Production of Elastin-Derived Peptides Contributesto the Development of Nonalcoholic SteatohepatitisBéatrice Romier,1 Corinne Ivaldi,1 Hervé Sartelet,1 Andrea Heinz,2,3 Christian E.H. Schmelzer,2,4

Roselyne Garnotel,1 Alexandre Guillot,1 Jessica Jonquet,1 Eric Bertin,5 Jean-Louis Guéant,6,7

Jean-Marc Alberto,6 Jean-Pierre Bronowicki,6,8 Johanne Amoyel,8 Thinhinane Hocine,1 Laurent Duca,1

Pascal Maurice,1 Amar Bennasroune,1 Laurent Martiny,1 Laurent Debelle,1 Vincent Durlach,1 andSébastien Blaise1

Diabetes 2018;67:1604–1615 | https://doi.org/10.2337/db17-0490

Affecting more than 30% of the Western population,nonalcoholic fatty liver disease (NAFLD) is the mostcommon liver disease and can lead to multiple compli-cations, including nonalcoholic steatohepatitis (NASH),cancer, hypertension, and atherosclerosis. Insulin resis-tance and obesity are described as potential causes ofNAFLD. However, we surmised that factors such asextracellular matrix remodeling of large blood vessels,skin, or lungs may also participate in the progression ofliver diseases. We studied the effects of elastin-derivedpeptides (EDPs), biomarkers of aging, on NAFLD pro-gression. We evaluated the consequences of EDP accu-mulation in mice and of elastin receptor complex (ERC)activation on lipid storage in hepatocytes, inflammation,and fibrosis development. The accumulation of EDPsinduces hepatic lipogenesis (i.e., SREBP1c and ACC),inflammation (i.e., Kupffer cells, IL-1b, and TGF-b), andfibrosis (collagen and elastin expression). These effectsare induced by inhibition of the LKB1-AMPK pathway byERC activation. In addition, pharmacological inhibitors ofEDPs demonstrate that this EDP-driven lipogenesis andfibrosis relies on engagement of the ERC. Our data reveala major role of EDPs in the development of NASH, andthey provide new clues for understanding the relation-ship between NAFLD and vascular aging.

Affecting 25% of the general population, 50% of patientswith type 2 diabetes, and 75% of obese patients (1), non-alcoholic fatty liver disease (NAFLD) is usually benign. Acombination of steatosis, hepatocyte injury, and inflam-mation give rise to a process called nonalcoholic steato-hepatitis (NASH), which can cause progressive fibrosis andcirrhosis. Although NASH is very closely linked to insulinresistance, obesity, and metabolic syndrome, the exactcause of this chronic disease has not been formally eluci-dated. Indeed, not all patients with obesity or insulinresistance exhibit NAFLD/NASH, and, conversely, not allpatients with NAFLD/NASH exhibit insulin resistance orobesity. Endogenous factors such as tissue aging and in-dependent lifestyle factors may contribute to the progres-sion of NAFLD into NASH (2). In addition, extracellularmatrix (ECM) remodeling such as elastin degradationduring aging and the production of elastin-derived pep-tides (EDPs) (found in systemic blood circulation) may playan important role in the development of NASH. Elastin,a major structural protein of skin, lung, and large bloodvessels, confers on them the physical properties of exten-sibility and elastic recoil and, thus, is essential for thephysiological function of these and other tissues. Poly-meric elastin is extraordinarily durable; in “inflamm-aging”

1UMR CNRS 7369 MEDyC, University of Reims Champagne-Ardenne, Reims, France2Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale),Germany3Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark4Fraunhofer Institute for Microstructure of Materials and Systems IMWS, Halle (Saale),Germany5Champagne Ardenne Specialized Center in Obesity, University Hospital Center,Reims, France6Institut National de la Santé et de la Recherche Médicale, U954, and UniversityHospital Center, Nancy University, Vandoeuvre lès Nancy, France7Department of Molecular Medicine and Personalized Therapeutics and Department ofBiochemistry, Molecular Biology, Nutrition, and Metabolism, University HospitalCenter, Nancy University, Vandoeuvre lès Nancy, France

8Department of Gastroenterology and Hepatology, University Hospital Center, NancyUniversity, Vandoeuvre lès Nancy, France

Corresponding author: Sébastien Blaise, sebastien.blaise@univ-reims.fr.

Received 25 April 2017 and accepted 14 May 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0490/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

1604 Diabetes Volume 67, August 2018

PATHOPHYSIO

LOGY

conditions, elastase secretion, such as the secretion ofneutrophil elastase (NE), is increased and the elastin under-goes degradation, releasing elastokines (3). Elastokinesare EDPs that exhibit biological activities and are con-sidered to be aging biomarkers. The typical motif of EDPis the hexapeptide VGVAPG, whose sequence is tan-demly repeated six times in human tropoelastin (4).Our laboratory has previously shown that EDPs bind tothe elastin receptor complex (ERC) and contribute to thedevelopment of several diseases, such as cancer, athero-sclerosis (5), and insulin resistance (6), in mice fed witha low-calorie diet. This widely expressed receptor complexcomprises a peripheral EDP-binding subunit, the so-calledelastin binding protein (EBP), a protective protein/cathepsinA, and neuraminidase-1 (Neu-1), whose enzymatic activityis required for ERC signaling (7). Elastin knockout micehave been shown to exhibit diabetes (8), suggesting thatelastic fiber aging may influence the development ofdiseases characteristic of the metabolic syndrome.Nevertheless, no study has yet investigated the role ofEDPs and their receptors in liver physiology and NASHdevelopment. The present work shows, for the first time,that ERC activated by EDPs contributes to lipid accumu-lation and inflammation by impairment of the AMPKpathway. In addition, EDPs induce a hepatic ECM remod-eling leading to strong fibrosis. With regard to humans,we suggest that plasma EDPs may serve as new non-invasive biomarkers to diagnose the risk of developingNASH.

