- Email: [email protected]
184 Endogenous cannabinoids and cardiac dysfunction in cirrhosis
02A. Cirrhosis' and complications assessed by morphometric evaluation of liver slides stained with Azan Mallory, hydroxyproline (Hyp) determination and mRNA the steady state levels of collagen I, TGF-~I, MMP-3, -13, -14, TIMP-1 and PAI-1 in the liver were quantified in total liver RNA by QRT-PCR using the TaqMan technique. The TAA treatment during 3 months induced micronodular liver fibrosis with expressed deposition of collagen fibers. The treatment with LOS, IFN, MMF, LOS+IFN, LOS+MMF significantly decreased the square of liver connective tissue stained by Aza~Mallory compared to the resolution untreated group. Only LOS decreased the total Hyp content, whereas MMF lowered relative Hyp in the liver. However, their combination did not change these parameters. IFN, LOS, MMF as well as LOS+IFN and LOS+MMF increased mRNA expression of MMP-14. The expression of MMP-13 was enhanced in rats treated with IFN, LOS+IFN and LOS+MMF, whereas SYL and LOS+PGZ decreased this parameter. The profibrogenic gene expression of TGF-[Jl in animals treated with MMF, SYL and LOS+SYL and the PAI-1 expression in rats treated with LOS+PGZ were significantly down-regulated. No effects on collagen I and TIMP-1 expression were found in all the experimental groups. The preliminary testing suggests that two combinations, LOS+MMF and LOS+IFN, are most effective in this experimental situation. These combinations, which are effective in TAA-induced rat fibrosis model in vivo, can be easily tested in a clinical setting, since single agents have a satisfactory clinical safety profile.
F•
E N D O G E N O U S C A N N A B I N O I D S AND CARDIAC DYSFUNCTION IN CIRRHOSIS
S. Ippolito, A.R. Mani, K.P. Moore. The UCL Institute of Hepatology, Royal Free & University College Medical School University College London, London, UK Cardiac function is abnormal in cirrhosis, with an increased cardiac output at rest, and subnormal cardiac responses to pharmacologic and physiological stimuli. Recent studies have shown that the endocannabinoid system is involved in the pathogenesis of the vasodilatation liver disease. The endogenous ligand for the endocannabinoid CB1 receptor is anandamide which can decrease cardiac contractility by activation of the CB1 receptors in cardiac tissue. The aim of this study was to determine whether a CB1 receptor antagonist (AM251) could improve cardiac function in a rat model of biliary cirrhosis, and to determine whether CB1 receptor blockade alters the cardiac autonomic regulation in vivo. Cardiac contractility was assessed in the isolated atria with electrical field stimulation. There was a significant 2.32-fold increase in EC50 of isoproterenol in stimulation of atria isolated from cirrhotic rats compared with controls (1.47• to 3.42• nM in control and cirrhotic rats respectively, P < 0.05). The impaired responsiveness of isolated atrium was completely normalized by treatment of cirrhotic animals with AM251 for three days (3 mg/kg). Autonomic regulation of cardiac function was assessed by analysis of heart rate variability in anesthetized rats using Fast Fourier Transformation (HRV software, Powerlab). Biliary cirrhosis in rats was associated with bradycardia (358 • 10 vs 301 • 13.3 beats/min in controls and cirrhotic rat s respectively, P < 0.01) which was completely reversed by 3 days administration of CB1 receptor antagonist AM251 (3 mg/kg). Linear analysis of heart rate variability represented impaired sympathovagal balance towards increase of cardiac sympathetic activity in rats with cirrhosis (P < 0.05). However the sympathovagal balance did not show any change following CB1 receptor blockade in both control and cirrhotic rats. We conclude that CB1 receptors might have a role in pathogenesis of cirrhosis-associated cardiac dysfunction in cirrhosis and this effect was independent of changes in sympathetic activity as determined by spectrum analysis of heart rate variability.
