Santiago Zalvidea

Lucas Andre

Xavier Loyer

Cécile Cassan

Yannis Sainte-Marie

Jérôme Thireau

Ivar Sjaastad

Christophe Heymes

Jean Luc Pasquié

Olivier Cazorla

L André

J-L Pasquié

F Aimond

Sylvain Richard
email: sylvain.richard@inserm.fr

ACE inhibition prevents diastolic Ca2+ overload and loss of myofilament

Keywords: Angiotensin-converting enzyme inhibitors, excitation-contraction coupling, heart failure, hypertrophy, myofilaments, sarcoplasmic reticulum Ca 2+ ATPase

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INTRODUCTION

Over-activation of the renin-angiotensin system (RAS) plays a critical role in the progression of cardiac remodeling after myocardial infarction (MI) [START_REF] Unger | The role of the renin-angiotensin system in the development of cardiovascular disease[END_REF]. Evidences for the cardiovascular benefits of angiotensin-converting enzyme inhibitors (ACE-I) have emerged from long-term studies (CONSENSUS, SAVE, AIRE, TRACE, SOLVD) [START_REF] Sleight | Angiotensin II and trials of cardiovascular outcomes[END_REF]. Indeed, ACE-I lower mortality rates and hospital admissions in patients with left ventricular (LV) dysfunction and progressive heart failure (HF) [START_REF] Flather | Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. ACE-Inhibitor Myocardial Infarction Collaborative Group[END_REF]. Benefits occur mainly via blood pressure reduction, yet there is evidence of blood pressure independent protective effects in coronary disease ( [START_REF] Turnbull | Blood pressure-dependent and independent effects of agents that inhibit the renin-angiotensin system[END_REF].

Experimental studies have shown that ACE-I therapy delays myocardial remodeling, thus inhibiting the transition to HF [START_REF] Litwin | Serial echocardiographic-Doppler assessment of left ventricular geometry and function in rats with pressure-overload hypertrophy. Chronic angiotensin-converting enzyme inhibition attenuates the transition to heart failure[END_REF][START_REF] Brooks | Effect of angiotensin-converting enzyme inhibition on myocardial fibrosis and function in hypertrophied and failing myocardium from the spontaneously hypertensive rat[END_REF][START_REF] Sakata | Temocapril prevents transition to diastolic heart failure in rats even if initiated after appearance of LV hypertrophy and diastolic dysfunction[END_REF]. At the clinical level, short-term studies (e.g., CONSENSUS-II, AIRE) suggest that early ACE-I therapy provides significant and rapid benefits [START_REF] Sleight | Angiotensin II and trials of cardiovascular outcomes[END_REF]. Long-term therapy attenuates morphological and functional alterations related to hypertrophy and fibrosis [START_REF] Aire | Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. The Acute Infarction Ramipril Efficacy (AIRE) Study Investigators[END_REF][START_REF] Kober | A clinical trial of the angiotensin-convertingenzyme inhibitor trandolapril in patients with left ventricular dysfunction after myocardial infarction. Trandolapril Cardiac Evaluation (TRACE) Study Group[END_REF]. In particular, ACE-I therapy blocks the fibrogenic action of angiotensin both in experimental models and in patients. However, the pure anti-remodeling effects of ACE-I through normalization (or prevention) of impaired intracellular Ca 2+ signaling and excitation-contraction coupling as observed in HF (i.e. independent of the blood pressure reduction) at the molecular level have been poorly explored.

After MI, the sustained activation of compensatory neurohormonal systems, coupled with the alteration of various cell signaling and gene expression pathways, triggers a domino effect that leads to severely compromised cardiac function. Changes in cardiomyocyte phenotype are responsible for significant alterations in excitation-contraction (E-C) coupling and proarrhythmogenicity, due to the modification of cellular ion currents and Ca 2+ handling [START_REF] Wehrens | Novel therapeutic approaches for heart failure by normalizing calcium cycling[END_REF][START_REF] Kaye | Reversing advanced heart failure by targeting Ca2+ cycling[END_REF][START_REF] Bers | Altered cardiac myocyte Ca regulation in heart failure[END_REF]. Specific therapeutic options to optimize healing and prevent adverse post-MI remodeling are currently lacking [START_REF] Jugdutt | Preventing adverse remodeling and rupture during healing after myocardial infarction in mice and humans[END_REF]. Several studies have shown that ACE-I modify the expression of genes coding for Ca 2+ handling proteins during HF [START_REF] Ren | Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction[END_REF][START_REF] Guo | Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan[END_REF][START_REF] Shao | Captopril treatment improves the sarcoplasmic reticular Ca(2+) transport in heart failure due to myocardial infarction[END_REF] but the functional consequences are not well-established. In addition, no study has evaluated the effects of ACE inhibition on myofilament Ca 2+ sensitivity, a key determinant of contraction, during HF.

The ACE-I delapril is an old molecule but still currently used, in combination with Ca 2+ antagonist manidipine, as a therapeutic option to control hypertension complicated by diabetes and microalbuminuria, and to reduce cardiovascular morbidity and mortality in high-risk patients [START_REF] Coca | Manidipine plus delapril in patients with Type 2 diabetes and hypertension: reducing cardiovascular risk and end-organ damage[END_REF]. Interestingly, all ACE-I do not have exactly the same molecular effects and pharmacological properties. The affinities of the enzymatic carboxyterminal and aminoterminal regions (C-site and N-site) of ACE for ACE-I drugs differ according to the organ involved and the two catalytic sites may control different functions [START_REF] Bevilacqua | Affinity of angiotensin I-converting enzyme (ACE) inhibitors for N-and C-binding sites of human ACE is different in heart, lung, arteries, and veins[END_REF][START_REF] Acharya | Ace revisited: a new target for structure-based drug design[END_REF]. Delaprilat, the active metabolite of delapril, binds with higher affinity to the C-site than to the N-site, in particular in the left ventricle (LV) and reduces cardiac hypertrophy [START_REF] Razzetti | Pharmacokinetic and pharmacologic properties of delapril, a lipophilic nonsulfhydryl angiotensin-converting enzyme inhibitor[END_REF]. By comparison, the ACE-I captopril displays a high affinity for the N-site and has less affinity for the myocardium [START_REF] Bevilacqua | Affinity of angiotensin I-converting enzyme (ACE) inhibitors for N-and C-binding sites of human ACE is different in heart, lung, arteries, and veins[END_REF][START_REF] Acharya | Ace revisited: a new target for structure-based drug design[END_REF]. In the present work, we focused on the independent blood pressure effects of a chronic treatment with delapril, started 24 hours after MI in mice, on surviving LV cardiomyocytes. The study focused on the molecular effects of delapril not only on Ca 2+ handling but also on Ca 2+ sensitivity of myofilaments which, to our best knowledge, has never been studied before. Delapril preserved the contractile properties of cardiomyocytes by reducing the Ca 2+ transient alterations and by preventing the alterations of myofilament contractile properties in HF after MI.

