Background/aims Placental growth factor (PlGF) is known for its role in pathological conditions to protect parenchymal cells of different organs from injury, whereas its presence in the liver and its potential importance in stimulating liver regeneration has never been described. This was investigated in this study using a rat model of partial hepatectomy (PH). Methods The rat model of 70, 80, and 90% PH was used. Liver samples were taken peroperatively, 1 h, 1, 2, 3, and 7 days after surgery. Liver regeneration was evaluated by liver weight/body weight ratio, liver regeneration rate (%), and proliferating cell marker Ki67. The expression of PlGF, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), VEGF receptor 2 (Flk-1), and hypoxia inducible factor-1α mRNA was measured by quantitative real-time PCR and localized by immunohistochemistry. Results The mRNA expression of PlGF was upregulated immediately after PH. Compared with 70 and 80% PH groups, the 90% PH group had a significantly lower PlGF and hypoxia inducible factor-1α mRNA expression, in parallel to a delayed liver weight/body weight ratio recovery. Only little differences were observed in VEGF, Flt-1, and Flk-1 mRNA expression among the PH groups. Conclusion This study shows for the first time the PlGF upregulation in regenerating livers, which is related to hypoxia stimulation and liver growth. The swift PlGF upregulation immediately after PH may indicate an important role for the PlGF/Flt-1 pathway in the early stage of liver regeneration.

BACKGROUND & AIMS:Portal hypertension is responsible for the major complications associated with cirrhosis. Angiogenesis has been associated with the pathophysiology of portal hypertension. We investigated the role of placental growth factor (PlGF) and tested the effects of monoclonal antibodies against PlGF (alphaPlGF) in a mouse model of portal hypertension.METHODS:Using a mouse model of prehepatic portal hypertension, we measured PlGF levels in the mesenteric tissue at different time points. We used knockout mice and alphaPlGF to determine the role of PlGF in the splanchnic hyperdynamic system and portosystemic collateral formation, examining its effects before and after portal hypertension was induced.RESULTS:PlGF was significantly up-regulated in the mesenteric tissue of mice with portal hypertension. Compared with wild-type animals, the vascular density in the mesentery was reduced in PlGF knockout hypertensive mice, preventing collateral formation and attenuation of mesenteric artery flow without affecting portal pressure. In the prevention study, alphaPlGF showed similar findings as in the knockout study. In mice with portal hypertension, administration of alphaPlGF resulted in a 32% decrease in portal pressure, compared with mice given immunoglobulin G(1) (control).CONCLUSIONS:Pathologic angiogenesis in the mesenteric tissues of mice with portal hypertension is mediated by PlGF. Blocking PlGF could be an effective strategy for reducing collateral formation and lowering portal pressure; further research into the effects in cirrhosis is warranted.

SummaryThe characterization of mice models of portal hypertension (PHT) is lacking in the literature. Therefore, the aim of the present study was to make a histological approach during development of PHT in two models of cirrhosis with PHT compared with one model of isolated PHT. The model of isolated PHT was developed by partial portal vein ligation (PPVL). Two portal hypertensive cirrhotic mice models were developed either by common bile duct ligation (CBDL) or administration of carbon tetrachloride (CCl4) subcutaneously (twice weekly, 1 ml/kg). These models represent, respectively, a secondary biliary cirrhosis and alcoholic cirrhosis. Mice were killed at several time points to evaluate liver changes by histological and ultrastructural methods. A correlation was made with portal pressure measurements. Histology revealed the absence of fibrosis or cirrhosis in PPVL mice. They developed an isolated portal hypertension. After CBDL induction, the mice developed the characteristics of cirrhosis after 6 weeks, with simultaneous increase in portal pressures. Fifty percent of the mice had ascites at that time point. Sixteen weeks after administration of CCl4, a micronodular cirrhotic aspect of the liver was seen associated with signs of portal hypertension. This is the first descriptive study of three widely used animal models in mice, allowing the study of pathophysiological changes in cirrhosis and portal hypertension. The PPVL in mice leads to a model of isolated portal hypertension. Secondary biliary cirrhosis developed after 6 weeks of common bile duct ligation in 50% of the mice that developed ascites. Subcutaneous injection of CCl4 for 16 weeks induces cirrhosis and poral hypertension, without ascites. Moreover, the present study is the first description of a cirrhotic model in mice developed by subcutaneous injections of CCl4. Well‐described mice models will facilitate use of knock‐out or transgenic mice and lead to a better understanding of the underlying molecular pathways in the field of portal hypertension and cirrhosis.