RESEARCH DESIGN AND METHODS

Animal ModelsAnimal treatments were performed according to the Amer-ican Physiological Society’s Guiding Principles for the Careand Use of Animals. Eight-week-old C57BL/6N, db/db, anddb/+ mice (Janvier, Saint-Berthevin, France) were main-tained continuously with normal food and water availablead libitum. Because several studies have shown that EDPclearance in vivo is extremely fast, we injected 10 mg/kgweekly for 8 weeks (6), i.e., less than observed in diabeticmice (15 mg/kg). To study the implications of the ERC, weinjected ERC inhibitors (V14 peptides [91 mg/kg/week],chondroitin sulfate [CS; 50 mg/kg/week], or 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [DANA; 10 g/L/week])(6) in parallel to kE (mixture of EDPs). We performeda treatment with metformin (35 mg/kg/day) (Sigma-Aldrich, Saint Quentin Fallavier, France) or resveratrol(2 mg/kg/day) (Sigma-Aldrich) in addition to the kEtreatment.

Cell CultureOnce reaching confluence, AML12 hepatocytes (CRL-2254;American Type Culture Collection, Manassas, VA), seededat a density of 7,500 cells/cm2, were exposed to the dif-ferent treatments and their carrier vehicles during a 24-hperiod, which was followed by mRNA or protein extrac-tion. The conditions of treatment were as follows: kE

(50 mg/mL) 6 CS sodium (200 mg/mL) or DANA, used asNeu-1 inhibitor (Merck, Molsheim, France), V14 peptide(purity.98%) (Genecust, Dudelange, Luxembourg), resver-atrol (50 mmol/L), or metformin hydrochloride (1 mmol/L).The data are the mean of three independent experiments,each performed in triplicate.

Clinical ApproachPlasma was collected from 48 obese French women andmen (BMI.40 kg/m2) (Supplementary Table 1) accordingto the ethical guidelines of the 1975 Declaration of Hel-sinki. The selection of patients is described in the Sup-plementary Materials and Methods. The patients weredivided into two groups: a group without diabetes (HOMAof insulin resistance [HOMA-IR],4.8) and a group withdiabetes (HOMA-IR .4.8). Two liver disease scores weredetermined: the fatty liver index (FLI) was calculatedaccording to Bedogni et al. (9) and the NAFLD fibrosisscore was calculated using the formula created by Anguloet al. (10). Among 48 patients, only 16 had been diagnosedwith NASH by biopsy. With regard to the function ofsteatosis, inflammation, and ballooning, this score rangesfrom 0 to 8. Plasma EDPs and NE activity were measuredusing the Fastin (Biocolor, Carrickfergus, U.K.) and NEActivity Assay (Abcam, Cambridge, U.K.) kits, respectively.After a 12-h overnight fast, venous blood samples weredrawn to determine the levels of transaminases (alanineaminotransferase [ALT], aspartate aminotransferase [AST],and g-glutamyltransferase), glucose, cholesterol, triglycerides(TGs), and insulin.

Biochemical AnalysesCpt1/2, medium-chain acyl-CoA dehydrogenase (MCAD),and long-chain acyl-CoA dehydrogenase (LCAD) activitywere analyzed from carnitine and acylcarnitine assaysdescribed by Pooya et al. (11). Malondialdehyde (MDA),superoxide dismutase (SOD), and glutathione peroxidase(GPX) were assayed as previously described by Dali-Youcefet al. (12). All elastin samples digested by NE were analyzedby nanoHPLC-nanoESI-QqTOF MS(/MS) (Thermo FisherScientific, Idstein, Germany and Waters/Micromass, Man-chester, U.K.). To measure liver fatty acid composition,lipid assays were performed using lipid samples extractedfrom mouse liver by Folch procedure.

Histological AnalysisMouse liver was fixed immediately after euthanasia inparaformaldehyde 4% and then paraffin embedded forhistological staining or frozen for Oil Red O staining. Sec-tions of 4 mm were stained with hematoxylin-eosin (H-E),picrosirius, or Oil Red O and observed under an Olympuslight microscope.

Serum Metabolic ParametersAssays of serum AST, ALT, total cholesterol, HDL choles-terol, LDL cholesterol, TGs, and free fatty acids (FFAs)assays were performed by the ICS metabolic platform (ICS-IGBMC, Illkirch, France) as previously described (12).

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Immunoprecipitation, Western Blotting, and GeneExpression AnalysesProtein lysate (150 mg) was immunoprecipitated using1 mg of c-Met (Cell Signaling) antibody overnight at 4°C,followed by 20% protein G–Sepharose (100 mL; Amer-sham Pharmacia Biotech) for 1 h. Western blotting wasperformed as previously described (6,11). The antibodiesused for Western blot were as follows: rabbit polyclonalanti-AMPK, p-AMPK (T172), acetyl CoA carboxylase (ACC),p-ACC (S79), and p-cMet (Y1234/1235) from Cell Signal-ing and LKB1, p-LK1 (S431), and anti-actin from SantaCruz Biotechnology. The sialic acid level was determinedby the DIG Glycan Differentiation Kit (Sigma-Aldrich, St.Louis, MO). The analysis for qPCR was performed aspreviously described (6,12), and specific primers are listedin Supplementary Table 3. 36B4 and RPS26 were used asinternal controls.

Statistical AnalysesFor correlation studies with human and mouse samples,P value and z test regressions were performed using theStatview software. Evaluations were assessed by Mann-Whitney U tests. For all analyses, at least three indepen-dent experiments were performed. Values of P, 0.05 wereconsidered significant (*P , 0.05). The error bar repre-sents the SEM.

