CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis

ORIGINAL ARTICLE

CCL2-dependent infiltrating macrophages promoteangiogenesis in progressive liver fibrosisJosef Ehling,1,2 Matthias Bartneck,3 Xiao Wei,3 Felix Gremse,1 Viktor Fech,3

Diana Möckel,1 Christer Baeck,3 Kanishka Hittatiya,4 Dirk Eulberg,5 Tom Luedde,3

Fabian Kiessling,1 Christian Trautwein,3 Twan Lammers,1,6,7 Frank Tacke3

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/gutjnl-2013-306294).1Department of ExperimentalMolecular Imaging, MedicalFaculty, Helmholtz Institute forBiomedical Engineering, RWTHUniversity, Aachen, Germany2Institute of Pathology, MedicalFaculty, RWTH University,Aachen, Germany3Department of Medicine III,Medical Faculty, RWTHUniversity, Aachen, Germany4Institute of Pathology,University Bonn, Bonn,Germany5NOXXON Pharma AG, Berlin,Germany6Department of TargetedTherapeutics, MIRA Institutefor Biomedical Technology andTechnical Medicine, Universityof Twente, Enschede,The Netherlands7Department of Pharmaceutics,Utrecht Institute forPharmaceutical Sciences,Utrecht University, Utrecht,The Netherlands

Correspondence toDr Frank Tacke, Department ofMedicine III, Medical Faculty ofthe RWTH Aachen, Pauwelsstr30, Aachen 52074, Germany;[email protected] Dr Twan Lammers,Department of ExperimentalMolecular Imaging, MedicalFaculty of the RWTH Aachen,Pauwelsstr. 30, 52074 Aachen,Germany;[email protected]

JE and MB contributed equally.

Received 17 October 2013Revised 11 January 2014Accepted 30 January 2014

To cite: Ehling J,Bartneck M, Wei X, et al.Gut Published Online First:[please include Day MonthYear] doi:10.1136/gutjnl-2013-306294

ABSTRACTObjectives In chronic liver injury, angiogenesis, theformation of new blood vessels from pre-existing ones,may contribute to progressive hepatic fibrosis and todevelopment of hepatocellular carcinoma. Althoughhypoxia-induced expression of vascular endothelialgrowth factor (VEGF) occurs in advanced fibrosis, wehypothesised that inflammation may endorse hepaticangiogenesis already at early stages of fibrosis.Design Angiogenesis in livers of c57BL/6 mice uponcarbon tetrachloride- or bile duct ligation-inducedchronic hepatic injury was non-invasively monitoredusing in vivo contrast-enhanced micro computedtomography (mCT) and ex vivo anatomical mCT afterhepatic Microfil perfusion. Functional contributions ofmonocyte-derived macrophage subsets for angiogenesiswere explored by pharmacological inhibition of CCL2using the Spiegelmer mNOX-E36.Results Contrast-enhanced in vivo mCT imagingallowed non-invasive monitoring of the close correlationof angiogenesis, reflected by functional hepatic bloodvessel expansion, with experimental fibrosis progression.On a cellular level, inflammatory monocyte-derivedmacrophages massively accumulated in injured livers,colocalised with newly formed vessels in portal tractsand exhibited pro-angiogenic gene profiles includingupregulated VEGF and MMP9. Functional in vivo andanatomical ex vivo mCT analyses demonstrated thatinhibition of monocyte infiltration by targeting thechemokine CCL2 prevented fibrosis-associatedangiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sproutingangiogenesis within the portal vein tract. Portal veindiameter as a measure of portal hypertension dependedon fibrosis, but not on angiogenesis.Conclusions Inflammation-associated angiogenesis ispromoted by CCL2-dependent monocytes during fibrosisprogression. Innovative in vivo mCT methodology canaccurately monitor angiogenesis and antiangiogenictherapy effects in experimental liver fibrosis.

INTRODUCTIONLiver disease progression is accompanied by patho-logical angiogenesis,1–3 which is likely a prerequis-ite favouring development of hepatocellularcarcinoma (HCC). However, although pathologicalangiogenesis is commonly observed in advancedfibrosis, it is currently unclear if and how angiogen-esis and fibrosis are linked during progression ofchronic liver diseases.4 Interestingly, antiangiogenic

multikinase inhibitors have demonstrated antifibro-tic potential in preclinical settings,3 5 andpro-inflammatory and pro-angiogenic factors areassumed to favour vessel sprouting, resulting inmicrostructural vascular changes and an increasedintrahepatic vascular resistance.6 Whereas several

Significance of this study

What is already known on this subject?▸ Although millions of people worldwide suffer

from liver fibrosis and cirrhosis, non-invasiveimaging techniques for quantitativelymonitoring the progression of liver fibrosis andpotential therapy effects in preclinical or clinicalsettings are currently limited.

▸ In advanced liver fibrosis, angiogenesis hasbeen observed in human patients as well as inexperimental animal models for chronic hepaticinjury.

▸ Although fibrosis-associated angiogenesis isregarded as a promoting factor for thetransition from chronic injury to hepatocellularcarcinoma, the mechanisms of fibrosis-associated angiogenesis remain largely obscure.

What are the new findings?▸ An innovative non-invasive contrast-enhanced

functional in vivo micro-CT approach wasestablished in experimental liver injury modelsin mice and validated by histological andanatomical ex vivo micro-CT-basedmicrostructural analyses.

▸ By using these innovative imaging techniques,we demonstrate that (1) even at the initialstages of liver fibrosis, angiogenesis is induced;(2) fibrosis stage correlates with extent ofhepatic neovascularisation; (3) infiltrating bonemarrow-derived inflammatory monocytesmediate fibrosis-associated angiogenesisinduction; (4) pharmacological inhibition ofCCL2 attenuates monocyte infiltration andangiogenesis, but not fibrosis progression;(5) these effects are primarily attributable tochanges in the portal vein system; and(6) portal hypertension is rather driven byextracellular collagen deposition than byfibrosis-associated pathological angiogenesis.

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studies have reported that in late stage liver fibrosis or cirrhosis,hypoxia-induced release of pro-angiogenic factors like vascularendothelial growth factor (VEGF) play an important role in thedevelopment of HCC,6 7 only few studies have investigated therole of inflammatory cells in mediating angiogenesis associatedwith HCC,8 9 non-alcoholic steatohepatitis10 or cirrhosis.3

Based on the pivotal role that (M2-polarised) macrophages playin tumour angiogenesis,11 we hypothesised that macrophagesare key mediators of fibrosis-associated angiogenesis as well.