MATERIALS AND METHODS

MI model and experimental groups

Experimental protocols were approved by the institutional standing committee for animal research and conformed to European directives (86/609/CEE) and the standards set forth in the Guide for the Care and Use of Laboratory Animals (published by the National Academy of Science, National Academy Press, Washington, D.C.). Male Swiss mice (Janvier, France), aged 8 weeks and weighing 34.5±1.5 g (n=102) were randomly assigned to one of three experimental groups: Sham-operated (Sham), subjected to MI, or subjected to MI and treated with delapril (MI-D). MI was induced by left coronary artery ligation [START_REF] Tarnavski | Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies[END_REF]. Briefly, a left thoracotomy was performed in anesthetized mice (2% inhaled isoflurane in O 2 , Aerrane®, Baxter, France). The artery was ligated 1-2 mm beyond the point of emergence from the top of the left atrium, using an 8-0 Sofsilk suture (Syneture, USA). A unique subcutaneous injection of 0.01 ml buprenorphine solution (0.3 mg.ml -1 ) for postoperative analgesia was administered. Sham animals were subjected to the same surgical procedure but without coronary artery ligation. The MI-D group received a daily dose of 6 mg.kg -1 of delapril (kindly supplied by Chiesi Farmaceutici, Parma, Italy) provided in drinking water, starting 24 hours after surgery and lasting for 6 weeks. This dose was chosen because delapril given for 3 weeks did not lower BP in conscious PMI rats [START_REF] Masson | Effects of a DA2/alpha2 agonist and a beta1blocker in combination with an ACE inhibitor on adrenergic activity and left ventricular remodeling in an experimental model of left ventricular dysfunction after coronary artery occlusion[END_REF]. A complete morphological and functional cardiac evaluation was performed at this stage as described [START_REF] Finsen | Echocardiographic parameters discriminating myocardial infarction with pulmonary congestion from myocardial infarction without congestion in the mouse[END_REF] before cellular and molecular studies.

Echocardiography

Longitudinal transthoracic echocardiography was carried out in awake mice using a sonographic apparatus equipped with an i13L 14 MHz linear array transducer designed for the examination of small rodents (Vivid 7, GE Medical systems, USA). Mice were trained for several days before the examination in order to decrease the stress due to handling. No difference in heart rate (bpm) was observed within the 3 groups (701±10, 702±10, 715±11, in Sham, Mi and MI-D respectively). Only mice with a large infarct and an atrial diameter of more than 1.9 mm were used to ensure the transition to HF.

Pressure-volume (P-V) analysis

Briefly, a 1F dual-field combination pressure-conductance catheter (model PVR-1045, Millar Instruments) connected to a pressure-conductance unit (MPVS-300, Millar) was inserted in the right carotid artery and advanced into the LV of anesthetized mice (isoflurane 1.5-2%).

Changes in LV function were visualized through the use of ventricular pressure-volume (P-V) loops. Measurements were acquired at rest and after dobutamine infusion (5 g.kg -1 .min -1 ).

Steady-state LV pressure levels were determined during suspended ventilation, and signalaveraged data from 5-10 consecutive beats were used. Parallel conductance from surrounding structures was calculated by injecting a 10 µL bolus of 15% NaCl through the femoral vein.

Load-independent parameters of contractility were derived from the P-V relationship as described [START_REF] Joho | Left ventricular pressure-volume relationship in conscious mice[END_REF]. The mean arterial pressure and the maximal (+dP/dt) and minimal (-dP/dt) first derivatives of LV pressure were calculated with IOX v1.8.9.4 software (EMKA Technologies, France).

Quantification of fibrosis

Interstitial fibrosis was measured in 10 μm thick transverse sections of mouse hearts in the peri-infarcted area [START_REF] Thireau | New insights into sexual dimorphism during progression of heart failure and rhythm disorders[END_REF] to assess the effect of delapril on this parameter. Briefly, after cervical dislocation, hearts were excised from mice, rapidly frozen in liquid nitrogen and stored at -80 °C. Collagen distribution was determined using picrosirius red-stained sections and expressed as a percentage of total LV surface area (Histolab, Gothenburg, Sweden).

Cell Contractility and Ca 2+ -Transient Measurements

LV myocytes were freshly isolated from the non-infarcted free wall and recorded as previously described [START_REF] Fauconnier | Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy[END_REF]. For measurements of intracellular Ca 2+ , confocal images were obtained by line scanning (1.5 ms/line) cells loaded with fluo-4-AM (4 µmol/L) [START_REF] Fauconnier | Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy[END_REF]. The time course of Ca 2+ transients was assessed by measuring the time constant (τ) of the exponential part of the decay phase. Sarcoplasmic reticulum (SR) Ca 2+ content was assessed by measuring the peak amplitude of the cytosolic Ca 2+ transient induced by the rapid application of caffeine (10 mmol/L). Sarcomere length (SL) and fluorescence (405 and 480 nm) were also simultaneously recorded using an IonOptix system (Milton, MA) and used specifically for the measurement of diastolic Ca 2+ . To do this, LV myocytes were incubated for 30 min with indo-1 AM (10 μmol/L Invitrogen inc. France) as previously described [START_REF] Flagg | Ca2+-independent alterations in diastolic sarcomere length and relaxation kinetics in a mouse model of lipotoxic diabetic cardiomyopathy[END_REF]. Cells were field stimulated to contract at 1 Hz. All experiments were performed at room temperature.

Force Measurements in Permeabilized Cardiomyocytes

Isometric force was measured in single permeabilized cardiomyocytes at different Ca 2+ concentrations at an SL of 1.9 µm as described [START_REF] Flagg | Ca2+-independent alterations in diastolic sarcomere length and relaxation kinetics in a mouse model of lipotoxic diabetic cardiomyopathy[END_REF]. Force was normalized to the crosssectional area measured from imaged cross-sections, and the force-pCa relation fitted to a Hill equation. In some experiments, myofilaments have been incubated with the catalytic domain of PKA (100 U/mL; Sigma-aldrich, Paris, France) or PKC (0.25 U/mL; Sigma) for 50 min at room temperature.

Real-time PCR

RNA was isolated and analyzed using standard methods. Tissue from the non-infarcted free wall of LV was dissected (n=6 in each group) and flash-frozen in liquid nitrogen. Gene expression levels were determined by RT-PCR (LightCycler, Roche, Meylan, France). The sequences of the specific primers for collagen I and collagen III were: Collagen I 5'-TGGTACATCAGCCCGAAC-3' (sense), and 5'-GTCAGCTGGATAGCGACA-3' (antisense); collagen III 5'-GACAGATTCTGGTGCAGAGA-3' (sense), and 5'-CATCAACGACATCTTCAGGAAT-3'(antisense). Results are expressed as a function of βtubulin expression level.

