Potential conflict of interest: Nothing to report.
In cirrhosis, increased RhoA/Rho-kinase signaling and decreased nitric oxide (NO) availability contribute to increased intrahepatic resistance and portal hypertension. Hepatic stellate cells (HSCs) regulate intrahepatic resistance. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) inhibit synthesis of isoprenoids, which are necessary for membrane translocation and activation of small GTPases like RhoA and Ras. Activated RhoA leads to Rho-kinase activation and NO synthase inhibition. We therefore investigated the effects of atorvastatin in cirrhotic rats and isolated HSCs. Rats with secondary biliary cirrhosis (bile duct ligation, BDL) were treated with atorvastatin (15 mg/kg per day for 7 days) or remained untreated. Hemodynamic parameters were determined in vivo (colored microspheres). Intrahepatic resistance was investigated in in situ perfused livers. Expression and phosphorylation of proteins were analyzed by RT-PCR and immunoblots. Three-dimensional stress-relaxed collagen lattice contractions of HSCs were performed after incubation with atorvastatin. Atorvastatin reduced portal pressure without affecting mean arterial pressure in vivo. This was associated with a reduction in intrahepatic resistance and reduced responsiveness of in situ–perfused cirrhotic livers to methoxamine. Furthermore, atorvastatin reduced the contraction of activated HSCs in a 3-dimensional stress-relaxed collagen lattice. In cirrhotic livers, atorvastatin significantly decreased Rho-kinase activity (moesin phosphorylation) without affecting expression of RhoA, Rho-kinase and Ras. In activated HSCs, atorvastatin inhibited the membrane association of RhoA and Ras. Furthermore, in BDL rats, atorvastatin significantly increased hepatic endothelial nitric oxide synthase (eNOS) mRNA and protein levels, phospho-eNOS, nitrite/nitrate, and the activity of the NO effector protein kinase G (PKG). Conclusion: In cirrhotic rats, atorvastatin inhibits hepatic RhoA/Rho-kinase signaling and activates the NO/PKG-pathway. This lowers intrahepatic resistance, resulting in decreased portal pressure. Statins might represent a therapeutic option for portal hypertension in cirrhosis. (HEPATOLOGY 2007;46:242–253.)
In cirrhosis, increased intrahepatic resistance to portal flow essentially contributes to portal hypertension and its complications.1–3 Apart from structural changes (i.e., fibrosis, capillarization of sinusoids, regeneratory nodules), increased vascular tone of the intrahepatic vasculature (mainly perisinusoidal hepatic stellate cells [HSC] and presinusoidal venules) is functionally responsible for this increased resistance.4, 5
Increased sensitivity of the intrahepatic microcirculation to vasoconstrictors4–7 and decreased levels of the vasodilator nitric oxide (NO) and its downstream signaling cyclic guanosine 3′,5′-monophosphate (cGMP)/protein kinase G (PKG) are responsible for this elevated contractile state.4, 8, 9 We previously showed that increased RhoA/Rho-kinase signaling essentially contributes to increased intrahepatic resistance as well as increased sensitivity to vasoconstrictors in rats with secondary biliary cirrhosis.10 The RhoA/Rho-kinase pathway is involved in vasoconstrictor-induced contraction of vascular smooth muscle. After activation of G-protein-coupled receptors by vasoconstrictors, RhoA activates Rho-kinase, which then phosphorylates and thereby inactivates myosin light chain phosphatase (MLCP), leading to increased myosin light chain phosphorylation and contraction.11, 12 Furthermore, in vascular tissue, RhoA/Rho-kinase down-regulates the expression and activity of endothelial nitric oxide synthase (eNOS).13–15 NO, in turn, via cGMP, activates its target, PKG, which then induces vasorelaxation by activation of MLCP.16 Thus, the RhoA/Rho-kinase pathway regulates vascular tone directly by inactivation of MLCP and indirectly by inhibition of eNOS/NO/PKG (Fig. 1).
An essential step in the activation of RhoA/Rho-kinase is the membrane translocation of RhoA. To translocate to the cell membrane, RhoA needs to be geranylgeranylated. Geranylgeranyl-pyrophosphate (GGPP) is a byproduct of endogenous cholesterol synthesis through 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA-R), which is inhibited by HMG-CoA-R inhibitors (statins).13, 17–20 For this reason, a lack of geranylgeranyl-pyrophosphate should lead to decreased activation of RhoA and Rho-kinase.13, 20 This should affect contractility as well as Rho-kinase-mediated contraction, eNOS expression and phosphorylation, and NO availability (Fig. 1).
