Liver International ISSN 1478-3223
CIRRHOSIS AND LIVER FAILURE
Role of gap junctions modulating hepatic vascular tone in cirrhosis ndez-Guerra1,2,*, Yanira Gonza lez-Me ndez1,*, Zaida A. de Ganzo1, Eduardo Salido3, Manuel Herna 4 1 n , Beatriz Abrante , Antonio M. Malago n5, Jaime Bosch3 and Enrique Quintero1,2 Juan C. Garcıa-Paga 1 2 3 4 5
Liver Unit, University Hospital of the Canary Islands, Tenerife, Spain Department of Internal Medicine, University of La Laguna, Tenerife, Spain Department of Pathology, University Hospital of the Canary Islands, Tenerife, Spain Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clinic, IDIBAPS and CIBERehd, Barcelona, Spain Surgery Department, University Hospital of the Canary Islands, Tenerife, Spain
Keywords chronic liver disease – cirrhosis – hepatic circulation – hepatic haemodynamics – portal hypertension
Correspondence Manuel Hern andez-Guerra, Liver Unit, University Hospital of the Canary Islands, s/n Ofra, 38320, La Laguna, Santa Cruz de Tenerife, Spain Tel/Fax: +0034 922 678557 e-mail: [email protected] Received 6 August 2013 Accepted 11 December 2013 DOI:10.1111/liv.12446 Liver Int. 2014: 34: 859–868
Abstract Background & Aims: Gap junctions are formed by connexins (Cx), a family of proteins that couple endothelial and smooth muscle cells in systemic vessels. In this context, Cx allow the transmission of signals modulating vascular tone. Recently, vascular Cx have been observed in liver cells implicated in liver blood flow regulation. Here, we investigated the role of Cx in the regulation of intrahepatic vascular tone in cirrhosis. Methods: Livers of Sprague– Dawley control and cirrhotic (common bile duct ligation-CBDL and CCl4) rats were perfused, and concentration–effect curves in response to acetylcholine (ACh) precontracted with methoxamine were obtained in the presence of the specific Cx inhibitor 18-alpha-glycyrrhetinic acid or vehicle. Cx expression was assessed by immunofluorescence, western blot and reversetranscription polymerase chain reaction in liver tissue, hepatic stellate cells, sinusoidal endothelial cells and hepatocytes isolated from control and cirrhotic rat livers. Cx protein expression was also determined in cirrhotic human tissue. Results: Gap junction blockade markedly attenuated relaxation of hepatic vasculature in response to ACh in control (maximal relaxation, 55 ± 10.5% vs. 95.3 ± 10% with vehicle; P < 0.01) and CBDL rats (50.9 ± 18.5% vs. 18.7 ± 5.5% with vehicle; P = 0.01). Livers from CBDL rats and patients with cirrhosis exhibited Cx overexpression. By contrast, CCl4-cirrhotic rats did not show attenuated relaxation of hepatic vasculature after blockade and Cx expression was significantly lower than in controls. Conclusions: Gap junctions may contribute to modulating portal pressure and intrahepatic vascular relaxation. Liver gap junctions may represent a new therapeutic target in cirrhotic portal hypertension.
A wide variety of pharmacodynamic substances released by endothelial cells, such as nitric oxide, arachidonic acid metabolites and endothelium-derived hyperpolarizing factors, help regulate vascular tone by inducing hyperpolarization and relaxation of vascular smooth muscle cells (1). In addition to this paracrine signalling, endothelium and vascular smooth muscle cells communicate via gap junctions in arteries and veins (2). These membrane structures are channels believed to form a web of interconnected cells that facilitate the spread of signals and cell-to-cell synchronization, allowing the free transfer of ions and molecules, including endothelium-derived hyperpolarizing factors, which may modify vessel diameter (3).
*Both authors contributed equally to this study.
Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Gap junction pores are formed by docking of two hexameric complexes of proteins called connexins (Cx), which are named according to their molecular weight (4). Characteristically, Cx43 and Cx40 are co-expressed connecting endothelial cells and endothelial-smooth muscle cells (myoendothelial gap junctions) in the vessels. Intercellular coupling and direct signalling through these subtypes of Cx has emerged as a novel pathway regulating vascular tone in systemic vessels (5). In fact, pharmacological inhibition of Cx attenuates endothelium-dependent vasorelaxation (6, 7). More recently, a study reported a contributing role of gap junctions in splanchnic vasodilation of cirrhotic rats with portal hypertension (8). In the liver, several Cx including Cx36, Cx32 and Cx26 have been found involved in hepatic homeostasis (9). In addition, Cx43 and Cx40 are also found particu-
859
Hernandez-Guerra et al.
