Experimental portal hypertension


175

Obeticholic acid, a farnesoid-X receptor agonist, improves portal hypertension by two distinct pathways in cirrhotic rats

Len D. Verbeke1,Ricard Farre2, Jonel Trebicka3, Mina Komuta4, Tania Roskams4, Sabine Klein3, Ingrid Vander Elst1, Petra Windmolders1, Tim Vanuytse2l, Frederik Nevens1, Wim Laleman1

1Department of Liver and Biliopancreatic disorders, University of Leuven, Leuven, Belgium; 2Translational Research Center for Gastrointestinal Disorders,University of Leuven, Leuven, Belgium; 3Department of Internal Medicine I, University of Bonn, Bonn, Germany; 4Departments of Morphology and Molecular Pathology, Translational Cell and Tissue Research, University hospitals Leuven, Leuven, Belgium

Study purpose: The farnesoid-X-receptor (FXR) is a nuclear bile acid receptor involved in bile acid homeostasis, hepatic and intestinal inflammation, liver fibrosis and cardiovascular disease. We studied the effect of short-term treatment with obeticholic acid (INT-747), a potent selective FXR agonist, on intrahepatic hemodynamic dysfunction and signaling pathways in different rat models of cirrhotic portal hypertension. Methods: Thioacetamide (TAA)-intoxicated and bile duct ligated (BDL) rats were used as models. After gavage of 2 doses of 30mg/kg INT-747 or vehicle within 24 hours, in vivo hemodynamics were assessed. Additionally, we evaluated the direct effect of INT-747 on total intrahepatic vascular resistance (IHVR) and intrahepatic vascular tone (endothelial dysfunction and hyperresponsiveness to methoxamine) by means of an in-situ liver perfusion system and on hepatic stellate cell contraction in vitro. FXR-expression and involved intrahepatic vasoactive pathways (e. g. endothelial nitric oxide synthase: eNOS, Rho-kinase, dimethylarginine dimethylaminohydrolase: DDAH) were analyzed by immunohistochemistry, RT-PCR or Western Blot. Results: In both cirrhotic models, FXR expression was heavily decreased. Immunohistochemically, this coincided with a disappearance of the typical FXR hepatocytic nuclear staining pattern seen in healthy livers. Treatment with INT-747 in TAA and BDL reactivated the FXR downstream signaling pathway and decreased portal pressure without deleterious systemic hypotension (portal pressure decreased from 13.8 ± 0, 6 mm Hg to 11.6 ± 0, 8 mm Hg in TAA, P=0.045, n=14 and from 12.3 ± 0, 8 mm Hg to 10.1 ± 0, 6 mm Hg in BDL, P=0.037, n=15). This was related to a decrease in total intrahepatic vascular resistance during liver perfusion (relative decrease in IHVR after INT747 treatment in TAA: −8.06 ± 0.69%, P<0.001, n=10 and in BDL: −21.8 ± 2.50%, P<0.001, n=10). In the perfused TAA and BDL cirrhotic liver, INT-747 improved endothelial vasorelaxation capacity but not hyperresponsiveness. In both groups, this was associated with an increased eNOS-activity which in TAA related to down-regulation of the Rho-kinase pathway and in BDL to up-regulation of DDAH-2.Furthermore, INT-747 induced a dose-dependent relaxation of cultured rat primary hepatic stellate cells in vitro. Conclusion: FXR-agonist INT-747 improves portal hypertension in 2 different rat models of cirrhosis by decreasing the IHVR. This hemodynamic effect relates to restoration of endothelial dysfunction by increased intrahep-atic eNOS-activity. The mechanisms of eNOS activity increase differ depending on the etiology of cirrhosis.

Disclosures:

Frederik Nevens - Grant/Research Support: Ipsen, Roche, MSD, Astellas, CAF

The following people have nothing to disclose: Len D. Verbeke, Ricard Farre, Jonel Trebicka, Mina Komuta, Tania Roskams, Sabine Klein, Ingrid Vander Elst, Petra Windmolders, Tim Vanuytsel, Wim Laleman

176

Metformin reduces hepatic resistance and portal pressure in CCl4 and BDL cirrhotic rats

Dinesh M. Tripathi1, 2, Eva Erice1, Jordi Gracia-Sancho1, Hector Garcia-Caldero1, Shiv K. Sarin2, Jaime Bosch1, Juan Carlos Garcia-Pagan1