I•
(a) Pathophysiology
$77
THY-1 EXPRESSION IN HUMAN LIVER CIRRHOSIS AND IN RAT MODELS OF LIVER DAMAGE AND REGENERATION
T. Mansuroglu, J. Dudas, B. Saile, D. Batusic, G. Ramadori. Department
of Gastroenterology and Endocrinology, Georg-August-University, Grttingen, Germany Background and Aims: Thy-1 is a glycophosphatidylinositol-linked outer membrane leaflet glycoprotein, belongs to the subset of the haematopoetic stem cell markers, and it was described as an oval cell marker before. In several organs (lung, kidney) Thy-1 was described in myofibroblastic mesenchymal cells. We investigated the Thy-1 expression in rat models of liver damage and regeneration, and in human liver cirrhosis. Methods: Thy-1 mRNA expression and immunolocalization were studied in different models of rat liver damage and regeneration (CC14-induced acute and chronic liver injury, 2-acetyl-aminofluorene/partial hepatectomy [AAF/PH] induced liver regeneration, liver regeneration without carcinogenic treatment) and in human cirrhotic liver samples. Investigations at RNA level were performed by real time RT-PCR and Northern blot analysis, at protein level by indirect immunofluorescent single stainings on sequential sections or double stainings with anti-Thy-1, anti-Laminin, anti-Fibulin-2, smooth muscle r actin (SMA) and anti LYVE-1. Results: In normal liver and in CC14 induced acute liver injury: Thy-1 positive cells were detected in the walls of the periportal vessels. Whereas in advanced stages of experimental fibrogenesis and in human cirrhotic liver samples it was detectable within the fibrotic tissue, throughout the full thickness of the massive scars infiltrating the liver parenchyma. In AAF/PH Thy-1 positive immunoreaction was found initially periportally and expanded into the parenchyma at later time points. In human cirrhotic liver samples Thy-1 positivity partly colocalized with Fibulin-2 and partly with LYVE-1. Thy-1 mRNA expression increased after acute and chronic CC14-induced liver damage. Interestingly, r Thy-1 and fibulin-2 had a similar regulation in AAF/PH, showing a peak of induction at day 7 after PH, which was followed by a decrease. Conclusions: Our results provide evidence that Thy-1 positive liver cells could be myofibroblasts and endothelial cells. The coordinated gene expression of Thy-1, r and fibulin-2 suggest that in addition to oval cells liver myofibroblasts are induced in the AAF/PH model.
I-~
THE INVOLVEMENT OF CB1 AND VR1 RECEPTORS IN CARDIOVASCULAR DISTURBANCES OF BILE-DUCT LIGATED RATS
L. Moezi 1'2, S.A. Gaskari 1, H. Liu 1, Y. Li 1, A.R. Dehpom2, S.S. Lee 1.
1Liver Unit, GI Research Group, University of Calgary, Calgary, Canada," 2pharmacology Department, School of Medicine, Tehran University of Medical Seiences, Tehran, Iran Background and Aims: Meseuteric arteriolar vasodilatation and increased portal inflow are involved in the pathogenesis of portal hypertension in cirrhosis. The endocannabinoid system has been shown to help regulate arterial pressure in cirrhotic rats. We aimed to examine the cardiovascular effects of AM251, AM630 and capsazepine, CB1, CB2 and VR1 receptor antagonists respectively, in cirrhotic rats. Methods: Cirrhosis was induced in rats by chronic bile-duct ligation (BDL), whereas controls underwent a sham operation. Four weeks after operation, diameter of meseuteric arteriole and venule were measured by intravital microscopy after AM251, AM630 and capsazepine intrajugular vein injection for 60 minutes. Mean arterial pressure, cardiac output, systemic vascular resistance and superior meseuteric artery flow were also measured after AM251, AM630 and capsazepine administration for 60 minutes. Expression of CB1, CB2 and VR1 receptors in superior mesenteric artery was measured by western blot and RT-PCR. Results: AM21 significantly increased the baseline of mean arterial pressure and systemic vascular resistance, while it decreased arteriole
F•
E N D O G E N O U S C A N N A B I N O I D S AND CARDIAC DYSFUNCTION IN CIRRHOSIS
S. Ippolito, A.R. Mani, K.P. Moore. The UCL Institute of Hepatology, Royal Free & University College Medical School University College London, London, UK Cardiac function is abnormal in cirrhosis, with an increased cardiac output at rest, and subnormal cardiac responses to pharmacologic and physiological stimuli. Recent studies have shown that the endocannabinoid system is involved in the pathogenesis of the vasodilatation liver disease. The endogenous ligand for the endocannabinoid CB1 receptor is anandamide which can decrease cardiac contractility by activation of the CB1 receptors in cardiac tissue. The aim of this study was to determine whether a CB1 receptor antagonist (AM251) could improve cardiac function in a rat model of biliary cirrhosis, and to determine whether CB1 receptor blockade alters the cardiac autonomic regulation in vivo. Cardiac contractility was assessed in the isolated atria with electrical field stimulation. There was a significant 2.32-fold increase in EC50 of isoproterenol in stimulation of atria isolated from cirrhotic rats compared with controls (1.47• to 3.42• nM in control and cirrhotic rats respectively, P < 0.05). The impaired responsiveness of isolated atrium was completely normalized by treatment of cirrhotic animals with AM251 for three days (3 mg/kg). Autonomic regulation of cardiac function was assessed by analysis of heart rate variability in anesthetized rats using Fast Fourier Transformation (HRV software, Powerlab). Biliary cirrhosis in rats was associated with bradycardia (358 • 10 vs 301 • 13.3 beats/min in controls and cirrhotic rat s respectively, P < 0.01) which was completely reversed by 3 days administration of CB1 receptor antagonist AM251 (3 mg/kg). Linear analysis of heart rate variability represented impaired sympathovagal balance towards increase of cardiac sympathetic activity in rats with cirrhosis (P < 0.05). However the sympathovagal balance did not show any change following CB1 receptor blockade in both control and cirrhotic rats. We conclude that CB1 receptors might have a role in pathogenesis of cirrhosis-associated cardiac dysfunction in cirrhosis and this effect was independent of changes in sympathetic activity as determined by spectrum analysis of heart rate variability.