Protein Analysis

Ca 2+ handling: Proteins extracted from the non-infarcted free wall of the LV were separated using 2-20 % SDS-PAGE and blotted onto a PVDF membrane (Protran, Schleichen and Schuele, Dassel, Germany). Membranes were incubated overnight at 4°C with the following primary antibodies: Anti-RyR2 (Affinity Bioreagents, Neshanic Station, NJ), NCX antibody (R3F1, SWANT, Switzerland), Phospho Ser 2809 -RyR2 antibody (A010-30, Badrilla, Leeds, UK), SERCA2a antibody (A010-20, Badrilla, UK), Ser 16 -PLB and Thr 17 -PLB and PLB antibody (Badrilla, UK). RyR2, SERCA2a and PLB levels were expressed relative to GAPDH (Chemicon International, CA, USA) on the same membrane. Immunodetection was carried out using the ECL Plus system (Amersham Pharmacia, Little Chalfont Buckinghamshire, UK).

Myofilament proteins: Myofibrillar proteins were analyzed in skinned muscle strips from the non infarcted area of the LV. The heart was pre-skinned by perfusion with a relaxing solution containing 1% Triton X-100 and protease inhibitors, for 10 min. Phosphorylated and nonphosphorylated forms of myosin light chain 2 (MLC-2) were separated on a 10% urea gel and detected with an antibody specific to cardiac MLC-2 (Coger SA, Paris, France). TnI and PKCα was separated by 15% SDS-PAGE and membranes were incubated with specific antibodies against total cardiac TnI (Cat#4T21, Hytest, Turku, Finland), the PKA-phosphorylated form of cardiac TnI (Cat#4T45, Hytest), PKC-α ((Upstate, Biotechnology, Milton Keynes, UK) or the phospho-Ser657 PKCα (Santacruz, Heidelberg,Germany). All gels were run in triplicate.

Statistical analysis

All data are expressed as means ± S.E.M. Statistical tests included the Mann-Whitney U test, a one-way analysis of variance (ANOVA) when more than two groups were compared, and a Newman-Keul's multiple comparison test for paired values. Differences were considered significant when the P-value was less than 0.05.

RESULTS

Survival, myocardial function and remodeling

We compared survival in Sham, MI and MI-D mice. Survival declined rapidly in the MI group compared to Sham, while Delapril (MI-D) prevented the high mortality observed in the MI group (Fig. 1A). Kaplan-Meier analysis showed that the survival of MI-D mice (87%, n=29) was higher than that of MI mice (62%, n= 49; P < 0.05) but not different from the survival of Sham animals (92%, n=24). The body weights (BW, g) of Sham, MI and MI-D mice were indistinguishable (Table 1). However, the heart weight (HW, mg) to BW ratio (HW/BW) was higher in MI than in Sham mice, confirming cardiac hypertrophy, which was reduced by delapril (Table 1). The mean arterial blood pressure was similar in Sham (89.3±2.6 9 mmHg, n=4) and MI (87.9±10.6 9 mmHg, n=4) mice, as reported before [START_REF] Quang | Role of T-type calcium channel subunits in postmyocardial infarction remodelling probed with genetically engineered mice[END_REF], and was not modified by delapril in MI-D animals (88.3±2.9 mmHg, n=4), suggesting that the cardiac effects of delapril were independent of blood pressure reduction.

In terms of cardiac morphology and function, 2D echocardiography in vivo (Fig. 1B) showed that MI mice had an augmented LVM (Fig. 1C) and LV dilation as assessed by the enddiastolic diameter (LVEdD) (Table 1). Posterior wall thickness (PWT) was reduced in MI and MI-D mice as compared to Sham mice (Table 1). Delapril treatment prevented the increase of the LVM (Fig. 1C) and attenuated LV dilation (Table 1). At the functional level, MI hearts exhibited a dramatic reduction of the shortening fraction (SF) (Table 1), reflecting a major alteration of ventricular function. Although MI-D mice exhibited signs of improved systolic function, the benefit was small (Table 1), consistent with the extensive cardiomyocyte necrosis caused by the large infarct of the LV in this model.

We further evaluated cardiac function based on LV pressure-volume relationships in vivo. In MI mice, the LV end-diastolic pressure (LVEdP) and LV end-systolic pressure (LVEsP) were, respectively, higher and lower than in Sham mice (Table 1). Furthermore, the maximal slope of the increment of systolic pressure (dP/dt max), an index of myocardial contractility, was decreased whereas the maximal slope of the increment of diastolic pressure (dP/dt min), an index of relaxation, was increased (Table 1). MI mice also exhibited larger LV cavity volumes (end systolic and diastolic) than Sham animals. Delapril treatment had no effect on LVSP, confirming that the dose of delapril used did not reduce blood pressure (Table 1). However, it attenuated the effect of MI on LV cavity volume with a remarkable benefit in terms of diastolic pressure (Table 1). Picrosirius red staining showed interstitial and scar fibrosis in the hearts of MI mice (vs. Sham) (Fig. 1D,E). The expression of both collagen I and collagen III mRNAs (Fig. 1F) was higher, consistent with the phenotype of HF at the molecular level.

Delapril treatment attenuated both fibrosis and collagen I mRNA expression.

Cell contraction and Ca 2+ transients

We next investigated the effect of delapril on cardiomyocytes isolated from the non-infarcted area comprised of viable cells subjected to cardiac remodeling following the MI. In unloaded intact single cardiomyocytes from MI mice (vs. Sham), sarcomere length (SL) shortening, reflecting cell contraction during field stimulation, was decreased (Fig. 2A,B). Contraction and relaxation velocities were also reduced (Fig. 2C). Delapril treatment prevented all of these changes. Notably, the diastolic SL was larger in MI-D than in MI or even Sham mice, consistent with the remarkable effect of delapril on cell relaxation (Fig. 2D).

We measured the Ca 2+ transients of field-stimulated cardiomyocytes loaded with fluo-4 AM.

The amplitude of Ca 2+ transients was smaller in MI mice (2.3±0.1, n=25) than in Sham mice (3.2±0.2, n=33; P < 0.05) (Fig. 3A). Their time to peak and decay (τ) were delayed in MI mice (Fig. 3A). In parallel, we determined that the Ca 2+ content of the SR, assessed by a challenge with caffeine leading to full Ca 2+ release, was lower in MI mice than in Sham mice (Fig. 3B), which could account for the decrease in the Ca 2+ -transient amplitude. All these alterations were partially prevented by treatment with delapril, which had a astonishing effect on Ca 2+transient decay kinetics (Fig. 3A). We further investigated diastolic Ca 2+ using the ratiometric indicator indo-1. Diastolic Ca 2+ was higher in MI mice than in Sham mice, an increase that was completely prevented by delapril (Fig. 3C). Delapril treatment thus, had a notable effect on cell relaxation, Ca 2+ -transient decay and diastolic Ca 2+ .

Expression of Ca 2+ handling proteins

We measured the expression level of Ca 2+ handling proteins involved in E-C coupling (Fig. 4).

The abundance of the voltage-gated L-type Ca 2+ channel (LTCC) protein, responsible for Ca 2+ entry into cardiomyocytes, and of the NCX protein was similar in Sham, MI and MI-D mice.

The abundance of the ryanodine receptor (RyR), responsible for Ca 2+ release from the SR, following Ca 2+ entry via LTCCs, was decreased in MI (vs. Sham) mice, a change that was prevented by delapril (Fig. 4A). In contrast, its phosphorylated form (P-RyR) was increased, and this increase was resistant to delapril treatment (Fig. 4A).

