilatação/fisiologia , Quinases Associadas a rho/fisiologiaRESUMO
BACKGROUND & AIMS: High oxidative stress plays a major role in increasing hepatic vascular resistance in cirrhosis, by facilitating liver fibrosis and by increasing hepatic vascular tone. This study is aimed at investigating whether the use of the novel isoform of recombinant human manganese superoxide dismutase (rMnSOD) could be a new therapeutic strategy to reduce oxidative stress and portal hypertension in cirrhotic rats. METHODS: In CCl(4)- and BDL-cirrhotic rats treated with rMnSOD (i.p. 15 µg/kg/day) or its vehicle for 7 days, mean arterial pressure (MAP), portal pressure (PP) and portal blood flow (PBF) or small mesenteric arterial flow (SMABF) were measured. In addition, in CCl(4)-cirrhotic rats, we evaluated the hepatic vasodilatory response to acetylcholine, liver fibrosis with Sirius red staining and hepatic stellate cell activation by α-smooth muscle actin (α-SMA) protein expression. RESULTS: rMnSOD treatment significantly reduced PP either in CCl(4)- or BDL-cirrhotic rats without significant changes in splanchnic blood flow, suggesting a reduction in hepatic vascular resistance. MAP was not modified. Reduction in PP was associated with a significant reduction in liver fibrosis, and α-SMA protein expression as well as with improved vasodilatory response to acetylcholine. CONCLUSIONS: Chronic rMnSOD administration to cirrhotic rats reduces portal pressure by reducing hepatic vascular resistance without deleterious effects on systemic hemodynamics, suggesting that it might constitute a new antioxidant to be considered as additional therapy for treating portal hypertension in cirrhosis.
Assuntos
Cirrose Hepática/complicações , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/etiologia , Pressão na Veia Porta/fisiologia , Superóxido Dismutase/uso terapêutico , Animais , Tetracloreto de Carbono/efeitos adversos , Modelos Animais de Doenças , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/etiologia , Hipertensão Portal/fisiopatologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pressão na Veia Porta/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Superóxido Dismutase/farmacologia , Resultado do Tratamento , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologiaRESUMO
UNLABELLED: Endothelial dysfunction drives vascular derangement and organ failure associated with sepsis. However, the consequences of sepsis on liver sinusoidal endothelial function are largely unknown. Statins might improve microvascular dysfunction in sepsis. The present study explores liver vascular abnormalities and the effects of statins in a rat model of endotoxemia. For this purpose, lipopolysaccharide (LPS) or saline was given to: (1) rats treated with placebo; (2) rats treated with simvastatin (25 mg/kg, orally), given at 3 and 23 hours after LPS/saline challenge; (3) rats treated with simvastatin (25 mg/kg/24 h, orally) from 3 days before LPS/saline injection. Livers were isolated and perfused and sinusoidal endothelial function was explored by testing the vasodilation of the liver circulation to increasing concentrations of acetylcholine. The phosphorylated endothelial nitric oxide synthase (PeNOS)/endothelial nitric oxide synthase (eNOS) ratio was measured as a marker of eNOS activation. LPS administration induced an increase in baseline portal perfusion pressure and a decrease in vasodilation to acetylcholine (sinusoidal endothelial dysfunction). This was associated with reduced eNOS phosphorylation and liver inflammation. Simvastatin after LPS challenge did not prevent the increase in baseline portal perfusion pressure, but attenuated the development of sinusoidal endothelial dysfunction. Treatment with simvastatin from 3 days before LPS prevented the increase in baseline perfusion pressure and totally normalized the vasodilating response of the liver vasculature to acetylcholine and reduced liver inflammation. Both protocols of treatment restored a physiologic PeNOS/eNOS ratio. CONCLUSION: LPS administration induces intrahepatic endothelial dysfunction that might be prevented by simvastatin, suggesting that statins might have potential for liver protection during endotoxemia.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Endotoxemia/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Sinvastatina/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Endotoxemia/induzido quimicamente , Endotoxemia/fisiopatologia , Lipopolissacarídeos/toxicidade , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Circulação Hepática/efeitos dos fármacos , Circulação Hepática/fisiologia , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Ratos , Ratos Wistar , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologiaRESUMO
BACKGROUND & AIMS: Resveratrol, a polyphenol found in a variety of fruits, exerts a wide range of beneficial effects on the endothelium, regulates multiple vasoactive substances and decreases oxidative stress, factors involved in the pathophysiology of portal hypertension. Our study aimed at evaluating the effects of resveratrol on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. METHODS: Resveratrol (10 and 20 mg/kg/day) or its vehicle was administered to cirrhotic rats for two weeks and hepatic and systemic hemodynamics were measured. Moreover, we evaluated endothelial function by dose-relaxation curves to acetylcholine, hepatic NO bioavailability and TXA2 production. We also evaluated liver fibrosis by Sirius Red staining of liver sections, collagen-1, NFκB, TGFß mRNA expression, and desmin and α-smooth muscle actin (α-SMA) protein expression, as a surrogate of hepatic stellate cell activation. RESULTS: Resveratrol administration significantly decreased portal pressure compared to vehicle (12.1 ± 0.9 vs. 14.3 ± 2.2 mmHg; p <0.05) without significant changes in systemic hemodynamics. Reduction in portal pressure was associated with an improved vasodilatory response to acetylcholine, with decreased TXA2 production, increased endothelial NO, and with a significant reduction in liver fibrosis. The decrease in hepatic fibrosis was associated with a reduced collagen-1, TGFß, NFκB mRNA expression and desmin and α-SMA protein expression. CONCLUSIONS: Resveratrol administration reduces portal pressure, hepatic stellate cell activation and liver fibrosis, and improves hepatic endothelial dysfunction in cirrhotic rats, suggesting it may be a useful dietary supplement in the treatment of portal hypertension in patients with cirrhosis.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Hipertensão Portal/prevenção & controle , Cirrose Hepática Experimental/fisiopatologia , Cirrose Hepática/prevenção & controle , Fígado/irrigação sanguínea , Pressão na Veia Porta/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Tetracloreto de Carbono/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/fisiopatologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/tratamento farmacológico , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Pressão na Veia Porta/fisiologia , Ratos , Ratos Wistar , Resveratrol , Estilbenos/uso terapêutico , Tromboxano A2/metabolismoRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Most morbidity associated with the metabolic syndrome is related to vascular complications, in which endothelial dysfunction is a major pathogenic factor. However, whether NAFLD is associated with endothelial dysfunction within the hepatic vasculature is unknown. The aims of this study were to explore, in a model of diet-induced overweight that expresses most features of the metabolic syndrome, whether early NAFLD is associated with liver endothelial dysfunction. Wistar Kyoto rats were fed a cafeteria diet (CafD; 65% of fat, mostly saturated) or a control diet (CD) for 1 month. CafD rats developed features of the metabolic syndrome (overweight, arterial hypertension, hypertryglyceridemia, hyperglucemia and insulin resistance) and liver steatosis without inflammation or fibrosis. CafD rats had a significantly higher in vivo hepatic vascular resistance than CD. In liver perfusion livers from CafD rats had an increased portal perfusion pressure and decreased endothelium-dependent vasodilation. This was associated with a decreased Akt-dependent eNOS phosphorylation and NOS activity. In summary, we demonstrate in a rat model of the metabolic syndrome that shows features of NAFLD, that liver endothelial dysfunction occurs before the development of fibrosis or inflammation.
