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

Angiotensin-II Type 1 Receptor-Mediated Janus Kinase2 Activation Induces Liver Fibrosis

Michaela Granzow,1* Robert Schierwagen,1* Sabine Klein,1 Benita Kowallick,1 Sebastian Huss,2 Markus

Linhart,3 Irela G. Reza Mazar,4 Jan G€ortzen,1 Annabelle Vogt,1 Frank A. Schildberg,5 Maria A. Gonzalez-

Carmona,1 Alexandra Wojtalla,1 Benjamin Kr€amer,1 Jacob Nattermann,1 S€oren V. Siegmund,1 Nikos Werner,3

Dieter O. F€urst,4 Wim Laleman,6 Percy Knolle,5 Vijay H. Shah,7 Tilman Sauerbruch,1 and Jonel Trebicka1

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 fibro-sis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intra-cellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodentsand 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 confocalmicroscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intox-ication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibitedby AG490. In vitro experiments were performed with primary rodent hepatic stellatecells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human andhuman-derived LX2 cells. JAK2 expression and activity were increased in experimentalrodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimula-tion in wild-type animals led to activation of HSCs and fibrosis in vivo through phos-phorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects wereprevented in AT1R2/2 mice. Pharmacological inhibition of JAK2 attenuated liver fibro-sis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showedincreased expression and activation in activated HSCs, when compared to quiescentHSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells,AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 ledto increased profibrotic gene expression in LX2 cells, which was blocked by AG490.Conclusion: Our study substantiates the important cell-intrinsic role of JAK2 in HSCsfor development of liver fibrosis. Inhibition of JAK2 might therefore offer a promisingtherapy for liver fibrosis. (HEPATOLOGY 2014;60:334-348)

Chronic liver diseases represent a major globalhealth problem with annually approximately800,000 deaths worldwide.1,2 Persistent liver

injury induces hepatic fibrosis defined as excessivehepatic production and deposition of extracellular

matrix (ECM) by myofibroblasts.3 In liver fibrosis,the main source of these myofibroblasts are activatedhepatic stellate cells (HSCs) located in the space ofDisse.3 These myofibroblastic HSCs express alpha-smooth muscle actin (a-SMA) as a marker of their

Abbreviations: Ab, antibody; ACE, angiotensin converting enzyme; AngII, angiotensin II; AT1R, angiotensin-II type I receptor; BDL, bile duct ligation; Col1,collagen I; DCFDA, dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemiluminescence; ECM, extracellular matrix;FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSCs, hepatic stellate cells; IHC, immunohistochemical; IP, intraperitoneal; I/R, ische-mia-reperfusion; JAK2, Janus kinase 2; KCs, Kupffer cells; mRNA, messenger RNA; RAS, renin angiotensin system; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; pJAK2, phosphorylated JAK2; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, standard error of themean; a-SMA, alpha-smooth muscle actin; STAT, signal transducers and activators of transcription; TAA, thioacetamide; WT, wild type.

From the 1Department of Internal Medicine I, University of Bonn, Bonn, Germany; 2Department of Pathology, University of Bonn, Bonn, Germany; 3Depart-ment of Internal Medicine II, University of Bonn, Bonn, Germany; 4Institute for Cell Biology, University of Bonn, Bonn, Germany; 5Institutes for Molecular Med-icine and Experimental Immunology, University of Bonn, Bonn, Germany; 6Department of Liver and Biliopancreatic disorders, University of Leuven, Leuven,Belgium; and 7Gastroenterology Research Unit and Cancer Cell Biology Program, Mayo Clinic, Rochester, MN.

Received August 28, 2013; accepted February 21, 2014.

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activation.3-5 Stimulation of the angiotensin-II (AngII)type 1 receptor (AT1R) by local or systemic activa-tion of the renin angiotensin system (RAS) plays acrucial role in HSC activation and fibrogenesis.6-13

Subsequent accumulation of ECM in the liver dis-turbs the intrahepatic angioarchitecture and thereforeleads to further complications.14 Activated HSCs playa pivotal role in the progression of fibrosis to de-compensated cirrhosis with high morbidity andmortality.14,15

AT1R is coupled to heterotrimeric G proteins (Gaq/11 and Ga12/13), allowing stimulation and activationof several signal pathways involved in cell contractionand ECM production.16 One of these pathways is theRhoA/Rho-kinase pathway, which is crucially involvedin fibrosis and portal hypertension as previously shownby our group.17-20 Recently, a link between AT1 recep-tor and RhoA/Rho-kinase pathway was established insmooth muscle cells showing the involvement of thetyrosine kinase, Janus kinase 2 (JAK2).21 AT1R stimula-tion activates JAK2, which, in turn, induces Arhgef1,the nucleotide exchange factor responsible for activationof RhoA, which subsequently activates Rho-kinase.21

JAK2 is involved in the intracellular signaling ofmany other receptors, for example, for hormones andcytokines toward transcription regulators of the signaltransducers and activators of transcription (STAT)family. In HSCs, JAK2 acts through the STAT path-way or independent of it.22,23 The role of these JAK2-induced pathways in hepatocytes has been investigatedfor hepatic steatosis, ischemia-reperfusion (I/R) injuryand cancer, whereas the role of JAK2 in fibrosis, espe-cially mediated by AT1R stimulation, has not beeninvestigated to date.

The present study showed that AT1 receptor-mediated JAK2 activation induces liver fibrosis. Conse-quently, inhibition of JAK2 blunts fibrosis. This activa-tion of the JAK2/Rho-kinase pathway—dependent onstimulation of AT1R and leading to activation ofHSC—was shown in different animal models and cellculture experiments. Furthermore, we confirmed theup-regulation of JAK2/Rho-kinase expression inhuman fibrosis.

Materials and Methods

Animals. We used 190 Sprague-Dawley wild-type(WT) rats and 155 mice (95 C57BL/6J WT and 60AT1aR2/2 mice) for our experiments. AT1aR-deficient mice were kindly provided by Nikos Werner(Department of Internal Medicine II, University ofBonn, Bonn, Germany). The responsible committeefor animal studies in North Rhine-Westphaliaapproved the study (LANUV 8.87-50.10.31.08.28).

