Martin Hennenberg

Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase.

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.)

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells

BACKGROUND & AIMS Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL). METHODS BDL rats were treated with atorvastatin (15 mg/kg/d) immediately after ligation (prophylactically) or in on-going fibrosis (therapeutically). Fibrosis was assessed by hydroxyproline content and Sirius-red staining. The activation of HSC was investigated by analysis of alphaSMA expression. mRNA levels of cytokines and procollagen were analyzed by RT-PCR, and MMP-2 activity by zymography. Proliferation was assessed by expression of cathepsins (B and D), proliferating cell nuclear antigen (PCNA), and Ki67-staining. Apoptosis was characterized by caspase-3 activity, cleavage of PARP-1, and TUNEL assay. Hepatic inflammation was investigated by serum parameters and liver histology. RESULTS Prophylactic and early therapy with atorvastatin significantly attenuated fibrosis and HSC activation. Later therapy lacked significant effects on fibrosis but reduced profibrotic cytokine expression and led to a more quiescent state of HSC with less proliferation and apoptosis, while hepatic inflammation did not change. CONCLUSIONS This study shows that very early atorvastatin treatment inhibits HSC activation and fibrosis in the BDL model in vivo, while late treatment reduces HSC turnover and activity. Our findings underline that long-term studies in humans are warranted.

Mechanisms of extrahepatic vasodilation in portal hypertension

In liver cirrhosis, abnormal persistent extrahepatic vasodilation leads to hyperdynamic circulatory dysfunction which essentially contributes to portal hypertension. Since portal hypertension is a major factor in the development of complications in cirrhosis, the mechanisms underlying this vasodilation are of paramount interest. Extensive studies performed in cirrhotic patients and animals revealed that this vasodilation is associated on the one hand with enhanced formation of vasodilators, and on the other hand with vascular hyporesponsiveness to vasoconstrictors. The latter phenomenon has been termed “vascular hypocontractility”. It is caused by a combination of different mechanisms and factors described in this review.

Intrahepatic upregulation of RhoA and Rho-kinase signalling contributes to increased hepatic vascular resistance in rats with secondary biliary cirrhosis

Background and aims: Portal hypertension in cirrhosis is mediated in part by increased intrahepatic resistance, reflecting an increased sensitivity of the hepatic microvasculature to vasoconstrictors. Activation of the RhoA/Rho-kinase pathway is essential for contraction of vascular smooth muscle. The aim of this study was to investigate RhoA/Rho-kinase mediated regulation of the intrahepatic vascular tone in cirrhotic rats. Methods: Cirrhosis was induced by bile duct ligation (BDL). Hepatic RhoA and Rho-kinase expressions were studied by real time reverse transcription polymerase chain reaction and western blot analysis. Hepatic Rho-kinase activity in rat and human livers was assessed as phosphorylation of the Rho-kinase substrate moesin. The effect of the Rho-kinase inhibitor Y-27632 on hepatic perfusion pressure was measured in livers perfused at constant flow. The in vivo effect of intravenous application of Y-27632 was studied by haemodynamic measurements. Results: Hepatic expressions of RhoA and Rho-kinase were increased at mRNA and protein level in BDL rats. Intrahepatic moesin phosphorylation was increased in livers from cirrhotic rats and patients with alcohol induced cirrhosis. Y-27632 reduced the basal perfusion pressure of in situ perfused livers in BDL rats but not in sham operated rats. Y-27632 reduced the sensitivity to methoxamine in isolated perfused livers in sham operated rats more than in BDL rats. In vivo, Y-27632 reduced portal pressure to a greater extent in BDL rats than in sham operated rats. Intrahepatic vascular resistance was decreased in response to bolus injection of Y-27632 in BDL rats but not in sham operated rats. Conclusions: Upregulation of RhoA and Rho-kinase contributes to increased intrahepatic resistance in cirrhotic rats and to an increased sensitivity of cirrhotic livers to vasoconstrictors.

Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats

In cirrhosis, splanchnic vasodilation contributes to portal hypertension, subsequent renal sodium retention, and formation of ascites. Urotensin II(U‐II) is a constrictor of large conductive vessels. Conversely, it relaxes mesenteric vessels, decreases glomerular filtration, and increases renal sodium retention. In patients with cirrhosis, U‐II plasma levels are increased. Thus, we investigated hemodynamic and renal effects of U‐II and its receptor antagonist, palosuran, in cirrhotic bile duct–ligated rats (BDL). In BDL and sham‐operated rats, we studied acute effects of U‐II (3 nmol/kg; intravenously) and palosuran (10 mg/kg; intravenously) and effects of oral administration of palosuran (30 mg/kg/day; 3 days) on hemodynamics and renal function. We localized U‐II and U‐II‐receptor (UTR) in livers and portal veins by immunostaining. We determined U‐II‐plasma levels by enzyme‐linked immunosorbent assay (ELISA), and mesenteric nitrite/nitrate‐levels by Griess‐reaction. RhoA/Rho‐kinase and endothelial nitric oxide synthase (eNOS) pathways were determined by western blot analysis and reverse transcription polymerase chain reaction (RT‐PCR) in mesenteric arteries. U‐II plasma levels, as well as U‐II and UTR‐receptor expression in livers and portal veins of cirrhotic rats were significantly increased. U‐II administration further augmented the increased portal pressure (PP) and decreased mean arterial pressure (MAP), whereas palosuran decreased PP without affecting MAP. The decrease in PP was associated with an increase in splanchnic vascular resistance. In mesenteric vessels, palosuran treatment up‐regulated expression of RhoA and Rho‐kinase, increased Rho‐kinase‐activity, and diminished nitric oxide (NO)/cyclic guanosine 3′,5′‐monophosphate (cGMP) signaling. Moreover, palosuran increased renal blood flow, sodium, and water excretion in BDL rats. Conclusion: In BDL rats, U‐II is a mediator of splanchnic vasodilation, portal hypertension and renal sodium retention. The U‐II‐receptor antagonist palosuran might represent a new therapeutic option in liver cirrhosis with portal hypertension. (HEPATOLOGY 2008.)

Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis

Background and purpose:  Extrahepatic vasodilation and increased intrahepatic vascular resistance represent attractive targets for the medical treatment of portal hypertension in liver cirrhosis. In both dysfunctions, dysregulation of the contraction‐mediating Rho kinase plays an important role as it contributes to altered vasoconstrictor responsiveness. However, the mechanisms of vascular Rho kinase dysregulation in cirrhosis are insufficiently understood. They possibly involve mitogen‐activated protein kinase/extracellular signal‐regulated kinase (ERK)‐dependent mechanisms in extrahepatic vessels. As the multikinase inhibitor sorafenib inhibits ERK, we tested the effect of sorafenib on haemodynamics and dysregulated vascular Rho kinase in rats with secondary biliary cirrhosis.

Vascular dysfunction in human and rat cirrhosis: role of receptor-desensitizing and calcium-sensitizing proteins.

In cirrhosis, vascular hypocontractility leads to vasodilation and contributes to portal hypertension. Impaired activation of contractile pathways contributes to vascular hypocontractility. Angiotensin II type 1 receptors (AT1‐Rs) are coupled to the contraction‐mediating RhoA/Rho‐kinase pathway and may be desensitized by phosphorylation through G‐protein‐coupled receptor kinases (GRKs) and binding of β‐arrestin‐2. In the present study, we analyzed vascular hypocontractility to angiotensin II in cirrhosis. Human hepatic arteries were obtained during liver transplantation. In rats, cirrhosis was induced by bile duct ligation (BDL). Contractility of rat aortic rings was measured myographically. Protein expression and phosphorylation were analyzed by Western blot analysis. Immunoprecipitation was performed with protein A–coupled Sepharose beads. Myosin light chain (MLC) phosphatase activity was assessed as dephosphorylation of MLCs. Aortas from BDL rats were hyporeactive to angiotensin II and extracellular Ca2+. Expression of AT1‐R and Gαq/11,12,13 remained unchanged in hypocontractile rat and human vessels, whereas GRK‐2 and β‐arrestin‐2 were up‐regulated. The binding of β‐arrestin‐2 to the AT1‐R was increased in hypocontractile rat and human vessels. Inhibition of angiotensin II–induced aortic contraction by the Rho‐kinase inhibitor Y‐27632 was pronounced in BDL rats. Basal phosphorylation of the ROK‐2 substrate moesin was reduced in vessels from rats and patients with cirrhosis. Analysis of the expression and phosphorylation of Ca2+‐sensitizing proteins (MYPT1 and CPI‐17) in vessels from rats and patients with cirrhosis suggested decreased Ca2+ sensitivity. Angiotensin II–stimulated moesin phosphorylation was decreased in aortas from BDL rats. MLC phosphatase activity was elevated in aortas from BDL rats. Conclusion: Vascular hypocontractility to angiotensin II in cirrhosis does not result from changes in expression of AT1‐Rs or G‐proteins. Our data suggest that in cirrhosis‐induced vasodilation, the AT1‐R is desensitized by GRK‐2 and β‐arrestin‐2 and that changed patterns of phosphorylated Ca2+‐sensitizing proteins decrease Ca2+ sensitivity. (HEPATOLOGY 2007;45:495–506.)

