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262 THE ALTERATIONS OF VON WILLEBRAND FACTOR AND ITS CLEAVING PROTEASE, ADAMTS-13 SHOW AN OPPOSITE CHANGE OF DIRECTION IN PATIENTS WITH LIVER CIRRHOSIS I. Tornai1 , M. Papp1 , M.L. Udvardy2 , P. Orosz3 , J. Harsfalvi2 . 1 Second Department of Medicine, Division of Gastroenterology, 2 Clinical Research Center, University of Debrecen, Hungary, 3 Second Department of Medicine, County Hospital, Miskolc, Hungary E-mail:
[email protected] 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. 263 EARLY IMMUNE SYSTEM ACTIVATION AT THE SYSTEMIC LEVEL INITIATES IN THE LIVER IN EXPERIMENTAL COMPENSATED CIRRHOSIS M. Ubeda1 , L. Munoz1,4 , M. Nieto1,4 , M. Lario1 , D. Diaz1 , L. Lledo1 , J. Monserrat1,4 , E. Reyes1,4 , E. Sanz1 , A. De-la-Hera1,4 , M. AlvarezMon1,2,4 , A. Albillos1,3,4 . 1 Laboratorio de Enfermedades del Sistema Inmune, Dept. Medicina, Unidad I+D Asociada Al CSIC, Universidad de Alcala, Alcala de Henares, Madrid, Spain; 2 Hospital Universitario Principe de Asturias, Alcala de Henares, Madrid, Spain, 3 Hospital Universitario Ramon y Cajal, Madrid, Spain, 4 Ciberehd, Barcelona, Spain E-mail:
[email protected] Background: Gut bacterial translocation leads to immune system activation in cirrhosis with ascites (Mu˜noz L et al. Hepatology 2005; 42: 411).
Enteric bacteria reach the mesenteric lymph nodes and activate resident immune-system cells; later, recirculation of these activated immune cells extends inflammation to the systemic circulation. However, in compensated cirrhosis, in which gut bacterial translocation is unlikely, it is unknown whether immune activation occurs at the systemic level, and the potential contribution of the liver, as the target organ of inflammation in cirrhosis, is also unclear. Aims: To determine in compensated cirrhosis (without ascites): (1) the activation status of the immune system in peripheral blood, liver, and liverdraining (LLN) and mesenteric lymph nodes (MLN), and (2) the contribution of these systems, if any, to systemic inflammation. Methods and Results: Protocol I: The activation status of immune cells was assessed by flow cytometry in rats with compensated cirrhosis (induced by gavage CCl4, 12 wk) and controls. Inflammation occurred at the systemic level in cirrhotic rats, as shown by the expansion (p < 0.05) in peripheral blood of infiltrating monocytes (1.9-fold controls), recently activated Th cells (Th-r) (5.9-fold) and non-terminated effector memory Th cells (Th-m) (1.7-fold). The cirrhotic liver showed expansion (p < 0.05) of infiltrating monocytes (1.4-fold) and Th-r cells (4.8-fold). The number of Th-r, Th-m and monocytes was elevated (p < 0.05) in the LLN and MLN of cirrhotic rats. Th-cells were locally activated in LLN and MLN, as shown by the expansion in these territories of CD62L–Th-cells. In cirrhotic rats, systemic inflammation, defined by the circulating number of TH-m cells, correlated with inflammation in LLN (r = 0.74, p < 0.05), but not in MLN. Protocol II: Cirrhotic rats were administered non-absorbable antibiotics or placebo to examine the contribution of enteric bacteria to systemic inflammation. Treatment normalized Th-m, inflammatory monocytes and dendritic cell numbers in MLN, but not in peripheral blood or LLN. Conclusion: Systemic activation of the immune system is an early event in experimental compensated cirrhosis. Such activation is initiated in the liver draining lymph nodes and, unlike the case in cirrhosis with ascites, is independent of gut bacterial translocation.