Assuntos
Endotélio Vascular/patologia , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Cirrose Hepática/complicações , Animais , Dieta/efeitos adversos , Fígado Gorduroso/complicações , Fígado Gorduroso/etiologia , Hemodinâmica , Inflamação/complicações , Fígado/irrigação sanguínea , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Microcirculação , Hepatopatia Gordurosa não Alcoólica , Obesidade/etiologia , Obesidade/patologia , Obesidade/fisiopatologia , Ratos , Ratos Wistar , Circulação EsplâncnicaRESUMO
Reduced intrahepatic nitric oxide (NO) bioavailability and increased cyclooxygenase-1 (COX-1)-derived vasoconstrictor prostanoids modulate the hepatic vascular tone in cirrhosis. We aimed at investigating the reciprocal interactions between NO and COX in the hepatic endothelium of control and cirrhotic rats. NO bioavailability (DAF-FM-DA staining), superoxide (O(2)(-)) content (DHE staining), prostanoid production (PGI(2) and TXA(2) by enzyme immunoassays) as well as COX expression (Western Blot), were determined in hepatic endothelial cells (HEC) from control and cirrhotic rats submitted to different experimental conditions: COX activation, COX inhibition, NO activation and NO inhibition. In control and cirrhotic HEC, COX activation with arachidonic acid reduced NO bioavailability and increased O(2)(-) levels. These effects were abolished by pre-treating HEC with the COX inhibitor indomethacin. In control, but not in cirrhotic HEC, scavenging of O(2)(-) by superoxide dismutase (SOD) incubation partially restored the decrease in NO bioavailability promoted by COX activation. NO supplementation produced a significant and parallel reduction in PGI(2) and TXA(2) production in control HEC, whereas it only reduced TXA(2) production in cirrhotic HEC. By contrast, in control and cirrhotic HEC, NO inhibition did not modify COX expression or activity. Our results demonstrate that NO and COX systems are closely interrelated in HEC. This is especially relevant in cirrhotic HEC where COX inhibition increases NO bioavailability and NO supplementation induces a reduction in TXA(2). These strategies may have beneficial effects ameliorating the vasoconstrictor/vasodilator imbalance of the intrahepatic circulation of cirrhotic livers.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/terapia , Fígado/citologia , Proteínas de Membrana/metabolismo , Óxido Nítrico/metabolismo , Animais , Ácido Araquidônico/metabolismo , Disponibilidade Biológica , Hepatócitos/citologia , Indometacina/farmacologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Superóxido Dismutase/metabolismo , Tromboxano-A Sintase/genética , Tromboxano-A Sintase/metabolismo , Vasoconstritores/metabolismoRESUMO
BACKGROUND & AIMS: Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor activated by ligands that regulates genes related to vascular tone, oxidative stress, and fibrogenesis, pathways implicated in the development of cirrhosis and portal hypertension. This study aims at evaluating the effects of PPARα activation with fenofibrate on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl(4)-cirrhotic rats. METHODS: Mean arterial pressure (MAP), portal pressure (PP), and portal blood flow (PBF) were measured in cirrhotic rats treated with oral fenofibrate (25mg/kg/day, n=10) or its vehicle (n=12) for 7 days. The liver was then perfused and dose-relaxation curves to acetylcholine (Ach) were performed. We also evaluated Sirius Red staining of liver sections, collagen-I mRNA expression, and smooth muscle actin (α-SMA) protein expression, cyclo-oxygenase-1 (COX-1) protein expression, and cGMP levels in liver homogenates, and TXB(2) production in perfusates. Nitric oxide (NO) bioavailability and eNOS activation were measured in hepatic endothelial cells (HEC) isolated from cirrhotic rat livers. RESULTS: CCl(4) cirrhotic rats treated with fenofibrate had a significantly lower PP (-29%) and higher MAP than those treated with vehicle. These effects were associated with a significant reduction in hepatic fibrosis and improved vasodilatory response to acetylcholine. Moreover, a reduction in COX-1 expression and TXB(2) production in rats receiving fenofibrate and a significant increase in NO bioavailability in HEC with fenofibrate were observed. CONCLUSIONS: PPARα activation markedly reduced PP and liver fibrosis and improved hepatic endothelial dysfunction in cirrhotic rats, suggesting it may represent a new therapeutic strategy for portal hypertension in cirrhosis.