Role of cannabinoid receptors in alcoholic hepatic injury: steatosis and fibrogenesis are increased in CB2 receptor-deficient mice and decreased in CB1 receptor knockouts

Background: Alcohol is a common cause of hepatic liver injury with steatosis and fibrosis. Cannabinoid receptors (CB) modulate steatosis, inflammation and fibrogenesis. To investigate the differences between CB1 and CB2 in the hepatic response to chronic alcohol intake, we examined CB knockout mice (CB1−/−, CB2−/−).

Role of β3‐adrenoceptors for intrahepatic resistance and portal hypertension in liver cirrhosis

Increased intrahepatic resistance and splanchnic blood flow cause portal hypertension in liver cirrhosis. Nonselective β‐adrenoceptor (β‐AR) antagonists have beneficial effects on hyperdynamic circulation and are in clinical use. In this context, the role of the β3‐AR is undefined. Here we investigated their expression and role in portal hypertension in patients and rats with liver cirrhosis. We analyzed cirrhotic human and rat tissues (liver, splanchnic vessels) and primary rat cells. Protein expression of β3‐AR was determined by western blot and messenger RNA (mRNA) levels by reverse‐transcription polymerase chain reaction (RT‐PCR). Activities of Rho‐kinase and the nitric oxide (NO) effector protein kinase G (PKG) were assessed by way of substrate phosphorylation (moesin, vasodilator‐stimulated phosphoprotein [VASP]). Cyclic 3′,5′ adenosine monophosphate (cAMP) accumulation was determined by an enzyme‐immunoassay kit. The effects of selective β3‐AR agonists (CGP12177A, BRL37344) and antagonist (SR59230A) were investigated by collagen matrix contraction of hepatic stellate cells (HSCs), in situ liver perfusions, and in vivo hemodynamic parameters in bile duct ligation and carbon tetrachloride intoxication in cirrhotic rats. In cirrhosis of humans and rats, β3‐AR expression is markedly increased in hepatic and in splanchnic tissues. Stimulation of β3‐AR leads to relaxation of HSCs by way of cAMP accumulation, and by inhibition of Rho‐kinase activity; any role of NO and its effector PKG was not observed. β3‐AR agonists decrease intrahepatic resistance and portal pressure in cirrhotic rats. Conclusion: There is a marked hepatic and mesenteric up‐regulation of β3‐ARs in human cirrhosis and in two different animal models of cirrhosis. The β3‐AR‐agonists should be further evaluated for therapy of portal hypertension. (HEPATOLOGY 2009.)

Hepatic and HSC-specific sorafenib effects in rats with established secondary biliary cirrhosis

Portal hypertension in cirrhosis depends on increased intrahepatic vascular resistance, which is explained by fibrosis and intrahepatic hyperresponsiveness to vasoconstrictors. Both are caused by activation and proliferation of hepatic stellate cells (HSCs). Portal hypertension of cirrhotic rats can be reduced by the multikinase inhibitor sorafenib, due to a reduction of intrahepatic vascular resistance. Therefore, the hepatic effects of sorafenib require further understanding. Here, we investigated hepatic and HSC-specific sorafenib effects in cirrhotic rats. Animal models of bile duct ligation-induced secondary biliary cirrhosis in rats were studied. The rats were treated with sorafenib (60 mg/kg/day) for 1 week, starting after established cirrhosis. Histological evaluation was carried out using hemalaun and eosin (HE) staining. Apoptosis was studied by PARP cleavage, colorimetric caspase-3 assay, and electrophoretic DNA detection. HSC activation was studied by hepatic Sirius red and immunohistochemical αSMA (α-smooth muscle actin) staining, and by in vitro experiments with culture-activated primary HSCs. Biochemical serum parameters suggested the occurrence of sorafenib-induced liver damage. HE staining revealed histological changes in livers of sham-operated and bile duct-ligated (BDL) rats in response to sorafenib, which were different in both groups. In BDL rats and isolated HSCs, the treatment with sorafenib reduced hepatic αSMA and procollagen-1α mRNA expression. As shown by immunohistochemical staining, perisinusoidal αSMA expression was reduced by sorafenib in BDL rats. This was associated with reduced perisinusoidal deposition of extracellular matrix, as revealed by Sirius red staining. Although no change in PARP cleavage and only a minor increase in hepatic caspase-3 activity were detected in BDL rats in response to sorafenib, livers of sorafenib-treated BDL rats contained small DNA fragments, which were not observed in untreated BDL rats. In conclusion, sorafenib treatment reduces the number of activated HSCs in cirrhotic livers. This leads to the decrease in intrahepatic vascular resistance, but also to liver damage in the dosage we used. Therefore, any translation to portal hypertensive patients who may profit from sorafenib should be done with particular care.