Introduction: Liver cirrhosis is one of the major causes of the acquired haemostatic disorders.Von Willebrand factor (VWF) is known to be elevated but very little is known about its cleaving protease, ADAMTS-13 in cirrhotic patients.Method: Patients with cirrhosis of different origin (n = 151; m/f:78/73; Child A/B/C: 32/33/35%) and healthy volunteers (n = 64) were examined.Plasma VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and collagen binding activity (CBA) were measured by ELISA.The multimeric structure of VWF was determined by SDS agarose electrophoresis, immunoblot and densitometric analysis.The activity and antigen levels of ADAMTS-13 have been measured by fluorescence resonance energy transfer assay and ELISA.Results: The antigen and activity levels of VWF were significantly higher in the cirrhotic patients than in the controls.Furthermore, all these parameters were significantly higher in Child C than in Child A patients: VWF:Ag: 301±122 vs. 236±59%, VWF:RCo: 198±94 vs. 146±49%, CBA: 243±107 vs. 160±38%, respectively (p < 0.01).The elevation of the functional activities was significantly less than that of the antigen level (p < 0.01).The multimeric distribution of VWF was characterized by the significant elevation of all molecular weight fractions with the significant predominance of the low molecular weight fractions.The activity and antigen levels of ADAMTS-13 showed a significant decrease in the Child C group as compared to patients with less severe disease.In patients with Child C vs. Child A disease, the ADAMTS-13 activity and antigen levels were 96±45 vs. 138±51% and 96±49 vs. 146±54%, respectively (p < 0.01).Discussion: Plasma VWF:Ag level and its functional activities were significantly elevated in patients with liver cirrhosis.However, the decreased activity/antigen ratios indicate a relative loss of function of the molecule.This is also suggested by the altered multimeric structure i.e. the reduced polymerization.These changes might be the consequences of endothelial perturbation in liver cirrhosis which results in the alteration of the endothelial synthesis of VWF.ADAMTS-13 is synthesized by the stellate cells and both the activity and antigen levels are significantly reduced in patients with more advanced liver disease.ADAMTS-13 has no role in the structural and functional alterations of VWF in patients with liver cirrhosis.

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (alpha-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl(4) and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl(4) mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and alpha-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl(4) and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.

There is growing interest in new therapeutic options for the treatment acute liver failure or to bridge liver function after extensive liver resections or small for size liver transplantation.We recently described a well differentiated human monocyte derived hepatocyte-like cell, which we can now generate form porcine monocytes.The porcine cells show similar function and are to be tested in an experimental model of extended liver resection, to bridge liver function.Methods: Monocytes were isolated from blood and after a six day treatment with MCSF and IL-3 were then exposed to a hepatocyte differentiation media containing FGF4 for 10 to 20 days.Neo-hepatocytes were analyzed for Albumin, Cytochrome P450 2D6 and 2C9, Connexin32, Actin and b-catenin.Urea formation was measured in vitro.Phase I biotransformation activity of neo-hepatocytes was verified using 7-ethoxycoumarin O-de-ethylase.The main phase II reactant, uridine di-phosphate (UDP)glucuronyltransferase was measured.1 x 107 human "neo-hepatocytes" were injected into the liver parenchyma of healthy mice to investigate albumin expression and integration into the liver parenchyma.Results: After 14-20 days cells displayed a typical hexagonal shape.Cells expressed, Cytochrome P450 2D6 and 2C9.Intermediate filaments were demonstrated by actin, intercellular connection characteristically by connexin 32.b-Catenin was expressed in some of the cells on the surface.Functional analysis of the neo-hepatocytes revealed a sustained albumin synthesis and a detoxifying capacity (urea production).Furthermore we found activity of 7-ethoxycoumarin O-deethylase in neohepatocytes.Phase II Metabolism was shown by UDP-glucuronidation.After injection of 1x107 Neo-hepatocytes we found integration into mice liver and excluded a fusion event.Cells expressed albumin.Our new findings indicate that porcine monocyte derived neo-hepatocytes have similar in vitro function with regards to synthesis and excretion aas well as detoxification and are now to be tested in a model of extended liver resection.