RESULTS

Inhibition of NE Activity Reduced NAFLD in DiabeticMiceNeutrophils through NE production play a crucial roleduring liver disease (13, 14). Accordingly, we observed anincrease in NE activity during the progression of metabolicsyndrome characterized by an accumulation of blood FFAs,liver TGs, and hepatic collagen concentrations in db/dbmice (Fig. 1A–D) as well as in mice fed with a high-fat diet(Supplementary Fig. 1). Since the deletion of the NEgene improves liver inflammation in the NAFLD model,we hypothesized that a pharmacological treatment fo-cused on NE activity could influence lipid metabolism inmice. Therefore, db/db mice (6 weeks) were injected withLY54439 (2 mg/kg/day for 2 weeks), which is reportedto be an NE inhibitor (Fig. 1E). We found a decrease inlipid droplet accumulation in the liver (Fig. 1F) and mRNAexpression coding for lipogenesis enzymes (Fig. 1G).LY544349 treatment restored activated AMPK signalinginitially decreased in db/db mice (Fig. 1H). According toMansuy-Aubert et al. (13) and Talukdar et al. (14), the datasuggested a role for neutrophils in modulating hepaticmetabolism through a mechanism involving NE, promot-ing an intense inflammatory response. Nevertheless, therelationship between NE and lipid metabolism/AMPKsignaling was not clearly identified in these studies.

Increase of NE Activity Is Responsible for ElastinFragmentation and EDP ProductionNE is a protease that cleaves ECM proteins such as elastinand leads to the formation of potentially active EDPs. We

demonstrated in vitro that potential bioactive peptides,bearing the GXXPG motif (Table 1 and Supplemen-tary Table 4, respectively), were released from both miceand human vascular elastin by NE. The sequences of thepeptides were of the GXXPG type. In parallel, we observeda high fragmentation of aortic elastic fibers in several obeseand diabetic mouse models, such as db/db mice (Fig. 2A)and mice fed with a high-fat diet (Supplementary Fig. 1).Plasma EDPs were significantly increased (Fig. 2B). In-terestingly, plasma EDP levels and NE activity werepositively correlated (Fig. 2C). The treatment of db/dbmice with NE inhibitors reduced aortic elastic fiber alter-ations (Fig. 2A) and plasma EDP levels (Fig. 2D) signif-icantly. Previously, several studies have demonstratedthat elastic fiber integrity is an important factor for glu-cose and lipid homeostasis (8,15). Therefore, we supposethat EDPs produced by NE may be involved in NAFLDprogression.

Inhibitors of ERC Reduced NAFLD Induced by EDPs inMiceWe tested an EDP known to be biologically active(VGVAPG), with its scramble (VVGPGA) known to be bi-ologically inactive, and a mixture of EDPs called kE (3)onmice. All analyses were performed after 2 months of EDPtreatment (6). The plasma assays showed an increase ofFFAs, TGs, and transaminases (Supplementary Fig. 2Aand B), suggesting hepatic impairment in mice with accu-mulated EDPs when compared with the control sample.Hepatic microvesicular steatosis (Fig. 3A) present in micetreated with EDPs was characterized by an accumulation oftotal lipid (Supplementary Fig. 2C), lipid peroxidation (Fig.3C), TGs (Fig. 3B and Supplementary Fig. 2D), and satu-rated fatty acids (Supplementary Fig. 2E), such as stearateand palmitate (Supplementary Fig. 2F). The higher densityof lipid droplets observed under the influence of kE (Fig.3A) can be explained by an increase in the expression oflipogenic genes (Fig. 3D and E). Conversely, the expressionof b-oxidation genes and the lipolytic gene were greatlyreduced (Fig. 3F and G). These data confirmed our hy-pothesis that EDPs are involved in NAFLD progression.Previously, we demonstrated that EDPs are biologicallyactive through their binding on the ERC, which induces themodulation of several signaling pathways. Based on theseconclusions, we used several inhibitors known to inhibitERC activity. Therefore, in parallel to kE injections, wetreated the mice with DANA, CS, or V14 once a week for8 weeks. Histological approaches (H-E and Oil Red Ostaining) showed that the three inhibitors reduced lipiddroplet accumulation in the liver (Fig. 3A) but also restoredthe normal expression of genes coding for lipogenesis orb-oxidation enzymes (Fig. 3D–G). Acylcarnitines wereanalyzed in livers from the different groups (Fig. 3H).The ratios of acylcarnitine C14/C16 and C8/C10 weresignificantly decreased, suggesting a reduction in the ac-tivity of LCAD and MCAD, respectively. Similarly, a sig-nificant decrease in the ratio of C0/(C16 + C18) and an

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increase in the ratio of (C16+C18:1)/C2 suggested thatcarnitine palmitoyltransferase I (CPT1) and CPT2 (12)were impaired by kE. When administrating ERC inhibitors,acylcarnitine levels were unaffected by kE. These lipidmetabolisms are under the control of the AMPK pathway(Fig. 3I). The addition of ERC inhibitors protected againsta decrease in AMPK phosphorylation induced by kE, thus

suggesting a specific role for EDPs in NAFLD. Otherwisein db/dbmice, we observed a decrease of AMPK phosphor-ylation associated with an increase of lipogenesis (Fig. 1),on the one hand, and an increase of endogenous EDPs (Fig.2) on the other. Accordingly, we treated the db/db micewith the ERC inhibitors (DANA or CS) for 4 weeks. Weobserved that these inhibitors partially restored the AMPK

Figure 1—Inhibition of NE (LY544349) slows down hepatic lipogenesis in db/dbmice (n = 5/group). A: Quantification of NE activity measuredin liver.B: Intrahepatic TG levels and correlation with NE activity.C: FFA assay and correlation with NE activity.D: Intrahepatic collagen assayand correlation with NE activity. E: NE activity quantification in db/dbmice treated for 2 weeks with LY544349 (2 mg/kg/day for 14 days). F:H-E or Oil Red O staining in liver of normal (db/+), db/db, and db/db mice treated with LY544349. G: qPCR analysis of peroxisomeproliferator–activated receptor (PPARg), ACC, and FAS expression (NE = LY544349). H: Western blot and semiquantification of AMPK andACC from liver of mice treated with LY544349 (NEi), metformin (Met), or resveratrol (Resv). SPSS was used to perform the Mann-Whitney Utest. *P , 0.05; **P , 0.01; ***P , 0.001. NEi, NE inhibitor.