Cholestatic Model of Fibrosis. Bile duct ligation(BDL) was performed in rats with an initial bodyweight of 180-200 g, as described previously.17,18

Experiments were carried out 2 weeks after BDL in 19rats, whereas 28 sham-operated rats served as controls,respectively. Ten rats undergoing BDL for 2 weeksreceived AG490 (1 mg/kg/day, intraperitoneally [IP])on the last 7 days before sacrifice. BDL and shamoperation was performed in 5 20-25 g WT mice,which were sacrificed after 2 weeks.

Toxic Models of Fibrosis. Twenty-three rats withan initial body weight of 100-120 g underwent twice-weekly inhalation of 1 L/min CCl4 for 14-16 weeksuntil ascites were present, as described previously.17,18

Twenty-one age-matched control rats did not receiveCCl4. Periodic CCl4 inhalation of 2 L/min was per-formed in 25 mice (15 WT and 10 AT1aR2/2) for 4weeks, as described previously,24 whereas 38 miceserved as controls (11 WT and 27 AT1aR2/2). Addi-tionally, 24 rats with an initial body weight of 200-250 g underwent weekly thioacetamide (TAA) admin-istration with adjusted dosing in their drinking waterfor 18 weeks, as described previously.25

AngII-Mediated Fibrosis. For continuous releaseof AngII, osmotic pumps (2ML2 for rats, 2002for mice, Alzet; Charles River Laboratories, Sulzfeld,Germany) were subcutaneously implanted in vivo in18 rats, 15 AT1aR2/2, and 19 WT littermates, asdescribed previously.26 Each pump released 0.7 mg/kg/day of AngII in rats as well as mice for 14 days, whichhas been shown to induce hepatic fibrosis.9 Pumpsreleasing saline were implanted in 5 rats and 24 miceas controls. Additionally, 9 of the rats received AG490(1 mg/kg/day, IP) for the last 7 days before sacrifice.

Address reprint requests to: Jonel Trebicka, M.D., Department of Internal Medicine I, University of Bonn, Sigmund-Freud Straß e 25, D-53105 Bonn,Germany. E-mail: [email protected]; Fax: 149 228 287 19718.

The study was supported by grants from Deutsche Forschungsgemeinschaft (SFB TRR57 projects 1, 10, 15, 18, and Q1).*These authors contributed equally.Copyright VC 2014 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.27117Potential conflict of interest: Nothing to report.

HEPATOLOGY, Vol. 60, No. 1, 2014 GRANZOW, SCHIERWAGEN, ET AL. 335

Human Liver Samples. The human ethics commit-tee of the University of Bonn (202/01) approved the useof human liver samples, obtained during liver transplan-tation from patients with alcohol-induced cirrhosis(n 5 16). Liver samples from patients without cirrhosisundergoing liver resection served as controls (n 5 10).None of the patients or donors received catecholamines,angiotensin converting enzyme (ACE) inhibitors, orangiotensin receptor antagonists before transplantation.Samples were snap-frozen after excision.

Western Blotting. Snap-frozen cells and liver sam-ples were processed as previously described usingsodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE) gels and nitrocellulose mem-branes.17,18 Ponceau S staining assured equal proteinloading. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) or b-actin served as endogenous controls.Membranes were incubated with the respective pri-mary antibody (Ab; Supporting Table 1) and corre-sponding secondary peroxidase-coupled Ab (Santa-Cruz-Biotechnology, Santa Cruz, CA). After enhancedchemiluminescence (ECL; Amersham, Bucks, UK),digital detection was evaluated using Chemi-Smart(PeqLab Biotechnologies GmbH, Erlangen, Germany).

Western blotting analysis was quantified by densi-tometry of all experiments (means 6 standard error ofthe mean [SEM]) with values of controls set to 100densitometric units (representative western blottingsare shown in the Supporting Figures). Expression ofphosphorylated JAK2 (pJAK2) at Tyr1007/1008 servedas a marker of JAK2 activation, and Rho-kinase activ-ity was analyzed as phosphorylation of its substratemoesin at Thr558 detected by phospho- and site-specific Abs.

Quantitative Real-Time Polymerase Chain Reac-tion. RNA isolation, reverse transcription, and detec-tion by real-time polymerase chain reaction (RT-PCR)were performed as described previously.17,18 Assaysprovided by Applied Biosystems (Foster City, CA) arelisted in Supporting Table 2. 18S ribosomal RNAserved as an endogenous control. The results of HSCand liver samples were expressed as 22DDCt, and thedata are reported as relative gene expression comparedto the control group.

Hepatic Hydroxyproline Content. Hepatichydroxyproline content was determined photometri-cally, as described previously.19

Sirius Red Staining. For the detection of collagenfibers, paraffin-embedded liver sections were stainedwith Sirius red using standard methods describedpreviously.19

Immunohistochemical Staining for JAK2, pJAK2,and a-SMA. Staining for JAK2, pJAK2, and a-SMAwas performed in cryosections from liver tissue (3 and7 mm). The detailed method is described in theSupporting Information. The amount of stainingwas evaluated by computational analysis (Histoquant;3DHistech, Budapest, Hungary), as describedpreviously.27,28

Quantification (% of stained area) of immunohisto-chemical (IHC) staining is expressed as mean 6 SEMof all experiments. For representative sections, pleasesee the Supporting Figures.

Coimmunofluoresence Stainings of pJAK2 anda-SMA. Colocalization of pJAK2 and a-SMA wasanalyzed by immunofluorescent staining of 7-mm cryo-sections, as described in the Supporting Information.

Isolation of Primary HSCs. Rat and mouse HSCswere isolated as described previously.18,29 Briefly, pri-mary HSCs were isolated in a two-step pronase-colla-genase perfusion from livers of healthy rats (n 5 36),as well as WT (n 5 8) or AT1aR2/2 mice (n 5 8),and fractionated by density-gradient centrifugation.Viability and purity were systematically over 95%.Cells were seeded on uncoated plastic culture dishes.Experiments were performed 7 days after isolation orafter the first passage (10 days) when HSCs were fullyactivated.