The macrophage pool of the liver is composed of residentimmune cells, traditionally termed Kupffer cells, and infiltratingmonocytes. In case of liver injury, bone marrow-derived mono-cytes massively accumulate in injured liver and differentiate intoinflammatory macrophages (iMΦ), dependent on interactions ofthe chemokine receptor CCR2 with its ligand CCL2.12 13 Theseinfiltrating monocytes are characterised by the surface markerLy-6C (Gr1) and a pro-inflammatory cytokine expressionprofile,14 15 while expression of angiogenic factors has not beensystematically addressed to date. We therefore aimed at investi-gating if iMΦ promote fibrosis-associated angiogenesis and, con-sequently, if the inhibition of monocyte migration into the liverresults in a reduced angiogenic activity in chronically injuredlivers.

In spite of this, no efficient in vivo imaging techniques arecurrently established, neither for non-invasively quantifyingfibrosis-associated pathological angiogenesis nor for monitoringtherapy effects of novel pharmacological treatments in (pre)clin-ical settings. At present, antiangiogenic treatment effects areusually assessed by quantifying CD31-positive vascular struc-tures using immunohistochemistry, and increased CD31+ areasare also observed in virtually all types of advanced hepatic dis-eases in liver biopsies from human patients.2 Though useful,this methodology does not allow longitudinal examinationsduring disease progression or quantifications of functional para-meters such as blood volume, blood flow and tissue perfusion.In contrast, due to its high spatial resolution, user-independencyand suitability for high-throughput analyses, mCT has become a(pre)clinically relevant diagnostic modality for the non-invasivevisualisation and quantification of functional blood vessels.16

The aim of this study was to establish novel mCT-based imagingprotocols to characterise the role of CCL2-dependent inflamma-tory monocytes in mediating pathological angiogenesis duringthe initiation and progression of liver fibrosis. Alterations in thehepatic relative blood volume (rBV) as well as changes in themicroarchitecture of liver blood vessels were visualised andquantified using functional in vivo mCT and anatomical ex vivomCT, convincingly demonstrating that CCL2-dependent inflam-matory monocyte-derived macrophages control angiogenesis inexperimental liver fibrogenesis in mice.

MATERIALS AND METHODSLiver injury models and pharmacological CCL2 inhibitionin miceC57bl/6 wild-type mice were housed in a specific pathogen-freeenvironment under ethical conditions approved by Germanlegal requirements. Chronic liver injury was induced in 8–12-week old C57bl/6 wild-type mice by repetitive carbon tetra-chloride (CCl4) administration (0.6 ml/kg body weight) or bysurgical ligation of the biliary duct (bile duct ligation; BDL), asdescribed earlier.17 To block CCL2-dependent monocyte migra-tion, mice were treated with PEGylated L-RNA SpiegelmermNOX-E36 (50 nucleotides long L-RNA oligonucleotide;50-GGC-GAC-AUU-GGU-UGG-GCA-UGA-GGC-GAG-GCC-C-UU-UGA-UGA-AUC-CGC-GGC-CA-30, 40 kDa PEG), kindlyprovided by NOXXON Pharma AG (Berlin, Germany), subcuta-neously three times per week at 20 mg/kg body weight.mNOX-E36 binds specifically to murine CCL2 (monocytechemoattractant protein-1 (MCP-1)) and inhibits its biologicaleffects in vitro and in vivo.12 Liver injury, fibrosis progressionand immune cell alterations were assessed as publishedearlier.12 17 Primary hepatocytes were isolated by conventionalmethodology, all other primary cell types (stellate cells, endothe-lial cells, Kupffer cells, iMΦ) after collagenase/pronase-perfusion, gradient centrifugation and ultrapure FACS sortingusing a BD Aria SORP equipped with an UV laser.18 Details onmethodology are provided as online supplementary methods.

In vivo mCTFor contrast-enhanced in vivo mCT imaging, a dual-energy flat-panel mCT scanner was employed (TomoScope 30 s Duo; CTImaging, Erlangen, Germany). Mice were scanned before andimmediately after intravenous injection of 100 mL eXIA160XL(Binitio Biomedical, Ottawa, Canada), an iodine-based bloodpool contrast agent optimised for in vivo mCT. Animals wereanaesthetised with 1.5% isoflurane in oxygen-enriched airduring the entire in vivo imaging process. For each mouse, adual-energy scan was performed at 41 and 65 kV (at 0.5 and1 mA), acquiring 2880 projections of size 1032×1024 over6 min of continuous rotation for each tube. A Feldkamp-typereconstruction algorithm (CT-Imaging, Erlangen, Germany) wasimplemented with a voxel size of 35×35×35 mm3, includingring artefact correction. The reconstructed data were visualisedand analysed with Imalytics Preclinical software (PhilipsResearch, Aachen, Germany).19 After 3D liver segmentation,which was performed by interactively delineating the liverboundaries in 10–20 slices, rBV values were determined basedon the mean brightness of the liver after contrast agent injec-tion, a large blood vessel after contrast agent injection (100%rBV) and the liver before contrast agent administration (0%rBV).20 Portal vein diameter was quantified on cross-sectionalimages in transversal planes 3–4 slices above the junction of thesuperior mesenteric and splenic veins.21

Ex vivo mCTAfter in vivo mCT imaging, mice were intracardially perfusedwith Microfil (Flow Tech, Carver, Massachusetts, USA), a lead-containing silicone rubber CT contrast agent for high-resolution3D investigation of the microarchitecture of blood vessels in theliver. Microfil replaces the blood volume and polymerises intra-vascularly 20 min after application, resulting in vascular casting.Perfusion was performed by direct infusion of Microfil into theleft ventricle (after incising the inferior vena cava) at physio-logical pressures by using a perfusion pump. After Microfil

Significance of this study

How might it impact on clinical practice in theforeseeable future?▸ The establishment of a novel non-invasive imaging

technique for monitoring fibrosis-associated angiogenesis inthe liver and the identification of CCL2-dependent infiltratingmonocytes as the driving force of inflammation-associatedhepatic blood vessel expansion during fibrosis progressionmay advance diagnostics and allow novel therapeuticapproaches in chronic liver diseases.