The slowing of Ca 2+ -transient decay, as seen in MI cells (Fig. 3), usually reflects impaired Ca 2+ reuptake due to the weakened activity of the SR Ca 2+ ATPase 2 (SERCA2a). Immunoblot analysis showed that SERCA2a expression was decreased in MI (vs. Sham) mice (Fig. 4B).

Since SERCA2a activity is regulated by the inhibitory protein phospholamban (PLB) [START_REF] Minamisawa | Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy[END_REF][START_REF] Schmitt | Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban[END_REF],

we also investigated the effect of MI on this protein. Both protein expression and phosphorylation of phospholamban (P-PLB) at the serine 16 and threonine 17 residues (Ser 16 -PLB and Thr 17 -PLB), were increased in MI hearts (Fig. 4B). Delapril treatment had no effect on the reduction of SERCA2a abundance but remarkably, prevented the increase of PLB protein and phosphorylation levels. Importantly, both the decrease in the P-PLB/PLB ratio (for both Ser 16 -PLB and Thr 17 -PLB) and the increase in the PLB/SERCA2a ratio induced by MI were abolished by delapril (Fig. 4C).

Ca 2+ sensitivity of myofilaments

Cardiomyocyte force development depends on both the amount of Ca 2+ released by the SR and the Ca 2+ sensitivity of the contractile machinery. To determine the effect of delapril on LV myofilament function, we measured the relationship between intracellular Ca 2+ concentration and maximal isometric tension normalized to the cross-sectional area in single permeabilized LV myocytes, as described previously [START_REF] Cazorla | The cAMP binding protein Epac regulates cardiac myofilament function[END_REF]. Cross-sectional area was greater in the MI group (248±16 µm 2 , n=20) than in Sham mice (203±18 µm 2 , n=16), consistent with cardiomyocyte hypertrophy. This effect was prevented by delapril (198±14 µm 2 , n=22, P<0.05). In permeabilized cardiomyocytes, the resting SL was unchanged in MI (1.88±0.01 µm) and MI-D (1.89±0.01 µm) mice when compared to the Sham group (1.90±0.01 µm). In contrast, in MI mice (when compared with Sham animals), the maximum Ca 2+ -saturated force (F max ) was decreased, and the curve representing the tension-pCa relationship was shifted to the right due to a reduction in myofilament Ca 2+ sensitivity (pCa 50 : 5.66±0.01, n=20 in MI, vs. pCa 50 :

5.72±0.02, n=16 in Sham mice, P<0.05; Fig. 5A,B). Delapril treatment had a powerful preventive effect on changes in both maximal active tension and myofilament Ca 2+ sensitivity (pCa 50 : 5.74±0.02, n=22; P<0.05 for MI-D vs. MI).

The Ca 2+ sensitivity of myofilaments is regulated by the phosphorylation status of sarcomeric proteins such as MLC2 and TnI [START_REF] Hamdani | Sarcomeric dysfunction in heart failure[END_REF]. The phosphorylation level of MLC2 was unchanged in the ventricles of MI mice when compared with Sham mice, as well as after delapril treatment (Fig. 5C). In contrast, the phosphorylation level of TnI at the protein kinase A (PKA) sites Ser 23/24 , known to result in decreased Ca 2+ sensitivity, was increased in MI hearts, an effect prevented by delapril (Fig. 5D) suggesting post-translational modifications of regulatory contractile proteins mediated by kinases. We next evaluated the contribution of kinases such as PKA and protein kinase C (PKC) to modulation of Ca 2+ sensitivity in MI and MI-D mice by incubating myofilaments with the two activated kinases. PKA had no effect on maximal tension in the three groups (Fig. 6A). PKA decreased myofilament Ca 2+ sensitivity in both Sham and MI mice by 0.09 and 0.06 pCa unit, respectively (Fig. 6B). In contrast, PKA had no significant effect in MI-D mice (decrease of 0.04 pCa unit). Overstimulation with PKA normalized myofilaments Ca 2+ sensitivity in Sham and MI myocytes but not in MI-D cells, where it remained higher than in Sham and MI, suggesting that delapril protects myofilament Ca 2+ sensitivity by a pathway independent of PKA signaling. Next, we explored the PKC signaling pathway known to be involved in angiotensin II effects through AT1R binding [START_REF] Joho | Left ventricular pressure-volume relationship in conscious mice[END_REF],

and to regulate the myofilament function during HF [START_REF] Belin | Augmented protein kinase C-alpha-induced myofilament protein phosphorylation contributes to myofilament dysfunction in experimental congestive heart failure[END_REF]. Incubation of the myofilaments with activated PKC decreased the maximal tension in the three groups to a similar level (Fig. 6A).

PKC also decreased the pCa 50 in Sham and MI-D mice by 0.18 and 0.12 pCa unit, respectively, but had no effect in MI mice with a non-significant decrease of pCa 50 of 0.03 pCa unit (Fig. 6B). Altogether the results indicate that myofilament function is affected by both PKA and PKC pathways in MI mice while PKC signaling is downregulated in MI-D mice. We thus determined the expression level of PKC-α and its activation evaluated by the level of phosphorylation at Ser 657 (Fig. 6C). The expression and activation of PKC-α increased in MI mice. Delapril treatment prevented the increase in PKC-α expression and did not affect the level of activation compared with MI animals (Fig. 6C).

DISCUSSION

ACE-I have been studied extensively over the last three decades and their clinical benefits are well-established. However, their molecular effects are complex and unclear. In particular, all cardiac therapeutical benefits are not supported exclusively by blood pressure reduction. Our study provides novel information about delapril effects at the cardiomyocytes level. We showed the strong benefits of delapril treatment on the contraction and relaxation of viable cardiomyocytes after MI in a model of cardiac remodeling in mice, which occurred independently of blood pressure reduction. Chronic treatment, started 24 hours after infarction, prevented the intracellular Ca 2+ overload that occurs following MI and preserved the relaxation of cardiomyocytes. We also show for the first time that, in addition, delapril maintained the Ca 2+ sensitivity of contractile myofilaments at normal levels. Both effects resulted in a remarkable preservation of cellular contraction and attenuated the typical transition toward HF after MI. Ours results were also consistent with the modulation of PKC expression.

Our experimental mice model reproduced critical features of post-ischemic HF, including depressed myocardial contraction and relaxation, chamber dilation, increased LV mass, and akinesia of the anterior wall due to a large infarcted area. Structural remodeling and functional alterations of the non necrosed tissue were accompanied by fibrosis and the increased expression of mRNAs for HF markers (Collagen I and III). At the cardiomyocyte level, contraction was depressed due to reduced SR Ca 2+ load and systolic Ca 2+ release, relaxation was slowed owing to blunted Ca 2+ reuptake that resulted in a diastolic Ca 2+ overload, and the Ca 2+ sensitivity of myofilaments was reduced [START_REF] Bers | Altered cardiac myocyte Ca regulation in heart failure[END_REF][START_REF] Hobai | Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure[END_REF][START_REF] Piacentino | Cellular basis of abnormal calcium transients of failing human ventricular myocytes[END_REF]. At the protein level, HF remodeling was associated with changes in the abundance and/or phosphorylation levels of Ca 2+ -handling proteins, including SERCA2a, PLB and RyR2, consistent with earlier reports [START_REF] Bers | Altered cardiac myocyte Ca regulation in heart failure[END_REF][START_REF] Hobai | Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure[END_REF][START_REF] Piacentino | Cellular basis of abnormal calcium transients of failing human ventricular myocytes[END_REF].