I•
(a) Pathophysiology
$77
THY-1 EXPRESSION IN HUMAN LIVER CIRRHOSIS AND IN RAT MODELS OF LIVER DAMAGE AND REGENERATION
T. Mansuroglu, J. Dudas, B. Saile, D. Batusic, G. Ramadori. Department
of Gastroenterology and Endocrinology, Georg-August-University, Grttingen, Germany Background and Aims: Thy-1 is a glycophosphatidylinositol-linked outer membrane leaflet glycoprotein, belongs to the subset of the haematopoetic stem cell markers, and it was described as an oval cell marker before. In several organs (lung, kidney) Thy-1 was described in myofibroblastic mesenchymal cells. We investigated the Thy-1 expression in rat models of liver damage and regeneration, and in human liver cirrhosis. Methods: Thy-1 mRNA expression and immunolocalization were studied in different models of rat liver damage and regeneration (CC14-induced acute and chronic liver injury, 2-acetyl-aminofluorene/partial hepatectomy [AAF/PH] induced liver regeneration, liver regeneration without carcinogenic treatment) and in human cirrhotic liver samples. Investigations at RNA level were performed by real time RT-PCR and Northern blot analysis, at protein level by indirect immunofluorescent single stainings on sequential sections or double stainings with anti-Thy-1, anti-Laminin, anti-Fibulin-2, smooth muscle r actin (SMA) and anti LYVE-1. Results: In normal liver and in CC14 induced acute liver injury: Thy-1 positive cells were detected in the walls of the periportal vessels. Whereas in advanced stages of experimental fibrogenesis and in human cirrhotic liver samples it was detectable within the fibrotic tissue, throughout the full thickness of the massive scars infiltrating the liver parenchyma. In AAF/PH Thy-1 positive immunoreaction was found initially periportally and expanded into the parenchyma at later time points. In human cirrhotic liver samples Thy-1 positivity partly colocalized with Fibulin-2 and partly with LYVE-1. Thy-1 mRNA expression increased after acute and chronic CC14-induced liver damage. Interestingly, r Thy-1 and fibulin-2 had a similar regulation in AAF/PH, showing a peak of induction at day 7 after PH, which was followed by a decrease. Conclusions: Our results provide evidence that Thy-1 positive liver cells could be myofibroblasts and endothelial cells. The coordinated gene expression of Thy-1, r and fibulin-2 suggest that in addition to oval cells liver myofibroblasts are induced in the AAF/PH model.
I-~
THE INVOLVEMENT OF CB1 AND VR1 RECEPTORS IN CARDIOVASCULAR DISTURBANCES OF BILE-DUCT LIGATED RATS
L. Moezi 1'2, S.A. Gaskari 1, H. Liu 1, Y. Li 1, A.R. Dehpom2, S.S. Lee 1.
1Liver Unit, GI Research Group, University of Calgary, Calgary, Canada," 2pharmacology Department, School of Medicine, Tehran University of Medical Seiences, Tehran, Iran Background and Aims: Meseuteric arteriolar vasodilatation and increased portal inflow are involved in the pathogenesis of portal hypertension in cirrhosis. The endocannabinoid system has been shown to help regulate arterial pressure in cirrhotic rats. We aimed to examine the cardiovascular effects of AM251, AM630 and capsazepine, CB1, CB2 and VR1 receptor antagonists respectively, in cirrhotic rats. Methods: Cirrhosis was induced in rats by chronic bile-duct ligation (BDL), whereas controls underwent a sham operation. Four weeks after operation, diameter of meseuteric arteriole and venule were measured by intravital microscopy after AM251, AM630 and capsazepine intrajugular vein injection for 60 minutes. Mean arterial pressure, cardiac output, systemic vascular resistance and superior meseuteric artery flow were also measured after AM251, AM630 and capsazepine administration for 60 minutes. Expression of CB1, CB2 and VR1 receptors in superior mesenteric artery was measured by western blot and RT-PCR. Results: AM21 significantly increased the baseline of mean arterial pressure and systemic vascular resistance, while it decreased arteriole