Primary human HSCs were obtained from ScienCell(San Diego, CA) and were cultured and harvested asdescribed previously.30-32

Isolation of Hepatocytes. Primary hepatocyteswere isolated from male WT rats (n 5 5) or WT mice(n 5 8), as described previously.29

Isolation of Kupffer Cells. Primary Kupffer cells(KCs) were isolated from WT mice (n 5 10), asdescribed previously.29

Incubation with AngII, AG490, and Losartan. LX2cells were provided by Vijay H. Shah (Mayo Clinic,Rochester, NY), originally established by Scott Fried-man. AngII (10 mM) and/or AG490 (1.5, 5, and25 mM) and/or Losartan (10 mM) was added to theculture medium of these cells, as indicated, for 3 days,or cells remained untreated.

Transfection With JAK2 Plasmid. Twenty-fourhours before transfection, 6 x 105 LX2 cells were incu-bated with transfection media (Dulbecco’s modifiedEagle’s medium [DMEM] with 10% fetal calf serum[FCS] without penicillin/streptomycin). The JAK2plasmid and the respective empty plasmid control wereisolated according to manufacturer instructions (Nucle-oBond Xtra Maxi kit; Machery, Nagel, Germany).

336 GRANZOW, SCHIERWAGEN, ET AL. HEPATOLOGY, July 2014

Plasmid (15 mL) and 37.5 mL of lipofectamine wereincubated for 20 minutes with a total volume of 3.6mL of media. This plasmid/lipofectamine mix wasadded drop-wise to cells after removal of media. After3-4 hours, cells were again incubated with media, con-taining 10% FCS, and harvested after 3 days. Efficacyof transfection was tested by RT-PCR and westernblotting.

Transduction With AdJAK2. The recombinantreplication-deficient adenoviral vector, AdJAK2, wasgenerated using the adenoviral backbone vector,pAdEasy-1, containing the sequence of human adeno-virus 5 with deletion of E1- and E3-genes with thetransfer vector, pShuttleCMV (Stratagene, La Jolla,CA). JAK2 transgene was obtained from the plasmid,pUNO1-mJAK2a (Invivogen, Toulouse, France).

LX2 cells (6 3 105) were cultured in 10-cm plates.After 24 hours, cells were transduced with AdJAK2and AdLacZ as a control at a transfection multiplicityof infection of 250 in DMEM supplemented with 2%FCS and 1% penicillin/streptomycin for 2 hours. Cellswere washed twice with media, and AdJAK2-transduced cells were treated with 10% FCS and 1%penicillin/streptomycin alone, or additionally with 5mM of AngII, or with 5 mM of AngII and AG490.AdLacZ-transduced cells were prepared in the sameway as AdJAK2-transduced cells. Efficacy of transfec-tion was tested by RT-PCR and western blotting.

Detection of Reactive Oxygen Species. Serum-starved LX2 cells (1.8 3 104 cells) plated in six-wellplates were loaded with the reagent, 2’,7’-dichloro-fluorescein diacetate (DCFDA), a fluorogenic dye thatmeasures hydroxyl, peroxyl, and other reactive oxygenspecies (ROS) activity within the cell (DCFDA-Cellu-lar Reactive Oxygen Species Detection Assay Kit, cata-log no. ab113851; Abcam, Cambridge, MA). After 20minutes at 37�C incubation with AngII (1025 M)with or without AG490 (5 mM), cells were washedand measured for the indicated time in a multiwellfluorescence plate reader using excitation and emissionfilters of 485 and 535 nm, respectively.

Apoptosis and Cycle Analysis. Analysis of apopto-sis (Annexin V Apoptosis Detection Kit; BD Bioscien-ces, Heidelberg, Germany) and cell-cycle analysis wasperformed as previously described.33

Statistical Analysis. Data are presented as mean-

6 SEM. The Student t test was used for comparison,

where appropriate. Mann-Whitney’s U test or analysis

of variance were used for comparison between groups

(minimum n 5 5/group). P values <0.05 were consid-

ered statistically significant.

Results

Increased Expression and Phosphorylation ofJAK2 in Experimental and Human Liver Fibrosis isFound Predominantly in Activated HSCs and Myofi-broblasts. In human and experimental fibrosis,hepatic expression of the components of RAS wasincreased (Fig. 1A), suggesting that RAS is activated inliver fibrosis and cirrhosis. Accordingly, hepatic expres-sion of AT1R and its downstream effectors (JAK2,Arhgef1, RhoA, and Rho-kinase) were increased inhuman liver fibrosis (Fig. 1B,D and Supporting Fig.S1A). We also observed that activation of JAK2, ana-lyzed as JAK2 phosphorylation at Tyr1007/1008(pJAK2),21 and of Rho-kinase, analyzed by the phos-phorylation of its substrate, moesin, at Thr558, wereincreased in human cirrhosis, when compared to non-fibrotic controls (Fig. 1B and Supporting Fig. S1A).This was confirmed by IHC staining. Similarly, usingtwo different models of fibrosis in mice and three dif-ferent models in rats, we found that AT1R-mediatedJAK2 phosphorylation at Tyr1007/100821 wasincreased in fibrotic livers, when compared to controllivers (Fig. 2A,B and Supporting Fig. S1B,C). Further-more, the JAK2 downstream effector, Arhgef1, its tar-get, RhoA, and the downstream effector, Rho-kinase,were strongly expressed and activated in liver fibrosis(Fig. 2A,B).

Interestingly, although faint JAK2 staining wasfound in most liver cells, it predominated in fibroticsepta as well as in perivascular and -sinusoidal regions(Supporting Figs. S3A-S5A). In contrast, pJAK2 wasonly found in fibrotic septa and around vessels andsmall sinusoids (Figs. 1C and 2C and Supporting Figs.S3B-S5B). Immunofluorescence costaining of pJAK2and a-SMA in fibrotic livers showed the expression ofpJAK2 in a-SMA-positive cells in fibrotic septa as wellas in perivascular and -sinusoidal areas (Fig. 1E andSupporting Fig. S3C). These data show, for the firsttime, that AT1R-dependent JAK2 phosphorylation atTyr1007/1008 occurs in hepatic myofibroblasts, whichderive mainly from activated HSCs.

Protein expression of JAK2-dependent STAT3/sup-pressor of cytokine signalling 3 signaling was also acti-vated in different fibrosis models of mice and rats(Supporting Fig. S2A,B), suggesting an increased activ-ity of JAK2.