Moreover, HMG-CoA-R inhibition might also affect posttranslational modifications of isoprenylates (GGPP, farnesylpyrophosphate), lipid anchors, and various other proteins, including small GTPases similar to RhoA. For example, the monomeric GTPase Ras needs to be farnesylated for activation.13, 17–22
We therefore investigated the effect of chronic atorvastatin treatment on intrahepatic RhoA/Rho-kinase and NO/PKG signaling as well as on hepatic and systemic hemodynamics.
BDL, bile duct ligation; eNOS, endothelial nitric oxide synthase; HSCs, hepatic stellate cells; L-NAME, Nω-nitro-l-arginine methyl ester; NO, nitric oxide; PKG, protein kinase G; VASP, vasodilator-stimulated phosphoprotein.
Materials and Methods
For our experiments, male Sprague-Dawley rats with an initial body weight of 180-200 g were used. One group (n = 32) underwent bile duct ligation (BDL) as previously described.23 The rats in the second group (n = 38) underwent BDL and 4 weeks after BDL were treated with atorvastatin (15 mg/kg bodyweight per day) for 1 week. This dosage was chosen according to the literature.24, 25 Sham-operated rats (n = 34) served as controls. In these rats, the common bile duct was exposed by median laparotomy, but no ligation or resection was performed. The study was approved by the local committee for animal studies (Bezirksregierung Köln, 50.203.2-BN22, 46/05).
Treatment with Atorvastatin and Biochemical Analyses.
Twenty-eight days after bile duct ligation, the animals were housed in individual cages (Techniplast, Buguggiate, Italy) and received atorvastatin-mixed chow and water ad libitum. The rats were weighed and administered mixed food daily. At the end of the experiment, the animals were killed, and blood was obtained to measure biochemical parameters (AST, ALT, gamma-glutamyltransferase, alkaline phosphatase, bilirubin, creatinine) using standard methods.
Hepatic Hydroxyproline Content.
Analog segments (200 mg) of snap-frozen livers were hydrolyzed in HCl (6 N). Hepatic hydroxyproline content was determined photometrically in liver hydrolysate as previously described.26
RNA was isolated from 30 mg of shock-frozen liver tissue with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines. RNA concentration was measured spectrophotometrically at 260 nm. For each sample, 1 μg of total RNA was used. Prior to reverse transcription, samples were DNA-digested by incubation with RQ1 RNase-free DNase (Promega, Madison, WI). Reverse transcription was performed using MMLV reverse transcriptase (Invitrogen, Karlsruhe, Germany) and random primers (250 ng; Microsynth, Balgach, Switzerland). Primers and probes for RT-PCR were designed using Primer Express Software (Applied Biosystems, Foster City, CA) and custom-synthesized by Microsynth (Balgach, Switzerland) and Applied Biosystems (Foster City, CA), respectively (Table 1). Primers and probes for the housekeeping gene (18SrRNA) were provided by Applied Biosystems (Foster City, CA) as a ready-to-use mix. RT-PCR was performed using an ABI 7300 Sequence detector (Applied Biosystems, Foster City, CA) as described elsewhere.27 The PCR reaction was performed in a 25-μL volume containing 12.5 μL of 2× TaqMan-PCR master mix (Roche Molecular Systems, Mannheim, Germany) and 2 μL (equivalent to 67 ng of total RNA) of cDNA. The final concentrations of the primers and probes are given in Table 1. 18SrRNA served as the endogenous control. The final concentrations were 100 nM for the primers and 200 nM for the probe. The ΔCT method was used to quantify the results. A validation experiment was performed according to the manufacturer's guidelines for each target gene and for the 18SrRNA. These experiments showed that RT-PCR was about equally efficient for the target gene and the endogenous control.
