Robert Schierwagen

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−/−).

HSC-specific inhibition of Rho-kinase reduces portal pressure in cirrhotic rats without major systemic effects

BACKGROUND & AIMS Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition is needed. The delivery of Rho-kinase inhibitor to activated hepatic stellate cells reduces fibrosis. It might also relax these contractile cells and therewith decrease intrahepatic resistance. We tested this hypothesis by performing acute experiments in cirrhotic rats. METHODS Cirrhosis models were CCl(4)-intoxication and bile duct ligation. Three hours after injection of the Rho-kinase inhibitor (Y26732) coupled with a carrier (mannose-6-phosphate modified human serum albumin), which targets activated hepatic stellate cells, hemodynamics were analyzed by the colored microsphere technique and direct pressure measurements. The delivery site and effect of Rho-kinase inhibitor were investigated by immunohistochemical stainings, as well as Western blot. Experiments with Rho-kinase inhibitor coupled with unmodified human serum albumin served as untargeted control. RESULTS In both models of cirrhosis, the carrier coupled Rho-kinase inhibitor lowered the portal pressure and decreased the hepatic-portal resistance. Immunohistochemical desmin-staining showed the carrier in hepatic stellate cells. The targeted therapy decreased the expression of the phosphorylated substrate of Rho-kinase (moesin) and abolished myosin light chains phosphorylation in fibrotic septae (collagen-staining). The targeted Rho-kinase inhibitor showed no major extrahepatic effects. By contrast, the untargeted Rho-kinase inhibitor elicited severe systemic hypotension. CONCLUSIONS Activated hepatic stellate cells are crucially involved in portal hypertension in cirrhosis. Targeting of Rho-kinase in hepatic stellate cells not only decreased fibrosis, as previously shown, but also lowers portal pressure acutely without major systemic effects as demonstrated in this study.

Angiotensin‐II type 1 receptor‐mediated Janus kinase 2 activation induces liver fibrosis

Activation of the renin angiotensin system resulting in stimulation of angiotensin‐II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real‐time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human‐derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild‐type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho‐kinase activation. These effects were prevented in AT1R−/− mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII‐induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Conclusion: Our study substantiates the important cell‐intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis. (Hepatology 2014;60:334–348)

Activation of the Mas Receptor by Angiotensin-(1-7) in the Renin-Angiotensin System Mediates Mesenteric Vasodilatation in Cirrhosis

BACKGROUND & AIMS Splanchnic vascular hypocontractility with subsequent increased portal venous inflow leads to portal hypertension. Although the renin-angiotensin system contributes to fibrogenesis and increased hepatic resistance in patients with cirrhosis, little is known about its effects in the splanchnic vasculature, particularly those of the alternate system in which angiotensin (Ang) II is cleaved by the Ang-converting enzyme-2 (ACE2) to Ang-(1-7), which activates the G-protein-coupled Mas receptor (MasR). We investigated whether this system contributes to splanchnic vasodilatation and portal hypertension in cirrhosis. METHODS We measured levels of renin-angiotensin system messenger RNA and proteins in splanchnic vessels from patients and rats with cirrhosis. Production of Ang-(1-7) and splanchnic vascular reactivity to Ang-(1-7) was measured in perfused mesenteric vascular beds from rats after bile-duct ligation. Ang-(1-7) and MasR were blocked in rats with cirrhosis to examine splanchnic vascular hemodynamics and portal pressure response. RESULTS Levels of ACE2 and MasR were increased in splanchnic vessels from cirrhotic patients and rats compared with healthy controls. We also observed an ACE2-dependent increase in Ang-(1-7) production. Ang-(1-7) mediated splanchnic vascular hypocontractility in ex vivo splanchnic vessels from rats with cirrhosis (but not control rats) via MasR stimulation. Identical effects were observed in the splanchnic circulation in vivo. MasR blockade reduced portal pressure, indicating that activation of this receptor in splanchnic vasculature promotes portal inflow to contribute to development of portal hypertension. In addition, the splanchnic effects of MasR required nitric oxide. Interestingly, Ang-(1-7) also decreased hepatic resistance. CONCLUSIONS In the splanchnic vessels of patients and rats with cirrhosis, increased levels of ACE2 appear to increase production of Ang-(1-7), which leads to activation of MasR and splanchnic vasodilatation in rats. This mechanism could cause vascular hypocontractility in patients with cirrhosis, and might be a therapeutic target for portal hypertension.

FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.

