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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., (© 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

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.

OBJECTIVES:Tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase, might have a role in the treatment of portal hypertension (PHT) as its administration improves endothelial nitric oxide generation and hepatic endothelial dysfunction, and reduces portal pressure in experimental models of cirrhosis. Sapropterin is an oral synthetic analogue of BH4 recently approved for the treatment of phenylketonuria. This study evaluated the safety and effects of sapropterin on hepatic and systemic hemodynamics in patients with cirrhosis and PHT.METHODS:Forty patients with cirrhosis and PHT (hepatic venous pressure gradient (HVPG) ≥10 mm Hg) were randomly allocated to receive sapropterin (n=19) for 2 weeks (5 mg/kg/day increased to 10 at day 8) or placebo (n=21) in a double-blind multicenter clinical trial. Randomization was stratified according to concomitant treatment with β-adrenergic blockers. We studied at baseline and post-treatment splanchnic (HVPG and hepatic blood flow (HBF)) and systemic hemodynamics, endothelial dysfunction and oxidative stress markers (von Willebrand factor and malondialdehyde), liver function tests, and safety variables.RESULTS:HVPG was not modified by either sapropterin (16.0±4.4 vs. 15.8±4.7 mm Hg) or placebo (16.0±4.6 vs. 15.5±4.9 mm Hg). HBF, systemic hemodynamics, endothelial dysfunction markers, and liver function tests remained unchanged. Sapropterin was well tolerated (no patient required dose adjustment or withdrawal), and adverse events were mild and similar between groups.CONCLUSIONS:Sapropterin, an oral synthetic analogue of BH4, at the used dose did not reduce portal pressure in patients with cirrhosis. Sapropterin was safe and no serious adverse effects or deleterious systemic hemodynamic effects were observed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Large-conductance calcium-activated potassium (BKCa) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BKCa channels. The aim of this study was to evaluate the role of BKCa 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 BKCa channel blocker), NS1619 (a BKCa channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (l-NNA) orl-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. BKCa mRNA expression was assessed in liver homogenates, and BKCa 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 α-subunit of BKCa. 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. [ABSTRACT FROM AUTHOR]

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 (O2 −). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O2 − 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 CCl4-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl4-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 O2 −, endothelial dysfunction, and increased vascular tone in cirrhotic livers. [Copyright &y& Elsevier]

Graphical abstract Highlights • Autophagy maintains liver endothelial cell homeostasis. • Autophagy deficiency in LSEC increases oxidative stress. • Autophagy regulates nitric oxide bioavailability and maintains LSEC phenotype. • Impairment of endothelial autophagy enhances endothelial dysfunction and exacerbates fibrosis. Background & Aims Endothelial dysfunction plays an essential role in liver injury, yet the phenotypic regulation of liver sinusoidal endothelial cells (LSECs) remains unknown. Autophagy is an endogenous protective system whose loss could undermine LSEC integrity and phenotype. The aim of our study was to investigate the role of autophagy in the regulation of endothelial dysfunction and the impact of its manipulation during liver injury. Methods We analyzed primary isolated LSECs from Atg7 control and Atg7 endo mice as well as rats after CCl 4 induced liver injury. Liver tissue and primary isolated stellate cells were used to analyze liver fibrosis. Autophagy flux, microvascular function, nitric oxide bioavailability, cellular superoxide content and the antioxidant response were evaluated in endothelial cells. Results Autophagy maintains LSEC homeostasis and is rapidly upregulated during capillarization in vitro and in vivo. Pharmacological and genetic downregulation of endothelial autophagy increases oxidative stress in vitro. During liver injury in vivo , the selective loss of endothelial autophagy leads to cellular dysfunction and reduced intrahepatic nitric oxide. The loss of autophagy also impairs LSECs ability to handle oxidative stress and aggravates fibrosis. Conclusions Autophagy contributes to maintaining endothelial phenotype and protecting LSECs from oxidative stress during early phases of liver disease. Selectively potentiating autophagy in LSECs during early stages of liver disease may be an attractive approach to modify the disease course and prevent fibrosis progression. Lay summary Liver endothelial cells are the first liver cell type affected after any kind of liver injury. The loss of their unique phenotype during injury amplifies liver damage by orchestrating the response of the liver microenvironment. Autophagy is a mechanism involved in the regulation of this initial response and its manipulation can modify the progression of liver damage. [ABSTRACT FROM AUTHOR]

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 20mg/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±2.2mmHg; 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. [Copyright &y& Elsevier]

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 CCl4-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 7days. 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 TXB2 production in perfusates. Nitric oxide (NO) bioavailability and eNOS activation were measured in hepatic endothelial cells (HEC) isolated from cirrhotic rat livers. Results: CCl4 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 TXB2 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. [ABSTRACT FROM AUTHOR]

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 CCl4-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. [ABSTRACT FROM AUTHOR]

Background/Aims: Albumin administration prevents renal failure and improves survival in spontaneous bacterial peritonitis. This study characterizes the mechanisms of action of albumin in this condition.Methods: Systemic and splanchnic hemodynamics, plasma renin activity and plasma concentration of interleukin-6, serum concentration of nitric oxide and ascitic fluid levels of nitric oxide and interleukin-6 were assessed at diagnosis and resolution of infection in 12 patients with spontaneous bacterial peritonitis treated with ceftriaxone plus albumin. At infection resolution there was a significant improvement in circulatory function, as indicated by a significant increase in mean arterial pressure (+8%, P=0.02), a fall in heart rate (-10%, P=0.01), a suppression of plasma renin activity (-67%, P=0.002) and a decrease in creatinine levels. These changes were related to both an increase in cardiac work (stroke work index: +18%, P=0.005) and in peripheral vascular resistance (+14%, P=0.05). The improvement in cardiac function was due to an increase in filling. No significant changes were observed in portal pressure or hepatic blood flow.Conclusions: These results indicate that the beneficial effects of albumin administration on systemic hemodynamics and renal function in spontaneous bacterial peritonitis are related to both an improvement in cardiac function and a decrease in the degree of arterial vasodilation. [Copyright &y& Elsevier]