264 DEFICIENCY IN PLACENTAL GROWTH FACTOR CAUSES A DECREASED PORTO-SYSTEMIC COLLATERAL VESSEL FORMATION IN PORTAL HYPERTENSIVE MICE C. Van Steenkiste1 , A. Geerts1 , E. Vanheule1 , H. Van Vlierberghe1 , F. De Vos2 , P. Carmeliet3 , M. De Vos1 , I. Colle1 . 1 Department of Gastroenterology and Hepatology, University Hospital Ghent, Ghent, Belgium; 2 Faculty of Pharmaceutical Sciences – Radiopharmacy, University Ghent, Ghent, Belgium, 3 Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Leuven, Belgium E-mail:
[email protected] Background: the major complications of portal hypertension (PHT) are the development of porto-systemic collaterals. Previously, we and others showed that angiogenesis is involved in the formation of this collateral circulation. We recently observed that the Placental Growth Factor (PlGF), a VEGF analogue responsible for angiogenesis in pathological circumstances, is upregulated in the splanchnic microvasculature of portal hypertensive mice. With the use of portal hypertensive PlGF knockout mice (PlGF −/ − ), we were able to demonstrate an inhibition of the mesenteric neo-angiogenesis compared to wild type portal hypertensive mice. Aim: to determine whether the development of porto-systemic collateral vessels in portal hypertension is a PlGF-dependent angiogenic process. Methods: A model for isolated PHT is induced in male (C57 Bl/6) wildtype (wt) mice (n = 6) or PlGF deficient mice (PlGF −/ − ) (n = 8) by partial portal vein ligation (PPVL). The extent of porto-systemic collateral vessels is assessed using radioactive Cr51-labeled microspheres injected into the spleen. The radioactivity in the liver and lungs was determined and the shunt fraction was calculated as [lungs radioactivity/(lungs radioactivity +
02A. CIRRHOSIS AND COMPLICATIONS – A) PATHOPHYSIOLOGY liver radioactivity)]. The study is performed 14 days after PPVL, when the portal hypertensive syndrome is fully established. Portal pressure and spleen weight are also measured. Results: Portal hypertensive PlGF−/ − mice had a significant (p < 0.05) 52% inhibition in the formation of porto-systemic collaterals compared with wild-type PPVL mice (42% vs 94% shunting; p = 0.005). The spleen volume was significantly lower in PlGF −/ − mice (g/10g) compared to the WT group (g/10g) (p < 0.003). There was a tendency to a lower portal pressure (p = 0.07) in PlGF−/ − portal hypertensive mice (10±0.7 mmHg) compared to wild type mice (11.2±0.8 mmHg) Conclusion: This study shows for the first time that deficiency in Placental Growth Factor partially prevents the formation of porto-systemic collaterals in portal hypertensive mice. However, there was no significant decrease in portal pressure which is consistent with previous studies with other anti-angiogenic substances. This study confirms the importance of angiogenesis in the pathophysiology of collateral vessels formation and provides a new potential anti-angiogenic target. 265 DEVELOPMENT OF CIRRHOSIS IS ASSOCIATED WITH INCREASED LEVELS OF PLACENTAL GROWTH FACTOR: A DESCRIPTIVE STUDY C. Van Steenkiste1 , B. Schroyen2 , A.M. Geerts1 , E. Vanheule1 , H. Van Vlierberghe1 , D. Laukens1 , K. Olievier1 , H. Reynaert2 , A. Geerts2 , P. Carmeliet3 , M. De Vos1 , I. Colle1 . 1 Department of Gastroenterology and Hepatology, University Hospital Ghent, Ghent, Belgium; 2 Laboratory for Molecular Liver Cell Biology, Free University of Brussels (VUB), Brussels, Belgium, 3 Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Leuven, Belgium E-mail:
[email protected] Background: The importance of angiogenesis during the development of cirrhosis is widely accepted. Vascular Endothelial Growth Factor is the most thoroughly described pro-angiogenic factor. Placental Growth Factor (PlGF) is a pleiotropic cytokine that stimulates pathological angiogenesis and several other processes. PlGF selectively binds to VEGFR-1. Aims: to evaluate 1. time-dependent PlGF, soluble (s) VEGFR-1 (anti-PlGF effect) and transmembrane VEGFR-1 protein expression in liver tissues of CCL4 induced cirrhotic mice. 2. expression of PlGF protein and mRNA by Hepatic Stellate Cells (HSC), isolated from normal mice livers. Methods: PlGF and sVEGFR-1 protein concentrations are determined by ELISA at different time points (1, 4, 8, 10, 12, 16 weeks after induction) on lysates of liver tissues of male Swiss mice, in which cirrhosis was induced by CCL4 SC injection (n = 7) and in control mice (n = 7). On the same time points, qPCR is performed for PlGF mRNA analysis. Transmembrane VEGFR-1 protein expression is assessed by immunohistochemistry. HSCs are isolated by collagenase/pronase digestion, followed by density gradient centrifugation and brought in culture. ELISA for PlGF on cell supernatans and qPCR for PlGF