Serum markers of the extracellular matrix remodeling reflect antifibrotic therapy in bile-duct ligated rats.

Background: Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins. Methods: Bile duct ligation (BDL) was performed on wild type rats, which received atorvastatin (15 mg/kg*d) for 1 week starting at 1, 2, 3, 4 and 5 weeks after BDL (T1–T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohistochemistry and hepatic hydroxyproline content. TGFβ levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of degradation MMP driven type I, III, IV and VI collagen degradation (C1M, C3M, C4M, and C6M) and type III and IV collagen formation (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes. Results: Serum markers of ECM neo-epitopes reflected significantly the deposition of ECM in the liver and were able to distinguish between early (T1–T3) and severe fibrosis (T4–T5). Statin treatment resulted in reduction of neo-epitope markers, especially when therapy was started in the stage of severe fibrosis (T4–T5). Furthermore, these markers correlated with hepatic expression of profibrotic cytokines TGFβ1 and TGFβ2. Formation markers of type III and IV collagen (PRO-C3 and P4NP7S) and degradation markers C4M and C6M correlated significantly with hepatic MMP-2 activity in rats with severe fibrosis. Conclusion: Determination of ECM remodeling turnover markers in serum allowed a distinction between mild and severe fibrosis. With respect to statin therapy, the markers may serve as read-out for efficacy of anti-fibrotic treatment.

Seven weeks of Western diet in apolipoprotein-E-deficient mice induce metabolic syndrome and non-alcoholic steatohepatitis with liver fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation and fibrosis, which might progress to cirrhosis. Human NASH is associated with metabolic syndrome (MS). Currently, rodent NASH models either lack significant fibrosis or MS. ApoE−/− mice are a MS model used in cardiovascular research. The aim of this work was to establish and characterise a novel mouse NASH model with significant fibrosis and MS. ApoE−/− and wild-type mice (wt) were fed either a western-diet (WD), methionine-choline-deficient-diet (MCD) or normal chow. Liver histology, RT-PCR, hepatic hydroxyproline content, triglycerides and cholesterol levels, and fasting glucose levels assessed hepatic steatosis, inflammation and fibrosis. Further, portal pressure was measured invasively, and kidney pathology was assessed by histology. ApoE−/− mice receiving WD showed abnormal glucose tolerance, hepatomegaly, weight gain and full spectrum of NASH including hepatic steatosis, fibrosis and inflammation, with no sign of renal damage. MCD-animals showed less severe liver fibrosis, but detectable renal pathological changes, besides weight loss and unchanged glucose tolerance. This study describes a murine NASH model with distinct hepatic steatosis, inflammation and fibrosis, without renal pathology. ApoE−/− mice receiving WD represent a novel and fast model with all characteristic features of NASH and MS well suitable for NASH research.

Circulating MicroRNAs as a marker for liver injury in human immunodeficiency virus patients

Human immunodeficiency virus (HIV) and hepatitis virus coinfection amplify and accelerate hepatic injury. MicroRNAs (miRNAs) are small regulatory RNAs suggested as biomarkers for liver injury. We analyzed the circulating levels of miRNAs in HIV patients with regard to the extent and etiology of liver injury. Total RNA was extracted from 335 serum samples of HIV patients and 22 healthy control participants using Qiazol. Comprehensive polymerase chain reaction (PCR) array analyses (768 miRNA) were performed in serum samples of eight HIV, eight HIV/HCV (hepatitis C virus), six HCV patients, and three healthy controls. Reverse transcription (RT)‐PCR measured levels of miRNA‐122, miRNA‐22, and miRNA‐34a in serum samples of 335 patients and 19 healthy control participants. Liver injury and fibrosis in these patients were defined using aspartate aminotransferase (AST) levels, fibrosis‐4 (FIB‐4) index and AST‐to‐platelet ratio index (APRI) score. The miRNA pattern of HIV/HCV samples showed altered expression of 57 and 33 miRNA compared to HCV and HIV infection, respectively. miRNA‐122, miRNA‐22, and miRNA‐34a were highly up‐regulated in HIV/HCV patients. Analyzing the entire cohort, these miRNAs were correlated with liver function tests and were independent predictors of liver injury (AST >2 × ULN). miRNA‐122 and miRNA‐22 were associated with relevant fibrosis (FIB‐4 >1.45; APRI >1). Circulating levels of miRNA‐122 were independent predictors for relevant fibrosis in HIV patients. Interestingly, miRNA‐122 and miRNA‐34a levels were higher in HIV/HCV patients, miRNA‐22 levels were highest in HIV/HBV patients, and circulating levels of miRNA‐34a correlated positively with illicit drug use and ethanol consumption. Conclusion: Circulating miRNA‐122, miRNA‐22, and miRNA‐34a correlates with the etiology of liver injury in HIV patients. These biomarkers not only mirror different mechanisms of hepatic injury, but also are independent predictors of liver injury in HIV patients. (Hepatology 2015;61:46–55)