ACE-I are among the most beneficial drugs used in HF patients. ACE-I therapy improves survival after MI, and large clinical trials point to the importance of initiating therapy as early as possible [START_REF] Flather | Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. ACE-Inhibitor Myocardial Infarction Collaborative Group[END_REF][START_REF] Latini | ACE inhibitor use in patients with myocardial infarction. Summary of evidence from clinical trials[END_REF]. In our MI mouse model, chronic treatment with a non-hypotensive dose of delapril (6 mg/kg) reproduced the expected cardiac effects of ACE-I, including attenuated LV chamber dilation, increased LVM, reduced myocardial fibrosis and collagen production [START_REF] Bartha | Effect of L-2286, a poly(ADP-ribose)polymerase inhibitor and enalapril on myocardial remodeling and heart failure[END_REF].

Similar beneficial effect has been described with perindopril, which prevents cardiac hypertrophy without affecting systemic blood pressure in the rat with HF after MI [START_REF] Chiba | Lack of correlation of hypotensive effects with prevention of cardiac hypertrophy by perindopril after ligation of rat coronary artery[END_REF]. The anti-fibrotic effects of delapril were also comparable to those of a non-hypotensive dose of fosinopril (1 mg/kg; compared to a 25 mg/kg antihypertensive dose), which has also been shown to restore the ß-adrenergic signal transduction pathway independently of the regression of hypertrophy [START_REF] Bohm | Dose-dependent dissociation of ACEinhibitor effects on blood pressure, cardiac hypertrophy, and beta-adrenergic signal transduction[END_REF]. Our data are consistent with ACE inhibition delaying the transition from hypertrophy to HF by targeting mechanisms intrinsic to cardiomyocytes, in addition to its antifibrotic effects [START_REF] Takeishi | Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure[END_REF].

A major finding of our study, focused on the post-MI remodeling of cardiomyocytes, was that delapril preserved the normal contraction/relaxation cycle and attenuated SR dysfunction in the surviving cells. Delapril stabilized the amplitude and the onset of contraction through the normalization of Ca 2+ -transient amplitude and kinetics, in line with reports showing improved cell shortening, Ca 2+ -transient amplitude and SERCA2a activity in aortic-banded rats and guinea-pigs treated with the ACE-I ramipril [START_REF] Takeishi | Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure[END_REF][START_REF] Boateng | Low-dose ramipril treatment improves relaxation and calcium cycling after established cardiac hypertrophy[END_REF]. In our model, delapril did not affect the level of expression of SERCA2a and NCX. We propose that delapril promotes efficient reuptake of Ca 2+ into the SR by maintaining SERCA2a activity through normal P-PLB/PLB (for both P S16 -PLB and P T17 -PLB) and PLB/SERCA2a ratios [START_REF] Minamisawa | Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy[END_REF][START_REF] Schmitt | Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban[END_REF][START_REF] Kadambi | Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice[END_REF]. In addition, delapril attenuated the reduction in RyR2 abundance in viable cardiomyocytes (see also [START_REF] Yamaguchi | Effects of long-term treatment with trandolapril on sarcoplasmic reticulum function of cardiac muscle in rats with chronic heart failure following myocardial infarction[END_REF]), thereby favoring Ca 2+ release in systole. Surprisingly, the higher PKA-phosphorylation level of RyR2, which has previously been associated with Ca 2+ leakage from the SR in HF [START_REF] Reiken | PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure[END_REF], was not prevented by delapril. Therefore, if we were to assume that RyR2 was still leaky in the presence of delapril, which might explain the lower SR Ca 2+ content, the beneficial effect of delapril on SERCA2a activity was enough to maintain the normal decay of the Ca 2+ transient (and relaxation) and normal diastolic cytoplasmic Ca 2+ . Several group proposed an increase of NCX expression as an important mechanism to rescue the compromised SERCA2a function in HF in both human [START_REF] Hasenfuss | Relationship between Na+-Ca2+exchanger protein levels and diastolic function of failing human myocardium[END_REF][START_REF] Studer | Gene expression of the cardiac Na(+)-Ca2+ exchanger in end-stage human heart failure[END_REF][START_REF] Flesch | Evidence for functional relevance of an enhanced expression of the Na(+)-Ca2+ exchanger in failing human myocardium[END_REF] and experimental models [START_REF] Gomez | Increased exchange current but normal Ca2+ transport via Na+-Ca2+ exchange during cardiac hypertrophy after myocardial infarction[END_REF][START_REF] Wasserstrom | Altered E-C coupling in rat ventricular myocytes from failing hearts 6 wk after MI[END_REF]. Surprisingly, we noted no change in the NCX expression in MI and MI-D mice, which may be in line with evidence for different phenotypes in HF (hearts with increased NCX and unchanged SERCA2a; and hearts with decreased SERCA2a and unchanged levels of NCX) [START_REF] Hasenfuss | Relationship between Na+-Ca2+exchanger protein levels and diastolic function of failing human myocardium[END_REF] although other groups reported a decrease of NCX during HF [START_REF] Dixon | Sarcolemmal Na(+)-K(+)-ATPase activity in congestive heart failure due to myocardial infarction[END_REF][START_REF] Makino | Regression of hypertrophy after myocardial infarction is produced by the chronic blockade of angiotensin type 1 receptor in rats[END_REF][START_REF] Sethi | Beneficial effects of propionyl L-carnitine on sarcolemmal changes in congestive heart failure due to myocardial infarction[END_REF].

Another key finding of our study was that delapril treatment preserved the maximum Ca 2+saturated active force and Ca 2+ sensitivity of myofilaments in surviving cardiomyocytes after MI. In MI mice, the altered Ca 2+ sensitivity of myofilaments involves the increased activity of both PKA and PKC (Fig. 6B), leading notably to the phosphorylation of cTnI, as previously shown in other rodents [START_REF] Belin | Augmented protein kinase C-alpha-induced myofilament protein phosphorylation contributes to myofilament dysfunction in experimental congestive heart failure[END_REF][START_REF] Belin | Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure[END_REF]. In the present study, the abnormal function of the troponin complex, related to the augmented phosphorylation of cTnI in MI, was prevented by delapril.