Assuntos
Endotélio Vascular/fisiopatologia , Hipertensão Portal/fisiopatologia , Cirrose Hepática/fisiopatologia , Fígado/patologia , PPAR alfa/fisiologia , Animais , Pressão Sanguínea/fisiologia , Tetracloreto de Carbono/efeitos adversos , Ciclo-Oxigenase 1/metabolismo , Modelos Animais de Doenças , Fibrose , Hipertensão Portal/metabolismo , Fígado/metabolismo , Fígado/fisiopatologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Ratos , Ratos Wistar , Tromboxano B2/metabolismoRESUMO
BACKGROUND & AIMS: Insulin contributes to vascular homeostasis in peripheral circulation, but the effects of insulin in liver microvasculature have never been explored. The aim of this study was to assess the vascular effects of insulin in the healthy and fatty liver. METHODS: Wistar rats were fed a control or a high fat diet (HFD) for 3days, while treated with a placebo, the insulin-sensitizer metformin, or the iNOS inhibitor 1400W. Vascular responses to insulin were evaluated in the isolated liver perfusion model. Insulin sensitivity at the sinusoidal endothelium was tested by endothelium-dependent vasodilation in response to acetylcholine in the presence or absence of insulin and by the level of liver P-eNOS after an insulin injection. RESULTS: Rats from the HFD groups developed liver steatosis. Livers from the control group showed a dose-dependent hepatic vasodilation in response to insulin, which was blunted in livers from HFD groups. Metformin restored liver vascular insulin-sensitivity. Pre-treatment with insulin enhanced endothelium-dependent vasodilation of the hepatic vasculature and induced hepatic eNOS phosphorylation in control rats but not in HFD rats. Treatment with metformin or 1400W restored the capacity of insulin to enhance endothelium dependent vasodilation and insulin induced eNOS phosphorylation in HFD rats. CONCLUSIONS: The administration of a HFD induces insulin resistance in the liver sinusoidal endothelium, which is mediated, at least in part, through iNOS upregulation and can be prevented by the administration of metformin. Insulin resistance at the hepatic vasculature can be detected earlier than inflammation or any other sign of advanced NALFD.
Assuntos
Fígado Gorduroso/fisiopatologia , Resistência à Insulina/fisiologia , Fígado/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Endotélio/enzimologia , Endotélio/fisiopatologia , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fígado/patologia , Fígado/fisiopatologia , Circulação Hepática , Masculino , Metformina/farmacologia , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Regulação para Cima , Vasodilatadores/farmacologiaRESUMO
BACKGROUND & AIMS: Increased superoxide in cirrhotic livers, by reducing nitric oxide bioavailability, contributes to increase intrahepatic vascular resistance to portal blood flow and as a consequence portal pressure. We aimed to evaluate whether a strategy directed to reduce superoxide using tempol, a small membrane permeable SOD-mimetic, is able to modulate intrahepatic nitric oxide content and reduce portal pressure in cirrhotic rats. METHODS: Superoxide and nitric oxide were evaluated in control sinusoidal endothelial cells (SEC) pre-treated with the pro-oxidant diethyldithiocarbamate (DDC) and in CCl(4)-cirrhotic rat livers treated with tempol or vehicle. Mean arterial pressure, portal pressure, and portal blood flow were measured in control and cirrhotic rats treated with tempol (180µmol/kg/h; via ileocholic vein) or vehicle. In a subset of animals, hemodynamic measurements were performed after NO-inhibition with l-NAME. RESULTS: Tempol reduced superoxide content and increased NO both in SEC and cirrhotic livers. In cirrhotic rats, but not in controls, tempol significantly reduced portal pressure, and increased portal blood flow, which most likely reflects a reduction in intrahepatic vascular resistance. Tempol significantly reduced mean arterial pressure. l-NAME prevented all these effects. CONCLUSIONS: Tempol reduces superoxide, increases nitric oxide, and reduces portal pressure in sinusoidal endothelial cells and in cirrhotic livers. These results confirm that oxidative stress has a role in the pathogenesis of portal hypertension and supports the use of antioxidants in its treatment. However, when considering the use of antioxidants as additional therapy to treat portal hypertension, the potential to produce deleterious effects on systemic hemodynamics needs to be carefully evaluated.