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perfusion and solidification of the contrast medium, the liver wasexcised, formalin-fixed and scanned using a high-resolutionSkyScan 1172 mCT system (SkyScan, Kontich, Belgium). Liverswere positioned on a computer-controlled rotation platform andscanned 180° around the vertical axis in rotation steps of 0.3° at100 kV and an electric current of 100 mA, resulting in 640acquired projections (4000×2096 pixels). Acquisition times forwhole livers ranged from 6 to 8 h. Reconstructions of a voxel sizeof 6×6×6 mm3 were performed using filtered backprojection(Feldkamp-type). The volume data were downsampled to a voxelsize of 12×12×12 mm3 for image processing. After 3D volumerendering of reconstructed high-resolution mCT data sets, bloodvessel branching and 3D micromorphology of vessels were sys-tematically and semiautomatically analysed using ImalyticsPreclinical software. For this approach, five representative vesselswere analysed, and the number of blood vessel branches perprimary vessel was quantified in total and relatively per risingbranching order.

RESULTSAngiogenesis, but not inflammation, correlates closely withprogressive fibrosis in chronic liver injuryIn order to delineate the association among progressive liverinjury, inflammation, fibrosis and angiogenesis, chronic toxicliver injury was induced by repetitive (twice weekly) injectionsof CCl4 in c57bl/6 wild-type mice. After 2, 4, 6 and 8 weeks, aprogressive liver damage including necrotic areas, characteristicfibrotic bridging, infiltration of CD45+ leucocytes and forma-tion of new CD31+ hepatic blood vessels was observed (figure1A). Interestingly, the kinetics between these principal featuresdiffered considerably: while liver cell injury as reflected by ALTactivity in serum remained stably elevated throughout the time-course of repetitive CCl4 injections, the infiltration of CD45+

leucocytes into injured livers was most pronounced at earlytime-points and slowly decreased over 8 weeks (figure 1B). Incontrast, the development of hepatic fibrosis as quantified bySirius red staining (figure 1A) and hydroxyproline concentra-tions in liver tissue (figure 1B) progressed almost linearly overtime. The formation of blood vessels, as analysed by immuno-fluorescent staining of CD31+ endothelial cells, was stronglyinduced in progressive liver injury and largely paralleled theprogression of fibrosis (figure 1B). These observations demon-strated that angiogenesis and fibrosis progression closely correl-ate in experimental liver injury.

Functional in vivo mCT imaging allows accurate assessmentof fibrosis-associated angiogenesisIn order to quantify hepatic blood vessels during fibrogenesis,we established a contrast-enhanced mCT approach for in vivoimaging. To this end, we visualised the 3D micromorphology ofhepatic blood vessels in animals treated repetitively with CCl4after 2, 4, 6 and 8 weeks and in healthy control animals fromlongitudinal mCT scan series, and we simultaneously quantifiednon-invasively the rBV in livers using an iodine-based contrastagent optimised for blood pool imaging.20 Via 2D mCT imagesin transversal, sagittal and coronal planes as well as via 3Dvolume renderings, dual-energy flat-panel in vivo mCT enabledthe non-invasive visualisation of blood vessels in the liver with aspatial resolution of 35 mm voxel side length (figure 2A).Representative images and movies demonstrated that chronicallyinjured livers showed a significantly higher amount of functionalblood vessels as compared with vehicle-treated controls (figure2A, see online supplementary videos 1 and 2). In line, hepaticrBV values were significantly higher for animals challenged with

CCl4 for 6 weeks (25.7±0.9%) than for control animals (19.4±0.6%, p<0.0005) (figure 2B), corroborating that fibrosis pro-gression is paralleled by an increasing hepatic blood volume.

To assess the accuracy of in vivo mCT (which considers vesselfunctionality) versus standard CD31-based immunohistochemis-try (which does not consider vessel functionality), rBV valueswere correlated with the area fraction of CD31+ endothelialcells (figure 2C). Whereas hepatic rBV values determined by invivo mCT ranged from 22.4±0.9% for animals exposed to CCl4for 2 weeks to 27.1±1.6% for animals challenged for 8 weeks(control animals: 19.4±0.6%), CD31+ area fractions deter-mined by ex vivo immunohistochemistry ranged from 2.4±0.6% for injured livers after 2 weeks of CCl4 to 6.0±1.0%after 8 weeks of CCl4 (control animals: 0.8±0.2%). A highlysignificant correlation between rBV values determined using invivo mCT and ex vivo immunohistochemistry was observed(R²=0.9572, p<0.0001, n=15), indicating that contrast-enhanced in vivo mCT is an accurate means for non-invasivelyassessing the hepatic blood volume during experimentalfibrogenesis.

The spatial resolution of this in vivo mCT-based imaging tech-nique, however, is limited to 35 mm voxel side length, andnewly formed blood vessels may partially be smaller. Therefore,we also perfused the animals with the lead-containing vascularcasting agent Microfil and performed high-resolution ex vivomCT scans. Whereas healthy livers showed a proper hierarchicvascular branching system, both for the central and portal veinsystem, high-resolution ex vivo mCT imaging of fibrotic liversrevealed areas with sprouting angiogenesis, in particular in theperiphery of chronically injured livers (figure 2D, see onlinesupplementary videos 3 and 4).

In order to exclude model-specific effects related to toxicCCl4-induced damage, we confirmed the close correlationbetween hepatic fibrosis and angiogenesis in a second mousemodel of chronic liver injury (surgical BDL, resulting in progres-sive cholestatic liver injury). Twenty-one days after BDL or shamoperation, mice were imaged using contrast-enhanced in vivomCT, followed by Microfil perfusion and high-resolution exvivo mCT imaging, and finally by histological validation. Liversfrom BDL-treated mice showed severe cholestatic damage withlarge necrotic areas, bridging fibrosis, leucocyte infiltration andneovascularisation (figure 3A). In line with the results obtainedfor CCl4, using functional in vivo (figure 3B, see online supple-mentary videos 5 and 6) and anatomical ex vivo (figure 3C, seeonline supplementary videos 7 and 8) mCT imaging, a signifi-cant increase in the formation of new blood vessels was detectedin livers with chronic cholestatic injury. Quantification of liverinjury by serum ALT, collagen deposition by hepatic hydroxy-proline concentrations and CD45+ leucocytes as well as CD31+

endothelial cells by immunohistochemistry confirmed the pres-ence of injury, inflammation, fibrosis and angiogenesis inBDL-induced liver damage (figure 3D). Accordingly, rBV valueswere significantly higher in fibrotic (26.3±1.3%) versus controllivers (19.6±1.0%, p<0.01) (figure 3D). Collectively, thesefindings from two independent experimental models for pro-gressive liver fibrosis showed a strong association between liverfibrosis and angiogenesis, and they exemplify that functional invivo and anatomical ex vivo mCT imaging can be used tomonitor fibrosis-associated angiogenesis.