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pathway (Fig. 3J and K), mRNA expression involved inlipogenesis (Fig. 3L), and lipid droplet accumulation (Fig.3M). These data suggest that EDPs produced by NE could

be an intermediate link in the relationship between NEand NAFLD.

Inhibitors of EDPs or ERC Reduced the LipogenesisPathway in HepatocytesTo determine the direct effect of EDPs on NAFLD, weincubated hepatocytes with kE with or without ERC inhib-itors (DANA, CS, or V14). In parallel, we compared theeffects induced by these inhibitors with cells treated withkE and metformin or resveratrol, both known to respec-tively stimulate lipolysis and block lipogenesis. Except forresveratrol, all tested drugs reversed the effects of kE, i.e.,they inhibited the induction of lipogenesis by the expres-sion of ACC, fatty acid synthase (FAS), or SREBP1c andrestored b-oxidation by expression of MCAD and CPT1a(Fig. 4A). Interestingly, these enzymes are targets ofAMPK signaling, which was activated in the presence ofERC inhibitors (Fig. 4B). The literature mentions thatAMPK phosphorylation can be induced, for instance, bymetformin (activation of AMPK by LKB1) or by resveratrol(which activates AMPK by SIRT1 stimulation). Despite thepresence of kE, metformin increased phosphorylation ofAMPK, whereas resveratrol had a limited effect, suggestingthat kE inhibited AMPK phosphorylation independentlyof SIRT1. These results were also observed in vivo (Figs.1H and 3J). We tested several stimuli to explain AMPKphosphorylation. Whereas the AMP-to-ATP ratio did notseem be affected (data not shown), AMPK could also beactivated by the phosphorylation of LKB1 (16).We observedthat kE alone inhibited LKB phosphorylation, which wasrestored by DANA, CS, V14, and metformin, whereasresveratrol had no effect in vitro or in vivo. Interestingly,the LKB1/AMPK cascade was regulated by hepatic growthfactor receptor (HGFR), also called c-Met (16), and thedecrease of c-Met activity was associated with NAFLDprogression (17–19). In our cell culture model (Fig. 4C), theEDP decrease of the phosphorylation of c-Met could beexplained by desialilation of this receptor.

ERC Inhibitors Reduced EDP-Induced NASHAny chronic liver aggression, such as lipid accumulation,induces oxidative stress and inflammation, which causesfibrous scarring of tissue according to the two-hit theory(19). First, the results showed in the EDP group an increaseof oxidative stress (decrease of SOD and GPX activity) (Fig.5A) and of inflammation (Fig. 5B), suggested by Kupffercell marker expressions (F4/80). The expression of cyto-kines (Fig. 3B), which increased in the group treated withkE, is known to induce fibrosis. Chronic injections of kE orVGVAPG increased the picrosirius staining (Fig. 5C andSupplementary Fig. 3A) and collagen (Fig. 5D and E andSupplementary Fig. 3B and C) and significantly decreasedprotease expressions (Fig. 5F and Supplementary Fig. 3D).In terms of matrix remodeling, the expression of elasticfiber components (tropoelastin, fibulin-5, and fibrillin-1)was significantly increased (Fig. 5E and SupplementaryFig. 3C). However, the absence of orcein staining failed