Connexins and intrahepatic vascular tone
larly in cells involved in regulating hepatic vascular tone, such as sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC) and endothelial cells of portal venules and arteries (10, 11). However, the possible role of gap junctions in modulating the physiological tone of intrahepatic circulation has not been explored, and whether this mechanism contributes to increase hepatic vascular tone in cirrhotic portal hypertension is unknown. This study addressed these issues. Material and methods Patients
Non-tumoral liver tissue from cirrhotic and non-cirrhotic patients was obtained during surgery for tumour resection after informed consent, according to the protocol approved by our hospital Clinical Research Ethics Committee in accordance with the Helsinki Declaration of 1975. Animals
We evaluated the functional role of Cx in control rats and two different models of cirrhosis in the rat: common bile duct ligation (CBDL) (12) and carbon tetrachloride (CCl4) (13). All rats were fed standard rat chow and provided drinking water ad libitum, and kept in environmentally controlled animal facilities. CBDL-cirrhotic animals
Male Sprague–Dawley rats weighing 230–280 g underwent bile duct ligation as described before. Briefly, rats were anaesthetized with ketamine (100 mg/kg body weight, Imalgene 1000; Merial, Lyon, France) plus midazolam (5 mg/kg body weight, Laboratorio Reig Jofre, S.A., Barcelona, Spain). Then, the abdomen was opened through a 2 cm midline incision and the common bile duct was exposed, ligated twice with 3-0 silk and resected between ligatures. The abdomen was closed and the animals allowed to recover. All CBDL animals were treated with antibiotics (100 mg/kg body weight, Amoxicillin/Clavulanic acid; Combino Pharm, S.L. Barcelona, Spain), buprenorphine (0.05 mg/kg body weight; Schering-Plough, Hertfordshire, UK) the day of surgery and thereafter during 72 h. Vitamin K was administrated weekly (0.05 mg/kg body weight; Roche, Madrid, Spain). After 5 days, animals without dark urine were discarded or otherwise continued until day 28 after ligation. CCl4-cirrhotic animals
Rats weighing 300–350 g underwent inhalation exposure to CCl4. Phenobarbital (0.3 g/L) was added to the drinking water 1 week before inhalation to shorten the time required to induce cirrhosis (approximately
860
12–17 weeks). When cirrhotic rats developed ascites, administration of phenobarbital was stopped, and subsequent experiments were performed 1 week later. Control animals received only phenobarbital. The experiments were approved by the University of La Laguna Ethics Committee for the Care and Use of Laboratory Animals and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1985). Isolated liver perfusion system
All rats were anaesthetized as for CBDL procedure. Then a flow-controlled perfusion system was used (14). Briefly, livers were perfused with Krebs’ solution in a closed circuit with 150 ml at a constant flow rate of 35 ml/min. An ultrasonic flow probe (T201; Transonic System, Ithaca, NY, USA) and a pressure transducer were placed in line, immediately ahead of the portal inlet cannula, to continuously monitor flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet cannula to measure outflow pressure. The flow probe and the two pressure transducers were connected to a Power Lab (4SP) linked to a computer using the Chart V5.5.6 for Windows software (ADInstruments, Mountain View, LA, USA). Mean portal flow, inflow and outflow pressures were sampled and recorded every second and exported to data management software for analysis. The perfused rat liver preparation was allowed to stabilize for 20 min before the vasoactive substances were added. The criteria of liver viability included gross appearance, stable perfusion pressure, bile production >0.4 ml/min/g liver (except for CBDL rats) and a stable buffer pH (7.4 ± 0.1) during the initial stabilization period. Hemodynamic measurements
Baseline portal pressure (PP) during perfusion was recorded before and 10 min after adding to the reservoir either the vehicle (DMSO, 0.01%) or the gap junction blocker 18-alpha-glycyrrhetinic acid (GLY) at increasing concentrations (20, 50 and 100 lM). GLY is a synthetic structural analogue of glycyrrhicic acid, belonging to the pentacyclic triterpenes family, and this compound inhibits all Cx including those specifically involved in vascular functions (Cx43 and Cx40) and thus gap junctions, without exerting any intrinsic vasopressor effect or any interference with nitric oxide activity (15–17). Intrahepatic microcirculation was preconstricted with the alpha-1-adrenergic agonist methoxamine (Mtx, 10 4 M). Then, dose–response curves to cumulative doses of acetylcholine (ACh, 10 8, 10 7 and 10 6 M) were evaluated. ACh concentration increased by 1 log unit every 1.5 min. Response to cumulative doses of ACh was calculated as a percentage change in PP (14). Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Hern andez-Guerra et al.