Interestingly, delapril limitedFigure 1 :

 1 ABBREVIATIONS

Figure 2 :

 2 Figure 2: Effects of delapril on cardiomyocyte sarcomere length

Figure 3 :

 3 Figure 3: Effects of delapril on Ca 2+ transients in LV myocytes A . Top left panel: Representative Ca 2+ transients from Sham (n=33), MI (n=27) and MI-D

Figure 4 :

 4 Figure 4: Effect of delapril on excitation-contraction coupling proteinsTop: Representative Western blot for LTCC: L-type Ca 2+ channel; NCX: Na + /Ca 2+ exchanger; RyR: ryanodine receptor 2; P-RyR: phosphorylated ryanodine receptor; SERCA2a: SR Ca 2+ ATPase 2a; Total PLB: total phospholamban; P S16 -PLB: PLB phosphorylated by PKA at Serine 16; P T17 -PLB: phospholamban phosphorylated by CaMKII at the threonine-17 site. A.

Figure 5 :

 5 Figure 5: Effects of delapril on contractile machinery in permeabilized cardiomyocytes A. Relationship between Ca 2+ -activated tension and intracellular Ca 2+ content measured at an

ACKNOWLEDGMENTS

We thank Dr. Roberta Razzetti for careful reading and comments on this manuscript, and Pr.

Ole Sejersted and Halvor Kjean Mork for helpful discussions during this study. This work was supported in part by ANR (GENOPAT N°RPV08141FSA).


Marie-Noële Chobert

Dominique Couchie

Agnès Fourcot

Elie-Serge Zafrani

Yannick Laperche

Philippe Mavier

PhD Arthur Brouillet
email: arthur.brouillet@inserm.fr

Marie-Noele Chobert

Liver precursor cells increase hepatic

Keywords: 1-Oral, Gastrointestinal, Hepatic & Pancreatic Systems 2-Blood, Lymphatics, Immune System, Stem Cells SMA,-smooth muscle actin, AAF, acetylaminofluorene, AFP, -fetoprotein, ALT, alanine aminotransferase, CCl 4 , carbon tetrachloride, CK19, cytokeratin 19, CCR2, chemokine C-C motif receptor 2, EMT, epithelial-mesenchymal transition, HSC, hepatic stellate cells, LPC, liver progenitor cells, TGFtransforming growth factor-β, TNFtumor necrosis factor- fibrosis, liver progenitor cells, TGFepithelial-mesenchymal transition

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Chobert MN et al

Hepatic fibrosis is the major complication of virtually all types of chronic liver damage (e.g. viral, alcoholic liver disease-or non-alcoholic fatty liver disease). In human, fibrosis that ultimately leads to cirrhosis initiates predominantly in the portal areas even though the primary targets of injury are hepatocytes within the lobule. [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF] Impairment of the replicative capacity of most remnant hepatocytes induces an alternate regenerative process from liver progenitor cells (LPC). These cells (also called oval cells in rodent) are located in the most peripheral branches of the biliary tree (canal of Hering). [START_REF] Paku | Origin and structural evolution of the early proliferating oval cells in rat liver[END_REF] Once activated, LPC proliferate in the portal region and migrate into the hepatic lobule where they undergo further differentiation into hepatocytes or bile duct cells to repopulate the hepatic parenchyma. [START_REF] Paku | 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies[END_REF][START_REF] Knight | Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell[END_REF] This proliferative response characterized by the appearance of bile-duct-like structures in human is referred to atypical ductular reaction. [START_REF] Sell | Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury[END_REF] LPC have been of increasing interest in latest years since they could play a role not only in regeneration but also in fibrogenesis and carcinogenesis. [START_REF] Bird | Activation of stem cells in hepatic diseases[END_REF] In experimental models and various human liver diseases, LPC expand in close proximity to -smooth muscle actin (SMA)-positive cells deriving from either sinusoidal hepatic stellate cells (HSC) or portal fibroblasts depending upon location of the injury in the lobule. [START_REF] Paku | Origin and structural evolution of the early proliferating oval cells in rat liver[END_REF][START_REF] Schotanus | Cross-species immunohistochemical investigation of the activation of the liver progenitor cell niche in different types of liver disease[END_REF] There are increasing data demonstrating an intimate cross-talk between LPC and SMA-positive myofibroblasts [START_REF] Paku | 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies[END_REF][START_REF] Friedman | Mechanisms of hepatic fibrogenesis[END_REF][START_REF] Fausto | Liver regeneration[END_REF][START_REF] Svegliati-Baroni | Hepatic fibrogenesis in response to chronic liver injury: novel insights on the role of cell-to-cell interaction and transition[END_REF][START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF] and several studies in rodent models and human diseases have pointed out a strong relationship between severity of fibrosis and intensity of the ductular reaction. [START_REF] Lowes | Oval cell numbers in human chronic liver diseases are directly related to disease severity[END_REF][START_REF] Clouston | Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis[END_REF][14][START_REF] Knight | Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice[END_REF] Interactions between LPC and sinusoidal HSC or portal fibroblasts are poorly understood but their activation could occur independently, successively or in tandem through similar stimuli. [START_REF] Clouston | Hepatic progenitor cell-mediated regeneration and fibrosis: chicken or egg?[END_REF] First, it can be considered that secretion of profibrogenic cytokines and growth factors by LPC could participate to portal fibrosis by inducing HSC or portal fibroblast activation, [START_REF] Pi | Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats[END_REF][START_REF] Akhurst | Differential lymphotoxin-beta and interferon gamma signaling during mouse liver regeneration induced by chronic and acute injury[END_REF] but this implication of LPC in fibrosis has not been clearly shown in vivo. On the opposite, activation of HSC or portal fibroblasts into myofibroblasts producing extracellular matrix and growth factors, precedes LPC expansion and differentiation in periportal regions along a porto-venous axis. [START_REF] Bird | Activation of stem cells in hepatic diseases[END_REF][START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF] Recently, it has been shown that inhibition of HSC activation greatly diminished LPC expansion. [START_REF] Pintilie | Hepatic stellate cells' involvement in progenitor-mediated liver regeneration[END_REF] Therefore, HSC or portal fibroblast activation seems a pre-requisite for LPC expansion, as already demonstrated in other stem cell niches. [START_REF] Lorenzini | Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver[END_REF] Additionally, myofibroblast-LPC cross-talk could indirectly enhance fibrogenesis by recruiting cells of innate immune response, such as leukocytes required for wound healing. [START_REF] Ruddell | Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury[END_REF] Among the large variety of Chobert MN et al factors secreted by myofibroblasts, transforming growth factor-TGF, the most potent fibrogenic cytokine, is also a well established mediator of epithelial-mesenchymal transition (EMT), which contributes to fibrosis following injury in several organs including the liver. [START_REF] Kaimori | Transforming growth factor-beta1 induces an epithelial-tomesenchymal transition state in mouse hepatocytes in vitro[END_REF][START_REF] Zeisberg | Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition[END_REF][START_REF] Robertson | Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis[END_REF][25][START_REF] Battaglia | Liver cancer-derived hepatitis C virus core proteins shift TGF-Beta responses from tumor suppression to epithelial-mesenchymal transition[END_REF] To investigate the fibrogenic potential of LPC, we established a new model in the rat in which persistent LPC expansion induced by a chronic 2-acetylaminofluorene (AAF) treatment was combined to the wellestablished model of liver fibrosis using chronic carbon tetrachloride (CCl 4 ) injection. [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF] Metabolism of CCl 4 into highly reactive CCl 3 radicals by cytochrome P450; is responsible for centrilobular hepatocellular necrosis which triggers matrix deposition starting around the central veins, with gradual formation of septa bridging neighboring central veins, without any ductular reaction. 25 In our model, administration of AAF prior to and during CCl 4 -treatment blocks the proliferative hepatocyte response caused by CCl 4 -induced necrosis [START_REF] Petersen | Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats[END_REF] and leads to the emergence of an alternate regenerative pathway with expansion of LPC in periportal regions as commonly reported in human fibrosis of various etiologies. 12- 14,28 Using this new model, we investigated the involvement of LPC activation in fibrosis development by comparing the fibrogenic response in rats treated with a combination of CCl 4 and AAF to that obtained in animals treated with either CCl 4 or AAF alone. We also investigated whether LPC may directly participate to fibrogenesis and serve as a source of myofibroblasts through EMT as reported for hepatocytes and biliary cells. [START_REF] Kaimori | Transforming growth factor-beta1 induces an epithelial-tomesenchymal transition state in mouse hepatocytes in vitro[END_REF][START_REF] Zeisberg | Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition[END_REF][START_REF] Robertson | Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis[END_REF][25][START_REF] Battaglia | Liver cancer-derived hepatitis C virus core proteins shift TGF-Beta responses from tumor suppression to epithelial-mesenchymal transition[END_REF]