on cell lysates are performed on different time points (day 2, 4, 6, 8 and 10 of culture). Results: PlGF protein levels increased rapidly (p < 0.01) after 1 week of CCL4 application and remained significantly higher compared to controls with a maximum after 1 week. The PlGF mRNA level on week 16 is significantly elevated (p < 0.05) compared with control livers. ELISA for sVEGFR-1 shows a significant downregulation on the different time points (p < 0.01) compared to controls. With immunohistochemistry, stronger staining was obtained for VEGFR-1 (compared to controls), localised on endothelial cells and bile ducts. Activated HSCs (on D10) have significant higher PlGF mRNA and secrete higher levels of PlGF protein (p < 0.05) in their supernatans and this is gradually increasing starting from D2 to D10. Conclusions: The present study shows an increased PlGF protein and mRNA level in murine cirrhotic liver. Activated HSC may contribute to
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this increased PlGF secretion. VEGFR-1 overexpression and sVEGFR-1 downregulation may reflect the increased angiogenesis in cirrhotic mice. 266 INFLUENCE OF BOSENTAN ON LIVER MICROCIRCULATION IN AN EXPERIMENTAL MOUSE MODEL OF CIRRHOSIS STUDIED BY INTRAVITAL MICROSCOPY E. Vanheule, F. de Clerck, C. Van Steenkiste, A.M. Geerts, H. Van Vlierberghe, M. De Vos, I. Colle. Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium E-mail:
[email protected] Background and Aims: Chronic liver damage causes hepatic stellate cell activation and contraction, leading to intrahepatic microvascular and structural changes. Bosentan, an endothelin-1 receptor A and B antagonist, decreases portal pressure, however intrahepatic in vivo effects have never been studied. We will study the in vivo effects of bosentan on hepatic microcirculation in a mouse model of cirrhosis by measuring sinusoidal diameter with intravital fluorescence microscopy (IVFM). Methods: In male Swiss mice (n = 4) cirrhosis was induced by subcutaneous (SC) injection of carbon tetrachloride (CCl4, 1:1 in olive oil; 1 ml/kg) twice weekly for 16 weeks. Control mice (n = 4) received pure olive oil (1 ml/kg) SC. Hepatic microcirculation was visualised in vivo by IVFM after intravenous (IV) infusion of sodium fluorescein. Sinusoidal diameters were measured after 0, 5, 10, 15, 20, 25, 30, 45 and 60 min of a continuous IV infusion of 10 mg/kg/h bosentan or placebo. Results: Baseline sinusoidal diameter in CCl4 mice (5.9 mm ± 0.3) was significantly smaller than in control mice (6.7 mm ± 0.2) (P < 0.05). Sinusoidal diameter increased in the CCl4 group after 5 minutes (6.0 mm ± 0.3) and increased further after 15 and 20 minutes (6.4 mm ± 0.3 and 6.3 mm ± 0.3 respectively) of a continuous bosentan IV infusion. This vasodilatory effect remains for 20 minutes and thereafter, sinusoidal diameter decreased to baseline, which demonstrates the presence of tachyphylaxis. In the control group, sinusoidal diameter did not increase. Placebo did not had an influence on sinusoidal diameters in both groups. Conclusions: Sinusoidal diameter in a cirrhotic mouse is significantly smaller than in a control mouse, which demonstrates in vivo the narrowing of liver sinusoids in a cirrhotic mouse model. Bosentan causes a significant sinusoidal vasodilation after 15 and 20 minutes of an IV administration in the CCl4 group. On the other hand, bosentan does not have an influence on liver sinusoids in the control group. These results demonstrate for the first time the in vivo vasodilatory effect of bosentan on liver sinusoids during cirrhosis, but there is tachyphylaxis after 20 minutes of IV infusion. 267 GASTRIC MYOELECTRICAL ACTIVITY IN PATIENTS WITH LIVER CIRRHOSIS G.B. Vladimirov1 , M.V. Boyadzhieva1,2 , S. Sulejman1,3 , R. Ivanova2 , J. Petrova3 , L. Mateva1,4 . 1 Clinic of Gastroenterology, University Hospital “St. I. Rilski”, Sofia, Bulgaria, 2 Clinic of Cardiology, 3 Clinic of Neurology, University Hospital, Sofia, Bulgaria E-mail:
[email protected] Background: Electrogastrography (EGG) is a noninvasive method for recording gastric myoelectrical activity and abnormal electrogastrogram show some correlation with gastric dysmotility. These abnormalities may play a role in malnutrition and portosystemic encephalopathy. There are few data on abnormal gastric myoelectrical activity in liver cirrhosis. Aims: The aim of this study was to evaluate gastric myoelectrical activity in different stage of liver cirrhosis. Methods: Fifty patients with liver cirrhosis (LC) and 70 healthy control persons (HC) were investigated. Assessment of gastric electrical activity and EGG abnormalitis were based on the surface EGG recording by DIGITPAPER EGG (Synectics Medical). Computer spectral analysis of