Western blot analysis of Cx43 and Cx40
Protein expression of Cx43 and Cx40 from control and cirrhotic human and rat livers (rat heart and aorta were used as a reference protein expression in rat samples) was assessed by Western blot. Freshly isolated tissue was homogenized, at 100 mg tissue per ml RIPA lysis buffer, containing 0.15 M NaCl; 0.01% NP40; 1% SDS; 0.5% DOC; 0.05 M TrisHCl pH 7.5, centrifuged at 16 300 g for 10 min, and conserved at 80°C until analyzed. Protein concentration was assessed by bicinchoninic acid assay, and 50 lg of protein were denatured in Laemmli’s buffer, separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were subsequently blocked for 3 h with phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBS-T), 5% (wt/vol) non-fat dry milk, and probed with either rabbit anti-rat Cx 43 antibody (Sigma, Madrid, Spain) diluted 1:1000, rabbit anti-rat C 940 (Chemicon, Millipore, UK) antibody diluted 1:500, or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody diluted 1:1000 (Santa Cruz Biotechnology, Dallas, TX, USA) in PBS-T, 2 mM azide, overnight at 4°C. After 3 washes in PBS-T, 5 min each, the membranes were incubated with HRP-conjugated anti-rabbit IgG (Jackson Immunoresearch, Philadelphia, PA, USA), diluted 1:10 000 in PBS-T, for 30 min at room temperature. Next, blots were washed in PBS-T again (3 changes, 5 min each), and a chemiluminiscence signal was developed with luminol and H2O2 (Pierce, Rockford, IL, USA). Final X-ray films were exposed for 5 and 20 min, developed, fixed and scanned. Densitometry of digital images was carried out with Melanie v.6 software. Quantitative densitometric values for Cx signals were shown and displayed as histograms. GAPDH bands were used as internal loading control. mRNA expression of Cx43 and Cx40
Samples were collected and immediately homogenized in solution D containing guanidium thiocyanate. Total RNA was isolated by the Chomczynski method (18). The purity and concentration of RNA was determined by Nanodrop 2000 (Thermo-Fisher, Wilmington, MA, USA). The quantification of relative mRNA abundance was carried out using quantitative PCR (qPCR) and SYBR Green detection method. Total RNA (5 lg) was reverse transcribed using a reverse transcription kit (Improm II; Promega, Madison, WI, USA), according to the manufacturer’s protocol, and the genes of interest were PCR amplified using gene-specific primers described in Table 1, using SYBR green master mix (Bio-Rad, Hercules, CA, USA) and a real-time PCR machine (Bio-Rad). The resulting increase in fluorescence during the PCR reaction was detected in the iQ5 system (Bio-Rad) and the cycle at which the fluorescence intensity reached the threshold (Ct) was used to compare each
Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Connexins and intrahepatic vascular tone
Table 1. Primers/control Cx rat genes used for real-time quantitative reverse transcriptase-polymerase chain reaction Gene Cx 43 Forward Reverse Cx 40 Forward Reverse RNA 18S Forward Reverse Beta-actin Forward Reverse
Primer sequence 5′-TGGGGGAAAGGCGTGAGGAAAG-3′ 5′-CACCTTCCCTCCAGCGGTGG-3′ 5′-CGGAGGAAAGGAAGCAGAAGGCTCA-3′ 5′-GAGCCAGACCTTGCCGATGACC-3′ 5′-GGCTACCACATCCAAGGAA-3′ 5′-GCTGGAATTACCGCGGCT-3′ 5′-AACGAGCGGTTCCGATGCC-3′ 5′-GGACAGTGAGGCCAGGATGGA-3′
gene with the reference genes (RNA18S or Betaactin). The data were analyzed using the qBASE software. All experiments were performed in duplicate, and the mean values were calculated. Temperature profiles included an initial stage at 95°C for 5 min, followed by 40 cycles of amplification at 94°C for 20 s, 60°C for 40 s and 72°C for 30 s. Melt curve analysis was performed by ramping products from 72 to 95°C, acquiring fluorescence readings for each 0.5°C change. The relative quantification of each gene was performed using the comparative method (ΔΔCt) using the expression of control genes (19). mRNA gene expression levels were reported as fold changes relative to the sample with the lowest mean Cx expression, used as a relative calibrator (arbitrarily set at 1). Immunostaining for Cx43 and Cx40
