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.
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. (Hepatology 2013;58:1424–1435)
In cirrhosis, increased intrahepatic vascular resistance (IHVR) is the primary factor for portal hypertension (PH) development. Hepatic stellate cells (HSCs) play a major role increasing IHVR ...because, when activated, they are contractile and promote fibrogenesis. Protease‐activated receptors (PARs) can activate HSCs through thrombin and factor Xa, which are known PAR agonists, and cause microthrombosis in liver microcirculation. This study investigates the effects of the oral anticoagulant, rivaroxaban (RVXB), a direct antifactor Xa, on HSC phenotype, liver fibrosis (LF), liver microthrombosis, and PH in cirrhotic rats. Hepatic and systemic hemodynamic, nitric oxide (NO) bioavailability, LF, HSC activation, and microthrombosis were evaluated in CCl4 and thioacetamide‐cirrhotic rats treated with RVXB (20 mg/kg/day) or its vehicle for 2 weeks. RVXB significantly decreased portal pressure (PP) in both models of cirrhosis without changes in portal blood flow, suggesting a reduction in IHVR. RVXB reduced oxidative stress, improved NO bioavailability, and ameliorated endothelial dysfunction. Rivaroxaban deactivated HSC, with decreased alpha‐smooth muscle actin and mRNA expression of other HSC activation markers. Despite this marked improvement in HSC phenotype, no significant changes in LF were identified. RVXB markedly reduced fibrin deposition, suggesting reduced intrahepatic microthrombosis. Conclusion: RVXB decreases PP in two rat models of cirrhosis. This effect is mostly associated with decreased IHVR, enhanced NO bioavailability, HSC deactivation, and reduced intrahepatic microthrombosis. Our findings suggest that RVXB deserves further evaluation as a potential treatment for cirrhotic PH. (Hepatology 2017;65:2031‐2044).
Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of ...the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl
and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl
and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression.
Increased hepatic vascular resistance is the primary factor in the development of portal hypertension. Metformin ameliorates vascular cells function in several vascular beds. Our study was aimed at ...evaluating the effects, and the underlying mechanisms, of metformin on hepatic and systemic hemodynamics in cirrhotic rats and its possible interaction with the effects of propranolol (Prop), the current standard treatment for portal hypertension. CCl4-cirrhotic rats received by gavage metformin 300 mg/kg or its vehicle once a day for 1 wk, before mean arterial pressure (MAP), portal pressure (PP), portal blood flow (PBF), hepatic vascular resistance, and putative molecular/cellular mechanisms were measured. In a subgroup of cirrhotic rats, the hemodynamic response to acute Prop (5 mg/kg iv) was assessed. Effects of metformin ± Prop on PP and MAP were validated in common bile duct ligated-cirrhotic rats. Metformin-treated CCl4-cirrhotic rats had lower PP and hepatic vascular resistance than vehicle-treated rats, without significant changes in MAP or PBF. Metformin caused a significant reduction in liver fibrosis (Sirius red), hepatic stellate cell activation (α-smooth muscle actin, platelet-derived growth factor receptor β polypeptide, transforming growth factor-βR1, and Rho kinase), hepatic inflammation (CD68 and CD163), superoxide (dihydroethidium staining), and nitric oxide scavenging (protein nitrotyrosination). Prop, by decreasing PBF, further reduced PP. Similar findings were observed in common bile duct ligated-cirrhotic rats. Metformin administration reduces PP by decreasing the structural and functional components of the elevated hepatic resistance of cirrhosis. This effect is additive to that of Prop. The potential impact of this pharmacological combination, otherwise commonly used in patients with cirrhosis and diabetes, needs clinical evaluation.
Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial ...dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel‐like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2‐inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold‐stored rat livers exhibit a time‐dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2‐dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction. Conclusion: Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation. (Hepatology 2012)
Preeclampsia is a multi-system disorder unique to pregnancy responsible for a great part of maternal and perinatal morbidity and mortality. The precise pathogenesis of this complex disorder is still ...unrevealed.
We examined the pathophysiological pathways involved in early-onset preeclampsia, a specific subgroup representing its most severe presentation, using LC-MS/MS metabolomic analysis based on multi-level extraction of lipids and small metabolites from maternal blood samples, collected at the time of diagnosis from 14 preeclamptic and six matched healthy pregnancies. Statistical analysis comprised multivariate and univariate approaches with the application of over representation analysis to identify differential pathways.
A clear difference between preeclamptic and control pregnancies was observed in principal component analysis. Supervised multivariate analysis using orthogonal partial least square discriminant analysis provided a robust model with goodness of fit (R
X = 0.91,
= 0.002) and predictive ability (Q
Y = 0.72,
< 0.001). Finally, univariate analysis followed by 5% false discovery rate correction indicated 82 metabolites significantly altered, corresponding to six overrepresented pathways: (1) aminoacyl-tRNA biosynthesis; (2) arginine biosynthesis; (3) alanine, aspartate and glutamate metabolism; (4) D-glutamine and D-glutamate metabolism; (5) arginine and proline metabolism; and (6) histidine metabolism.
Metabolomic analysis focusing specifically on the early-onset severe form of preeclampsia reveals the interplay between pathophysiological pathways involved in this form. Future studies are required to explore new therapeutic approaches targeting these altered metabolic pathways in early-onset preeclampsia.
Background & Aims
In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that ...cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria‐targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension.
Methods
Ex vivo: Hepatic stellate cells phenotype was analysed in human precision‐cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl4‐ and thioacetamide‐cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation.
Results
Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl4‐cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide‐cirrhotic model.
Conclusion
We propose mitochondria‐targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis.
See Editorial on Page 963
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.)
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 CCl4 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.
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