Table 1—Peptides identified from a 48-h digest of mouseaortic elastin with NE

Residue

Mice aorta Mr (Da)Start End

37 46 GLPGGVPGGV 808.44

80 93 FGAGPGGLGGAGPG 1,070.51

80 94 FGAGPGGLGGAGPGA 1,141.55

80 96 FGAGPGGLGGAGPGAGL 1,311.66

80 97 FGAGPGGLGGAGPGAGLG 1,368.68

81 94 GAGPGGLGGAGPGA 994.48

81 96 GAGPGGLGGAGPGAGL 1,164.59

136 144 GGVPGGVGV 697.38

181 191 STGAVVPQVGA 984.52

205 213 VGLPGVYPG 873.46

210 218 VYPGGVLPG 857.46

251 264 GIPGVGPFGGQQPG 1,282.63

251 267 GIPGVGPFGGQQPGVPL 1,591.83

325 342 GGAGVLPGVGGGGIPGGA 1,348.71

328 342 GVLPGVGGGGIPGGA 1,179.62

328 342 GVLPGVGGGGIPGGA 1,163.63

453 462 GAGGFPGYGV 880.41

455 462 GGFPGYGV 752.35

493 503 LGGLVPGAVPG 935.54

512 525 VPGAGGVPGAGTPA 1,106.57

540 552 GLGPGVGGVPGGV 1,021.56

542 552 GPGVGGVPGGV 851.45

558 575 PGGVGVGGVPGGVGPGGV 1,390.72

558 575 PGGVGVGGVPGGVGPGGV 1,390.72

564 575 GGVPGGVGPGGV 908.47

564 575 GGVPGGVGPGGV 924.47

576 588 TGIGAGPGGLGGA 983.50

576 592 TGIGAGPGGLGGAGSPA 1,295.65

576 592 TGIGAGPGGLGGAGSPA 1,311.64

577 588 GIGAGPGGLGGA 882.46

577 592 GIGAGPGGLGGAGSPA 1,194.60

579 592 GAGPGGLGGAGSPA 1,024.49

611 621 GLGAGVPGFGA 901.47

611 621 GLGAGVPGFGA 917.46

Bioactive motifs are highlighted in bold. Hydroxylated prolineresidues are shown in italic/underline. Digests of the mouseaortic samples were analyzed by nanoHPLC-nanoESI-QqTOF-MS/MS. In brief, chromatographic separation was performedusing a binary reverse-phase gradient, and peptides in the mass-to-charge ratio range 300–2,500 were selected for MS/MS indata-directed acquisition mode. All fragment spectra were pre-processed using proprietary software, and the resultant peaklists were subjected to automated de novo sequencing usingPeaks Studio 7.0 (Bioinformatics Solutions, Waterloo, ON,Canada) with subsequent database matching using a SwissProtdatabase.

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to demonstrate elastic fiber formation (data not shown).Interestingly, ERC inhibitors used in parallel with kEinjections restored the expression of proteases, collagen,and elastin in a similar way to those observed in the controlmice (Fig. 5). These data suggest that the inhibition of theEDP-ERC pathway may limit NASH progression. The fibro-sis observed with EDP injections is interesting because themodels of NAFLD commonly used, such as mice fed witha high-fat diet or db/db, do not show spontaneous fibrosis.Therefore, we decided to induce a moderate NASH with db/db mice, which were fed either a methionine- and choline-deficient (MCD) diet for 4 weeks or treated with thioacet-amide (TAA; 200 mg/kg body weight three times per week)for 3 weeks to determine whether EDPs could potentializethe NASH and whether treatment with an ERC inhibitorcould be sufficient to block the onset of fibrosis. Unfortu-nately, CS, which has previously given the best effects, hasonly partially reduced inflammation markers such as F4/80(Fig. 5G) as well as fibrosis labeling (Fig. 5H) and collagenexpression (Fig. 5I). We suppose that this decrease wasassociated with fibrosis induced by EDPs and the majoritywas associated with either diet or TAA. Nevertheless, inparallel with TAA injection, db/db mice treated with an NEinhibitor dramatically reduced fibrosis and inflammationmarkers (Supplementary Fig. 4).

Clinical Proof of ConceptPlasma EDPs are positively correlated with the degree ofhepatic steatosis in patients with NASH. In order totranspose the findings obtained in mice to human pathol-ogy, we analyzed 48 French patients with or without type 2diabetes. The individual characteristics of the patientsare listed in Supplementary Table 1. Although the NEactivity was not significantly modified, we observed, asin the diabetic mice, that EDP concentrations were in-creased in patients with diabetes when compared with

patients without diabetes (Fig. 6A). Plasma EDP concen-trations had a 60.2% correlation with NE activity (Fig. 6B).A positive correlation was observed between NE activity(Supplementary Fig. 5) and plasma EDPs (Fig. 6C) andBMI, glycemia, HOMA-IR, plasma TGs, FFAs, C-reactiveprotein (CRP), and neutrophil concentrations. Obesity,hyperglycemia, and high levels of FFAs and CRP encom-pass an alteration of the hepatic metabolism. There-fore, the FLI (9) and the NAFLD fibrosis score (10) werecalculated. They correlated with both NE activity (Supple-mentary Fig. 5) and plasma EDP levels (Fig. 6C). However,multiple regression analyses showed that EDPs were a de-terminant of FLI (P = 0.0041) and NAFLD fibrosis (P =0.0024), suggesting that EDP levels may be a better plasmabiomarker of liver diseases than NE activity. Amongour cohort, 16 patients had been diagnosed with NASHafter liver biopsies (Supplementary Table 2). Although nodifferences in NE activity were observed, multiple regres-sion analyses showed that EDPs are determinants of thedegree of fibrosis (P , 0.0001). Indeed, the plasma EDPlevel was increased in tandem with the degree of steatosis(Fig. 6D) and hepatic fibrosis (Fig. 6E). These histopathol-ogy data confirm that endogenous EDPs might be consid-ered as plasma biomarkers to facilitate the diagnosis ofliver injuries.

DISCUSSION

Several studies have shown that metabolism, in particu-lar hepatic metabolism, not only depends on a normalgenetic/epigenetic status of the genes regulating fat andglucide use but also involves ECM homeostasis. Elastin,found in large blood vessels, skin, and lungs, represents90% of the composition of the elastic fibers (17). Duringaging, mechanical vascular stresses and local actions of pro-teases such as NE lead to degradation of the elastic fibers, i.e.,

Figure 2—NE inhibitor reduces elastin fragmentation and EDP production (n. 5/group).A: Autofluorescence of aortic elastic fibers in normal(control [Ctrl], db/+), db/db (8 weeks old), or db/db mice treated with kE and LY544349 (NE inhibitor [NEi]). Arrowheads correspond tofractures of elastic fibers.B: Quantification of plasma EDP in age function of db/dbmice.C: Correlation betweenNE activity and plasma EDP.D: Plasma EDP quantification in db/db mice treated with LY544349 (1 mg/kg/day, 14 days). SPSS was used to perform the Mann-WhitneyU test. *P , 0.05; **P , 0.01. L, lumen.