AT1R Stimulation Induced Fibrosis Through theJAK2 Pathway In Vivo. We assessed the effect ofJAK2 inhibition on hepatic fibrogenesis in rats andmice. AG490, injected daily for 1 week before killingof BDL rats (14 days of BDL in total) attenuated


fibrogenesis, as assessed by hydroxyproline content andSirius red staining. This treatment attenuated the acti-vation of HSCs, as mirrored by a-SMA immunostain-ing as a result of inhibition of JAK2 phosphorylation

at Tyr 1007/1008, as assessed by IHC (Fig. 3A,B andSupporting Fig. S6).

Transcription of the JAK2 downstream proteins, aswell as HSC activation documented by elevated a-

Fig. 1. Expression and activity of AT1R and its downstream effectors in human fibrosis were up-regulated in activated HSCs. (A) mRNA levels ofangiotensinogen, ACE, Renin, AT1R, JAK2, Arhgef1, RhoA, and Rho-kinase were analyzed by RT-PCR in liver samples of patients with cirrhosis andcompared to noncirrhotic liver samples. (B) Protein expression of AT1R, JAK2, pJAK2, Arhgef1, RhoA, Rho-kinase, and pMoesin were analyzed in liversamples of patients with cirrhosis and compared to noncirrhotic liver samples. (C) Expression and distribution of pJAK2 were investigated by IHC (seerepresentative section in Supporting Fig. S3A,B). (D) Scheme of the AT1R signaling pathway, elucidating the downstream effectors and their relationto each other. (E) Staining for pJAK2 in green, a-SMA in red and overlay with Hoechst33258 elucidate the localization of pJAK2 in a-SMA-positivecells in human liver fibrosis sections (see also Supporting Fig. S3C). Scale bar, 20 mm.


SMA levels and collagen production, was significantlyup-regulated at the messenger RNA (mRNA) levelafter BDL. This effect was attenuated by JAK2 inhibi-tion with AG490 (Fig. 3C).

To further underline the crucial role of AT1R, con-tinuous stimulation of AT1R was performed usingAngII infusion in rats and mice for 14 days. Indeed,this treatment induced expression and activation ofJAK2 pathway components (Fig. 3D), resulting inmild fibrosis.

Interestingly, concomitant JAK2 inhibition duringthe last 7 days before sacrifice of rats receiving AngIIinfusion significantly decreased fibrosis, as assessed byhydroxyproline content and Sirius red staining, as wellas inhibition of JAK2 phosphorylation at Tyr 1007/1008 and JAK2 downstream signaling (Fig. 4 andSupporting Fig. S7). These findings demonstrate thatAngII injection induced fibrosis as a result of AT1R-mediated activation of JAK2 and its downstreameffectors.

Fig. 2. Expression and activity ofAT1R and its downstream effectors inexperimental fibrosis were up-regulated.(A) Protein expression of AT1R, JAK2,pJAK2, Arhgef1, RhoA, Rho-kinase, andpMoesin, analyzed by western blotting,was increased in liver samples fromrats with BDL and CCl4-induced fibrosis,compared to their respective controls.(B) Significantly increased protein levelsof AT1R, JAK2, pJAK2, Arhgef1, RhoA,Rho-kinase, and pMoesin were detectedusing western blotting in liver samplesfrom mice after 2 weeks of BDL or 4weeks of CCl4 treatment, compared totheir respective controls. (C) Expressionand distribution of pJAK2 were investi-gated by IHC for rats and mice (seerepresentative section in SupportingFigs. S4 and S5).


Fig. 3. Stimulation of AT1R activated JAK2, but JAK2 inhibition attenuated hepatic fibrosis. (A) Quantification of Sirius red, a-SMA, and pJAK2IHC staining is shown along with representative stained sections of rat liver after BDL for 14 days, with or without AG490 treatment (1 mg/kg/day) 7 days before sacrifice (see quantifications of Sirius red and a-SMA stainings, as well as representative sections of pJAK2 staining, in Sup-porting Fig. S6). (B) Less hepatic hydroxyproline content as a marker for fibrosis is shown. (C) mRNA levels of JAK2, Arhgef1, RhoA, Rho-kinase,a-SMA, and Col1 were analyzed by RT-PCR in liver samples of rats after BDL for 14 days, with or without AG490 treatment (1 mg/kg/day) 7days before sacrifice. (D) Protein expression of AT1R, JAK2, pJAK2, Arhgef1, RhoA, Rho-kinase, pMoesin, and a-SMA was analyzed by westernblotting in liver samples of rats receiving AngII infusion (0.7 mg/kg/day by osmotic pumps for 14 days) or solvent. Expression and activation ofAT1R downstream effectors were increased after AT1 receptor stimulation.


In these models, we could not detect any differenceregarding thrombogenic or angiogenic effects of theJAK2 inhibitor, as assessed by histology.

Inhibition of JAK2 Pathway or Absence of AT1RWith Consecutive Down-Regulation of JAK2 Path-way In Vivo Attenuated Fibrosis. To further assessthe role of AT1R in vivo, we induced another form of

chronic liver injury by means of CCl4 inhalation inAT1aR2/2 mice and their respective WT littermates.(AT1aR is the subtype of AT1R in mice responsiblefor contraction of smooth muscle cells and importantfor development of liver fibrosis,10,16 further abbrevi-ated as AT1R). Expression of JAK2 and its down-stream effectors was significantly down-regulated in

Fig. 4. Stimulation of AT1R with AngII activated JAK2, but JAK2 inhibition attenuated hepatic fibrosis. (A) Hepatic hydroxyproline content andquantification of Sirius red and pJAK2 IHC staining are shown in rats after AngII infusion (0.7 mg/kg/day) for 14 days with or without additionalAG490 (1 mg/kg/day) 7 days before sacrifice. JAK2 inhibition with AG490 blunted fibrosis development in these rats (for representative sec-tions, see Supporting Fig. S7). (B) Protein levels of AT1R, JAK2, pJAK2, Arhgef1, RhoA, Rho-kinase, and pMoesin were analyzed using westernblotting, and mRNA levels of JAK2, Arhgef1, RhoA, Rho-kinase, a-SMA, and Col1 were analyzed by RT-PCR in liver samples of rats after AngIIinfusion (0.7 mg/kg/day) for 14 days with or without additional AG490 (1 mg/kg/day) 7 days before sacrifice. (C) Hepatic protein levels of a-SMA are shown in rats after AngII infusion (0.7 mg/kg/day) for 14 days with or without additional AG490 (1 mg/kg/day) 7 days beforesacrifice.


these mice (Fig. 5 and Supporting Fig. S8). This wasassociated with reduced hepatic fibrosis after challenge(Fig. 5 and Supporting Fig. S8). AngII induces fibrosis

and activation of JAK2 and its downstream effectors inwt mice, but not in AT1R2/2 mice. (Fig. 6A,B andSupporting Fig. S9).

