The data presented in this study provide evidence that elevated portal venous pressure following PH can be reduced by a pharmacological approach and improves parenchymal regeneration in mice. Importantly, terlipressin improved hepatocellular proliferation in mice after 80% hepatectomy and increased survival in mice with liver steatosis indicating a therapeutic potential for terlipressin in situations with a high risk of postoperative liver insufficiency. Mechanistically, hepatoprotection from terlipressin may be explained by reduced cellular stress as reflected by decreased levels of p21 and GADD45 and altered cytokine response. Interestingly, terlipressin modulated regeneration but had no effect on liver injury.
An increase in portal venous pressure correlated with the extent of PH and was detected immediately following resection of liver lobes. Minor elevations of portal venous pressure after PH have been shown to be pivotal for the initiation of liver regeneration (20). Conversely, extended liver resections or small-for-size liver transplantations that are associated with a marked increase of portal venous pressure, beyond yet undefined levels, may impair the regenerative capacity of the liver and lead to liver failure (21). To be clinically applicable and to avoid side effects in animals with normal portal venous pressure, the administration of the drug was performed immediately after PH. Therefore, the very first elevation of portal venous pressure was not altered. Despite this delayed injection, a positive effect on liver regeneration and reduced cellular stress after 80% PH has been observed. The reduction of portal venous pressure at 8 hr post-PH reflects early changes of hepatic remodeling and adaptation of the sinusoids to the alterations of pressure and flow in response to PH (22). Thus, maintaining optimal levels of postoperative portal venous pressure seems to be crucial for successful outcomes.
Administration of terlipressin was only protective after 80% PH. Thus, nonsignificant changes in portal venous pressure after 60% PH after treatment with terlipressin are not sufficient to impact on liver regeneration. This finding is supported by increasing evidence that not the size alone but the flow in resected or transplanted livers play an important role in liver regeneration and “small for flow” rather than “small for size” seems to be critical (23).
Our report now shows for the first time that modulation of portal venous pressure by terlipressin is beneficial in the absence of liver cirrhosis. Up to now, vasopressin agonists have been shown to be clinically effective in lowering portal venous pressure in cirrhotic livers and terlipressin has been shown to improve short-term survival of patients with hepatorenal syndrome (24). Interestingly, a reduction of portal venous pressure by terlipressin occurs only after 80% PH that critically impact not just on portal venous pressure but also on splanchnic blood flow and systemic metabolism (25). The impact of physiological release of vasopressin on liver regeneration via Ca2+ signaling has been previously described (5, 26). These reports have shown neuroendocrine secretion of vasopressin from the hypothalamus in response to PH that protects the liver. We now support these mechanistic findings at the target organ and show that pharmacological activation of vasopressin receptors reduces portal venous pressure and is associated with an increase in liver regeneration.
There is little evidence whether terlipressin has direct stimulative potential on hepatocyte proliferation. No effect of terlipressin on hepatocellular proliferation or on portal venous pressure has been observed after minor or standard PH. Thus, the main pathway of inducing hepatocyte proliferation is probably by means of lowering the portal venous pressure and consecutive reduced cellular stress rather than via direct hepatocyte V1a vasopressin receptors.
Terlipressin mainly acts through a vasoconstriction in the splanchnic vessels and therefore reduces the portal flow and portal venous pressure in cirrhosis and consecutive splanchnic vasodilatation. Hypothetically, terlipressin also counteracts a potential hepatic buffer response that may occur post-PH. The doses of terlipressin used in this study were comparable to previous reports in cirrhotic mice with effective reduction of portal pressure mainly after 80% PH and without negative side effects on, for example, intestinal perfusion. In patients undergoing living liver transplantation, terlipressin administration was associated with an effective reduction of portal venous pressure and improvement of renal function without having negative systemic side effects (14).
Hepatic steatosis is a frequent parenchymal liver disease in Western countries. Liver transplantation of livers with modest steatosis is a potential clinical scenario, and it has been shown clinically and experimentally that it is associated with an increased risk for complications (15, 27). There was no mortality after 80% PH, but after 60% PH in steatotic mice. Thus, a blunted response to this critical extent of PH may explain the effect of terlipressin at 60% PH in steatotic mice.
In addition to the hemodynamic changes in response to terlipressin administration, we demonstrated reduced cellular stress (GADD45) and cell cycle inhibition (p21) after 80% PH. Both parameters were already significantly up-regulated in steatotic livers before surgery, which explains the effect already at 60% PH. These results confirm recent studies that demonstrated a critical impact of both p21 and GADD45 on the initiation and the kinetics of liver regeneration (28–30).