Distinct subsets of hepatic macrophages induceangiogenesis during fibrogenesisBased on the pivotal role of monocytes and macrophages in theprogression of liver inflammation and fibrosis,15 we

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hypothesised that hepatic macrophages could be the drivingforce for the formation of new blood vessels during fibrogenesis.Indeed, when mice were challenged repetitively with CCl4 twiceper week for up to 8 weeks, a continuous accumulation of F4/80+ macrophages could be observed in injured livers (figure4A). Upon quantifying the F4/80 area fraction (three differentsections per liver, n=15 mice), a massive increase of hepaticmacrophages could be detected already at early stages of fibrosisafter exposure to CCl4 for 2 weeks (3.9±0.4% vs 0.5±0.2% in

healthy livers; p<0.001) (figure 4B). The amount of infiltratedmacrophages steadily increased upon prolonged exposure toCCl4 (from 4.7±0.3% after 4 weeks to 6.9±0.8% after8 weeks). In both models of progressive liver fibrosis, F4/80+

macrophages were localised in close vicinity of newly formedand mainly periportally localised CD31+ blood vessels in chron-ically injured livers (figure 4C).

A further dissection of iMΦ and Kupffer cells via FACS,based on their differential expression of F4/80 and CD11b,

Figure 1 Association among chronicliver injury, inflammation, hepaticfibrosis and angiogenesis. (A) Chronictoxic liver injury was induced byrepetitive intraperitoneal injections ofcarbon tetrachloride (CCl4) in c57BL/6mice, and mice were sacrificed 48 hafter the last injection of CCl4. Controlmice received corn oil for 6 weeks.Representative H&E staining, Sirius red(fibrotic fibres in red), CD45immunohistochemistry (leucocytes),and CD31 immunofluorescence (bloodvessels) of controls and after 4 or8 weeks CCl4. (B) Serum ALT activity(liver injury), hepatic hydroxyprolinecontent (collagen deposition), CD45+

cells in liver sections (hepaticinflammation) and area fraction ofCD31+ endothelial cells (quantificationof neovascularisation). Data are shownas mean±SD (n=15 mice).***p<0.001, **p<0.01 and *p<0.05(Student t test).

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revealed a strong increase of CD11b+F4/80+ inflammatorymonocyte-derived macrophages already very early in chronicliver injury, and iMΦ remained the predominant leucocytepopulation infiltrating throughout the progression of chronicliver diseases (figure 4D).

To determine the pro-angiogenic activity of hepatic macro-phage subsets during fibrogenesis, gene expression analyses ofpurely isolated iMΦ (CD11b+F4/80+), Kupffer cells (defined asCD11blowF4/80++ cells), primary hepatocytes, endothelial cells(CD45−CD146+) and hepatic stellate cells (CD45−UV+) frominjured and healthy control livers were performed afterFACS-based cell sorting using quantitative real-time PCR.Although Kupffer cells and stellate cells were an importantsource of angiogenic factors in homeostasis (figure 4E,F), onlyiMΦ showed a strongly increased expression of pro-angiogenicfactors like VEGF-A (10.2-fold higher expression in injured vs

control livers) or MMP9 (3.5-fold higher expression in injuredvs control livers) upon liver injury. On the contrary, VEGF-Aand MMP9 expression was strongly reduced in Kupffer cells,hepatocytes, endothelial cells and hepatic stellate cells inCCl4-treated livers (figure 4E,F). Collectively, these findingsstrongly indicated that the distinct subset of monocyte-derivediMΦ actively promotes hepatic neovascularisation duringfibrogenesis.

Angiogenesis, but not fibrosis progression, is controlled byCCL2-dependent monocyte infiltrationFindings obtained both in mice and in humans demonstrated thatthe accumulation of inflammatory monocyte-derived macro-phages in injured livers is critically controlled by the chemokineCCL2 (also termed MCP-1).15 In order to provide functional evi-dence that infiltrating monocytes drive angiogenesis in liver

Figure 2 Functional and anatomicalmCT imaging of angiogenesis incarbon tetrachloride (CCl4)-inducedliver fibrosis. (A) Visualisation ofhepatic blood vessels by in vivo mCTusing an iodine-based blood poolcontrast agent (eXIA 160XL), resultingin a spatial resolution of 35 mm voxelside length (2D cross-sectional imagesin transversal (I), sagittal (II) andcoronal (III) planes, as well asrepresentative pictures of 3D volumerenderings). (B) Non-invasivemCT-based quantification of therelative blood volume (rBV) in fibroticand healthy livers. (C) A highlysignificant correlation was foundbetween hepatic rBV determined usingin vivo mCT and area fraction of CD31determined using ex vivoimmunofluorescence (IF) staining. Dataare shown as mean±SD; n=15 mice;***p<0.001 (Student t test).Correlation analyses were performedby calculating R² (square of Pearsoncorrelation coefficient). (D)High-resolution ex vivo mCT imaging(after perfusion with Microfil, alead-containing radiopaque contrastagent) enables a detailed 3Dexamination of vascularmicroarchitecture of healthy liver (left)and after 6 weeks CCl4 (right). Spatialresolution: 12 mm voxel side length.

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fibrosis, we investigated the effect of specific pharmacologicalinhibition of CCL2 on fibrosis-associated angiogenesis by usingthe ‘Spiegelmer’ mNOX-E36.12 In fact, administration ofmNOX-E36 during CCl4-induced chronic liver injury signifi-cantly reduced infiltrating leucocytes, particularly macrophages(as evidenced by F4/80 immunohistochemistry, figure 5A). In linewith prior observations,12 mNOX-E36 treatment did neither sig-nificantly diminish fibrosis progression nor ALT or AST serumactivity in CCl4 injured livers (figure 5A, see online

supplementary figure S1). However, blood vessel formation wassignificantly reduced in mNOX-E36-treated animals (figure 5A,B), and immunofluorescence co-stainings revealed a simultaneousreduction of F4/80+ liver macrophages and CD31+ blood vesselsover 2–8 weeks of CCl4 injury in case of mNOX-E36 administra-tion (figure 5A). Importantly, administration of the CCL2 inhibi-tor mNOX-E36 specifically reduced the amount of CD11b+F4/80+ iMΦ as assessed by FACS analysis from intrahepatic leuco-cytes (figure 5C), in line with prior findings from our group.12