Materials and Methods

Animals and experimental design

200g male Sprague Dawley rats (Janvier Animal Center) received intraperitoneal injection of CCl 4 (Sigma-Aldrich) diluted 1/1 in olive oil twice a week in combination with 2-acetylaminofluorene (0.02% AAF in pellet form, Genestyl) treatment (AAF/CCl 4 group, n=27) for 6 weeks. AAF was given five days before the first CCl 4 injection. In a preliminary study, a continuous AAF exposition in association with a commonly used CCl 4 dose (2ml/kg of body weight) was not well tolerated leading to a severe weight loss after 4 weeks of treatment and a high rate of animal mortality. For these reasons, we used 2ml/kg of body weight for the initial CCl 4 injection and half of this dose for subsequent injections (i.e., 1ml/kg body Chobert MN et al weight). From the second week, the AAF treatment was discontinued 3 days a week (replaced by standard chow). Rats were sacrificed 2, 4 or 6 weeks after the first CCl 4 injection and 48 hours after the last CCl 4 injection and due to significant mortality in this group, 7 to 9 animals were analyzed per time point. Two other animal groups received either CCl 4 only (CCl 4 group, n=27) or AAF only (AAF group, n=13). . An additional group of untreated rats was used as a control and fed a standard rat chow ad libitum (control group, n=7). The livers were removed from animals under anaesthesia and processed as previously described. [START_REF] Lafdil | Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice[END_REF] Blood samples were collected to measure serum alanine aminotransferase (ALT) activity by using an Advia 1650 automate (Bayer Diasys). Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Caspase-3 activity.

Liver lysates were prepared by homogenization in hypotonic buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1% NP40, 1mM EGTA pH8, 1 mM DTT, protease inhibitor cocktail (Roche)). Homogenates were centrifuged at 10 000 g. for 15 min, and extracted proteins (50 µg) were tested in duplicate experiments by measuring the proteolytic cleavage of Ac-DEVD-AFC fluorogenic substrate for caspase-3 (Biomol) on microplate reader TriStar LB 941 (Berthold) as previously described. [START_REF] Lafdil | Induction of Gas6 protein in CCl4-induced rat liver injury and anti-apoptotic effect on hepatic stellate cells[END_REF] .

Histopathological and immunohistochemical methods

Liver injury and fibrosis were assessed on 4 µm-thick paraffin-embedded liver sections stained with hematoxylin and eosin (H&E) and picrosirius red, respectively. Immunohistochemistry was carried out on paraffin-embedded or 5 µm-thick frozen sections, as previously described. [START_REF] Lafdil | Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice[END_REF] Immunodetection of -SMA on paraffin-embedded sections used the streptavidin-alkaline phosphatase conjugate (Serotec) and the FastRed substrate systems (Dako). Double-staining experiments were performed on 6 µm frozen sections. Immunofluorescence double staining of collagen1 and CK19 was revealed with the secondary goat anti-rabbit IgG Alexa fluor 555 and anti-mouse IgG Alexa fluor 488, and double staining of TGF and CK19 with the secondary goat anti-rabbit IgG Alexa fluor 488 and antimouse IgG Alexa fluor 555, respectively. When mouse primary antibodies directed against SMA or desmin were used, they were incubated alone and detected with goat anti-mouse IgG Alexa fluor 555.

After a 20 minute step of permeabilization by 0.2% triton in PBS, sections were incubated with an anti-CK19 antibody directly labeled with mouse IgG Alexa fluor 488, using Zenon® labeling technology (Invitrogen).

In situ hybridization

Complementary RNA probe specific for -fetoprotein (AFP) and TGF were synthesized using a digoxigenin RNA labeling kit (Roche Diagnostics). The cRNA AFP probes were obtained from a rat cDNA plasmid (gift of Dr JL. Danan) digested with XmaI (antisense) or SphI (sense) probe) and transcribed from the SP6 or T7 promoters, respectively. TGF probes were transcribed from a cDNA fragment obtained by RT-PCR from rat liver at day 9 in the AAF/partial hepatectomy model using forward 5'-TACGTCAGACATTCGGGAAGCAGT-3' and reverse 5'-TGTACTGTGTGTCCAGGCTCCAAA-3' primers which map to positions 553-576 and 1143-1131 of the rat TGF sequence (GI: 148747597) and subcloned into pCR4-TOPO-plasmid. The cRNA TGF probes were transcribed from T7 and T3 promoters of the plasmid digested with SpeI (antisense) or NotI (sense) and labeling, hybridization and revelation procedures were performed in paraffin sections as previously described. [START_REF] Mavier | Expression of stromal cell-derived factor-1 and of its receptor CXCR4 in liver regeneration from oval cells in rat[END_REF] Detection of AFP or TGF mRNA in consecutive liver sections stained with immunofluorescent anti-CK19 as describe above, were realized with a standard in situ hybridization protocol on frozen serial sections cut at 5µm thickness (Supplemental materials).

Chobert MN et al

Quantitative real-time PCR

Total RNA was isolated from frozen hepatic tissue using the RNeasy mini kit (Qiagen). Two µg were reverse-transcribed from random hexamers using a first-strand synthesis kit (Fermentas Life Sciences) and specific cDNA amplifications were performed as previously described [START_REF] Lafdil | Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice[END_REF] using primers listed in Table 1. Relative quantification of gene expression was performed using the 2 -CT method and ribosomal 18S RNA normalization and results were expressed as fold induction over values obtained from control animals.