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elastolysis, and production of EDPs (18). Although theseelastolysis mechanisms are still poorly understood, re-cruitment of elastases, such as the NE observed in ourpresent study, could explain EDP production in mu-rine models of obesity and also in obese patients. EDPs,

recognized as aging markers (20), can accelerate metabolicsyndrome features such as NAFLD/NASH (Fig. 6F). Wedemonstrate here, for the first time, that EDPs, potentialproducts of the increase of NE activity, allow the accumu-lation of hepatic TGs. In contrast, the inhibition of NE

Figure 3—Inhibitors of ERC activation reduce NAFLD induced by EDP (n. 5/group). A–I: Analysis frommice injected or not injected with kEand ERC inhibitors (DANA [green bar], CS [purple bar], and V14 [orange bar]). A: H-E and Oil Red O staining. B: Intrahepatic TG level.C: Lipidintrahepatic peroxidation by MDA assay. qRT-PCR analysis of genes involved in the lipogenic pathway (ACC, FAS, SREBP1c, andperoxisome proliferator–activated receptor [PPARg]) (D), the TG synthesis pathway (diacylglycerol O-acyltransferase [Dgat]) (E), b-oxidation(MCAD, LCAD, CPT1, and PPARa) (F ), lipolysis (hormone-sensitive lipase [HSL], lipoprotein lipase [LPL], and adipose triglyceride lipase[ATGL]) (G).H: Evaluation of b-oxidation enzyme activities (CPT1/2, MCAD, and LCAD) bymeasures of ratio plasma acylcarnitines. I: Westernblot (phosphorylated or not) and semiquantification (ImageJ) of AMPK and ACC in mice treated or not with ERC inhibitors, metformin (Met),or resveratrol (Resv). J–M: Analysis from db/db mice treated or not with DANA or CS. Western blot (J) and semiquantification (K) of AMPKand ACC. L: qRT-PCR analysis of genes involved in the lipogenic pathway. M: H-E. SPSS was used to perform the Mann-Whitney U test.*P , 0.05.

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activity protects mice from accumulating fat. From a clin-ical perspective, our study shows that EDPs, rather thanNE activity, are determinants of the degree of hepaticsteatosis and fibrosis. During the last decade, our teamhas deciphered the signaling pathways triggered by theERC upon its binding to the canonical human sequence

VGVAPG (21). This receptor has a very singular compo-sition that comprises three subunits: a peripheral subunitcalled EBP and two membrane-associated proteins, a pro-tective protein/cathepsin A (PPCA) and a neuraminidase(Neu-1), which is essential for signal transduction by theERC. ERC activity can be inhibited by several molecules or

Figure 4—Impact of kE and ERC inhibitors on hepatocytes (AML12) (n = 3 independent cell cultures). A: qRT-PCR analysis of genes involvedin the lipogenic pathway (ACC, FAS, and SREBP1c) and b-oxidation (MCAD and CTP1). B: Western blot (phosphorylated or not) andsemiquantification (by ImageJ) of LKB1, AMPK, and ACC. C: Immunoprecipitation (IP) of c-MET and Western blot of phosphorylated c-METand sialylation level. Semiquantification by ImageJ of Input and IP. SPSS was used to perform ANOVA. *P , 0.05. Met, metformin; Resv,resveratrol.

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peptides. V14 blocks EDP binding with EBP, CS inhibitsEBP function, and DANA is an inhibitor of Neu-1 activity(21). Interestingly, based on our current data, we identifiedthat these ERC inhibitors restore the normal expression ofmRNA coding for lipogenesis, b-oxidation, and lipolysisimpaired by EDPs. Futhermore, the LKB1-AMPK path-way, dramatically reduced by EDPs in hepatocytes, canbe restored by the presence of an ERC inhibitor similarly

to metformin treatments described in the literature (22).This alteration of the AMPK pathway associated withhepatic lipid accumulation has been demonstrated inseveral studies (22) but is still poorly understood. Severalstimuli can be involved in AMPK activation, such as theactivation of the leptin receptor, the reduced AMP-to-ATPratio, or SIRT1 activation. In our study, the data obtainedwith db/db mice (leptin receptor deficient), whether or

Figure 5—Inhibitors of ERC activation reduce hepatic fibrosis induced by EDPs (n = 5/group).A–F: Analysis frommice injected or not injectedwith kE (blue bar) and ERC inhibitors (DANA [green bar], CS [purple bar], and V14 [orange bar]). A: Measurements of SOD and GPX. B: qRT-PCR analysis of genes involved in the inflammation pathway (F4/80, IL-1b, TNF-a, and TGF-b). C: Picrosirius staining. D: Total intrahepaticcollagen assay. E: mRNA expression of type I collagen (a1) and tropoelastin and microfibrils of elastases (fibrillin 1 and fibulin 5). F: qRT-PCRanalysis of genes coding for proteases (MMP 2/9/12, NE, and TIMP1). G–I: Analysis from db/+ (white bar) and db/db (gray bar) mice injectedwith TAA or fed with an MCD diet and treated or not with CS. G: mRNA expression of F4/80, TNF-a, and TGF-b. H: Picrosirius staining. I:mRNA expression of type I collagen (a1) and tropoelastin. SPSS was used to perform the Mann-Whitney U test. *P , 0.05.

