Aina Rodríguez-Vilarrupla

Increased oxidative stress in cirrhotic rat livers: A potential mechanism contributing to reduced nitric oxide bioavailability.

In cirrhotic livers, decreased nitric oxide (NO) bioavailability is a major factor increasing intrahepatic vascular tone. In several vascular disorders, an increase in superoxide (O2−) 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 O2−, 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 O2− 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 O2− and the role of superoxide dismutase (SOD) enzymatic activity as an O2− scavenger were determined in liver homogenates. Protein‐nitrotyrosination, a marker of the NO‐O2− reaction, was evaluated in liver homogenates. Furthermore, in control SEC and bovine aortic endothelial cells, NO modulation by O2− was evaluated. Cirrhotic livers exhibited increased O2− 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 O2− was associated with a significant reduction in NO bioavailability and increased nitrotyrosinated proteins. In endothelial cells, an inverse relationship between O2− 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 O2− reduction as a potential mechanism to restore NO content. (HEPATOLOGY 2008.)

The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis

In cirrhosis, intrahepatic endothelial dysfunction is one of the mechanisms involved in the increased resistance to portal blood flow and therefore in the development of portal hypertension. Endothelial nitric oxide synthase (eNOS) uncoupling due to deficiency of tetrahydrobiopterin (BH4) results in decreased production of NO and plays a major role in endothelial dysfunction in other conditions. We examined whether eNOS uncoupling is involved in the pathogenesis of endothelial dysfunction of livers with cirrhosis. Basal levels of tetrahydrobiopterin and guanosine triphosphate (GTP)‐cyclohydrolase (BH4 rate‐limiting enzyme) expression and activity were determined in liver homogenates of control and rats with CCl4 cirrhosis. Thereafter, rats were treated with tetrahydrobiopterin, and eNOS activity, NO bioavailability, assessed with a functional assay, and the vasodilator response to acetylcholine (endothelial function) were evaluated. Livers with cirrhosis showed reduced BH4 levels and decreased GTP‐cyclohydrolase activity and expression, which were associated with impaired vasorelaxation to acetylcholine. Tetrahydrobiopterin supplementation increased BH4hepatic levels and eNOS activity and significantly improved the vasodilator response to acetylcholine in rats with cirrhosis. In conclusion, the impaired response to acetylcholine of livers with cirrhosis is modulated by a reduced availability of the eNOS cofactor, tetrahydrobiopterin. Tetrahydrobiopterin supplementation improved the endothelial dysfunction of cirrhotic livers. (HEPATOLOGY 2006;44:44–52.)

Sinusoidal Endothelial Dysfunction Precedes Inflammation and Fibrosis in a Model of NAFLD

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.

The Protein SET Regulates the Inhibitory Effect of p21Cip1on Cyclin E-Cyclin-dependent Kinase 2 Activity

The cyclin-dependent kinase (CDK) inhibitor p21Cip1 has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21Cip1-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21Cip1 were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21Cip1 antibodies. We found that SET bound directly to p21Cip1 in vitro by the carboxyl-terminal region of p21Cip1. SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21Cip1. This result is specific for p21Cip1, since SET neither bound to p27Kip1 nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21Cip1 inhibitory function. These results suggest that SET can regulate G1/S transition by modulating the activity of cyclin E-CDK2.

Superoxide dismutase gene transfer reduces portal pressure in ccl4 cirrhotic rats with portal hypertension

Background: Increased intrahepatic vascular tone in cirrhosis has been attributed to a decrease of hepatic nitric oxide (NO) secondary to disturbances in the post-translational regulation of the enzyme eNOS. NO scavenging by superoxide (O2−) further contributes to a reduction of NO bioavailability in cirrhotic livers. Aim: To investigate whether removing increased O2− levels could be a new therapeutic strategy to increase intrahepatic NO, improve endothelial dysfunction and reduce portal pressure in cirrhotic rats with portal hypertension. Methods: Adenoviral vectors expressing extracellular superoxide dismutase (SOD) (AdECSOD) or β-galactosidase (Adβgal) were injected intravenously in control and CCl4-induced cirrhotic rats. After 3 days, liver O2− levels were determined by dihydroethidium staining, NO bioavailability by hepatic cGMP levels, nitrotyrosinated proteins by immunohistochemistry and western blot, and endothelial function by responses to acetylcholine in perfused rat livers. Mean arterial pressure (MAP) and portal pressure were evaluated in vivo. Results: Transfection of cirrhotic livers with AdECSOD produced a significant reduction in O2− levels, a significant increase in hepatic cGMP, and a decrease in liver nitrotyrosinated proteins which were associated with a significant improvement in the endothelium-dependent vasodilatation to acetylcholine. In addition, in cirrhotic livers AdECSOD transfection produced a significant reduction in portal pressure (17.3 (SD 2) mm Hg vs 15 (SD 1.6) mm Hg; p<0.05) without significant changes in MAP. In control rats, AdECSOD transfection prevents the increase in portal perfusion pressure promoted by an ROS-generating system. Conclusions: In cirrhotic rats, reduction of O2− by AdECSOD increases NO bioavailability, improves intrahepatic endothelial function and reduces portal pressure. These findings suggest that scavenging of O2− might be a new therapeutic strategy in the management of portal hypertension.

Identification of the nuclear localization signal of p21cip1 and consequences of its mutation on cell proliferation

Overexpression of p21cip1 induces cell cycle arrest. Although this ability has been correlated with its nuclear localization, the evidence is not conclusive. The mutants that were used to inhibit its nuclear translocation could no longer bind to several proteins known to interact with the last 25 amino acids of p21cip1. Here we used point mutation analysis and fusion of the proteins to DsRed to identify which amino acids are essential for the nuclear localization of p21cip1. We conclude that amino acids RKR140–142 are essential for nuclear translocation of p21cip1. While wild‐type DsRed‐p21 induces cell cycle arrest in 95% of transfected cells, overexpression of cytoplasmatic p21AAA140–142 arrested only 20% of transfected cells. We conclude that cytoplasmatic p21, with no deletion in the C‐terminal region, had a much lower capacity to arrest the cell cycle.

Resveratrol improves intrahepatic endothelial dysfunction and reduces hepatic fibrosis and portal pressure in cirrhotic rats

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, TGFβ 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, TGFβ, 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.

Insulin resistance and liver microcirculation in a rat model of early NAFLD.

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.

Effects of simvastatin administration on rodents with lipopolysaccharide-induced liver microvascular dysfunction

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. (HEPATOLOGY 2013)

Recombinant human manganese superoxide dismutase reduces liver fibrosis and portal pressure in CCl4-cirrhotic rats

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.