1Barcelona Hepatic Hemodynamic Lab, Hospital Clinic de Barcelona, IDIBAPS, CIBEREHD, Barcelona, Spain; 2Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India

Increased hepatic vascular resistance is the primary factor in the development of portal hypertension (PH). In other vascular beds, metformin has been shown to ameliorate vascular cells phenotype and function. Our study evaluated the effects of metformin on hepatic and systemic hemodynamics and the underlying mechanisms in cirrhotic rats. In addition, we studied the possible interaction between metformin and propranolol (Prop), the current standard treatment of PH. Methods: CCl4-cirrhotic rats received by gavage metformin 300mg/kg or its vehicle (n=12 per group) once a day for 1 week, before measuring hemodynamic parameters (MAP, Portal Pressure-PP, Portal Blood Flow-PBF, Hepatic Vascular Resistance-HVR), and molecular/cellular potential mechanisms (liver fibrosis, HSC activation, Rho-Kinase activity, oxidative stress, eN〇S and AMPK pathways). In 10 rats per group, the PP and MAP response to acute Prop (5mg/kg i. v.) was assessed. Effects of metformin±Prop on PP and MAP were further evaluated in BDLcirrhotic rats (n=8 per group). Results: Metformin-treated CCl4 cirrhotic rats had lower PP (10.2±0.8 vs 13.9±0.8 mmHg; 27%; p<0.01) and HVR (0.8±0.1 vs 1.3±0.2 mmHg*mL- 1*min; −40%; p<0.01) than vehicle-treated rats, without significant changes in MAP or PBF. Prop further reduced PP in metformin (−26%) and vehicle treated rats (−14%), being the additional effect greater in the metformin group (p<0.01). As a result, the final PP was much lower in the metformin group (38%; p<0.01) with no significant differences in MAP (79±7 vs 80±7 mmHg). Metformin treatment caused a significant reduction in liver fibrosis (−41%), HSC-activation (a-SMA −72% and desmin −46%) and Rho-kinase activity (−55%) (all p<0.01). In addition, hepatic oxidative stress (O2-: −76%) and oxidative stress-mediated N〇-scavenging (nitrotyrosine: −43%) was also reduced in livers from metformin rats. No significant changes in AMPK or eN〇S pathways were observed. CBDL-cirrhosis: Metformin-treated rats also had significantly lower PP than vehicle (17.2±2.3 vs 19.1±2.7 mmHg; p<0.01) without changes in MAP. Prop further reduced PP in both groups of BDL rats, resulting in significantly lower PP in the metformin group (14, 8±1, 7 vs 17, 5±1, 4; p<0.01). Conclusions: Our study demonstrates that in cirrhotic rats metformin administration reduces PP by decreasing HVR; probably due to an amelioration of the structural and functional components of the elevated hepatic resistance of cirrhosis. This effect is additive to that obtained with propranolol. The potential impact of this pharmacological combination, otherwise commonly used in patients with cirrhosis and diabetes, needs further clinical evaluation.

Disclosures:

Jaime Bosch - Advisory Committees or Review Panels: Intercept pharma; Consulting: chiasma, Gilead Science, Norgine, ONO-USA; Grant/Research Support: GOre

Juan Carlos Garcia-Pagan - Grant/Research Support: GORE

The following people have nothing to disclose: Dinesh M. Tripathi, Eva Erice, Jordi Gracia-Sancho, Hector Garcia-Caldero, Shiv K. Sarin

177

Vasopressin la (Vla) Receptor Partial Agonism Increases Sodium Excretion And Reduces Portal Hypertension And Ascites In Cirrhotic Rats

Guillermo Fernández-Varo1, 3, Denise Oro1, Edward Cable4, Vedrana Reichenbach1, Silvia Carvajal1, Bernadino Gonzalez de la Presa1, Kazimierz Wisniewski4, Pere Gines2, Geoffrey Harris4, Wladimiro Jiménez1, 3

1 Biochemistry and Molecular Genetics Service, Hospital Clίnic i Provincial de Barcelona, IDIBAPS, CBERehd, University of Barcelona, Barcelona, Spain; 2Liver Unit, Hospital Clίnic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain; 3Department of Physiology I, University of Barcelona, Barcelona, Spain; 4Ferring Research Institute, San Diego, CA