Control rats (n = 3) were anaesthetized as previously detailed and liver tissues were cryopreserved in optimal cutting temperature compound, and snap frozen in liquid nitrogen. Liver sections were cryosectionned at 8 lm, fixed and rehydrated in PBS for 10 min. Permeabilization was performed in PBS containing 0.1% (vol/vol) Triton X-100, 4% goat serum for 1 h at room temperature. The sections were incubated overnight with rabbit polyclonal antibodies raised against Cx43 (Sigma) dilution 1/100 and Cx40 (Alpha Diagnostics, San Antonio, TX, USA) dilution 1/25. After five, 3 min each, PBS rinses, sections were incubated with biotinylated goat anti-rabbit (Jackson Immunoresearch), 1/200 in PBS for 2 h at room temperature. Next, five, 3 min each, PBS rinses were performed and the sections were incubated with Cy2conjugated streptavidin (Jackson Immunoresearch), dilution 1/1000 1 h, in the dark. Slides were washed again and mounted with PBS-glycerol. Finally, slides were photographed and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan) with a 940-20-10 epifluorescence objective.
861
Hernandez-Guerra et al.
Connexins and intrahepatic vascular tone
Liver cell isolation Parenchymal cells (Hepatocytes)
Control (n = 3) and CCl4-cirrhotic (n = 3) rats were anaesthetized as previously described and hepatocytes were isolated by in situ collagenase perfusion through the portal vein, as described by Munthe-Kaas et al. (20) with minor modifications (21). Briefly, livers were perfused for 10 min at a flow rate of 20 ml/min at 37°C with Hanks’ balanced salt solution. For cell isolations from CH animals, the concentration of collagenase was increased by 25%. The cells were passed through nylon filters (100 lm; Becton-Dickinson Labware, Franklin Lakes, NJ, USA), collected in cold Krebs buffer, and centrifuged at 50g for 3 min. The obtained pellet contains the hepatocytes, whereas the supernatant is enriched in non-parenchymal cells. Sinusoidal endothelial cells
Sinusoidal endothelial cells were obtained from the supernatant of control and CCl4-cirrhotic rat livers (21). Briefly, supernatant was centrifuged at 800g for 10 min at 4°C, and the obtained pellet was resuspended in Dulbecco’s PBS and centrifuged at 800g for 20 min through a 25–50% Percoll gradient at room temperature. The interface of the gradient containing Kupffer cells and SEC was seeded on 37-mm tissue culture plates and incubated at 37°C for 1 h. Cell monolayers adhered to the dishes were characterized as Kupffer cells and the non-attached cells as SEC. Hepatic stellate cells
Hepatic stellate cells were isolated from control (n = 3) and CCl4-cirrhotic (n = 3) rat livers as described above. After perfusion, the resultant digested liver was excised, and in vitro digestion was performed as previously described (21). Cells were grown in Iscove’s modified Dulbecco’s medium, and kept until experimentation; they were highly viable and 95% pure. Drugs and reagents
Glycyrrhetinic, Mtx and ACh were purchased from Sigma (Tres Cantos, Madrid, Spain), mouse anti-RECA monoclonal antibody from Serotec (Oxford, UK), collagenase A from Roche Diagnostics (Mannheim, Germany), Percoll from Amersham Biosciences (Uppsala, Sweden), and RPMI medium and culture complements were from Biological Industries (Kibbutz Beit Haemek, Israel). Statistical analysis
Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). All data are reported as
862
mean ± SEM. Comparisons between groups were performed with Student’s t-test for unpaired data or the Mann–Whitney t-test when adequate. Differences with a P value
CIRRHOSIS AND LIVER FAILURE
Role of gap junctions modulating hepatic vascular tone in cirrhosis ndez-Guerra1,2,*, Yanira Gonza lez-Me ndez1,*, Zaida A. de Ganzo1, Eduardo Salido3, Manuel Herna 4 1 n , Beatriz Abrante , Antonio M. Malago n5, Jaime Bosch3 and Enrique Quintero1,2 Juan C. Garcıa-Paga 1 2 3 4 5
Liver Unit, University Hospital of the Canary Islands, Tenerife, Spain Department of Internal Medicine, University of La Laguna, Tenerife, Spain Department of Pathology, University Hospital of the Canary Islands, Tenerife, Spain Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clinic, IDIBAPS and CIBERehd, Barcelona, Spain Surgery Department, University Hospital of the Canary Islands, Tenerife, Spain