Statins activate the canonical hedgehog-signaling and aggravate non-cirrhotic portal hypertension, but inhibit the non-canonical hedgehog signaling and cirrhotic portal hypertension.

Liver cirrhosis but also portal vein obstruction cause portal hypertension (PHT) and angiogenesis. This study investigated the differences of angiogenesis in cirrhotic and non-cirrhotic PHT with special emphasis on the canonical (Shh/Gli) and non-canonical (Shh/RhoA) hedgehog pathway. Cirrhotic (bile duct ligation/BDL; CCl4 intoxication) and non-cirrhotic (partial portal vein ligation/PPVL) rats received either atorvastatin (15 mg/kg; 7d) or control chow before sacrifice. Invasive hemodynamic measurement and Matrigel implantation assessed angiogenesis in vivo. Angiogenesis in vitro was analysed using migration and tube formation assay. In liver and vessel samples from animals and humans, transcript expression was analyzed using RT-PCR and protein expression using Western blot. Atorvastatin decreased portal pressure, shunt flow and angiogenesis in cirrhosis, whereas atorvastatin increased these parameters in PPVL rats. Non-canonical Hh was upregulated in experimental and human liver cirrhosis and was blunted by atorvastatin. Moreover, atorvastatin blocked the non-canonical Hh-pathway RhoA dependently in activated hepatic steallate cells (HSCs). Interestingly, hepatic and extrahepatic Hh-pathway was enhanced in PPVL rats, which resulted in increased angiogenesis. In summary, statins caused contrary effects in cirrhotic and non-cirrhotic portal hypertension. Atorvastatin inhibited the non-canonical Hh-pathway and angiogenesis in cirrhosis. In portal vein obstruction, statins enhanced the canonical Hh-pathway and aggravated PHT and angiogenesis.

Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis.

Objective Angiotensin II (AngII) activates via angiotensin-II-type-I receptor (AT1R) Janus-kinase-2 (JAK2)/Arhgef1 pathway and subsequently RHOA/Rho-kinase (ROCK), which induces experimental and probably human liver fibrosis. This study investigated the relationship of JAK2 to experimental and human portal hypertension. Design The mRNA and protein levels of JAK2/ARHGEF1 signalling components were analysed in 49 human liver samples and correlated with clinical parameters of portal hypertension in these patients. Correspondingly, liver fibrosis (bile duct ligation (BDL), carbon tetrachloride (CCl4)) was induced in floxed-Jak2 knock-out mice with SM22-promotor (SM22Cre+-Jak2f/f). Transcription and contraction of primary myofibroblasts from healthy and fibrotic mice and rats were analysed. In two different cirrhosis models (BDL, CCl4) in rats, the acute haemodynamic effect of the JAK2 inhibitor AG490 was assessed using microsphere technique and isolated liver perfusion experiments. Results Hepatic transcription of JAK2/ARHGEF1 pathway components was upregulated in liver cirrhosis dependent on aetiology, severity and complications of human liver cirrhosis (Model for End-stage Liver disease (MELD) score, Child score as well as ascites, high-risk varices, spontaneous bacterial peritonitis). SM22Cre+- Jak2f/f mice lacking Jak2 developed less fibrosis and lower portal pressure (PP) than SM22Cre−-Jak2f/f upon fibrosis induction. Myofibroblasts from SM22Cre+-Jak2f/f mice expressed less collagen and profibrotic markers upon activation. AG490 relaxed activated hepatic stellate cells in vitro. In cirrhotic rats, AG490 decreased hepatic vascular resistance and consequently the PP in vivo and in situ. Conclusions Hepatic JAK2/ARHGEF1/ROCK expression is associated with portal hypertension and decompensation in human cirrhosis. The deletion of Jak2 in myofibroblasts attenuated experimental fibrosis and acute inhibition of JAK2 decreased PP. Thus, JAK2 inhibitors, already in clinical use for other indications, might be a new approach to treat cirrhosis with portal hypertension.