Assuntos
Óxidos N-Cíclicos/farmacologia , Cirrose Hepática Experimental/tratamento farmacológico , Cirrose Hepática Experimental/fisiopatologia , Superóxido Dismutase/metabolismo , Animais , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/fisiopatologia , Ditiocarb/toxicidade , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Masculino , Óxido Nítrico/metabolismo , Pressão na Veia Porta/efeitos dos fármacos , Ratos , Ratos Wistar , Marcadores de Spin , Superóxido Dismutase/antagonistas & inibidores , Superóxidos/metabolismo , Resistência Vascular/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Tetrahydrobiopterin is an essential cofactor for NOS enzymes to synthesize NO. It has been suggested that reduced intrahepatic tetrahydrobiopterin decreases intrahepatic NO and contributes to increase hepatic vascular resistance and portal pressure in cirrhosis. The main aim of the study was to evaluate the effect of tetrahydrobiopterin supplementation in portal pressure in CCl4 cirrhotic rats. METHODS: Cirrhotic rats received vehicle or tetrahydrobiopterin (10mg/kg/day i.p.) for 3 days. Hepatic and systemic hemodynamics and hepatic tetrahydrobiopterin, NOS activity and cGMP levels were measured. In addition, hepatic and systemic hemodynamics were evaluated in normal rats in which tetrahydrobiopterin deficiency was induced by administrating 2,4-diamino-6-hydroxy-pyrimidine (DAHP) for 8h. RESULTS: In cirrhotic rats, tetrahydrobiopterin administration increased liver NOS activity and cGMP levels and markedly and significantly reduced portal pressure. Amelioration of portal hypertension was associated with a normalization of arterial pressure. In normal rats DAHP decreased hepatic tetrahydrobiopterin and NOS activity and increased hepatic vascular tone. These effects of DAHP administration were corrected by tetrahydrobiopterin supplementation. CONCLUSIONS: The present study shows that tetrahydrobiopterin markedly reduces portal hypertension and improves systemic hemodynamics in cirrhotic rats. These data support the concept that tetrahydrobiopterin supplementation may represent a new therapeutic strategy for portal hypertension.
Assuntos
Biopterinas/análogos & derivados , Hipertensão Portal/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Fígado/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Biopterinas/deficiência , Biopterinas/farmacologia , Tetracloreto de Carbono/toxicidade , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Hipertensão Portal/induzido quimicamente , Hipertensão Portal/metabolismo , Hipoxantinas/farmacologia , Fígado/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Ratos , Ratos Wistar , Circulação Esplâncnica/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Splanchnic vasodilation initiates the hyperdynamic syndrome in portal hypertension. We aimed to explore molecular mechanisms involved in the development of mesenteric vasodilation in portal hypertension. METHODS: Superior mesenteric artery (SMA) samples from portal vein ligated (PVL) and sham rats were compared in a time course experiment using DNA microarrays. Selected genes were quantified by qRT-PCR in PVL and cirrhotic rats. Inmunohistochemistry of tyrosine hydroxylase (Th) and norepinephrine was assessed in SMA sections of PVL and sham rats. Western blot analysis of Th, dopamine beta-hydroxylase (Dbh) and synaptosome-associated protein (Snap-25) was performed in SMA and jejunum samples from the animal models. RESULTS: Fifty differentially expressed genes implicated in neurotransmission, especially adrenergic, were detected in SMA samples from PVL rats. Sequential analysis showed a profound down-regulation at 14 days in PVL rats. These down-regulated genes were confirmed by RT-PCR in SMA from PVL and cirrhotic rats. Th and NE detection by immunohistochemistry was reduced in PVL compared to sham. Th, Dbh and Snap-25 expression was lower in SMA from 14-day PVL and cirrhotic rats compared to sham and control rats, respectively. CONCLUSIONS: Genetic down-regulation of genes related to the adrenergic system might have a role in splanchnic vasodilation of portal hypertension.