Our study also revealed that terlipressin reduced disruptions of hepatic sinusoids, which were associated with extended PH. As previously shown in a transgenic animal model, portal hypertension is associated with a morphological change of sinusoidal fenestrations, and PH is characterized by a disappearance of the sieve-plate arrangement, endothelial fenestration, and dilatation of bile canaliculi (31, 32). The sinusoidal disruption as observed in the present study is potentially associated with an increased intrahepatic blood shunting between portal venous and systemic circulation. Therefore, metabolites of the liver such as bile acids might be found in higher concentrations in the systemic blood circulation because of increased blood shunting after extended PH. It remains unclear if coincidence of reduced bile acids and reduced sinusoidal disruption in response to terlipressin administration is the consequence or the cause of improved outcome for the liver regeneration.
In summary, our results show pharmacological reduction of portal venous pressure with terlipressin leads to an improvement of liver regeneration, maintenance of microstructural hepatic tissue anatomy, a reduction of stress response genes, and, most importantly, a better survival.
MATERIALS AND METHODS
All procedures were carried out in accordance with the Swiss National Institutes of Health guidelines for the care and use of experimental animals, and the experimental protocol received approval by the Animal Care Committee of the Canton of Bern, Switzerland.
Animal Preparation and Experimental Setting
Experiments were performed on 8- to 12-week-old adult wild-type C57BL/6J mice obtained from Harlan Animal Research Laboratories (Boxmeer, The Netherlands). The animals were housed in the University Animal Facility with a 12 hr light/dark cycle at 22°C, and fed either with normal chow (fat: 4.5%, protein: 18.5%, fibers: 4.5%) or with a high-fat diet (Ssniff, Soest, Germany) for 6 weeks (HFD—fat: 16.6% [50% lard and 50% cacao butter], protein: 15.7%, fibers: 4.5%). Surgical procedures were performed under general anesthesia using isoflurane (Nicholas Piramal (I) Limited, London, UK). During the procedure, the intestine was rinsed with saline to avoid drying-out and resuscitation with saline of the intraoperative fluid loss was given at the end of the operation. Postoperative analgesia with buprenorphin (Reckitt Benckiser AG, Switzerland) was regularly administered subcutaneously during the postoperative course. At the time of sacrifice, mice were anesthetized with isoflurane inhalation, lethal blood samples were taken from the inferior vena cava, and livers were collected for further analyses.
Partial Hepatectomy Model and Treatment With Terlipressin
The details of the PH model in mice were previously published (33). In brief, resection of the median and left lobe equals a standard 60% PH, whereas for minor PH (30%) only the left superior lobe was excised. For extended PH (80%), the right inferior lobe was resected in addition to the standard 60% PH. Directly after performing PH and before closure of the abdomen, the animals received terlipressin (0.05 μg/g mouse, Glypressin; Ferring Pharmaceuticals, Switzerland) or the vehicle phosphate-buffered saline (PBS; Gibco, Invitrogen, Auckland, New Zealand) as control (5 μL/g mouse) intravenously injected into the inferior vena cava. After 8 hr, this administration was repeated by injections into the femoral vein under general anesthesia with isoflurane. Two hours before sacrifice, 50 mg/kg mouse of bromodeoxyuridine (BrdU, #16880; Fluka, Sigma Aldrich, Switzerland) was injected intraperitoneally. For each extent of PH with or without HFD, there was a treatment and control group of each 8 to 12 animals per group and time point.
Measurement of Portal Venous Pressure
Portal venous pressure measurements were terminal experiments and were not used for survival analysis. After laparotomy the ileocecal vein was identified and a 26 G catheter was inserted and the catheter tip placed close to the portal confluence and finally fixed with histoacryl. Baseline portal venous pressure was measured by a computer-based program (FlowChart 7.0; AD Instruments, Spechbach, Germany) during 1 min. Portal venous pressure was measured at specific time points before and after (5, 10, 15, 30 min and 8 hr) PH and always required anesthesia and laparotomy.
Histological and Immunohistochemical Analysis of the Liver
Fresh liver tissue was fixed overnight in 4% paraformaldehyde in PBS and embedded in paraffin. Liver sections (5 μm) were deparaffinized with xylol and counterstained with hematoxylin-eosin and with reticulin stain (Ag) for histological assessment. For immunohistochemistry, paraffin-embedded tissue sections were dried for 24 hr, deparaffinized, and rehydrated, followed by blocking of endogenous peroxidase with 3% H2O2 (Sigma H-1009; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in PBS. Antigen was retrieved by heating the slides for 10 min. Diluted biotinylated anti-BrdU antibody (BrdU In-Situ Detection Kit [#550803]; BD Biosciences, San Diego, CA) or anti-Ki-67 antibody (Ki-67 clone-Tec 3 [#M7249]; Dako, Glostrup, Denmark) were then applied and slides incubated for 1 hr at 48°C in a humidified chamber. Then, ready-to-use streptavidin horseradish peroxidase complex (#550803; BD Biosciences) was added, followed by a brief incubation with 3,3′-diaminobenzidine substrate (DAB, D-4293, Sigmafast; Sigma-Aldrich Chemie GmbH). The tissue sections were counterstained in hematoxylin. Finally, BrdU and Ki-67-positive cells on representative slides were counted on four high-power fields for each animal.