Fig. 5. AT1R deficiency attenuated hepatic fibrosis. (A) Decreased hepatic transcription of JAK2, Arhgef1, RhoA, Rho-kinase, a-SMA, and Col1,(B) reduced hepatic hydroxyproline content, and (C) decreased protein expression of JAK2, pJAK2, Arhgef1, RhoA, Rho-kinase, and pMoesin inAT1R-deficient mice after CCl4-induced fibrosis, compared to CCl4-treated WT mice. (D) Representative sections of IHC stains for a-SMA, JAK2,and pJAK2 in AT1R-deficient mice versus WT mice after CCl4 treatment are shown (for quantification, see Supporting Fig. S8B).


Thus, JAK2 inhibition upon fibrosis inductionblunted fibrosis. Similarly, AT1R-deficient animalsshowed attenuated fibrogenesis and lack of activationof JAK2 and its downstream effectors.

AT1R-Dependent Activation of JAK2 Pathway-InducedActivation of HSC and Collagen Production. In vitroexperiments confirmed the results from IHC staining.The components of the JAK2 pathway (JAK2,pJAK2, Arhgef-1, Rho-kinase, and pMoesin) werehighly expressed and activated in HSCs after theiractivation (day 7 in culture), when compared to hepa-tocytes and KCs (Fig. 6C,D), confirming that pJAK2was localized in activated HSCs (as shown inFig. 6C,D).

Activated HSCs showed increased expression andactivation of components of the AT1R-mediated JAK2pathway. To further decipher the role of JAK2, specifi-cally for fibrogenesis, we overexpressed JAK2 in animmortalized human HSC line, namely, LX2 cells,using a JAK2 plasmid (Fig. 7A). We observed elevatedtranscription of its downstream effectors, RhoA andRho-kinase, together with higher protein expression ofa-SMA and collagen I (Col1) as markers for profi-brotic activity of HSCs (Fig. 7B). Efficacy of JAK2transfection is shown in Supporting Fig. S10.

Additional stimulation of these cells with AngII fur-ther increased the transcription of downstream effectorsof JAK2 and their profibrotic activity (a-SMA andCol1; Fig. 7B). Again, the JAK2 inhibitor, AG490,blunted the AngII effect (Fig. 7B). In order to excludea vector-specific effect, we repeated these experimentsusing adenoviral vectors containing JAK2 or LacZ (as acontrol) and obtained similar results (Fig. 7B). AT1Rdependence was shown in further experiments usingHSCs isolated from AT1R-deficient mice. In thesemice, neither AngII nor AG490 elicited any significanteffect on the transcription of AT1R/JAK2 pathwayeffectors (Fig. 7C). Efficacy of JAK2 transduction withthe adenoviral vector is shown in Supporting Fig. S10.

In Vitro JAK2 Inhibition and Blocking Experi-ments. AT1R stimulation, assessed by incubation ofhuman immortalized HSCs (LX2 cells) with AngII,induced JAK2 phosphorylation at Tyr 1007/1008 (Sup-porting Fig. S10). The pharmacologic inhibitor ofJAK2, AG490, dose dependently blocked phosphoryla-tion of JAK2 and expression of the downstream effec-tors, RhoA and Rho-kinase (Supporting Fig. S10). Also,the elevated Arhgef1 expression and Rho-kinase activa-tion was blunted after AG490 coincubation (Fig. 8A).Experiments with human primary HSCs confirmed thefindings in LX2 cells (Fig. 8B,C). AngII treatment ofLX2 cells increased ROS, but after coincubation with

AG490, ROS dropped to normal levels (Fig. 8E). Block-ing experiments using AG490 (JAK2 inhibitor) or Los-artan (AT1R blocker) demonstrated blunting of proteinexpression of downstream effectors of JAK2 (Arhgef1and Rho-kinase) and a-SMA (activation marker) in pri-mary HSCs similarly to LX2 cells (Fig. 8D). Interest-ingly, blockade of AT1R or inhibition of JAK2 had noeffect on apoptosis or cell cycle (Supporting Fig. S10).

In summary, our in vitro data demonstrated thatAT1R-dependent JAK2 induced activation and profi-brotic activity of HSCs though its downstream effec-tors. Inhibition of JAK2 blunted AT1R-mediatedactivation, profibrotic properties, migration, and con-traction of HSCs.

Discussion

Our in vivo and in vitro findings show the impor-tance of AT1R-dependent JAK2 activation in hepaticfibrosis in mice, rats, and humans. These findings areuniformly supported by different approaches using sev-eral animal models of liver fibrosis as well as by phar-macological and genetic modulations. JAK2 expressionand activation was found to occur mainly in activatedHSCs, underlining the pivotal role of these cells forcollagen production.