Background and Aims: Patients and rats with cirrhosis and ascites present with portal hypertension and circulatory dysfunction. Synthetic vasopressin V1a- receptor agonists able to induce systemic and mesenteric vasoconstriction have shown their usefulness in reducing portal pressure (PP) in this condition. We assessed the potential therapeutic value of a new V1a receptor partial agonist with a preferential splachnic vasoconstrictor effect (FE 204038) in rats with cirrhosis with ascites (rCHA). Methods: The hemodynamic effects of continuous administration of cumulative intravenous doses of FE 204038, terlipressin or vehicle were investigated in 28 rCHA. Mean arterial pressure (MAP) and PP were continuously recorded and cardiac output (CO) and systemic vascular resistance (SVR) assessed at 30-min intervals for 90 min. Changes in urine volume (UV) and urinary excretion of sodium, osmolality and creatinine (UNaV, Um〇smV and UCreatV, respectively) were measured in basal conditions and following the administration of twice daily subcutaneous doses of FE 204038 or vehicle to an additional group of 16 rCHA. PP, MAP, CO, SVR and ascites volume (AsV) were also measured at the end of the 6day treatment period. Moreover, in order to establish the rationale for the preferential vasoconstrictor effect of the V1a- agonist on the splanchnic vascular bed, the expression of an array of vasoactive genes was assessed in the thoracic aorta and the mesenteric circulation of 4 control and 7 rCHA animals. Results: Intravenous administration of FE 204038 dose-dependently decreased PP, did not modify MAP and increased SVR. The beneficial effect of FE 204038 on PP was associated with a marked improvement in UV and UNaV during the first day of chronic treatment. At the end of the study, SVR was higher and C〇 and AsV were markedly lower in rCHA treated with the V1a partial agonist than in those receiving vehicle. As anticipated, significant differences in the expression of vasoactive genes between rCHA and control rats were observed. 〇f note however, was that V1a receptor expression in rCH rats was markedly enhanced in the mesenteric vasculature as compared to the thoracic aorta. Conclusions:. The V1a receptor partial agonist FE 204038 increases sodium excretion and reduces portal hypertension and ascites in experimental cirrhosis. These results indicate that V1a receptor partial agonism could be a useful pharmacological treatment in decompensated cirrhotic patients.

Disclosures:

Guillermo Fernández-Varo - Grant/Research Support: Ferring Research Institute Denise Oro - Grant/Research Support: Ferring Research Institute Edward Cable - Employment: Ferring Research Institute Vedrana Reichenbach - Grant/Research Support: Ferring Research Institute

Silvia Carvajal - Grant/Research Support: Ferring Research Institute

Bernadino Gonzalez de la Presa - Grant/Research Support: Ferring Research Institute

Kazimierz Wisniewski - Employment: Ferring Research Institute

Pere Gines - Advisory Committees or Review Panels: Ferring; Grant/Research Support: Sequana Medical, Grifols

Geoffrey Harris - Employment: Ferring Pharmaceuticals

Wladimiro Jiménez - Grant/Research Support: Ferring Research Institute

178

Key role of the enzyme chymase in the hepatic production of angiotensin II and in the development of liver cirrhosis: animal and human data

Giovanni Sansoe1, Manuela Aragno2, Raffaella Mastrocola2, Giulio Mengozzi3, Claudia Paternostro2, Maurizio Parola2

1 Gastroenterology Unit, Gradenigo Hospital, Torino, Italy; 2Dpt. of Clinical and Biological Sciences, University of Torino, Torino, Italy; 3Clinical Biochemistry Laboratory, San Giovanni Battista Hospital, Torino, Italy