Assuntos
Perfilação da Expressão Gênica , Hipertensão Portal/genética , Hipertensão Portal/fisiopatologia , Norepinefrina/fisiologia , Circulação Esplâncnica/fisiologia , Vasodilatação/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Modelos Animais de Doenças , Dopamina beta-Hidroxilase/genética , Dopamina beta-Hidroxilase/metabolismo , Regulação para Baixo/genética , Hipertensão Portal/induzido quimicamente , Imuno-Histoquímica , Ligadura , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/fisiopatologia , Masculino , Artéria Mesentérica Superior/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Veia Porta , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
BACKGROUND: Large-conductance calcium-activated potassium (BK(Ca)) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BK(Ca) channels. The aim of this study was to evaluate the role of BK(Ca) channels in the regulation of vascular tone in control (CT) and carbon tetrachloride-cirrhotic (CH) rat livers. METHODS: Changes in intrahepatic vascular resistance were assessed by evaluating the portal perfusion pressure (PP) response to methoxamine (Mtx) in the presence of Iberiotoxin (Ibtx; a BK(Ca) channel blocker), NS1619 (a BK(Ca) channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine (L-NNA) or L-NNA alone. In addition, in CH livers, PP dose-response curves to the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), were performed after pre-incubation with Ibtx or its vehicle. BK(Ca) mRNA expression was assessed in liver homogenates, and BK(Ca) protein expression in HSC isolated from CT and CH livers. RESULTS: In CH livers, Ibtx significantly increased baseline PP and exacerbated the PP response to Mtx. Conversely, NS1619 induced a mild nonsignificant decrease of baseline PP and attenuated the hyperresponse to Mtx. CH livers exhibited an upregulation of both mRNA and protein of the alpha-subunit of BK(Ca). CONCLUSION: Large-conductance calcium-activated potassium channels are overexpressed in CH livers and might represent a compensatory mechanism modulating the increased hepatic vascular tone of cirrhosis.
Assuntos
Hepatócitos/citologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Cirrose Hepática/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Animais , Western Blotting , Tetracloreto de Carbono , Células Cultivadas , Modelos Animais de Doenças , Hepatócitos/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/análise , Cirrose Hepática/patologia , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Distribuição Aleatória , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Resistência VascularRESUMO
UNLABELLED: In cirrhotic livers, decreased nitric oxide (NO) bioavailability is a major factor increasing intrahepatic vascular tone. In several vascular disorders, an increase in superoxide (O(2) (-)) has been shown to contribute to reduced NO bioavailability through its reaction with NO to form peroxynitrite. This study was aimed to test the hypothesis that, in cirrhotic livers, increased O(2) (-), by reacting with NO, reduces NO bioavailability. In control and cirrhotic rat livers, NO bioavailability was evaluated by the measurement of cyclic guanosine monophosphate in liver tissue and by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM-DA) fluorescence in isolated sinusoidal endothelial cells (SEC); the O(2) (-) content was determined by dihydroethidium staining in fresh liver sections. In addition, the role of endothelial nitric oxide synthase (eNOS), xanthine oxidase (XO), and cyclooxygenase (COX) as possible sources of O(2) (-) and the role of superoxide dismutase (SOD) enzymatic activity as an O(2) (-) scavenger were determined in liver homogenates. Protein-nitrotyrosination, a marker of the NO-O(2) (-) reaction, was evaluated in liver homogenates. Furthermore, in control SEC and bovine aortic endothelial cells, NO modulation by O(2) (-) was evaluated. Cirrhotic livers exhibited increased O(2) (-) levels. This was due, at least in part, to increased production by COX and XO but not eNOS and to reduced scavenging by SOD. Increased O(2) (-) was associated with a significant reduction in NO bioavailability and increased nitrotyrosinated proteins. In endothelial cells, an inverse relationship between O(2) (-) levels and NO bioavailability was observed. CONCLUSION: Our data show that oxidative stress may contribute to reduced NO bioavailability in cirrhotic livers, supporting the evaluation of O(2) (-) reduction as a potential mechanism to restore NO content.