Keywords chronic liver disease – cirrhosis – hepatic circulation – hepatic haemodynamics – portal hypertension
Correspondence Manuel Hern andez-Guerra, Liver Unit, University Hospital of the Canary Islands, s/n Ofra, 38320, La Laguna, Santa Cruz de Tenerife, Spain Tel/Fax: +0034 922 678557 e-mail: [email protected] Received 6 August 2013 Accepted 11 December 2013 DOI:10.1111/liv.12446 Liver Int. 2014: 34: 859–868
Abstract Background & Aims: Gap junctions are formed by connexins (Cx), a family of proteins that couple endothelial and smooth muscle cells in systemic vessels. In this context, Cx allow the transmission of signals modulating vascular tone. Recently, vascular Cx have been observed in liver cells implicated in liver blood flow regulation. Here, we investigated the role of Cx in the regulation of intrahepatic vascular tone in cirrhosis. Methods: Livers of Sprague– Dawley control and cirrhotic (common bile duct ligation-CBDL and CCl4) rats were perfused, and concentration–effect curves in response to acetylcholine (ACh) precontracted with methoxamine were obtained in the presence of the specific Cx inhibitor 18-alpha-glycyrrhetinic acid or vehicle. Cx expression was assessed by immunofluorescence, western blot and reversetranscription polymerase chain reaction in liver tissue, hepatic stellate cells, sinusoidal endothelial cells and hepatocytes isolated from control and cirrhotic rat livers. Cx protein expression was also determined in cirrhotic human tissue. Results: Gap junction blockade markedly attenuated relaxation of hepatic vasculature in response to ACh in control (maximal relaxation, 55 ± 10.5% vs. 95.3 ± 10% with vehicle; P < 0.01) and CBDL rats (50.9 ± 18.5% vs. 18.7 ± 5.5% with vehicle; P = 0.01). Livers from CBDL rats and patients with cirrhosis exhibited Cx overexpression. By contrast, CCl4-cirrhotic rats did not show attenuated relaxation of hepatic vasculature after blockade and Cx expression was significantly lower than in controls. Conclusions: Gap junctions may contribute to modulating portal pressure and intrahepatic vascular relaxation. Liver gap junctions may represent a new therapeutic target in cirrhotic portal hypertension.
A wide variety of pharmacodynamic substances released by endothelial cells, such as nitric oxide, arachidonic acid metabolites and endothelium-derived hyperpolarizing factors, help regulate vascular tone by inducing hyperpolarization and relaxation of vascular smooth muscle cells (1). In addition to this paracrine signalling, endothelium and vascular smooth muscle cells communicate via gap junctions in arteries and veins (2). These membrane structures are channels believed to form a web of interconnected cells that facilitate the spread of signals and cell-to-cell synchronization, allowing the free transfer of ions and molecules, including endothelium-derived hyperpolarizing factors, which may modify vessel diameter (3).
*Both authors contributed equally to this study.
Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Gap junction pores are formed by docking of two hexameric complexes of proteins called connexins (Cx), which are named according to their molecular weight (4). Characteristically, Cx43 and Cx40 are co-expressed connecting endothelial cells and endothelial-smooth muscle cells (myoendothelial gap junctions) in the vessels. Intercellular coupling and direct signalling through these subtypes of Cx has emerged as a novel pathway regulating vascular tone in systemic vessels (5). In fact, pharmacological inhibition of Cx attenuates endothelium-dependent vasorelaxation (6, 7). More recently, a study reported a contributing role of gap junctions in splanchnic vasodilation of cirrhotic rats with portal hypertension (8). In the liver, several Cx including Cx36, Cx32 and Cx26 have been found involved in hepatic homeostasis (9). In addition, Cx43 and Cx40 are also found particu-
859
Hernandez-Guerra et al.