RAS is critically involved in the development ofprogressive fibrosis in different tissues (heart, kidney,and lung) as well as in the liver, mainly as a result ofAT1R stimulation.6,34-36 In chronic liver injury, activa-tion of RAS is a hallmark of progressive liver dis-ease.2,7,14,15 RAS activation leads to sodium retentionwith formation of ascites in later stages of liver disease.But, it also leads to initiation, perpetuation, and aug-mentation of inflammatory and fibrogenic processeswithin the liver, where HSCs are key players.3

It has been shown only recently that AngII exerts itsAT1R-dependent contractile effect in the vascularsmooth muscle cells through phosphorylation of JAK2and the nucleotide exchange factor for RhoA, Arhgef1,which then activates the RhoA cascade toward Rho-kinase, finally resulting in contraction of these cells.21

Importantly, JAK2-mediated signaling in fibrosis iscaused by both increased JAK2 expression andincreased JAK2 activation. In this landmark study, theresearchers described the missing link between AT1Rstimulation and the RhoA/Rho-kinase pathway in arte-rial smooth muscle cells as a potential mechanism forarterial hypertension.21 Here, we demonstrate that thesame holds true for myofibroblastic-activated HSCs.We show that HSCs—but not hepatocytes or KCs—harbor an activated JAK2/RhoA/Rho-kinase signaling


Fig. 6. AT1R deficiency attenuated hepatic fibrosis. AT1R-mediated activation of JAK2 signaling is predominant in primary HSCs and inducedcollagen production. (A) Hepatic hydroxyproline content, and protein expression of AT1R, JAK2, pJAK2, Arhgef1, RhoA, Rho-kinase, and pMoesin,were analyzed in liver samples of WT and AT1R-deficient mice after AngII infusion (0.7 mg/kg/day) or solvent infusion. Quantification of IHCstaining for pJAK2 is also shown for these mice (for representative sections, see Supporting Fig. S9A). In AT1R-deficient mice, pJAK2 remainedunchanged after AngII infusion and mice did not develop fibrosis. (B) Hepatic transcription levels of JAK2, Arhgef1, RhoA, Rho-kinase, a-SMA,and Col1 in liver samples of WT and AT1R-deficient mice after AngII infusion (0.7 mg/kg/day) or solvent infusion. (C) Protein expression ofJAK2, pJAK2, Arhgef1, Rho-kinase, and pMoesin was analyzed by western blotting in activated (day 7) primary mouse HSCs, compared to quies-cent (day 0) cells, hepatocytes, and KCs. Of note, expression of loading control proteins b-actin and GAPDH was higher in HSCs, compared tohepatocytes, despite the fact that the same amount of protein was used for each lane, whereas Ponceau S staining revealed equal loading. (D)Protein transcription level of JAK2, Arhgef1, RhoA, Rho-kinase, a-SMA, and Col1 in mouse primary HSCs, hepatocytes, and KCs. For cell cultureexperiments, minimum duplicates from a minimum of 3 different animals (primary cells) or samples (cell lines) were used.


Fig. 7. AT1R-mediated JAK2 stimulation activated and induced collagen production, which is attenuated by JAK2 inhibition. (A) LX2 cells trans-fected with a JAK2 plasmid (1 mg/mL) for 48 hours showed increased protein expression of JAK2 and a-SMA detected by western blotting, aswell as increased transcription of JAK2, RhoA, Rho-kinase, a-SMA, and Col1 analyzed by RT-PCR. (B) Transfected JAK2-overexpressing LX2 cellsincubated with AngII (10 mM) for 3 days showed further augmented mRNA levels of Arhgef1, RhoA, Rho-kinase, a-SMA, and Col1 measured byRT-PCR, but this was blunted after AG490 (5 mM) coincubation. LX2 cells transduced by a JAK2 adenovirus were incubated with AngII (10 mM)for 3 days and showed increased mRNA levels of Arhgef1, RhoA, Rho-kinase, a-SMA, and Col1 measured by RT-PCR, which was blunted afterAG490 (5 mM) coincubation. (C) Primary HSCs isolated from AT1R2/2 mice showed no effect of AngII or AngII and AG490. For cell cultureexperiments, minimum duplicates from a minimum of 3 different animals (primary cells) or samples (cell lines) were used.


cascade that can be further stimulated by AngII andwhich is blocked or blunted by an AT1R blocker,AT1aR, deletion in mice (the subtype of AT1R inmice responsible for contraction of smooth muscle

cells and important for development of liver fibro-sis10,16) or JAK2 inhibition, respectively. This alteredsignaling clearly leads to functional changes withincreased a-SMA and collagen expression of these cells.

Fig. 8. In vitro AT1R-mediated JAK2 activation and JAK2 inhibition in rodent and human HSCs. (A) Protein levels of human-derived LX2 cellsafter AngII treatment with or without JAK2 inhibition. The increased expression after AngII incubation of Arhgef1 and Rho-kinase as well as Rho-kinase activation, measured by phosphorylation of moesin, was blunted after coincubation with AG490. (B) mRNA levels of JAK2, Arhgef1, RhoA,Rho-kinase, Col1, and a-SMA and (C) protein levels of Rho-kinase, pMoesin, and a-SMA in human primary HSCs after AngII incubation, with orwithout JAK2 inhibition with AG490, compared to untreated control cells. (D) Primary HSCs from healthy rats were incubated with AG490 (1.5mM) or AT1R blocker Losartan (10 mM) for 3 days, and expression of Arhgef1, Rho-kinase, and a-SMA was analyzed using western blotting. (E)ROS detection of LX2 cells incubated with AngII (10 mM) for 3 days with or without AG490 (5 mM), compared to untreated cells. For cell cultureexperiments, minimum duplicates from a minimum of 3 different animals (primary cells) or samples (cell lines) were used.


Of note, no effects on survival or cell-cycle progresswere observed in these cells.

In line with these in vitro findings, we show thatfibrosis in different animal models and in humans isassociated with increased JAK2 expression and activa-tion. Furthermore, fibrosis is attenuated by applicationof JAK2 inhibitors, whereas continuous exogenousAngII infusion9,37 increases collagen formation andHSC activation. In addition, we demonstrated in invivo experiments that cells showing JAK2 phosphoryla-tion were, in fact, activated myofibroblastic HSCs,because they were located in fibrotic septa and werepositive for a-SMA, whereas other a-SMA-negativeliver cells (e.g., hepatocytes and inflammatory cells)lacked evidence of JAK2 phosphorylation.