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not treated with resveratrol (SIRT activator) or metformin(LKB1-AMPK activator), suggest that EDPs influence theactivation of LKB1 before AMPK phosphorylation (Fig.6F). In the context of metabolic disorders, the activation ofc-Met/LKB1/AMPK signaling has been shown in NASHprogression (23) and to promote hepatocyte proliferationand viability (16). Upon HGF binding, Tyr-1234/1235residues in the catalytic domain of c-Met are phosphory-lated and the receptor is activated. Once activated, c-Metactivates multiple downstream signaling pathways, includ-ing the PI3K/Akt and MEK/ERK pathways. Through theseintermediary pathways, HGF/c-Met governs a diversity of

important cellular responses, such as protective actionsagainst lipid accumulation, lipotoxicity, oxidative stress, orECM remodeling in the liver (17–19). Nevertheless, asdescribed for the insulin receptor (6), EGFR, and PDGFR(24), c-Met activity may be dependent on N-glycan chainsfixed on the receptor ectodomain (25,26). HoweverN-glycosylation modifications regulating the enzymaticproperties of c-Met are unknown. In our study, we ob-served that the ERC activated by EDP is able to cleave sialicacids on the N-glycosylation chain of c-Met, thus decreas-ing the c-Met activity (Fig. 6F). Previously, we demon-strated (27) that removing sialic acid impairs the N-glycan

Figure 6—EDPs produced by elastin fragmentation are positively correlated with plasma markers of NAFLD in obese patients (n = 48). A: NEactivity and plasma EDP levels in obese patients with type 2 diabetes (black bar, n = 20) or without type 2 diabetes (white bar, n = 28).B: Positivecorrelation between NE activity and plasma EDPs. C: Correlation of plasma EDP level with HOMA-IR, glycemia, BMI, plasma FFA, plasma TG,plasma CRP, neutrophil number, FLI, and NALFD fibrosis score. D: NE activity and plasma EDP quantification in patients without (,NAS 5) orwith (.NAS 5) steatosis. E: NE activity and plasma EDP quantification in patients with fibrosis. SPSS was used to perform the Mann-WhitneyU test. *P , 0.05; **P , 0.01; ***P , 0.001. F: Diagram summarizing the EDP effects on NASH development. NAS, NAFLD activity score.

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chain dynamic and might impair receptor behavior with itsligands or catalytic domain.

The c-Met defect was involved with lipotoxicity, oxida-tive stress, and inflammation, which are the principal factorsmentioned in the transition from NAFLD to NASH (19). Inour study, we demonstrate for the first time that EDPsinduce the accumulation of nonesterified saturated FFAsknown to be hepatotoxic (28,29), an increase in oxidativestress and cytokine expression. These parameters areknown to be involved in ECM remodeling. Thus, weshow that EDPs essentially induce an accumulation oftype I collagen and, surprisingly, an expression of tropo-elastin, fibulin-5, and fibrillin-1, as mentioned by Pellicoroet al. (30). Oxidative stress, inflammation, and ECM remod-eling demonstrate that EDPs contribute to the NAFLD-NASH transition but also that ERC inhibitors are able torestore normal hepatic phenotypes. To find out whetherERC inhibitors such as CS can reduce fibrosis, we used twomouse models of NASH in which db/db mice were eitherinjected with TAA or fed with an MCD diet for 3 weeks.Unfortunately, we did not observe any real benefit after theadministration of CS. We believe that the fibrosis inducedby these procedures is extremely important and may maskthe fibrosis induced by endogenous peptides in db/db mice.Finally, it is rather difficult to estimate the effectiveness ofthe treatments because of the heterogeneity of the studies(population and objectives) and their lack of power. Severalmolecules have been evaluated but the results are incon-clusive to date. We think that reducing elastin aging, EDPproduction (i.e., by inhibition of NE), or the activation ofERC may be a new strategy to limit NASH progression.

In conclusion, a vicious cycle induced by blood vesseldefects may be responsible for NASH and NASH progression.Indeed, we show for the first time that the EDPs found inblood circulation have a systemic effect and play an importantrole in lipidmetabolism and hepatic ECM remodeling. Accord-ing to our clinical results, the identification of plasma EDPs isa new determinant of NASH. Finally, the inhibitors of elastinfragmentation, EDPs and ERC, which were used in this studyto demonstrate the role of EDPs on hepatic lipid metabolismand fibrosis, suggest that aging and preservation of elasticfibers may be a potential therapeutic target for NASH.

Acknowledgments. The authors thank Aude Bressonot-Marchal (HistologyDepartment, University Hospital, Reims, France) and Dr. Christian Garbar (GodinotInstitute, Reims, France) for excellent help in histology and Fanja Rabenoelina, OlivierBocquet, Annie Carlier, and Marie-Line Sowa (CNRS/URCA UMR7369 MEDyC) fortechnical help. The authors thank Bart Staels and Réjane Paumelle-Lestrelin (INSERMU545, Institut Pasteur, Lille, France) for help with the AML12 cell culture. The authorsthank Bart Staels, Philippe Valet (INSERM U1048, Toulouse, France), and CatherinePostic (INSERM 1016, Institut Cochin, Paris, France) for comments and Dr. JacquelineVan de Walle (Institut des Sciences de la Vie, Louvain-la-Neuve, Belgium) and Scribendi(Chatham-Kent, Canada) for reviewing the article for English grammar.Funding. This work was supported by funding from the CNRS and theUniversity of Reims Champagne-Ardenne as well as the German ResearchFoundation (DFG) (HE 6190/1-2 to A.H.), the LEO Foundation Center for CutaneousDrug Delivery (2016-11-01 to A.H.), and the Fraunhofer Internal Program (Attract

grant 069-608203 to C.E.H.S.). A.G. was supported by research grants from theRégion Champagne Ardenne.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. B.R. and S.B. wrote the manuscript andresearched data. C.I., H.S., A.H., C.E.H.S., E.B., and T.H. researched data andreviewed the manuscript. R.G., A.G., J.-L.G., J.-M.A., J.-P.B., and J.A. researcheddata. J.J., L.Du., P.M., A.B., and L.De. reviewed the manuscript. L.M. and V.D.contributed to discussion. S.B. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