Connexins and intrahepatic vascular tone
larly in cells involved in regulating hepatic vascular tone, such as sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC) and endothelial cells of portal venules and arteries (10, 11). However, the possible role of gap junctions in modulating the physiological tone of intrahepatic circulation has not been explored, and whether this mechanism contributes to increase hepatic vascular tone in cirrhotic portal hypertension is unknown. This study addressed these issues. Material and methods Patients
Non-tumoral liver tissue from cirrhotic and non-cirrhotic patients was obtained during surgery for tumour resection after informed consent, according to the protocol approved by our hospital Clinical Research Ethics Committee in accordance with the Helsinki Declaration of 1975. Animals
We evaluated the functional role of Cx in control rats and two different models of cirrhosis in the rat: common bile duct ligation (CBDL) (12) and carbon tetrachloride (CCl4) (13). All rats were fed standard rat chow and provided drinking water ad libitum, and kept in environmentally controlled animal facilities. CBDL-cirrhotic animals
Male Sprague–Dawley rats weighing 230–280 g underwent bile duct ligation as described before. Briefly, rats were anaesthetized with ketamine (100 mg/kg body weight, Imalgene 1000; Merial, Lyon, France) plus midazolam (5 mg/kg body weight, Laboratorio Reig Jofre, S.A., Barcelona, Spain). Then, the abdomen was opened through a 2 cm midline incision and the common bile duct was exposed, ligated twice with 3-0 silk and resected between ligatures. The abdomen was closed and the animals allowed to recover. All CBDL animals were treated with antibiotics (100 mg/kg body weight, Amoxicillin/Clavulanic acid; Combino Pharm, S.L. Barcelona, Spain), buprenorphine (0.05 mg/kg body weight; Schering-Plough, Hertfordshire, UK) the day of surgery and thereafter during 72 h. Vitamin K was administrated weekly (0.05 mg/kg body weight; Roche, Madrid, Spain). After 5 days, animals without dark urine were discarded or otherwise continued until day 28 after ligation. CCl4-cirrhotic animals
Rats weighing 300–350 g underwent inhalation exposure to CCl4. Phenobarbital (0.3 g/L) was added to the drinking water 1 week before inhalation to shorten the time required to induce cirrhosis (approximately
860
12–17 weeks). When cirrhotic rats developed ascites, administration of phenobarbital was stopped, and subsequent experiments were performed 1 week later. Control animals received only phenobarbital. The experiments were approved by the University of La Laguna Ethics Committee for the Care and Use of Laboratory Animals and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1985). Isolated liver perfusion system
All rats were anaesthetized as for CBDL procedure. Then a flow-controlled perfusion system was used (14). Briefly, livers were perfused with Krebs’ solution in a closed circuit with 150 ml at a constant flow rate of 35 ml/min. An ultrasonic flow probe (T201; Transonic System, Ithaca, NY, USA) and a pressure transducer were placed in line, immediately ahead of the portal inlet cannula, to continuously monitor flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet cannula to measure outflow pressure. The flow probe and the two pressure transducers were connected to a Power Lab (4SP) linked to a computer using the Chart V5.5.6 for Windows software (ADInstruments, Mountain View, LA, USA). Mean portal flow, inflow and outflow pressures were sampled and recorded every second and exported to data management software for analysis. The perfused rat liver preparation was allowed to stabilize for 20 min before the vasoactive substances were added. The criteria of liver viability included gross appearance, stable perfusion pressure, bile production >0.4 ml/min/g liver (except for CBDL rats) and a stable buffer pH (7.4 ± 0.1) during the initial stabilization period. Hemodynamic measurements
Baseline portal pressure (PP) during perfusion was recorded before and 10 min after adding to the reservoir either the vehicle (DMSO, 0.01%) or the gap junction blocker 18-alpha-glycyrrhetinic acid (GLY) at increasing concentrations (20, 50 and 100 lM). GLY is a synthetic structural analogue of glycyrrhicic acid, belonging to the pentacyclic triterpenes family, and this compound inhibits all Cx including those specifically involved in vascular functions (Cx43 and Cx40) and thus gap junctions, without exerting any intrinsic vasopressor effect or any interference with nitric oxide activity (15–17). Intrahepatic microcirculation was preconstricted with the alpha-1-adrenergic agonist methoxamine (Mtx, 10 4 M). Then, dose–response curves to cumulative doses of acetylcholine (ACh, 10 8, 10 7 and 10 6 M) were evaluated. ACh concentration increased by 1 log unit every 1.5 min. Response to cumulative doses of ACh was calculated as a percentage change in PP (14). Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Hern andez-Guerra et al.
