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Atorvastatin (Lipitor) lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase
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Hepatology July 2007
Jonel Trebicka 1 *, Martin Hennenberg 1, Wim Laleman 2, Nataliya Shelest 1, Erwin Biecker 1, Michael Schepke 1, Frederik Nevens 2, Tilman Sauerbruch 1, Jorg Heller 1
1Department of Internal Medicine I, University of Bonn, Bonn, Germany
2Department of Hepatology, University Hospital Gasthuisberg, Leuven, Belgium
Funded by:
Bonfor-Stiftung; Grant Number: O-107.0084
Deutsche Forschungsgemeinschaft; Grant Number: HE 2402/5-3
"....Statins might represent a therapeutic option for portal hypertension in cirrhosis..... Atorvastatin reduced portal pressure without affecting mean arterial pressure in vivo. ....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."
Abstract
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.
Article Text
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 vasoconstrictors[4-7] 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.
Discussion
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 nonspecific[41] and liver-specific NO donors[42] 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 -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.
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