References1. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiologyand natural history of non-alcoholic fatty liver disease and non-alcoholic steato-hepatitis in adults. Aliment Pharmacol Ther 2011;34:274–2852. Fontana L, Zhao E, Amir M, Dong H, Tanaka K, Czaja MJ. Aging promotes thedevelopment of diet-induced murine steatohepatitis but not steatosis. Hepatology2013;57:995–10043. Maurice P, Blaise S, Gayral S, et al. Elastin fragmentation and atherosclerosisprogression: the elastokine concept. Trends Cardiovasc Med 2013;23:211–2214. Debelle L, Tamburro AM. Elastin: molecular description and function. Int JBiochem Cell Biol 1999;31:261–2725. Gayral S, Garnotel R, Castaing-Berthou A, et al. Elastin-derived peptidespotentiate atherosclerosis through the immune Neu1-PI3Kg pathway. CardiovascRes 2014;102:118–1276. Blaise S, Romier B, Kawecki C, et al. Elastin-derived peptides are new reg-ulators of insulin resistance development in mice. Diabetes 2013;62:3807–38167. Duca L, Blanchevoye C, Cantarelli B, et al. The elastin receptor complextransduces signals through the catalytic activity of its Neu-1 subunit. J Biol Chem2007;282:12484–124918. DeMarsilis AJ, Walji TA, Maedeker JA, et al. Elastin insufficiency predisposesmice to impaired glucose metabolism. J Mol Genet Med 2014;8:129–1359. Bedogni G, Bellentani S, Miglioli L, et al. The Fatty Liver Index: a simple andaccurate predictor of hepatic steatosis in the general population. BMC Gastro-enterol 2006;6:3310. Angulo P, Hui JM, Marchesini G, et al. The NAFLD fibrosis score: a noninvasivesystem that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45:846–85411. Pooya S, Blaise S, Moreno Garcia M, et al. Methyl donor deficiency impairsfatty acid oxidation through PGC-1a hypomethylation and decreased ER-a, ERR-a,and HNF-4a in the rat liver. J Hepatol 2012;57:344–35112. Dali-Youcef N, Hnia K, Blaise S, et al. Matrix metalloproteinase 11 protectsfrom diabesity and promotes metabolic switch. Sci Rep 2016;6:2514013. Mansuy-Aubert V, Zhou QL, Xie X, et al. Imbalance between neutrophilelastase and its inhibitor a1-antitrypsin in obesity alters insulin sensitivity, in-flammation, and energy expenditure. Cell Metab 2013;17:534–54814. Talukdar S, Oh DY, Bandyopadhyay G, et al. Neutrophils mediate insulinresistance in mice fed a high-fat diet through secreted elastase. Nat Med 2012;18:1407–141215. Craft CS, Pietka TA, Schappe T, et al. The extracellular matrix protein MAGP1supports thermogenesis and protects against obesity and diabetes throughregulation of TGF-b. Diabetes 2014;63:1920–193216. Vázquez-Chantada M, Ariz U, Varela-Rey M, et al. Evidence for LKB1/AMP-activated protein kinase/endothelial nitric oxide synthase cascade regulated byhepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyteproliferation. Hepatology 2009;49:608–61717. Kroy DC, Schumacher F, Ramadori P, et al. Hepatocyte specific deletion ofc-Met leads to the development of severe non-alcoholic steatohepatitis in mice. JHepatol 2014;61:883–89018. Gomez-Quiroz LE, Seo D, Lee YH, et al. Loss of c-Met signaling sensitizeshepatocytes to lipotoxicity and induces cholestatic liver damage by aggravatingoxidative stress. Toxicology 2016;361–362:39–48

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19. Marquardt JU, Seo D, Gómez-Quiroz LE, et al. Loss of c-Met acceleratesdevelopment of liver fibrosis in response to CCl(4) exposure through deregu-lation of multiple molecular pathways. Biochim Biophys Acta 2012;1822:942–95120. Mecham RP, Gibson MA. The microfibril-associated glycoproteins (MAGPs)and the microfibrillar niche. Matrix Biol 2015;47:13–3321. Duca L, Blaise S, Romier B, et al. Matrix ageing and vascular impacts: focuson elastin fragmentation. Cardiovasc Res 2016;110:298–30822. You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW. The role of AMP-activated protein kinase in the action of ethanol in the liver. Gastroenterology 2004;127:1798–180823. Martínez-López N, Varela-Rey M, Fernández-Ramos D, et al. Activation ofLKB1-Akt pathway independent of phosphoinositide 3-kinase plays a critical role inthe proliferation of hepatocellular carcinoma from nonalcoholic steatohepatitis.Hepatology 2010;52:1621–163124. Arabkhari M, Bunda S, Wang Y, Wang A, Pshezhetsky AV, Hinek A.Desialylation of insulin receptors and IGF-1 receptors by neuraminidase-1 controls

the net proliferative response of L6 myoblasts to insulin. Glycobiology 2010;20:603–61625. Chen R, Li J, Feng CH, et al. c-Met function requires N-linked glycosylationmodification of pro-Met. J Cell Biochem 2013;114:816–82226. Qian J, Zhu CH, Tang S, et al. alpha2,6-hyposialylation of c-Met abolishes cellmotility of ST6Gal-I-knockdown HCT116 cells. Acta Pharmacol Sin 2009;30:1039–104527. Guillot A, Dauchez M, Belloy N, et al. Impact of sialic acids on the moleculardynamic of bi-antennary and tri-antennary glycans. Sci Rep 2016;6:3566628. Feldstein AE, Werneburg NW, Canbay A, et al. Free fatty acids promotehepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway.Hepatology 2004;40:185–19429. Malhi H, Barreyro FJ, Isomoto H, Bronk SF, Gores GJ. Free fatty acidssensitise hepatocytes to TRAIL mediated cytotoxicity. Gut 2007;56:1124–113130. Pellicoro A, Aucott RL, Ramachandran P, et al. Elastin accumulation isregulated at the level of degradation by macrophage metalloelastase (MMP-12)during experimental liver fibrosis. Hepatology 2012;55:1965–1975

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