Figure 3 Association between fibrosis and angiogenesis in bile duct ligation (BDL)-induced cholestatic liver injury. Chronic cholestatic liver injurywas induced by surgical BDL in c57BL/6 mice. Control animals received sham operations. Mice were imaged and sacrificed 21 days after surgery. (A)H&E staining, Sirius red staining (fibrosis), CD45 immunohistochemistry (inflammation) and CD31 immunofluorescence (blood vessel formation). (B,C) Functional in vivo (B) and morphological high-resolution ex vivo mCT imaging (C) of liver blood vessels from control and from BDL-treated mice(I: transversal, II: sagittal, III: coronal 2D cross-sectional images). Segmented gall bladders (green) illustrate gall bladder hydrops and cholestasisafter BDL. (D) Quantification of liver injury via ALT activity in serum, of liver fibrosis by hepatic hydroxyproline levels, of hepatic inflammation byquantifying CD45+ cells, of hepatic blood vessels by determining the CD31 area fraction and of the hepatic relative blood volume determinednon-invasively using contrast-enhanced in vivo mCT. Results are shown as mean±SD (n=8 mice). ***p<0.001, **p<0.01 and *p<0.05 (Student ttest).

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In order to confirm and to non-invasively monitor antiangio-genic therapy effects of mNOX-E36 in mice suffering fromCCl4-induced liver fibrosis under in vivo hepatic blood flowconditions, functional mCT imaging was performed.Contrast-enhanced in vivo mCT scans demonstrated a significantreduction of fibrosis-associated hepatic blood vessels as a thera-peutic effect of mNOX-E36 treatment (figure 5D, see onlinesupplementary video 9). Whereas rBV values rose continuouslyduring the course of CCl4 administration (+15.5%, +20.1%,+32.5% and +39.7% after 2, 4, 6 and 8 weeks, respectively;p<0.01) as compared with control animals, the CCl4-inducedincrease in rBV was almost completely blocked by mNOX-E36treatment (figure 5E). At each time-point evaluated, rBV valuesin the mNOX-E36 therapy group were significantly lower thanthose in the untreated CCl4 group (−18.8%, −19.4%, −25.9%and −24.4% after 2, 4, 6 and 8 weeks, respectively; p<0.05).

Importantly, the effect of CCL2 inhibition was specific toinjured livers, because no differences in splenic rBV values wereobserved in the corresponding animals (figure 5E). Gene expres-sion profiling of livers from mice treated with CCl4 andCCl4+mNOX-E36 for 6 weeks revealed that the reducednumbers of macrophages were reflected by lower expression ofmacrophage-associated genes such as cytokines, M1/2 prolifer-ation markers and pro-angiogenic factors (see online supplemen-tary figure S1).

In order to exclude that CCL2 or mNOX-E36 itself haddirect effects on blood vessel formation, two in vitro experi-ments were performed. First, primary endothelial cells were iso-lated from livers of healthy mice, cultured in VEGF-containingmedium with or without the presence of mNOX-E36, andeither stimulated with CCL2 or left unstimulated (see onlinesupplementary figure S2A,B). Second, using similar culture

Figure 4 Role of hepatic macrophage subsets in fibrosis-associated angiogenesis. Chronic toxic liver injury was induced by repetitiveintraperitoneal administrations of carbon tetrachloride (CCl4) in c57BL/6 mice; control mice received corn oil. Mice were sacrificed 48 h after the lastinjection of CCl4 (n=6 mice per condition and time-point). (A) Representative microscopy images of F4/80 immunohistochemistry (macrophages). (B)Quantification of total F4/80+ macrophages in sections from control and chronically injured livers. (C) Representative F4/80 (red) and CD31 (green)co-stainings, demonstrating periportal localisation of inflammatory macrophages (iMΦ) and colocalisation of macrophages with newly formed smallblood vessels in progressive CCl4 or BDL injury. (D) The relative amount of intrahepatic CD11b

+ F4/80+ iMΦ isolated by FACS sorting is early andpersistently increased in chronic liver injury. (E, F) Expression of VEGF-A (E) and MMP9 (F) by primary murine iMΦ, Kupffer cells, hepatocytes,endothelial cells and hepatic stellate cells. Cells were isolated from injured (n=12) and control (n=12) livers using FACS sorting, and expressionlevels were normalised to iMΦ isolated from corn oil-treated control livers. Data are shown as mean±SD. ***p<0.001 and **p<0.01 (Student ttest).

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Figure 5 Effect of pharmacological inhibition of CCL2-dependent inflammatory monocytes on fibrosis-associated angiogenesis. Chronic toxic liverinjury was induced by repetitive intraperitoneal administrations of carbon tetrachloride (CCl4) in c57BL/6 mice, and half of these animals receivedthrice weekly subcutaneous injections of the specific CCL2 inhibitor mNOX-E36, to block the CCL2-dependent infiltration of inflammatorymonocytes. Analyses were performed 48 h after the last CCl4 injection. Control mice received corn oil for 6 weeks. (A) Representative H&E staining,Sirius red, F4/80 immunohistochemistry and F4/80-CD31 immunofluorescence co-stainings. (B) Quantification of F4/80+ macrophages and CD31+

blood vessels in livers of chronically injured and mNOX-E36-treated mice. (C) Representative FACS plots and statistical analysis showing the increaseof intrahepatic inflammatory macrophages (iMΦ) in chronically injured livers and their significant reduction in mNOX-E36-treated livers. iMΦ wereseparated from Kupffer cells on the basis of differential expression of F4/80 and CD11b. (D) Liver vascularisation visualised by contrast-enhanced invivo mCT. (E) Quantification of the relative blood volume in livers and spleens using functional in vivo mCT imaging. Data are shown as mean±SD.***p<0.001, **p<0.01 and *p<0.05 for comparing CCl4 versus CCl4+mNOX-E36; ###p<0.001, ##p<0.01 and #p<0.05 for comparing CCl4 orCCl4+mNOX-E36 versus corresponding control groups (ie, 6 weeks oil or 6 weeks oil+mNOX-E36) (n=30 mice; Student t test).

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conditions, an angiogenic sprouting assay with dissected 0.5 mmthin aortic rings of c57bl/6 wild-type mice was performed, ana-lysing the number of sprouts after 10 days (see online supple-mentary figure S2C,D). Importantly, in both cases of CCL2stimulation or unstimulated controls, mNOX-E36 treatment didneither affect proliferation and viability of the hepatic endothe-lium nor the sprouting of aortic rings (see online supplementaryfigure S2). Taken together, our results demonstrate that thepharmacological inhibition of the chemokine CCL2 effectivelyblocks fibrosis-associated angiogenesis in chronically injuredlivers by inhibiting infiltrating CCL2-dependent monocytes.