Background. Most of renal and cardiac interstitial angiotensin || (Ang-II) is produced locally through non-ACE-dependent pathways, i. e. by chymase and other proteases. In the cirrhotic liver, Ang-II stimulates fibrogenesis, but chymase function is unknown. Aims & Methods. To assess hepatic content, localization, and function of chymase in advanced cirrhosis, we studied five groups of 10 rats: healthy controls (group G1), controls receiving three times a week for 9 weeks the oral chymase inhibitor SF2809E (10 mg/kg b. w.) (G2), rats with cirrhosis induced by 13 weeks of oral CCI4 (g3), rats receiving C C I 4 for 13 weeks but receiving also SF2809E 10 or 20 mg/kg b. w. three times a week between 4th and 13th week of CCI4 treatment (G4 and G5). All rats were then submitted to the assessment of portal pressure, plasma bilirubin and albumin levels, hormonal status, liver tissue content of chymase and Ang- ||,liver immunolocalization of chymase (indirect immunofluorescence staining), liver fibrosis (a-SMA immunohistochemistry, Masson trichrome, and Sirius red staining). Moreover, chymase was investigated by means of immunohistochemistry in resected samples of normal human liver and in livers removed from patients transplanted for cirrhosis and end-stage liver disease. Finally, chymase mRNA transcripts (real time PCR) were evaluated in vitro in human HepG2 cells and in human activated, myofibroblast-like, hepatic stellate cells (HSC/MFs), with and without stimulation by fibrogenic cytokines. Results. G3 rats had liver cirrhosis and ascites. G5 Rats were devoid of ascites and their livers had just fibrotic septa focally linking portal tracts, but no cirrhosis. Compared to G3, in G5 portal pressure, plasma renin activity, bilirubin, and norepinephrine, and liver contents of Ang-II were lower, and plasma albumin higher (all P<0.01). Liver content of chymase was higher in cirrhotic than in control rats (P<0.01). In rat cirrhotic liver, chymase was mainly detected in a-SMA-positive myofibroblasts in fibrotic septa. In human cirrhotic liver, chymase was detected both in parenchymal cells of regenerative nodules and in HSC/MFs of fibrotic septa. In vitro, both HepG2 cells and human HSC/MFs responded to recombinant TGF-β by significantly up-regulating transcription of chymase mRNA. Conclusions. Inhibition of hepatic chymase, which is expressed in both hepatocytes and HSC/MFs of rat and human cirrhotic liver, results in significant decrease in extracellular matrix deposition and portal pressure, and in the improvement of liver function.

Disclosures:

Giovanni Sansoe - Consulting: Shire Pharmaceuticals Ltd., Basingstoke, Hampshire,, UK.

Maurizio Parola - Independent Contractor: Shire Pharmaceutical Ltd, Basingstoke, UK

The following people have nothing to disclose: Manuela Aragno, Raffaella Mastrocola, Giulio Mengozzi, Claudia Patenostro

179

The mechanisms of arterial thinning observed in cirrhotic rats with portal hypertension

Hui-Chun Huang3, 4, Keitaro Tashiro2, 1, Teruo Utsumi1, Yasuko jwakiri1

1 Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT; 2Osaka Medical College, Takatsuki, Japan; 3Medicine/Gastroenterology, Taipei Veterans General Hospital, Taipei, Taiwan; 4National Yang-Ming University, Taipei, Taiwan

[Background] Thinning of arterial walls has been observed in the splanchnic and systemic circulations of cirrhotic rats with portal hypertension. This arterial thinning sustains arterial vasodilation and worsens portal hypertension. The mechanisms leading to arterial thinning remain to be elucidated. [Methods] Male SD rats were exposed to carbon tetrachloride inhalation for 12 weeks to generate cirrhosis with portal hypertension. Age-matched normal rats were used as controls. Hemodynamic measurements were performed. Superior mesenteric arteries (SMAs) were isolated and used for analyses. [Results] Arterial wall thickness of SMAs was significantly decreased in cirrhotic rats with portal hypertension, compared with controls (25% decrease, p<0.0005). Cirrhotic rats exhibited decreased mean arterial pressure (88.0+/-8.6 vs. 121.3+/-6.3 mmHg, p=0.02), increased SMA blood flow (8.2+/-1.6 vs. 3.4+/-0.6 ml/min/100gBW, p=0.03) and decreased blood viscosity. Computational modeling of arterial biomechanics indicated that thinning of SMAs in cirrhotic rats was a consequence of the vessel̉'s adaptation to these hemodynamic changes. In cirrhotic rats with portal hypertension, apoptosis was significantly increased in cells comprising all three compartments of the vessel wall of SMAs: endothelial cells (2.5-fold, p<0.01), smooth muscle cells (4-fold, p<0.01) and adventitia (5-fold, p<0.01). Matrix metalloproteinase-2 (MMP-2) activity was increased 5fold in SMAs of cirrhotic rats (p<0.001). Further, arterial thinning was associated with changes in the levels of proteins important for the maintenance of arterial integrity and function. The SMAs of cirrhotic rats showed a 3-fold decrease in caldesmon (p<0.05), an indicator of the vessel's contractility, and a 3-fold decrease in elastin (p<0.05), a contributor of the vessel's structural integrity. In contrast, collagen type I levels were significantly elevated (2.5-fold, p<0.05), suggesting increased matrix remodeling and a wound-healing response. Finally, the SMAs of cirrhotic rats showed a decrease in CD31 levels in endothelial cell junctions, indicating SMA's diminished flow-sensing ability, since CD31 is essential for a vessel's flowmediated response. [Conclusion] Arterial thinning is a consequence of hemodynamic changes caused by cirrhosis with portal hypertension. It is mechanistically linked to increased apoptosis in the cells constituting the arterial wall, particularly smooth muscle cells. This is accompanied by reduced protein levels necessary for arterial integrity and function, which may contribute to sustained arterial vasodilation in cirrhotic rats with portal hypertension.