Table 1. Primers and Probes Used for Quantitative RT-PCR for RhoA, Rho-kinase, and eNOS
Samples of shock-frozen livers were homogenized in a buffer containing 25 mM Tris/HCl, 5 mM ethylenediamine tetraacetic acid, 10 μM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 10 μg/mL leupeptin. Samples were diluted with sample buffer. Determination of proteins in the homogenates was performed with the DC Protein Assay kit (Bio-Rad, Munich, Germany). Samples (20 μg of protein/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 15% gels for RhoA and Ras; 8% gels for Rho-kinase, eNOS, and p-eNOS; 10% gels for moesin, p-moesin, vasodilator-stimulated phosphoprotein [VASP], p-VASP, and α-smooth muscle actin [α-SMA]), and proteins were blotted on nitrocellulose membranes. Ponceau-S staining was performed to assure equal protein loading. The membranes were blocked, incubated with primary antibodies (RhoA119, Rock-2 H-85, NOS3 [N-20]), moesin clone E10, p-moesin, VASP (Santa Cruz Biotechnology, Santa Cruz, CA), p-eNOS (Ser1177), Ras (New England Biolabs GmbH, Frankfurt, Germany); α-SMA (Abcam, Cambridge, UK), p-VASP clone 16C2 (Calbiochem, San Diego, CA), and thereafter with corresponding secondary peroxidase-coupled antibodies (Calbiochem, San Diego, CA). Blots were developed with enhanced chemiluminescence (ECL, Amersham, UK). Intensities of the resulting bands on each blot were compared densitometrically with a FLA-3000 phosphoimager (Fuji-Film, Düsseldorf, Germany).
Assessment of PKG and Rho-kinase Activity.
PKG activity was assessed as phosphorylation of the endogenous PKG substrate, VASP, at Ser-239. The phosphorylation state of VASP serves as a marker for PKG activity.28 Rho-kinase activity was assessed as phosphorylation of the endogenous Rho-kinase substrate, moesin, at Thr-558.10, 29 This was done by Western blot analysis using site- and phospho-specific antibodies.
Hepatic Nitrite/Nitrate Concentration.
The nitrite/nitrate (NOx) content of the shock-frozen liver samples was determined via the Griess reaction30 using a Nitralyzer-II kit (WPI, Berlin, Germany) according to the manufacturer's guidelines.
Cell Isolation and Culture.
Hepatic stellate cells were isolated as described previously.31 In brief, after in situ perfusion of the liver samples with sequential collagenase type IV (Sigma, St. Louis, MO) and pronase E (Merck, Darmstadt, Germany) solutions, the dispersed cells were fractionated by density gradient centrifugation using Optiprep (Nycomed, Sweden). Cells were harvested at densities less than 1.053 (9% Optiprep) as previously described.32 Viability and purity were systematically greater than 95%, as determined by Trypan blue exclusion and morphological characterization. The cells were seeded on uncoated plastic culture dishes and cultured in William's E medium supplemented with 10% fetal bovine serum, 0.6 IU/mL insulin, 2 mM glutamine, and 1% antibiotic–antimycotic solution (Invitrogen, Merelbeke, Belgium). The medium was renewed every 48-72 hours. Characterization of the rat liver–derived myofibroblast-like cultures, established by culturing enriched hepatic stellate cell (HSC) fractions on plastic, was performed by staining with anti-α-SMA, antidesmin, and synaptophysin as previously described.33 Experiments were performed between the first and third passages (1:3 split ratio) using 3 independent cell lines.
HSC Proliferation Assay.
Cell viability and proliferation of HSCs were determined using a 3-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) assay (Cell Proliferation Kit II; Roche Molecular Biochemicals, Indianapolis, IN), as described previously.34
Analysis of RhoA and Ras Activity in HSCs.
RhoA and Ras activity was determined by membrane translocation.29 In brief, culture-activated HSCs were incubated with or without atorvastatin (24 h, 10−4 M) and then shock-frozen. These samples were homogenized in Tris-buffer. To determine membrane translocation of RhoA and Ras, homogenates were subjected to ultracentrifugation (100,000g, 4°C, 30 min). The resulting pellet (membrane fraction) was resuspended in 60 μL of Tris buffer. After protein determination, the resuspended pellets and the supernatants (cytosolic fraction) were boiled with SDS-sample-buffer subjected to SDS-PAGE (membrane fraction, 10 μg of protein/lane; cytosolic fraction, 20 μg of protein/lane), blotted on nitrocellulose membranes, and analyzed with anti-Ras, anti-RhoA, and appropriate secondary antibodies (Ras 3965, New England Biolabs, Frankfurt, Germany; peroxidase-coupled antirabbit IgG, Calbiochem, San Diego, CA; RhoA 26C4, Santa Cruz Biotechnology, Santa Cruz, CA; peroxidase-coupled antimouse IgG, Calbiochem, San Diego, CA). The intensities of the resulting bands were determined densitometrically with a phosphoimager (FLA-3000; Fuji-Film). Intensity of a stimulated sample is expressed as the percentage of the corresponding unstimulated sample.