Assuntos
Cirrose Hepática/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Superóxidos/metabolismo , Animais , Células Endoteliais/metabolismo , Masculino , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Xantina Oxidase/metabolismoRESUMO
BACKGROUND & AIMS: Increased hepatic vascular resistance in cirrhosis is in part due to reduced nitric oxide (NO) bioavailability. This is related to insufficient NO synthesis from endothelial nitric oxide synthase and to enhanced NO scavenging by superoxide radicals (O(2)(-)). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O(2)(-) that increases vascular tone in different cardiovascular disorders. Thus, our aims were to study the molecular and biochemical state of NADPH-oxidase in cirrhotic livers and to investigate its possible role in modulating hepatic vascular tone in cirrhosis. METHODS: NADPH-oxidase expression and enzymatic activity were determined in control (n = 8) and CCl(4)-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl(4)-cirrhotic (n = 10) rats were treated with apocynin (a selective NADPH-oxidase inhibitor) or its vehicle. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow were measured in vivo. Moreover, hepatic endothelial function was evaluated in isolated and perfused rat livers by dose-response curves to acetylcholine. In addition, in 6 control and 6 cirrhotic human livers NADPH-oxidase activity and expression were evaluated. RESULTS: Rat cirrhotic livers had no increased NADPH-oxidase protein expression or activity in relation to control livers. NADPH-oxidase inhibition did not modify splanchnic or systemic hemodynamics in control or cirrhotic rats and did not improve the impaired endothelial-dependent vasodilatory response to acetylcholine of cirrhotic livers. Human cirrhotic livers also did not exhibit increased NADPH-oxidase expression or activity. CONCLUSIONS: Our study shows that NADPH-oxidase activity is decreased in the cirrhotic livers and therefore cannot explain increased hepatic O(2)(-), endothelial dysfunction, and increased vascular tone in cirrhotic livers.
Assuntos
Artéria Hepática/fisiopatologia , Cirrose Hepática/fisiopatologia , NADPH Oxidases/metabolismo , Acetofenonas/farmacologia , Adulto , Idoso , Animais , Antioxidantes/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Tetracloreto de Carbono , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Artéria Hepática/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Pessoa de Meia-Idade , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Superóxidos/metabolismoRESUMO
BACKGROUND/AIMS: Cyclooxygenase-1 (COX-1) is overexpressed in sinusoidal endothelial cells (SEC) of cirrhotic rat livers, and through an enhanced production of vasoconstrictor prostanoids contributes to increase intrahepatic resistance. Our study was aimed at investigating the role of enhanced AA bioavailability modulating the hepatic vascular tone of cirrhotic livers and identifying which prostanoid is involved. METHODS: SEC isolated from control and cirrhotic rat livers were incubated with AA, methoxamine or vehicle. TXA(2) was quantified. In addition, portal perfusion pressure (PP) response curves to AA were performed in rat livers pre-incubated with vehicle, SC-560 (COX-1 inhibitor), Furegrelate (inhibitor of TXA(2) synthesis) and SQ-29548 (PGH(2)/TXA(2) receptor blocker). cPLA2 activity was determined in control and cirrhotic livers. RESULTS: AA and methoxamine incubation promoted a significant increase in TXA(2) release by Cirrhotic-SEC, but not in Control-SEC. AA produced a dose-dependent increase in the PP, associated with increased TXA(2) release. These responses were significantly greater in cirrhotic livers. COX-1 inhibition and PGH(2)/TXA(2) receptor blockade, but not TXA(2) synthase inhibition, markedly attenuated the PP response to AA of cirrhotic livers. Additionally, cirrhotic livers exhibited significantly increased cPLA2 activity. CONCLUSIONS: An enhanced production of vasoconstrictor prostanoids, probably PGH(2), by SEC contributes to increase vascular tone of cirrhotic livers.