Disclosures:

The following people have nothing to disclose: Hui-Chun Huang, Keitaro Tashiro, Teruo Utsumi, Yasuko Iwakiri

180

Intrahepatic Microvascular Thrombosis contributes to Fibrogenesis and Portal Hypertension in Congestive Hepatopathy

Douglas A. Simonetto1, Hui-yin Yang1, Thiago de Assuncao1, Shuchong Pan2, Robert Simari2, Vijay Shah1

1 Gastroenterology Research Unit, Mayo Clinic, Rochester, MN; 2Cardiovascular Diseases, Mayo Clinic, Rochester, MN

Purpose: Congestive hepatopathy (CH), a result of passive congestion of the liver, leads to hepatic cirrhosis. Microvascular thrombosis has been implicated in fibrosis development and may be particularly important in chronic liver congestion. Tissue factor is the main initiator of the extrinsic coagulation cascade, which can be regulated by endogenous Tissue Factor Pathway Inhibitor (TFPI). In this study we demonstrate the potential role of thrombosis in fibrogenesis and the effect of TFPI overexpression and warfarin treatment in mice with congestive hepatopathy. Methods: Partial inferior vena cava ligation (pIVCL) was used to induce liver congestion as we previously described. pIVCL or SHAM surgery was performed in wild-type (WT) and transgenic mice overexpressing TFPI (SM22-TFPI). In some experiments wild-type mice were treated with vehicle or warfarin in drinking water for 6 weeks postoperatively. Portal pressure was measured and animals were sacrificed at 6 weeks after pIVCL or SHAM surgery. Liver sections were stained for H&E and Sirius red. Immunofluorescence for a-smooth muscle actin (αSMA)-a marker of hepatic stellate cell activation, aquaporin-1-a marker of angiogenesis, and fibrinogen-a marker of intravascular thrombosis, was performed. Hydroxyproline level and Western blot for fibrinogen, α-SMA and GAPDH were performed from liver lysates. Results: Fibrosis was significantly increased at 6 weeks after pIVCL compared to SHAM-operated mice, as measured by Sirius red staining (p<0.05) and hydroxyproline assay (p<0.001). Intrahepatic thrombosis was present after pIVCL as evidenced by increased fibrinogen immunostaining in the liver (p=0.0122). SM22-TFPI mice had reduced fibrosis after pIVCL compared to WT mice based on reduced hydroxyproline content and a-SMA expression (p<0.01 and p<0.05, respectively). Fibrinogen level was also reduced in SM22-TFPI mice after pIVCL compared to WT (p<0.05). WT mice administered warfarin after pIVCL exhibited a significant increase in INR compared to vehicle-treated WT mice (p<0.0001). Hydroxyproline content from liver lysates and a-SMA expression, by Western blot and immunofluorescence were significantly reduced in mice receiving warfarin (p<0.001 and p<0.05, respectively) compared with vehicle after pIVCL. Warfarin-treated mice also exhibited a significant reduction of portal pressure (p<0.05) after pIVCL compared to vehicle (p<0.05). Conclusion: Warfarin treatment and TFPI overexpression are associated with reduced liver fibrosis and portal pressure elevation during congestive hepatopathy. These studies highlight the importance of intrahepatic thrombosis during congestive hepatopathy associated fibrosis.

Disclosures:

The following people have nothing to disclose: Douglas A. Simonetto, Hui-yin Yang, Thiago de Assuncao, Shuchong Pan, Robert Simari, Vijay Shah

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