The ability of HSCs to contract 3-dimensional collagen matrices was measured as previously described with slight modifications.35 In brief, hydrated collagen gels were prepared using rat tail tendon collagen I (Becton Dickinson Labware, Bedford, MA) and adjusted to physiological strength and pH with 1 N NaOH and 10× PBS, respectively, at 4°C. The collagen solution was mixed with a HSC suspension, so that the final solution resulted in a collagen concentration of 1.5 mg/mL and 250,000 cells/mL. A 500-μL aliquot of the collagen solution was cast into each well of a 24-well tissue-culture plate (Falcon, Meylan, France) and after 1 hour covered with complete culture medium (1mL/well) in order to ascertain that gelation was adequate. The polymerized collagen matrix containing HSCs remained attached to the culture dish for 24 hours, leading to mechanical loading (the condition of being “stressed”). After 24 hours, stabilized lattices were washed twice with 1× PBS, followed by the addition of 1 mL/well serum-free culture medium containing 1 μCi3H2O (Amersham Biosciences, Roosendaal, Netherlands). Then atorvastatin at different concentrations (10−7-10−4 M) was added. To initiate matrix contraction, the mechanically stressed matrices were released by gentle circumferential dislodgment of the lattice using a micropipette tip (the condition of being “relaxed”). Cell-mediated contraction was measured by determining the relative partitioning of3H2O between the gel phase and the surrounding medium after 24 hours of contraction, thereby allowing estimation of gel-phase volume. More specifically, the separate tritium activity of the medium and the gel phase were measured in 10 mL of oscillation fluid (Perkin Elmer) using a Beckmann liquid scintillation spectrometer. Control cell-free gels provided estimates of the precontraction volume and of the relative change in volume (% contraction). All data are from experiments using at least 3 sets of 3 collagen lattices using culture-activated HSCs from 3 rat HSC isolations.
Hemodynamic studies were performed under ketamine anesthesia (60 mg/kg intramuscular). This condition has been shown to most closely approximate the conscious state in cardiac output and regional blood flow36 and has been used extensively to investigate the hemodynamic effects of portal-pressure-lowering drugs in animal models of portal hypertension.2, 23 Median laparotomy was performed, and a PE-50 catheter was introduced into a small ileocoecal vein and advanced to the portal vein for measurement of portal pressure. The left femoral artery was cannulated with PE-50 catheters for measurement of arterial pressure and blood withdrawal. Via the right carotid artery, another PE-50 catheter was advanced into the left ventricle under pulse curve control. This catheter was used for application of the microspheres. The catheters in the femoral artery and the portal vein were connected to a pressure transducer (Hugo Sachs Elektronik, March-Hugstetten, Germany) for blood pressure measurement. The zero point was 1 cm above the operating table. After insertion of all catheters, rats were allowed to stabilize hemodynamically for 30 minutes. Then mean arterial pressure (MAP) was monitored continuously for another 45 minutes, followed by measurement of the cardiac index.
The cardiac index was measured using the colored microsphere method as previously described.23, 37 The colored microsphere technique was validated by the more frequently used radioactive microsphere method.37 It has the advantage of being nonradioactive. A reference sample was obtained for 1 minute at a rate of 0.65 mL/min, using a continuous withdrawal pump (Hugo Sachs Elektronik, March-Hugstetten, Germany). In 0.3 mL of saline containing 0.05% Tween, 300,000 yellow microspheres (15 μm in diameter; Triton Technologies, San Diego, CA) were suspended and injected in the left ventricle 10 seconds after the withdrawal pump had been started. Portosystemic shunting was estimated after injection of 150,000 blue microspheres in 0.3 mL of saline containing 0.05% Tween in an ileocoecal vein within 30 seconds.38
The blood reference probe was digested by the addition of 3.8 mL of 5.3 M KOH and 0.5 mL of Tween 80 and subsequent boiling for 1 hour. The digested tissues and blood samples were vortexed and filtered using Whatman Nucleopore filters (Whatman International Limited, Madison, UK). The color of the filtered microspheres was dissolved in 0.2 mL of dimethylformamide, and absorption was measured by spectrophotometry. Thereafter, the cardiac index and portosystemic shunting were calculated using software from Triton Technologies and expressed per 100 g of body weight. Hepatic vascular resistance was calculated as the portal pressure (PP) divided by the sum of gastrointestinal and splenic perfusion minus shunt flow. Systemic vascular resistance was estimated as the ratio between mean arterial pressure and cardiac index.