Background: Chronic liver damage causes hepatic stellate cell (HSC) activation and contraction, leading to intrahepatic microvascular and structural changes. In vitro endothelin-1 (ET-1)-induced contraction of HSCs can be reduced by somatostatin (SST); however, intrahepatic in vivo effects have never been studied. Methods: Sinusoidal diameter was measured by intravital fluorescence microscopy in carbon tetrachloride (CCl4) and control mice before and after an intravenous (IV) bolus and after 0, 5, 10 and 15 min of an IV infusion of saline, 8 mu g/kg/h SST or 8 mu g/kg/h octreotide. Results: The baseline sinusoidal diameter in CCl4 mice (3.01 +/- 0.05 mu m) was significantly smaller than that in controls (4.37 +/- 0.06 mu m). The sinusoidal diameter increased significantly in both groups after a bolus (27, 16% respectively) and following 5 min of SST IV infusion (28, 14% respectively). The percentage increase was significantly higher in CCl4 mice as compared with controls. This dilatory effect continued for at least 15 min. SST did not influence the mean arterial blood pressure (MAP) and portal venous inflow. In none of the groups did octreotide or saline have any influence on sinusoidal diameters, MAP and portal venous inflow. Conclusions: Sinusoidal diameter in cirrhotic mice is significantly smaller than that in controls. SST causes significant sinusoidal dilation following a bolus and for at least 15 min of IV infusion. Octreotide does not have any influence on liver sinusoids. These results demonstrate for the first time the in vivo dilatory effect of SST on liver sinusoids.

BACKGROUND AND AIMS The main cells responsible for liver fibrosis are hepatic stellate cells (HSC), which undergo activation, proliferation and increased production of extracellular matrix following liver injury.Focal adhesion kinase (FAK) plays an important role in integrinmediated signal transduction pathways and its C-terminal noncatalytic domain FAK-related non-kinase (FRNK), which is autonomously expressed, acts as an inhibitor of FAK phosphorylation.In the present study, we therefore investigated the effects of FRNK on proliferation, apoptosis and cell cycle in HSC stimulated by fibronectin (FN).The signal transduction molecules-extracellular signal-regulated kinase 1/2(ERK1/2) was also investigated.METHODS We have used rat immortalized hepatic stellate cell lines CFSC, and cells were divided into 5 groups as below: blank control group; FN-treated group; liposome group; empty plasmid group and FRNK plasmid group.FRNK plasmids were transfected into HSCs mediated by lipofectamine 2000 reagent.Improved MTT assay was used to determine cell proliferation, and flow cytometry (FCM)was used to detect cell cycle phase.Apoptosis was examined by Annexin-V/PI double-labelled FCM, gel electrophoresis and transmission electron microscope.The expression of FRNK, FAK, P-FAK (Tyr397), ERK1/2 and cell cycle related protein (Cyclin D1 and CDK4) were detected by Western blot and RT-PCR.RESULTS Transfection of FRNK decreased FAK phosphorylation on Tyr397 without changing total FAK level, coincident with a large increase in FRNK expression.After FRNK transfection, the proliferation inhibition rates in HSC were 20.07%, 26.16% and 29.77% at 12 h, 24 h, and 48 h, respectively.Meanwhile, the apoptotic rate in HSC exposed to FRNK plasmid for 48 h was higher than that in the empty plasmid group (25.37%±1.92%vs 9.28%±1.05%),P<0.01.The morphology changes of HSC observed by transmission electron microscopy also revealed that the apoptosis of HSC was increased by FRNK.Consistent with this, apoptotic DNA Ladder was detected in HSC exposed to FRNK.The percentage of cells at G0/G1 phase was significantly higher in FRNK plasmid group than that in empty plasmid group (71.4±2.81 vs 48.9±1.66),P<0.01, accompanied by down-regulation of Cyclin D1 and CDK4.Additionally, we found that FRNK attenuated ERK1/2 phosphorylation and resulted in a down-regulation of ERK1/2 mRNA.CONCLUSION FRNK may inhibit the proliferation and induce the apoptosis of HSC and arrested HSC at G0/G1 phase via decreasing phosphorylation levels at FAK and ERK1/2.The inhibition of FAK/ERK1/2 pathway may be a potential therapeutic strategy for preventing liver fibrogenesis.