Assuntos
Células Endoteliais/metabolismo , Cirrose Hepática/fisiopatologia , Fígado/irrigação sanguínea , Sistema Porta/fisiopatologia , Prostaglandinas/biossíntese , Vasoconstritores/metabolismo , Animais , Ácido Araquidônico/administração & dosagem , Ácido Araquidônico/farmacocinética , Ácido Araquidônico/farmacologia , Disponibilidade Biológica , Ciclo-Oxigenase 1/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Perfusão , Fosfolipases A/metabolismo , Sistema Porta/efeitos dos fármacos , Pressão , Ratos , Ratos Wistar , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Tromboxano B2/biossíntese , Tromboxano-A Sintase/antagonistas & inibidores , VasoconstriçãoRESUMO
BACKGROUND/AIMS: Sinusoidal endothelial dysfunction with decreased nitric oxide (NO) production contributes to increased hepatic resistance in cirrhosis. Statins improve endothelial dysfunction in peripheral vasculature. This study was designed to characterize the hemodynamic and molecular effects of statins in cirrhotic rats. METHODS: Systemic and splanchnic hemodynamics were evaluated in CCl(4) ascitic cirrhotic rats treated with placebo or simvastatin (25 mg/kg/day, for 3 days), at baseline and after volume expansion. Vascular responses of liver vasculature were evaluated after isolation and perfusion of the liver. RESULTS: There were no differences in baseline hemodynamics in rats treated with simvastatin or placebo. However, in rats treated with simvastatin the increase in portal pressure induced by volume expansion was significantly attenuated. In isolated and perfused cirrhotic livers simvastatin pre-treatment significantly attenuated the pressure response to methoxamine, and significantly improved paradoxical vasoconstriction induced by acetylcholine. These effects were not observed in the presence of a nitric oxide synthase inhibitor. Simvastatin increased eNOS expression, Akt-dependent eNOS phosphorylation and cGMP liver content. CONCLUSIONS: The administration of simvastatin might constitute a new way to selectively increase NO availability in the cirrhotic liver circulation and, therefore improve the vascular disturbances that contribute to portal hypertension.
Assuntos
Anticolesterolemiantes/farmacologia , Tetracloreto de Carbono/toxicidade , Cirrose Hepática/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Fígado/patologia , Sinvastatina/farmacologia , Animais , GMP Cíclico/metabolismo , Fígado/metabolismo , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Perfusão , Fosforilação , Ratos , Ratos WistarRESUMO
Cirrhosis of the liver is by far the most common cause of portal hypertension in the western world. Portal hypertension is a frequent clinical syndrome, defined by a pathological increase in the portal venous pressure. When the portal pressure gradient (the difference between pressures in the portal and the inferior vena cava veins: normal value below 6 mmHg) increases above 10-12 mmHg, complications of portal hypertension can occur. Increased resistance to portal blood flow, the primary factor in the pathophysiology of portal hypertension, is in great part due to morphological changes occurring in chronic liver diseases. However, more recently a graded and reversible contraction of different elements of the porto-hepatic bed have been shown to play a role modulating intrahepatic vascular resistance which provides a rationale for the intention to reduce intrahepatic resistance and portal pressure by means of pharmacological agents. The subsequent increase in portal blood flow, as a result of the arteriolar vasodilatation of the splanchnic organs, plays a contributory role maintaining and aggravating the portal hypertensive syndrome. This splanchnic arteriolar vasodilatation is a multifactorial phenomenon, which may involve neurogenic, humoral and local mechanisms.