In situ Liver Perfusion.
In situ liver perfusion was performed in a recirculating system as described previously.10 Briefly, rats were fasted overnight but allowed free access to water. After anesthesia with ketamine hydrochloride (60 mg/kg), the abdomen was opened, and the bile duct was cannulated with a polyethylene tube to monitor bile flow. Loose ligatures were placed around the portal vein, the common hepatic artery, the splenic vein, and the posterior caval vein just cranially to the confluence of the right renal vein. A 500-U dose of heparin was injected into the posterior caval vein. The portal vein was cannulated with a 14-gauge catheter, initiating liver exsanguination by infusion (30 mL/min) of Krebs-Henseleit solution containing heparin (2 IU/mL) and oxygenated with carbogen (95% O2, 5% CO2). The posterior caval vein was immediately cut caudally to the loose ligature, allowing the perfusate to escape. Then the thorax was opened, and the right atrium was cut. Another catheter was introduced in the right atrium and pushed forward into the inferior caval vein. Next, all ligatures were pulled tight. At a constant flow (30 mL/min), perfusion pressure was continuously monitored and recorded with the strain-gauge transducer connected to the portal inflow and venous outflow cannula. The pressure gradient divided by the flow was defined as hepatic resistance.
Viability and Stability of Liver Perfusion Preparation.
The criteria used to determine liver viability were gross appearance, stable perfusion, bile production > 0.4 μL/(min × g,), and stable buffer pH (7.4 ± 0.1) during the initial 20-minute stabilization period. If one of the viability criteria was not met, the experiment was discarded.
Effect of the α1-adrenoceptor Agonist Methoxamine on Portal Perfusion Pressure.
In one set of experiments, livers were initially perfused at a constant flow (30 mL/min) for 20 minutes without any interference in order to stabilize the entire system. Then cumulative concentration response curves with the α1-adrenoceptor agonist methoxamine (10−6-10−3μM) were obtained by adding the agonist to the perfusate. Changes in perfusion pressure were expressed as absolute perfusion pressure after administration of methoxamine.
Effect of the NOS Inhibitor L-NAME on Methoxamine-Induced Resistance to Hepatic Flow.
In another set of experiments, Nω-nitro-l-arginine methyl ester (L-NAME) was added (1 mM) to the perfusate 10 minutes before the addition of the first dose of methoxamine. Then cumulative concentration response curves for methoxamine were constructed as already described.
Response of Perfusion Pressure to Acetylcholine.
In another set of experiments, methoxamine (10−4 M) was added to the perfusate 5 minutes before the addition of cumulative doses of acetylcholine (10−7-10−5M). The concentration of acetylcholine was increased by 1 log unit every 1.5 minutes. Thereafter, cumulative concentration response curves for acetylcholine were calculated as change in the percentage of perfusion pressure, as previously described.39
Data are presented as means ± SEMs. ANOVA followed by the Bonferroni/Dunn or Mann-Whitney U test was used for comparisons between groups (SPSS 10 for Windows, SPSS Inc., Chicago, IL). A P value < 0.05 was considered statistically significant. For the analysis of concentration response curves, data were fitted by nonlinear regression using the Prism computer program (Graph Pad Software Inc., San Diego, CA). Emax (maximum contraction) and pEC50 (negative logarithm of the concentration producing half the maximum effect) values were calculated from the fitted curves.
Mean body weight did not differ between treated and untreated cirrhotic rats. Liver and spleen weight was significantly increased in cirrhotic animals compared to sham-operated rats. There was no difference between untreated and atorvastatin-treated cirrhotic rats in liver weight, but the spleen weight of treated rats was significantly decreased (Table 2). Mortality was less than 15% and did not differ among all BDL animals.
Table 2. General Characteristics of Different Groups of Rats
All analyzed biochemical parameters of BDL rats except creatinine increased compared to those in sham-operated control animals (Table 3). This elevation remained unchanged after treatment with atorvastatin.
Table 3. Biochemical Parameters of the Different Groups
NOTE. Data are expressed as means ± SEMs (n = 6 each group
In livers of BDL rats the content of hydroxyproline and α-SMA was significantly increased compared with that in sham-operated rats. Treatment of BDL rats with atorvastatin did not affect these fibrosis parameters (Fig. 2).