Background/Aims: Patients with liver cirrhosis are predisposed to develop bacterial infections. An essential process in inflammatory responses is the recruitment of circulating leukocytes through the activation of adhesion molecules. Interfer-on-alpha2a is a cytokine reported to influence the expression of adhesion molecules. We investigated the effect of peginterferon-alpha2a (PegIFN-alpha(2a))in vivo on the leukocyte recruitment in the mesenteric microcirculation of cirrhotic rats after lipopolysaccharide exposure.Methods: Leukocyte rolling, adhesion and extravasation were visualized by intravital microscopy in sham-operated and common bile duct ligated (CBDL) rats. PegIFN-alpha(2a) was administered to influence leukocyte recruitment. Endothelial P-selectin, E-selectin and ICAM-1 expression were studied by immunohistochernistry.Results: CBDL placebo rats showed significantly impaired rolling, adhesion and extravasation of leukocytes compared to Sham-operated placebo rats. Endothelial P-selectin, E-selectin and ICAM-1 expressions in CBDL placebo rats were significantly reduced compared to Sham-operated placebo rats. PegIFN-alpha(2a) 18 mu g normalized number of rolling leukocytes in CBDL rats, without influencing on adhering and extravasated leukocytes. PegIFN-alpha(2a) upregulates the expression of P-selectin and E-selectin in CBDL rats, but ICAM-1 expression remained significantly lower than in Sham rats.Conclusions: Leukocyte recruitment is significantly impaired in the mesenteric microcirculation of cirrhotic rats. This deficiency appears to result from a reduced endothelial P-selectin, E-selectin and ICAM-1 expression. Peginterferon-alpha(2a) treatment normalizes rolling of leukocytes in cirrhotic rats by upregulation of P-selectin and E-selectin expressions, but has no influence on adhesion and extravasation possibly due to the absence of effect on ICAM-1 expression. (C) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

AIM:Increased angiogenesis in the mesenteric microvasculature of portal hypertensive animals may contribute to the development of splanchnic vasodilation associated with portal hypertension (PHT). Experimental data suggest that rapamycin may reduce angiogenesis and tumour growth by inhibiting the vascular endothelial growth factor (VEGF) pathway. This study determines whether rapamycin can prevent the neoangiogenesis in the mesentery of portal hypertensive mice and may influence the splanchnic vasodilation.METHODS:PHT was induced by partial portal vein ligation (PPVL). PPVL and Sham mice were treated daily with rapamycin or placebo for 2 weeks. Protein expressions of VEGF, CD 31, Akt and p70S6 kinase (mTOR signalling pathway) were evaluated. Mesenteric blood flow (MBF) was measured by a perivascular flow probe.RESULTS:Increased mesenteric angiogenesis and VEGF protein levels were observed in PPVL(placebo) mice compared to Sham(placebo) mice. Rapamycin treatment caused significant reduction in CD 31 positive endothelial cells and VEGF protein in the PPVL(rapamycine) group compared to the PPVL(placebo) group, to levels comparable with Sham(placebo) and Sham(rapamycine) groups. MBF was significantly higher in PPVL(placebo) mice compared to the Sham(placebo) mice. Rapamycin decreased significantly the MBF in PPVL(rapamycine) mice compared to PPVL(placebo) mice. Phospo-Akt and p70S6 kinase protein levels were increased in the mesenteric tissue of PPVL(placebo) mice compared to Sham(placebo) mice, which were also prevented by treatment with rapamycin.CONCLUSIONS:An increased VEGF dependent neo-angiogenesis is present in the mesentery of portal hypertensive mice. Rapamycin prevents angiogenesis in the mesenteric tissue and decreases the mesenteric blood flow in portal hypertensive mice, at least in part through an anti-VEGF activity and influence on the mTOR signalling pathway.

Background: In vivo evidence for angiogenesis in the splanchnic vasodilation in portal hypertension (PHT) and cirrhosis is lacking. Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are mediators of angiogenesis. The present study visualises in vivo structural changes (angiogenesis and vascular hyperpermeability) and examines the presence of VEGF and eNOS in the mesenteric microvasculature of animal models of PHT with and without cirrhosis. Methods: Portal hypertension was induced by partial portal vein ligation (PPVL) and cirrhosis was induced by common bile duct ligation (CBDL) in rats. The mesenteric microcirculation was examined by intravital microscopy. Expression of VEGF, eNOS and CD31 in mesenteric tissue were studied by immunohistochemistry. Results: An increased mesenteric angiogenesis was observed in PPVL and CBDL rats compared with Sham-operated and control rats, as shown by intravital microscopy and CD 31 staining. VEGF and eNOS expression was higher in CBDL and PPVL rats compared with control groups and correlated positively with vascular density. Macromolecular leakage was increased in cirrhotic rats compared with control and PPVL rats. Conclusion: Our study provides in vivo evidence of an increased angiogenesis in the mesenteric microvasculature of animal models of PHT and cirrhosis. Increased VEGF and eNOS expression in the mesentery of PPVL and CBDL rats may suggest their contribution. Microvascular permeability in the mesenteric vessels was only increased in cirrhotic rats.

Conclusion: Hepatic damage involves changes of the hepatic iron metabolism pathway due to induction of different acute phase cytokines.IL-6 is the mediator of the oppositely regulated hepcidin and Hjv gene expression, which indicates the presence of two independent regulatory mechanisms for these genes.