Activation of RAS is a uniform finding in advancedcirrhosis.14,15,38 One possible major reason for this couldbe a systemic hemodynamic derangement with splanch-nic and peripheral vasodilation, leading to “underfilling”in the central intrathoracic compartment and reactivesecretion of vasoconstrictors. However, the response fromthe peripheral and splanchnic vessels to AngII is inad-equate, whereas the hepatic vascular bed overreacts.Although this has been shown repeatedly in human andanimal models, it is only partially understood.14,15,38 It isquestionable whether systemic RAS activation aloneaccounts for hepatic JAK2 activation, as shown here. Cer-tainly, there must be a systemic effect because we wereable to show that AngII infusion enhanced hepatic colla-gen formation and activated the AT1R/JAK2/Rho-kinaseaxis in the liver. However, there are probably also para-crine intrahepatic factors involved that lead to up-regulation of AT1R expression on activated HSCs andlocal formation of AngII with subsequent phosphoryla-tion of JAK2, as shown for oxidative stress and the Ikappa B kinase/RelA pathway.6,39 Furthermore, JAK2may also be activated by cytokines or several concomitantpathological conditions in the liver.40-44

In our animal models, neither inflammatory infil-trates nor isolated KCs expressed pJAK2. Thombogenicand angiogenic properties of JAK2 were not observed inour models, as assessed by histology. We focused on theAT1R-dependent JAK2/Arhgef1/Rho-kinase pathway.Effects that might be driven by the JAK/STAT pathway,as shown for the transdifferentiation of HSC, I/R, ische-mia/reperfusion liver injury, hepatocellular carcinoma,and steatohepatitis, could also play a role in liver cirrho-sis.22,40,44 Indeed, these effects were present in our ani-mal models as well.

In summary, in the present work, we showed thatJAK2 expression and activation are increased in acti-vated HSCs in fibrosis and inhibition of JAK2

decreased liver fibrosis. Recently, new JAK2 inhibitorshave been released for human use in malignant disor-ders,45,46 which, based on these findings, might alsobe considered for treatment of fibrosis in a clinical set-ting. In this case, liver-specific targeting is warranted.

Acknowledgment: The authors thank G. Hack and S.Bellinghausen for their excellent technical assistance, as wellas U.B. Kaupp and S. Dentler for their critical reading.

References

1. WHO. The world health report 2002: reducing risks, promotinghealthy life. Geneva: World Health Organisation; 2002.

2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-218.

3. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology2008;134:1655-1669.

4. Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liverfibrosis suggest stellate cells and TGF-beta as major players and thera-peutic targets. J Cell Mol Med 2006;10:76-99.

5. Parola M, Marra F, Pinzani M. Myofibroblast-like cells and liver fibro-genesis: Emerging concepts in a rapidly moving scenario. Mol AspectsMed 2008;29:58-66.

6. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J,et al. NADPH oxidase signal transduces angiotensin II in hepatic stel-late cells and is critical in hepatic fibrosis. J Clin Invest 2003;112:1383-1394.

7. Bataller R, Gines P, Nicolas JM, Gorbig MN, Garcia-Ramallo E,Gasull X, et al. Angiotensin II induces contraction and proliferation ofhuman hepatic stellate cells. Gastroenterology 2000;118:1149-1156.

8. Croquet V, Moal F, Veal N, Wang J, Oberti F, Roux J, et al. Hemody-namic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension. J Hepatol 2002;37:773-780.

9. Bataller R, Gabele E, Parsons CJ, Morris T, Yang L, Schoonhoven R,et al. Systemic infusion of angiotensin II exacerbates liver fibrosis inbile duct-ligated rats. HEPATOLOGY 2005;41:1046-1055.

10. Yang L, Bataller R, Dulyx J, Coffman TM, Gines P, Rippe RA,Brenner DA. Attenuated hepatic inflammation and fibrosis in angioten-sin type 1a receptor deficient mice. J Hepatol 2005;43:317-323.

11. Sookoian S, Fernandez MA, Castano G. Effects of six months losartanadministration on liver fibrosis in chronic hepatitis C patients: a pilotstudy. World J Gastroenterol 2005;11:7560-7563.

12. Heller J, Shiozawa T, Trebicka J, Hennenberg M, Schepke M, Neef M,Sauerbruch T. Acute haemodynamic effects of losartan in anaesthetizedcirrhotic rats. Eur J Clin Invest 2003;33:1006-1012.

13. Heller J, Trebicka J, Shiozawa T, Schepke M, Neef M, Hennenberg M,Sauerbruch T. Vascular, hemodynamic and renal effects of low-dose los-artan in rats with secondary biliary cirrhosis. Liver Int 2005;25:657-666.

14. Bosch J, Garcia-Pagan JC. Complications of cirrhosis. I. Portal hyper-tension. J Hepatol 2000;32:141-156.

15. Benvegnu L, Gios M, Boccato S, Alberti A. Natural history of compen-sated viral cirrhosis: a prospective study on the incidence and hierarchyof major complications. Gut 2004;53:744-749.

16. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiologicaland pathological effects in the cardiovascular system. Am J Physiol CellPhysiol 2007;292:C82-C97.

17. Trebicka J, Hennenberg M, Laleman W, Shelest N, Biecker E, SchepkeM, et al. Atorvastatin lowers portal pressure in cirrhotic rats by inhibi-tion of RhoA/Rho-kinase and activation of endothelial nitric oxide syn-thase. HEPATOLOGY 2007;46:242-253.

18. Trebicka J, Hennenberg M, Schulze Probsting A, Laleman W, Klein S,Granzow M, et al. Role of beta3-adrenoceptors for intrahepatic


resistance and portal hypertension in liver cirrhosis. HEPATOLOGY 2009;50:1924-1935.

19. Trebicka J, Hennenberg M, Odenthal M, Shir K, Klein S, GranzowM, et al. Atorvastatin attenuates hepatic fibrosis in rats after bile ductligation via decreased turnover of hepatic stellate cells. J Hepatol 2010;53:702-712.

20. Klein S, Van Beuge MM, Granzow M, Beljaars L, Schierwagen R,Kilic S, et al. HSC-specific inhibition of Rho-kinase reduces portalpressure in cirrhotic rats without major systemic effects. J Hepatol2012;57:1220-1227.

21. Guilluy C, Bregeon J, Toumaniantz G, Rolli-Derkinderen M,Retailleau K, Loufrani L, et al. The Rho exchange factor Arhgef1 medi-ates the effects of angiotensin II on vascular tone and blood pressure.Nat Med 2010;16:183-190.

22. Lakner AM, Moore CC, Gulledge AA, Schrum LW. Daily geneticprofiling indicates JAK/STAT signaling promotes early hepatic stellatecell transdifferentiation. World J Gastroenterol 2010;16:5047-5056.