RhoA, Rho-kinase, and eNOS mRNA Levels.
mRNA levels of both RhoA and Rho-kinase were significantly increased in the livers of cirrhotic rats compared with those of sham-operated noncirrhotic rats. In cirrhotic rats treated with atorvastatin, RhoA and Rho-kinase mRNA levels remained unchanged when compared with those of untreated BDL rats (Fig. 3A-B). In contrast, in BDL rats, expression of hepatic eNOS mRNA increased in response to treatment with atorvastatin (Fig. 3C).
Expression of RhoA, Rho-kinase, and Ras Proteins.
Western blot analysis revealed increased intrahepatic protein expression of RhoA, Rho-kinase, and Ras in BDL rats compared to that in sham-operated rats. Administration of atorvastatin did not affect the expression of RhoA, Rho-kinase, and Ras proteins (Fig. 4A-C).
We determined the phosphorylation state of the Rho-kinase substrate moesin as a marker of Rho-kinase activity. Moesin is phosphorylated at Thr-558 by Rho-kinase. Western blot analysis with phospho- and site-specific (Thr-558) moesin antibody showed enhanced intrahepatic phosphorylation of moesin in cirrhotic rats when compared with that in sham-operated rats. Chronic administration of atorvastatin in cirrhotic rats significantly decreased the intrahepatic phosphorylation of moesin (Fig. 4D). Thus, chronic treatment with atorvastatin inhibits Rho-kinase activity in the livers of cirrhotic rats.
Expression and Phosphorylation of eNOS Protein.
Western blot analysis revealed no effect on intrahepatic eNOS expression, but a decrease in intrahepatic eNOS phosphorylation in BDL rats when compared to sham-operated rats. Treatment with atorvastatin increased the eNOS and p-eNOS content in livers of cirrhotic BDL rats (Fig. 5A,B).
As a marker of PKG activity, we determined the phosphorylation of the PKG substrate VASP, as described previously.28 VASP is phosphorylated at Ser-239 by PKG. Western blot analysis with phospho- and site-specific (Ser-239) VASP antibodies revealed increased intrahepatic phosphorylation of VASP in cirrhotic rats when compared to sham-operated rats. The administration of atorvastatin in cirrhotic rats significantly enhanced intrahepatic phosphorylation of VASP (Fig. 5C). Thus, atorvastatin enhances PKG activity in the livers of cirrhotic rats.
Hepatic NOx Concentration.
Measurement of NOx concentration revealed that it was significantly higher in atorvastatin-treated than in untreated cirrhotic BDL rats (Fig. 5D).
HSC Proliferation Assay.
Incubation with different doses of atorvastatin for 24 hours did not influence HSC proliferation (Fig. 6).
RhoA and Ras Activation in HSCs.
RhoA and Ras activity was determined by their membrane association. Incubation with atorvastatin (10−4M, 24 hours) induced a significant decrease in membrane-associated RhoA and Ras in activated HSCs (Fig. 7A,C). RhoA was increased in the cytosolic fraction after incubation with atorvastatin (Fig. 7B). In contrast, cytosolic Ras was hardly detectable and showed no difference after atorvastatin treatment (Fig. 7D).
Isolated and culture-activated HSCs were loaded into collagen lattices as previously described.31–33, 35 Incubation with atorvastatin dose-dependently relaxed the contraction of HSCs in the 3-dimensional stress-relaxed collagen lattice model (Fig. 8). Each atorvastatin concentration differed from the others and from the controls, indicating the effect of atorvastatin in HSCs.
The basal MAP was significantly lower in cirrhotic rats than in sham-operated rats. The chronic administration of atorvastatin caused no changes in MAP in cirrhotic rats (Fig. 9A).
Systemic vascular resistance was significantly decreased in BDL rats compared with that in sham-operated rats, and it remained unaffected by chronic treatment with atorvastatin (Fig. 9B). The cardiac index was significantly increased in BDL rats compared with that in sham-operated rats, but again, no change was observed after treatment with atorvastatin in cirrhotic rats (Fig. 9C).
As expected, the PP was markedly higher in BDL rats than in sham-operated rats (Fig. 10A). This was accompanied by an increase in hepatic vascular resistance, a decrease in splanchnic vascular resistance, and increased portal tributary blood flow (Fig. 10B-D). In cirrhotic rats, atorvastatin caused a significant decrease in PP (Fig. 10A) via a decrease in hepatic vascular resistance (Fig. 10B). Splanchnic vascular resistance and portal tributary blood flow remained unchanged in response to atorvastatin (Fig. 10C-D).