For CD31 staining, 2- to 3-μm liver sections were dewaxed, rehydrated, and pretreated by boiling in 10 mM citrate buffer, pH 6.0, in a microwave oven. Endogenous peroxidase activity was blocked with 0.5% H2O2 and 0.1% NaN3. Sections were then (and following all subsequent steps) washed in Tris-buffered saline (TBS) and incubated for 60 min at room temperature with a rat-anti-mouse CD31 antibody (clone MEC 14.7; Abcam, Cambridge, UK), diluted 1:100 in TBS with 0.5% casein and 5% normal goat serum. In negative controls, the primary antibody was replaced with antibody dilution buffer. A rabbit-anti-rat Ig secondary antibody (Dako) was then applied, followed by a polymer-based visualization system (Envision+; Dako), each for 30 min. Finally, sections were developed in 0.02% 3,3′-diaminobenzidine (Sigma, St. Louis, MO) with 0.01% H2O2, counterstained with hematoxylin, and mounted. Known positive controls were stained in parallel with each series.
To visualize liver steatosis, cryosections of 8-μm thickness from liver tissue frozen immediately after removal at −120°C were fixed in 4% paraformaldehyde. After dipping in 70% ethanol, sections were placed in Sudan Black solution for 20 min and rinsed afterwards in 70% ethanol and H2O. Finally, sections were stained with nuclear fast red vector (H-3403; Sigma-Aldrich Chemie GmbH).
Measurement of Liver Injury
ALT and AST levels were measured by a photometric UV test measuring the oxidation of NADH to NAD (Roche Modular P800). Bile acids levels in the serum after 48 hr post-PH were measured enzymatically using a Mira plus chemistry analyzer (Roche Diagnostics) with reagents from Trinity Biotech as previously described (34). Briefly, during oxidation of the bile acids to 3-oxo bile acids, equimolar quantity of NAD is reduced to NADH, which subsequently is oxidized to NAD. Nitroblue tetrazolium salt is then reduced to formazan, which has an absorbance maximum at 530 nm. The concentration of bile acids in the sample is directly proportional to the intensity of the produced color.
Quantification of hepatic hydroxyproline was measured as previously described (35). Briefly, after hydrolyzation of frozen liver tissue in 6 M HCl at 100°C for 16 hr, 50 μL was incubated with chloramine T (2.5 mM) for 5 min and Ehrlich reagent (410 mM) for 30 min at 60°C. Finally, absorption at 560 nm was measured and results expressed as micrograms per gram of wet liver tissue.
Quantitative TaqMan PCR
RNA was isolated from snap-frozen liver samples by Trizol Reagent according to the manufacturer’s protocol (Life Technologies). cDNA was synthesized by using Omniscript RT kit 200 (cat. no. 205113; Qiagen) and mRNA analyzed by RT-qPCR (ABI 7900, SDS 2.3 software). Primers and probes sequences were ready-to-use kits from Applied Biosystems (Rotkreutz, Switzerland), reference gene control (RG) beta actin (#Mm00607939_s1), IL-6 (#Mm00446190_m1), TNF-α (#Mm00443258_m1), GADD45 (#Mm00432802_m1), and CDKN1a (=p21) (#Mm00432448_m1). Relative changes in mRNA were calculated with the ΔΔΔCt method. Ct values of target gene expression (TG) was calculated relative to a RG using the following formula ΔCtTG=CtTG−CtRG. Experimental groups (TG) were normalized to control group (CG): ΔΔCt=ΔCtTG−ΔCtCG, fold increase=2−ΔΔCt.
Transmission Electron Microscopy
To assess a putative effect of terlipressin treatment on hepatic sinusoids, respective cellular structures liver tissues were imaged by transmission electron microscopy. For that, liver samples were fixed in 5% glutaraldehyde in PBS, postfixed in osmium tetroxide, stained en bloc in uranyl acetate, dehydrated, and embedded in epoxy resin. Ultrathin sections (50–100 nm) were analyzed with an EM12 transmission electron microscope (Philips, Eindhoven, Netherlands) equipped with a digital camera (Morada; SIS, Münster, Germany). Sinusoidal structures were analyzed blinded by an experienced liver pathologist (M.M.) for ultrastructural changes.
All data are expressed as geometric means±standard deviations unless stated otherwise. For statistical analysis, Student t test or two-way ANOVA test was used. Statistical analysis was performed with GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA). Data with P less than 0.05 was considered as statistically significant.
The authors would like to thank Anita Born and Cynthia Furer for technical support throughout the project.
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Liver regeneration; Portal hypertension; Partial hepatectomy; Liver steatosis; Terlipressin
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