Western blot analysis of Cx43 and Cx40
Protein expression of Cx43 and Cx40 from control and cirrhotic human and rat livers (rat heart and aorta were used as a reference protein expression in rat samples) was assessed by Western blot. Freshly isolated tissue was homogenized, at 100 mg tissue per ml RIPA lysis buffer, containing 0.15 M NaCl; 0.01% NP40; 1% SDS; 0.5% DOC; 0.05 M TrisHCl pH 7.5, centrifuged at 16 300 g for 10 min, and conserved at 80°C until analyzed. Protein concentration was assessed by bicinchoninic acid assay, and 50 lg of protein were denatured in Laemmli’s buffer, separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were subsequently blocked for 3 h with phosphate-buffered saline containing 0.1% (v/v) Tween 20 (PBS-T), 5% (wt/vol) non-fat dry milk, and probed with either rabbit anti-rat Cx 43 antibody (Sigma, Madrid, Spain) diluted 1:1000, rabbit anti-rat C 940 (Chemicon, Millipore, UK) antibody diluted 1:500, or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody diluted 1:1000 (Santa Cruz Biotechnology, Dallas, TX, USA) in PBS-T, 2 mM azide, overnight at 4°C. After 3 washes in PBS-T, 5 min each, the membranes were incubated with HRP-conjugated anti-rabbit IgG (Jackson Immunoresearch, Philadelphia, PA, USA), diluted 1:10 000 in PBS-T, for 30 min at room temperature. Next, blots were washed in PBS-T again (3 changes, 5 min each), and a chemiluminiscence signal was developed with luminol and H2O2 (Pierce, Rockford, IL, USA). Final X-ray films were exposed for 5 and 20 min, developed, fixed and scanned. Densitometry of digital images was carried out with Melanie v.6 software. Quantitative densitometric values for Cx signals were shown and displayed as histograms. GAPDH bands were used as internal loading control. mRNA expression of Cx43 and Cx40
Samples were collected and immediately homogenized in solution D containing guanidium thiocyanate. Total RNA was isolated by the Chomczynski method (18). The purity and concentration of RNA was determined by Nanodrop 2000 (Thermo-Fisher, Wilmington, MA, USA). The quantification of relative mRNA abundance was carried out using quantitative PCR (qPCR) and SYBR Green detection method. Total RNA (5 lg) was reverse transcribed using a reverse transcription kit (Improm II; Promega, Madison, WI, USA), according to the manufacturer’s protocol, and the genes of interest were PCR amplified using gene-specific primers described in Table 1, using SYBR green master mix (Bio-Rad, Hercules, CA, USA) and a real-time PCR machine (Bio-Rad). The resulting increase in fluorescence during the PCR reaction was detected in the iQ5 system (Bio-Rad) and the cycle at which the fluorescence intensity reached the threshold (Ct) was used to compare each
Liver International (2014) © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Connexins and intrahepatic vascular tone
Table 1. Primers/control Cx rat genes used for real-time quantitative reverse transcriptase-polymerase chain reaction Gene Cx 43 Forward Reverse Cx 40 Forward Reverse RNA 18S Forward Reverse Beta-actin Forward Reverse
Primer sequence 5′-TGGGGGAAAGGCGTGAGGAAAG-3′ 5′-CACCTTCCCTCCAGCGGTGG-3′ 5′-CGGAGGAAAGGAAGCAGAAGGCTCA-3′ 5′-GAGCCAGACCTTGCCGATGACC-3′ 5′-GGCTACCACATCCAAGGAA-3′ 5′-GCTGGAATTACCGCGGCT-3′ 5′-AACGAGCGGTTCCGATGCC-3′ 5′-GGACAGTGAGGCCAGGATGGA-3′
gene with the reference genes (RNA18S or Betaactin). The data were analyzed using the qBASE software. All experiments were performed in duplicate, and the mean values were calculated. Temperature profiles included an initial stage at 95°C for 5 min, followed by 40 cycles of amplification at 94°C for 20 s, 60°C for 40 s and 72°C for 30 s. Melt curve analysis was performed by ramping products from 72 to 95°C, acquiring fluorescence readings for each 0.5°C change. The relative quantification of each gene was performed using the comparative method (ΔΔCt) using the expression of control genes (19). mRNA gene expression levels were reported as fold changes relative to the sample with the lowest mean Cx expression, used as a relative calibrator (arbitrarily set at 1). Immunostaining for Cx43 and Cx40