Furthermore, arterial hepatic flow was significantly increased in untreated cirrhotic BDL rats compared with that in sham-operated rats. Atorvastatin did not change hepatic arterial flow in cirrhotic rats (Fig. 10E). Finally, treating cirrhotic rats with atorvastatin significantly reduced shunting in these animals. No shunting was observed in sham-operated rats (Fig. 10F).
Isolated Perfused Livers.
Hepatic vascular resistance was increased in cirrhotic rats compared to sham-operated rat livers. In livers from BDL rats atorvastatin significantly decreased hepatic resistance (Fig. 11A).
To further investigate the effect of atorvastatin on hepatic resistance of cirrhotic rats, we studied the response of hepatic resistance to the α1-adrenoceptor agonist methoxamine. As shown in Fig. 11C, the addition of methoxamine to the perfusate elicited dose-dependent increases in resistance in all groups. As expected, the responsiveness of BDL rat livers to methoxamine was significantly higher than that in sham-operated rats, as shown by the increase in hepatic vascular resistance (Fig. 11C). Atorvastatin significantly decreased the responsiveness and the maximal response to methoxamine of livers from cirrhotic BDL rats (Fig. 11B,C).
As we have explained, atorvastatin possibly increases eNOS expression and NO production. We therefore tested the effect of the NOS inhibitor L-NAME on dose-dependent methoxamine-induced elevation of hepatic resistance. The addition of L-NAME elicited a significant decrease in the EC50 (concentration producing half the maximum effect) to methoxamine in treated BDL rats (Table 4). Furthermore, L-NAME significantly increased response of hepatic resistance at every dose of methoxamine used (Figs. 11D and 12A-C).
Table 4. pEC50 for Methoxamine of Hepatic Resistance in In Situ Liver Perfusion
Sham + L-NAME
BDL + L-NAME
BDL + atorvastatin
BDL + atorvastatin + L-NAME
NOTE. Data are expressed as means ± SEMs. Comparisons between 2 groups were performed with Mann-Whitney U test
The response of perfusion pressure to acetylcholine was significantly different among all groups at each tested dose. Whereas a dose-dependent decrease in perfusion pressure was observed in sham-operated rats, acetylcholine caused only a reduction in perfusion pressure in cirrhotic BDL rats after the first dose. Atorvastatin treatment of cirrhotic BDL rats partially corrected the response to acetylcholine in perfusion pressure (Fig. 12D).
In this study we showed that atorvastatin lowers portal pressure and intrahepatic resistance in rats with secondary biliary cirrhosis, an established cirrhosis model for hemodynamic investigation.40 Furthermore atorvastatin reduces in vitro contraction of activated hepatic stellate cells and inhibits hepatic RhoA/Rho-kinase and activates hepatic NO/cGMP/PKG signaling.
Current innovative treatment strategies attempt to reduce portal pressure in cirrhosis by increasing hepatic NO availability either by nonspecific41 and liver-specific NO donors42 or experimentally by adenoviral hepatic gene transfer of eNOS or eNOS-regulating genes.43, 44 Furthermore, clinical trials have been performed to decrease intrahepatic vascular tone by inhibition of α-adrenoceptor-mediated pathways.45 However, all these treatment strategies either have not been shown to sufficiently decrease portal pressure or are not applicable in a clinical setting. By contrast, patients may be easily treated with atorvastatin, and therefore atorvastatin should be tested as an option for treating portal hypertension.
Statins can inhibit membrane association and activation of small GTPases including RhoA and Ras by inhibition of isoprenoid synthesis. Inhibition of GGPP synthesis inhibits RhoA activation and consequently activation of the downstream Rho-kinase pathway, resulting in vasorelaxation. Furthermore, inhibition of this pathway in endothelial cells can increase expression of eNOS and its activation via Ser1177 phosphorylation by Akt.13–15, 43, 46 Recently, we have shown that intrahepatic vascular RhoA/Rho-kinase signaling is enhanced in rats with secondary biliary cirrhosis.10 Thus, our aim was to investigate the effect of atorvastatin on intrahepatic resistance, RhoA/Rho-kinase signaling, and eNOS expression and activity in rats with secondary biliary cirrhosis.
In vivo, atorvastatin decreased portal pressure, intrahepatic resistance, and portosystemic shunt flow in cirrhotic rats without altering systemic hemodynamics (Figs. 9 and 10). Furthermore, in cirrhotic rat livers, atorvastatin decreased the response of intrahepatic resistance to the vasoconstrictor methoxamine as shown in the in situ perfused liver model (Fig. 11).