Macrophage-dependent angiogenesis during chronic liverinjury is primarily important for portal vein sproutingIn comparison with functional in vivo mCT, which spatial reso-lution ends at ∼35 mm, anatomical high-resolution ex vivo mCTimaging, allowing a spatial resolution of ∼12 mm, indicated aneven stronger antiangiogenic effect of CCL2 inhibition, espe-cially in the periphery of mNOX-E36-treated livers (figure 6A,see online supplementary video 10). After semiautomatic vesseltree segmentation of the central and portal vein systems, thetotal number of branching points as well as the number of

branching points per rising branching order was quantified(figure 6B). Whereas CCl4-induced toxic liver injury caused asignificant increase in branching points in both the central(+59.5%) and portal (+64.0%) vein systems, theCCL2-dependent inhibition of macrophage migration resultedin a clear reduction of newly formed vessels, primarily thoseassociated with the portal vein; as compared with the CCl4group, 17.6% less branching points of the central vein, and29.5% less branching points of the portal vein were detectedupon CCL2-inhibition (p<0.05 for both; figure 6C).Comparing the distribution of branching points per risingbranching order in both tracts, healthy livers demonstrated analmost equal distribution of branching points from the 1st tothe 9th order (figure 6D). In progressing liver fibrosis (6 weeksof repetitive CCl4 injections), the formation of new hepaticblood vessels was morphologically linked to a strong increase ofbranching points in the periphery of both the central and theportal veins: the relative number of 9th order branching pointsincreased significantly (5.6-fold, p<0.0001) and, moreover,10th and 11th order branching points were newly generated.Conversely, in livers exposed to CCl4 and treated withmNOX-E36, the number of 9th order branching points was

Figure 6 Vascular branching analysis of sprouting angiogenesis in the central and portal vein system in progressive liver fibrosis and uponpharmacological inhibition of CCL2. (A) Representative high-resolution ex vivo mCT images of chronically injured and mNOX-E36-treated livers aftersystematic Microfil perfusion. (B) Overview and magnification of segmented blood vessels of the liver after semiautomated discrimination betweenvessels related to the central (blue) or portal vein (red) system. Arrows schematically depict the order of rising branching points along the course ofblood vessels, from centre to periphery. (C) mCT-based quantification of the mean total number of branching points in livers from corn oil-, carbontetrachloride (CCl4)- and CCl4+mNOX-E36-treated mice (all for 6 weeks). Branching points from five representative blood vessels were quantified forboth the central and the portal vein system. (D) mCT-based quantification of the percentage of branching points per increasing order (1st to 11thbranching order) for livers from corn oil-, CCl4- and CCl4+mNOX-E36-treated mice. Data are shown as mean±SD. ***p<0.001, **p<0.005 and*p<0.05 (Student t test) for comparing CCl4 versus CCl4+mNOX-E36; ###p<0.001, and #p<0.05 for comparing CCl4 or CCl4+mNOX-E36 versuscontrol (Student t test) (D).

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again reduced, and this effect was much more prominent in theportal vein (−52.8%) than in the central vein (−25.5%)(p<0.0001; figure 6D). These findings were confirmed using

the BDL model (see online supplementary figure S3).Collectively, these highly detailed 3D micromorphological ana-lyses demonstrated that the macrophage-dependent angiogenesis

Figure 7 In vivo quantification of the portal vein diameter using contrast-enhanced mCT imaging. (A) Cross-sectional images in transversal planesexemplifying the determination of the portal vein diameter 3–4 slices above the junction of the superior mesenteric and splenic veins in control,chronically injured and mNOX-E36-treated livers. (B) Quantification of the portal vein diameter using functional mCT imaging in combination with aniodine-based large molecular weight blood pool contrast agent. (C) A highly significant correlation was found between in vivo quantified portal veindiameters and ex vivo determined hydroxyproline content. (D) No significant correlation was found for the comparison between portal veindiameters and relative blood volumes in carbon tetrachloride (CCl4)-treated, CCl4+mNOX-E36-treated or control mice. Data are shown as mean±SD.***p<0.001, ** p<0.01 for comparing CCl4 or mNOX-E36 versus corresponding control groups (ie, 6 weeks oil or 6 weeks oil+mNOX-E36) (n=30mice; Student t test).

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during chronic liver injury is largely confined to portal veinsand that pharmacological inhibition of CCL2-mediated inflam-matory monocyte infiltration primarily reduces angiogenicvessel sprouting in the portal vein system.

Differential impact of fibrosis and angiogenesis on portalhypertensionIn order to further investigate the association among fibrosisprogression, portal vein-related vessel sprouting and portalhypertension, we used contrast-enhanced mCT to quantify theportal vein diameter, as a measure of portal hypertension,21–23

3–4 transversal slices above the junction of the superior mesen-teric and splenic veins (figure 7A). Interestingly, portal veindiameter increased significantly during progression of liverfibrosis (eg, 1.68±0.07 mm for animals challenged with CCl4for 6 weeks vs 1.13±0.06 mm for control animals), but was notsignificantly influenced by mNOX-E36 treatment (eg, 1.69±0.05 mm for animals treated with CCl4 and mNOX-E36 for6 weeks; figure 7B). In detail, a highly significant correlationbetween portal vein diameters determined using in vivo mCTand ex vivo quantified hydroxyproline content was observed(R2=0.8194, p<0.001, n=30; figure 7C). In contrast, no sig-nificant correlation was found between portal vein diametersand in vivo quantified rBVs (R²=0.3795, p=0.0579, n=30;figure 7D). These findings strongly indicate that portal hyper-tension may be more directly caused by the progressive fibrosisitself (extracellular deposition of collagens causing increasedliver stiffness) than by pathological fibrosis-associated angiogen-esis (sprouting blood vessels causing additional micro-shunts).

DISCUSSIONChronic hepatic inflammation is regarded as a key requirementfor the progression of liver fibrosis, but its role in promotingfibrosis-associated angiogenesis has not yet been elucidated.Moreover, although angiogenesis is commonly observed inpatients with hepatic cirrhosis,2 the functional implications ofpathological angiogenesis in progressive liver disease haveremained largely obscure. Inflammation-associated angiogenesismight contribute to the initiation of liver fibrosis, and in theprogression from fibrosis into cirrhosis, and from cirrhosis intoHCC.6 7 9 Thus far, however, studies on the role of angiogenesisin progressive liver disease have been largely restricted to immu-nohistochemical endpoint analyses. We have here establishedand employed in vivo and ex vivo mCT-based imaging techni-ques, alongside conventional molecular methodologies, to dem-onstrate that (1) even at the initial stages of liver fibrosis,angiogenesis is induced; (2) fibrosis stage correlates with extentof hepatic neovascularisation; (3) infiltrating bone marrow-derived inflammatory monocytes mediate fibrosis-associatedangiogenesis induction; (4) pharmacological inhibition of CCL2attenuates monocyte infiltration and angiogenesis, but not fibro-sis progression; (5) these effects are primarily attributable tochanges in the portal vein system; and (6) portal hypertension israther driven by extracellular collagen deposition than byfibrosis-associated pathological angiogenesis.

