Expression of placental growth factor in regenerating livers after partial hepatectomy in the rat.

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.

Role of placental growth factor in mesenteric neoangiogenesis in a mouse model of portal hypertension.

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.

Comparison of three research models of portal hypertension in mice: macroscopic, histological and portal pressure evaluation.

The 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 (CCl(4)) 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 CCl(4), 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 CCl(4) for 16 weeks induces cirrhosis and portal hypertension, without ascites. Moreover, the present study is the first description of a cirrhotic model in mice developed by subcutaneous injections of CCl(4). 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.

Rapamycin prevents mesenteric neo-angiogenesis and reduces splanchnic blood flow in portal hypertensive mice.

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.

Influence of somatostatin and octreotide on liver microcirculation in an experimental mouse model of cirrhosis studied by intravital fluorescence microscopy.

BACKGROUND AND AIMS: 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 (CCl(4)) 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 microg/kg/h SST or 8 microg/kg/h octreotide. RESULTS: The baseline sinusoidal diameter in CCl(4) mice (3.01+/-0.05 microm) was significantly smaller than that in controls (4.37+/-0.06 microm). 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 CCl(4) 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.

An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis.

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.

Decreased leukocyte recruitment in the mesenteric microcirculation of rats with cirrhosis is partially restored by treatment with peginterferon: an in vivo study.

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. Interferon-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 immunohistochemistry. 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 microg 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.

Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.

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.