The decreased intrahepatic resistance might be a consequence of decreased contraction of activated HSCs and portal myofibroblasts. Our data support this hypothesis as in vitro atorvastatin significantly decreased contraction of activated HSCs. This might indeed be a result of the effect of atorvastatin on RhoA signaling because atorvastatin inhibits RhoA translocation to the membrane (Figs. 7 and 8).
Because in our experimental conditions atorvastatin did not affect fibrosis and proliferation of HSCs, any contribution of a structural component to the improvement in liver hemodynamics can be widely excluded (Figs. 2, 6, and 7).
As we have already described, hepatic phosphorylation of Rho-kinase target protein moesin was enhanced in cirrhotic livers. We have previously shown that moesin phosphorylation is an adequate measure of Rho-kinase activity.10, 29 Atorvastatin significantly decreased hepatic phospho-moesin levels. Hepatic expression of RhoA and Rho-kinase not being affected by atorvastatin indicates that atorvastatin decreased hepatic Rho-kinase activity. Thus, atorvastatin might directly decrease intrahepatic vasoconstrictor response and thus decrease intrahepatic vascular tone. Atorvastatin did not have systemic and splanchnic effects, which can be explained by the defective RhoA/Rho-kinase pathway in cirrhotic arteries as previously described.29
To distinguish between this direct effect on Rho-kinase and NO-mediated effects, we performed in situ perfusion experiments using L-NAME, an inhibitor of NOS. L-NAME only partially corrected the effect of atorvastatin on intrahepatic resistance (Fig. 11D). This indicates that the effect of atorvastatin is partially NO dependent and partially NO independent. Thus, by inhibition of RhoA, atorvastatin prevents activation of RhoA-dependent contraction cascades independently of NO and simultaneously increases NO production.
Because RhoA negatively regulates the stability of eNOS mRNA in vascular tissue, inhibition of RhoA with statins might increase eNOS expression and subsequently NOS activity (Fig. 1). Indeed, we have shown that in cirrhotic livers, atorvastatin increased expression of eNOS mRNA and protein (Figs. 3C and 5A). This was accompanied by an increase in hepatic content of p-eNOS (Ser-1177). Nevertheless, our experimental data support several lines of evidence that atorvastatin increases intrahepatic NO production. First, atorvastatin raised the intrahepatic phosphorylation state of VASP. VASP is a substrate of PKG, which in turn mediates NO-induced vasorelaxation. Second, increased NO availability was indicated by the elevated hepatic nitrite/nitrate content in response to atorvastatin. Third, treatment of BDL rats with atorvastatin partially restored the effect of the endothelium-dependent vasodilator acetylcholine. Finally, the effect of the NOS inhibitor L-NAME on the methoxamine-induced increase in intrahepatic vascular tone was more pronounced in untreated than in atorvastatin-treated BDL rats.
Last, there is evidence for PKG-dependent RhoA deactivation, which would lead to a perpetuating loop in the effect of statins on RhoA/Rho-kinase activity (Fig. 1).47
The present study is the first to investigate the effect of chronic administration of atorvastatin on cirrhosis with portal hypertension. A previous study investigated the acute effect on portal hypertension of simvastatin, a prodrug activated after the liver first pass.48 Patients with cirrhosis showed decreased intrahepatic resistance.48 Another experimental study investigated the effect of simvastatin in BDL rats.49 In this study, simvastatin (2.5 mg/kg body weight) administered for 4 weeks had no effect on the hemodynamics and fibrosis of cirrhotic rats. Simvastatin is a prodrug activated in the liver. We cannot explain the different results obtained for atorvastatin and simvastatin. According to the literature, the simvastatin dose chosen was very low and might have been ineffective.24, 25 In the present study we used atorvastatin, a very lipophilic statin, in a median dose for rats (15 mg/kg body weight) according to the literature.24, 25 For this dose and duration of treatment, no liver toxicity was observed (Table 3). Furthermore, human use of statins has been shown to be safe and not hepatotoxic.50
In summary, chronic treatment with atorvastatin lowered portal pressure without affecting systemic hemodynamics in cirrhotic bile duct ligated rats and portal hypertension. This effect was mediated by decreasing intrahepatic resistance via hepatic inhibition of the RhoA/Rho-kinase pathway and activation eNOS/NO signaling. These findings warrant further investigations in other cirrhosis models such as CCl4. Furthermore, the potentially beneficial effects of statins on human liver disease should be evaluated more thoroughly.
The authors thank G. Hack and A. Kang for excellent technical assistance.