Prior studies have indicated that hypoxia increases the expres-sion of the prototypic pro-angiogenic factor VEGF, thereby pro-moting angiogenesis and potentially also the progression fromfibrosis to cirrhosis and to HCC.1 3 7 VEGF enhances endothe-lial cell proliferation, promotes vessel sprouting and branching,and increases microvessel permeability.24 Besides classicalhypoxia-induced and HIF1-mediated VEGF expression, inflam-matory cells might also elevate the levels of pro-angiogenicfactors in injured livers.10 25 In experimental models of cancer

progression, infiltrating iMΦ are a major mediator of tumourangiogenesis.26 In line, inflammatory cells migrating into injuredlivers have been implicated in HCC-associated angiogenesis.8 9

Detailed analyses on the molecular link among inflammatorymonocyte infiltration, angiogenesis and liver fibrosis progres-sion, however, have not yet been performed to date, which canbe at least partially explained by the relative lack of suitableimaging techniques to non-invasively, longitudinally and quanti-tatively monitor angiogenesis.

The chemokine-dependent accumulation of monocyte-derivedmacrophages has been identified as an important mechanism forperpetuating hepatic inflammation and promoting fibrogenesis inexperimental mouse models as well as in human liver diseases.15

Upon experimental organ injury in mice, the chemokine receptorCCR2 and its ligand CCL2 (MCP-1) promote monocyte subsetaccumulation in the liver, in particular of Ly6C+ (Gr1+)monocytes. These monocyte-derived macrophages releasepro-inflammatory cytokines and can directly activate pro-fibrogenic hepatic stellate cells, the main collagen-producing cellsin the liver.15 Our study significantly extends these previous find-ings by demonstrating that infiltrating CCL2-dependent inflamma-tory monocytes also provide pro-angiogenic signals, likely via theproduction and release of VEGF-A and other factors like MMP9.

To test our hypothesis that these infiltrating monocytes areessential in mediating fibrosis-associated angiogenesis, theCCL2/CCR2-dependent migration of inflammatory monocytesinto injured livers was inhibited using a novel CCL2 inhibitor,the ‘Spiegelmer’ mNOX-E36.12 Findings from quantitative invivo and ex vivo mCT imaging of functional liver blood vessels,coupled with FACS and gene expression analyses, revealed thatthe inhibition of CCL2-dependent inflammatory monocytes bymNOX-E36 significantly reduced the formation of new bloodvessels in progressive liver fibrosis. Thus, mNOX-E36, whosehuman equivalent is currently being evaluated in a phase II clin-ical trial for the treatment of diabetic nephropathy (http://www.clinicaltrials.gov, NCT01547897), appears to be an attractivetherapeutic approach for reducing the inflammation-associatedangiogenesis in progressive liver fibrosis. Strikingly, the inhib-ition of CCL2-dependent monocyte infiltration did not affectliver fibrosis progression per se, in line with prior work fromour group.12 This further demonstrates that the inhibition ofmacrophage-mediated angiogenesis induction did not attenuatethe progression from early-to-late stage liver fibrosis, indicatingthat initially angiogenesis is a consequence of inflammation,rather than a cause for disease progression. However, at laterstages, the contribution of pathological angiogenesis to chronicliver disorders is anticipated to become more and more causal,in particular during the transition from cirrhosis to HCC, andthe subsequent progression and spread of HCC.

Besides identifying a yet unrecognised cellular and molecularmechanism of fibrosis-associated angiogenesis, our study mayalso be of interest for developing future imaging strategies in themanagement of patients with chronic liver disease. The current‘gold standard’ of fibrosis assessment is needle biopsy of theliver, carrying the risk of bleeding complications as well as sam-pling errors.27 In the last couple of years, only few ultrasoundand MR-based methods have been evaluated for non-invasivelymeasuring liver stiffness.4 28 29 Some studies reported on thenon-invasive quantification of the portal vein diameter via MRor ultrasound as a measure of portal hypertension.21–23 In add-ition, very few studies described molecular imaging approachesfor the in vivo assessment of liver fibrosis via collagen- orelastin-specific contrast agents in experimental settings.30 31

However, due to the fact that no antifibrotic drugs have been

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approved up to date,4 only limited data exist for these imagingapproaches for therapy monitoring. In contrast, functionalimaging approaches like quantitative contrast-enhanced CTenable the visualisation of liver blood vessels, non-invasivequantification of the hepatic rBV, which correlates significantlywith the amount of newly formed blood vessels during fibrosisprogression, and quantification of the portal vein diameter,which correlates with degree of portal hypertension. Withrespect to pathophysiological circulation conditions in fibroticlivers, mCT imaging can assess microstructural and functionalchanges in the hepatic vascular network and provides in add-ition the unique opportunity to monitor antiangiogenic therapyeffects. We show here that mCT imaging is highly useful forquantitatively assessing fibrosis-associated angiogenesis, and formonitoring anti-inflammatory and antiangiogenic therapy effectsin progressive liver disease, indicating that anatomical and func-tional contrast-enhanced CT imaging might hold great potentialfor facilitating the bench-to-bedside translation of novel targetedtreatments and therapeutic interventions.

Acknowledgements The authors thank Aline Roggenkamp, Carmen Tag andSibille Sauer-Lehnen for excellent technical assistance and NOXXON Pharma AG(Berlin, Germany) for providing mNOX-E36.

Contributors JE, MB, XW, FG, VF and DM: performed experiments, acquired dataand analysed results; CB and DE: contributed methodology for CCL2 inhibition; KH:performed immunohistochemistry and analysed results; TLu, FK and CT: helped indata interpretation and provided important intellectual content; JE, MB, TL and FT:designed the study, analysed data and wrote the manuscript.

Funding This work was supported by German Research Foundation (DFG; SFB/TRR57, TA434/2-1, EH412/1-1 and LA2937/1-2), Interdisciplinary Center for ClinicalResearch (IZKF Aachen) and European Research Council (ERC-StG-309495-NeoNaNo).