Control rats (n = 3) were anaesthetized as previously detailed and liver tissues were cryopreserved in optimal cutting temperature compound, and snap frozen in liquid nitrogen. Liver sections were cryosectionned at 8 lm, fixed and rehydrated in PBS for 10 min. Permeabilization was performed in PBS containing 0.1% (vol/vol) Triton X-100, 4% goat serum for 1 h at room temperature. The sections were incubated overnight with rabbit polyclonal antibodies raised against Cx43 (Sigma) dilution 1/100 and Cx40 (Alpha Diagnostics, San Antonio, TX, USA) dilution 1/25. After five, 3 min each, PBS rinses, sections were incubated with biotinylated goat anti-rabbit (Jackson Immunoresearch), 1/200 in PBS for 2 h at room temperature. Next, five, 3 min each, PBS rinses were performed and the sections were incubated with Cy2conjugated streptavidin (Jackson Immunoresearch), dilution 1/1000 1 h, in the dark. Slides were washed again and mounted with PBS-glycerol. Finally, slides were photographed and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan) with a 940-20-10 epifluorescence objective.
861
Hernandez-Guerra et al.
Connexins and intrahepatic vascular tone
Liver cell isolation Parenchymal cells (Hepatocytes)
Control (n = 3) and CCl4-cirrhotic (n = 3) rats were anaesthetized as previously described and hepatocytes were isolated by in situ collagenase perfusion through the portal vein, as described by Munthe-Kaas et al. (20) with minor modifications (21). Briefly, livers were perfused for 10 min at a flow rate of 20 ml/min at 37°C with Hanks’ balanced salt solution. For cell isolations from CH animals, the concentration of collagenase was increased by 25%. The cells were passed through nylon filters (100 lm; Becton-Dickinson Labware, Franklin Lakes, NJ, USA), collected in cold Krebs buffer, and centrifuged at 50g for 3 min. The obtained pellet contains the hepatocytes, whereas the supernatant is enriched in non-parenchymal cells. Sinusoidal endothelial cells
Sinusoidal endothelial cells were obtained from the supernatant of control and CCl4-cirrhotic rat livers (21). Briefly, supernatant was centrifuged at 800g for 10 min at 4°C, and the obtained pellet was resuspended in Dulbecco’s PBS and centrifuged at 800g for 20 min through a 25–50% Percoll gradient at room temperature. The interface of the gradient containing Kupffer cells and SEC was seeded on 37-mm tissue culture plates and incubated at 37°C for 1 h. Cell monolayers adhered to the dishes were characterized as Kupffer cells and the non-attached cells as SEC. Hepatic stellate cells
Hepatic stellate cells were isolated from control (n = 3) and CCl4-cirrhotic (n = 3) rat livers as described above. After perfusion, the resultant digested liver was excised, and in vitro digestion was performed as previously described (21). Cells were grown in Iscove’s modified Dulbecco’s medium, and kept until experimentation; they were highly viable and 95% pure. Drugs and reagents
Glycyrrhetinic, Mtx and ACh were purchased from Sigma (Tres Cantos, Madrid, Spain), mouse anti-RECA monoclonal antibody from Serotec (Oxford, UK), collagenase A from Roche Diagnostics (Mannheim, Germany), Percoll from Amersham Biosciences (Uppsala, Sweden), and RPMI medium and culture complements were from Biological Industries (Kibbutz Beit Haemek, Israel). Statistical analysis
Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). All data are reported as
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mean ± SEM. Comparisons between groups were performed with Student’s t-test for unpaired data or the Mann–Whitney t-test when adequate. Differences with a P value