23. De Minicis S, Seki E, Oesterreicher C, Schnabl B, Schwabe RF,Brenner DA. Reduced nicotinamide adenine dinucleotide phosphateoxidase mediates fibrotic and inflammatory effects of leptin on hepaticstellate cells. HEPATOLOGY 2008;48:2016-2026.

24. Domenicali M, Caraceni P, Giannone F, Baldassarre M, Lucchetti G,Quarta C, et al. A novel model of CCl4-induced cirrhosis with ascitesin the mouse. J Hepatol 2009;51:991-999.

25. Laleman W, Vander Elst I, Zeegers M, Servaes R, Libbrecht L,Roskams T, et al. A stable model of cirrhotic portal hypertension inthe rat: thioacetamide revisited. Eur J Clin Invest 2006;36:242-249.

26. Rudolph V, Andrie RP, Rudolph TK, Friedrichs K, Klinke A, Hirsch-Hoffmann B, et al. Myeloperoxidase acts as a profibrotic mediator ofatrial fibrillation. Nat Med 2010;16:470-474.

27. Huss S, Schmitz J, Goltz D, Fischer HP, Buttner R, Weiskirchen R.Development and evaluation of an open source Delphi-based softwarefor morphometric quantification of liver fibrosis. Fibrogenesis TissueRepair 2011;3:10.

28. Trebicka J, Racz I, Siegmund SV, Cara E, Granzow M, Schierwagen R,et al. Role of cannabinoid receptors in alcoholic hepatic injury: steatosisand fibrogenesis are increased in CB(2) receptor-deficient mice anddecreased in CB(1) receptor knockouts. Liver Int 2011;31:862-872.

29. Wojtalla A, Herweck F, Granzow M, Klein S, Trebicka J, Huss S, et al. Theendocannabinoid N-arachidonoyl dopamine (NADA) selectively inducesoxidative stress-mediated cell death in hepatic stellate cells, but not in hepa-tocytes. Am J Physiol Gastrointest Liver Physiol 2012;302:G873-G887.

30. Coenen M, Nischalke HD, Kramer B, Langhans B, Glassner A,Schulte D, et al. Hepatitis C virus core protein induces fibrogenicactions of hepatic stellate cells via toll-like receptor 2. Lab Invest 2011;91:1375-1382.

31. Glassner A, Eisenhardt M, Kramer B, Korner C, Coenen M, SauerbruchT, et al. NK cells from HCV-infected patients effectively induce apopto-sis of activated primary human hepatic stellate cells in a TRAIL-, FasL-and NKG2D-dependent manner. Lab Invest 2012;92:967-977.

32. Semela D, Das A, Langer D, Kang N, Leof E, Shah V. Platelet-derivedgrowth factor signaling through ephrin-b2 regulates hepatic vascularstructure and function. Gastroenterology 2008;135:671-679.

33. Klein S, Kl€osel J, Schierwagen R, K€orner C, Granzow M, Huss S,et al. Atorvastatin induces senescence in activated hepatic stellate cellsand attenuates hepatic fibrosis in rats. Lab Invest 2012;92:1440-1450.

34. Ma TK, Kam KK, Yan BP, Lam YY. Renin-angiotensin-aldosterone sys-tem blockade for cardiovascular diseases: current status. Br J Pharmacol2010;160:1273-1292.

35. Wolf G. Novel aspects of the renin-angiotensin-aldosterone-system.Front Biosci 2008;13:4993-5005.

36. Uhal BD, Li X, Piasecki CC, Molina-Molina M. Angiotensin signallingin pulmonary fibrosis. Int J Biochem Cell Biol 2012;44:465-468.

37. Moreno M, Ramalho LN, Sancho-Bru P, Ruiz-Ortega M, Ramalho F,Abraldes JG, et al. Atorvastatin attenuates angiotensin II-inducedinflammatory actions in the liver. Am J Physiol Gastrointest LiverPhysiol 2009;296:G147-G156.

38. Hennenberg M, Trebicka J, Sauerbruch T, Heller J. Mechanisms ofextrahepatic vasodilation in portal hypertension. Gut 2008;57:1300-1314.

39. Oakley F, Teoh V, Ching ASG, Bataller R, Colmenero J, Jonsson JR,et al. Angiotensin II activates I kappaB kinase phosphorylation of RelAat Ser 536 to promote myofibroblast survival and liver fibrosis. Gastro-enterology 2009;136:2334-2344.e1.

40. Shi SY, Martin RG, Duncan RE, Choi D, Lu SY, Schroer SA, et al.Hepatocyte-specific deletion of Janus kinase 2 (JAK2) protects againstdiet-induced steatohepatitis and glucose intolerance. J Biol Chem 2012;287:10277-10288.

41. Sos BC, Harris C, Nordstrom SM, Tran JL, Balazs M, Caplazi P, et al.Abrogation of growth hormone secretion rescues fatty liver in micewith hepatocyte-specific deletion of JAK2. J Clin Invest 2011;121:1412-1423.

42. Gu FM, Li QL, Gao Q, Jiang JH, Zhu K, Huang XY, et al. IL-17induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor pro-gression in hepatocellular carcinoma. Mol Cancer 2011;10:150.

43. Yu HC, Qin HY, He F, Wang L, Fu W, Liu D, et al. Canonical notchpathway protects hepatocytes from ischemia/reperfusion injury in miceby repressing reactive oxygen species production through JAK2/STAT3signaling. HEPATOLOGY 2011;54:979-988.

44. Freitas MC, Uchida Y, Zhao D, Ke B, Busuttil RW, Kupiec-Weglinski JW. Blockade of Janus kinase-2 signaling ameliorates mouseliver damage due to ischemia and reperfusion. Liver Transpl 2010;16:600-610.

45. Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-FrancoJ, Thomas DA, et al. Safety and efficacy of INCB018424, a JAK1and JAK2 inhibitor, in myelofibrosis. N Engl J Med 2010;363:1117-1127.

46. Lafave LM, Levine RL. JAK2 the future: therapeutic strategies for JAK-dependent malignancies. Trends Pharmacol Sci 2012;33:574-582.

Supporting Information

Additional Supporting Information may be foundin the online version of this article at the publisher’swebsite.


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