Competing interests Dirk Eulberg is an employee of Noxxon Pharma AG. Allother authors have nothing to disclose.

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement We agree to share all primary data and protocolsemployed in this study.

REFERENCES1 Corpechot C, Barbu V, Wendum D, et al. Hypoxia-induced VEGF and collagen I

expressions are associated with angiogenesis and fibrogenesis in experimentalcirrhosis. Hepatology 2002;35:1010–21.

2 Medina J, Arroyo AG, Sanchez-Madrid F, et al. Angiogenesis in chronicinflammatory liver disease. Hepatology 2004;39:1185–95.

3 Tugues S, Fernandez-Varo G, Munoz-Luque J, et al. Antiangiogenic treatment withsunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhoticrats. Hepatology 2007;46:1919–26.

4 Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest2013;123:1887–901.

5 Hong F, Chou H, Fiel MI, et al. Antifibrotic activity of sorafenib in experimentalhepatic fibrosis: refinement of inhibitory targets, dosing, and window of efficacy invivo. Dig Dis Sci 2013;58:257–64.

6 Coulon S, Heindryckx F, Geerts A, et al. Angiogenesis in chronic liver disease andits complications. Liver Int 2011;31:146–62.

7 Rosmorduc O, Housset C. Hypoxia: a link between fibrogenesis, angiogenesis, andcarcinogenesis in liver disease. Semin Liver Dis 2010;30:258–70.

8 Capece D, Fischietti M, Verzella D, et al. The inflammatory microenvironment inhepatocellular carcinoma: a pivotal role for tumor-associated macrophages. BiomedRes Int 2013;2013:187204.

9 Matsubara T, Kanto T, Kuroda S, et al. TIE2-expressing monocytes as a diagnosticmarker for hepatocellular carcinoma correlates with angiogenesis. Hepatology2013;57:1416–25.

10 Coulon S, Legry V, Heindryckx F, et al. Role of vascular endothelial growth factor inthe pathophysiology of nonalcoholic steatohepatitis in two rodent models.Hepatology 2013;57:1793–805.

11 De Palma M, Lewis CE. Macrophage regulation of tumor responses to anticancertherapies. Cancer cell 2013;23:277–86.

12 Baeck C, Wehr A, Karlmark KR, et al. Pharmacological inhibition of the chemokineCCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronichepatic injury. Gut 2012;61:416–26.

13 Karlmark KR, Weiskirchen R, Zimmermann HW, et al. Hepatic recruitment of theinflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.Hepatology 2009;50:261–74.

14 Ramachandran P, Pellicoro A, Vernon MA, et al. Differential Ly-6C expressionidentifies the recruited macrophage phenotype, which orchestrates the regression ofmurine liver fibrosis. Proc Natl Acad Sci U S A 2012;109:E3186–95.

15 Tacke F. Functional role of intrahepatic monocyte subsets for the progressionof liver inflammation and liver fibrosis in vivo. Fibrogenesis Tissue Repair 2012;5(Suppl 1):S27.

16 Ehling J, Lammers T, Kiessling F. Non-invasive imaging for studying anti-angiogenictherapy effects. Thromb Haemost 2013;109:375–90.

17 Karlmark KR, Zimmermann HW, Roderburg C, et al. The fractalkine receptor CX(3)CR1 protects against liver fibrosis by controlling differentiation and survival ofinfiltrating hepatic monocytes. Hepatology 2010;52:1769–82.

18 Hammerich L, Bangen JM, Govaere O, et al. Chemokine receptor CCR6-dependentaccumulation of gammadelta T cells in injured liver restricts hepatic inflammationand fibrosis. Hepatology 2014;59:630–42.

19 Gremse F, Grouls C, Palmowski M, et al. Virtual elastic sphere processing enablesreproducible quantification of vessel stenosis at CT and MR angiography. Radiology2011;260:709–17.

20 Ehling J, Theek B, Gremse F, et al. Micro-CT Imaging of Tumor Angiogenesis:Quantitative Measures Describing Micromorphology and Vascularization. Am JPathol 2014;184:431–41.

21 Zhang J, Tao R, You Z, et al. Gamna-Gandy bodies of the spleen detected withsusceptibility weighted imaging: maybe a new potential non-invasive marker ofesophageal varices. PloS one 2013;8:e55626.

22 Lessa AS, Paredes BD, Dias JV, et al. Ultrasound imaging in an experimental modelof fatty liver disease and cirrhosis in rats. BMC Vet Res 2010;6:6.

23 O’Donohue J, Ng C, Catnach S, et al. Diagnostic value of Doppler assessment ofthe hepatic and portal vessels and ultrasound of the spleen in liver disease. Eur JGastroenterol Hepatol 2004;16:147–55.

24 Carmeliet P, Jain RK. Molecular mechanisms and clinical applications ofangiogenesis. Nature 2011;473:298–307.

25 Avraham-Davidi I, Yona S, Grunewald M, et al. On-site education of VEGF-recruitedmonocytes improves their performance as angiogenic and arteriogenic accessorycells. J Exp Med 2013;210:2611–25.

26 Solinas G, Germano G, Mantovani A, et al. Tumor-associated macrophages (TAM)as major players of the cancer-related inflammation. J Leukoc Biol2009;86:1065–73.

27 Bedossa P, Dargere D, Paradis V. Sampling variability of liver fibrosis in chronichepatitis C. Hepatology 2003;38:1449–57.

28 Castera L. Noninvasive methods to assess liver disease in patients with hepatitis Bor C. Gastroenterology 2012;142:1293–302. e4.

29 Huwart L, Sempoux C, Vicaut E, et al. Magnetic resonance elastography for thenoninvasive staging of liver fibrosis. Gastroenterology 2008;135:32–40.

30 Ehling J, Bartneck M, Fech V, et al. Elastin-based molecular MRI of liver fibrosis.Hepatology 2013;58:1517–18.

31 Polasek M, Fuchs BC, Uppal R, et al. Molecular MR imaging of liver fibrosis:a feasibility study using rat and mouse models. J Hepatol 2012;57:549–55.

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fibrosispromote angiogenesis in progressive liver CCL2-dependent infiltrating macrophages

TackeLuedde, Fabian Kiessling, Christian Trautwein, Twan Lammers and FrankDiana Möckel, Christer Baeck, Kanishka Hittatiya, Dirk Eulberg, Tom Josef Ehling, Matthias Bartneck, Xiao Wei, Felix Gremse, Viktor Fech,

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