Liver collagen content

Hydroxyproline assay was performed as previously described, [START_REF] Julien | Antifibrogenic role of the cannabinoid receptor CB2 in the liver[END_REF] using small fragments of different lobes that were pooled, lyophilized and hydrolyzed in 6N HCl at 110°C overnight. Hepatic hydroxyproline content was spectrophotometrically measured using freshly prepared Ehrlich's reagent and results were expressed as µg/mg of tissue (dry weight).

Statistical analysis : Liver weight, mRNA and protein analysis (ALT, caspase-3, hydroxyproline) were determined in all animals in duplicate experiments and values are mean ± SEM of 4-9 separate rats from each group and time point Two-way ANOVA was employed to assess the effect of each treatment over the weeks using Bonferroni post-test. The student's t-test was used to compare the differences between 2 groups. Statistical analyses were performed with PRISM (GraphPad) and P-value of less than or equal to 0.05 indicated a significant difference between groups (* P<0.05, ** P<0.01 and *** P<0,001).

Results

Liver injury

CCl 4 treatment induced hepatocellular necrosis revealed by a gradually increase of serum ALT activity which reached their maximal values (238±33 U/L) at week 6 in group CCl 4 (Figure 1A). CCl 4 treatment also provoked hepatic apoptosis as revealed by measurement of active caspase-3 in liver extracts (Figure 1A). No significant differences in necrosis and apoptosis were observed between the CCl 4 and AAF/ CCl 4

Chobert MN et al animals by two-way ANOVA, indicating similar liver injury in both groups. Interestingly, AAF treated animals exhibited low ALT activities but values were significantly higher (52± 8 U/L; P< 0.05) than control rats (37±5 U/L). In contrast, AAF treatment produced the highest caspase-3 activity in all liver extracts tested, indicating that chronic AAF exposure alone induced marked hepatic apoptosis associated with a moderate hepatocellular necrosis.

After 6 weeks of treatment, animals from AAF/CCl 4 group had a body weight (262 ±20 g) significantly (P< 0,001) lower than in groups CCl 4 and AAF (370±28 g and 367±16 g respectively). At the end of the treatment, the ratio of the liver weight to the body weight was increased in both groups of animals treated by CCl 4 as compared to untreated rats and induction in the AAF/ CCl 4 group was significantly higher than in CCl 4 group (Figure 1B).

Livers from group CCl 4 exhibited gradual hepatic parenchymal alterations in centrilobular areas with the length of the treatment (Figure 1C), in accordance with increased serum ALT. After 2 weeks of CCl 4 treatment, focal necrosis and steatotic hepatocytes were seen around the central veins (data not shown).

Two weeks later, livers showed mild to moderate steatosis, often associated with inflammatory cell accumulation and bands of damaged tissue connecting central veins of adjacent lobules. After 6 weeks, the hepatic lobular structure was disorganized with moderate hepatocellular steatosis and ballooning in close proximity to inflammatory cells, leading in some cases to complete interconnection between central veins and thus dividing the parenchyma into pseudo-lobules centered by a portal tract. In the AAF/CCl 4 group after 2 weeks of treatment, hepatic centrilobular injury was similar to that observed in animals treated with CCl 4 alone, except for an expansion of basophilic cells with a high nuclear to cytoplasmic ratio detected in periportal areas (data not shown). After 4 weeks, basophilic cell infiltration extended more deeply into the lobule of AAF/CCl 4 -treated rats and formed porto-portal and porto-central bridges.

Two weeks later, large areas of basophilic cells delineated multiple nodules in the liver, and hepatic steatosis was localized around these connecting areas. Unexpectedly in the AAF group, we also observed periportal expansion of basophilic cells with small ovoid nuclei, progressively linking adjacent portal areas after 6 weeks of treatment, although there was neither steatosis nor centrilobular necrosis (Figure 1C).

Chobert MN et al

Liver precursor cell expansion

In groups AAF and AAF/CCl 4 after 2 weeks of treatment, small basophilic cells arising from portal areas expressed cytokeratin 19 (CK19), a liver precursor cell marker (data not shown). In these two groups, CK19-positive cells increased in number with time and expanded inside the lobule, where they formed duct-like structures (Figure 2A). After 6 weeks in both groups, there was an extensive arborization of CK19-positive cells, which extended across the liver lobule, forming bridges between portal tracts. In addition, in group AAF/CCl 4 , expansion of CK19-positive cells connected portal and centrilobular areas, dividing the parenchyma into smaller pseudo-lobules. As expected, such an accumulation of CK19positive cells was not detected in CCl 4 -treated rats, where labeling was restricted to bile duct-cells in portal tracts (Figure 2A), as in normal liver (data not shown). These results were confirmed by real-time PCR analysis, which revealed an increase of hepatic CK19 mRNA expression only in AAF and AAF/CCl 4 groups, peaking at 93-fold over the basal level at week 4 and 6 in AAF/CCl 4 -treated rats (Figure 2B). No significant difference was observed between the AAF and AAF/CCl 4 groups, suggesting that CCl 4 treatment did not contribute to CK19-positive cell expansion induced by AAF. These latter cells organized in ductular structures also expressed AFP mRNA detected by in situ hybridization, confirming their precursor phenotype (Figure 2C). Similar results were obtained on consecutive liver section stained with immunofluorescent antibody against CK19 (red) and in situ hybridization for AFP mRNA (Supplementary Figure S1). In summary, chronic AAF treatment activated the hepatic stem cell compartment leading to LPC proliferation, which was similar in AAF and AAF/CCl 4 groups. Portocentral connections formed by proliferating LPC and only observed in the AAF/CCl 4 group, seemed to be related to CCl 4 -induced lesions.

Liver fibrosis

As expected, rats treated with chronic CCl 4 injection displayed progressive liver fibrosis detected by picrosirius red-staining (Figure 3A). At week 2 in group CCl weeks, thin collagen deposition arose from portal tracts and progressed into the lobule in close proximity to the LPC expansion at week 6 (Figure 2A), suggesting that the faint picrosirius red-signal might correspond to type 4 collagen accumulation, 33 a major component of basement membrane associated to LPC activation. [START_REF] Paku | Origin and structural evolution of the early proliferating oval cells in rat liver[END_REF] Immunofluorescence detection of collagen 1 revealed an accumulation of this major component of liver fibrosis in group CCl 4 (Figure 3B), whereas a faint fluorescent signal was detected in group AAF. In accordance with these observations, real-time PCR analysis (Figure 3C) demonstrated an induction of hepatic mRNA expression of collagen type 1 and 3 in group CCl4 and collagen 4 in group AAF. Thus, the 2-times induction of the hydroxyproline content (Figure 3D) can be accounted by the accumulation of fibrotic collagens in group CCl 4 and the extension of basement membrane associated with LPC expansion in group AAF.

In animals treated by a combination of AAF and CCl 4 , disease progression and the fibrotic pattern corresponded to the responses above described in both CCl 4 and AAF groups (Figure 3A). After 4 weeks, picrosirius red-staining revealed thin fibrous septa from the portal tracts interconnected with larger fibrous scars from central veins, leading to porto-portal and porto-central fibrosis. After 6 weeks, fibrosis was more extensive, delineating multiple micronodules and forming complete cirrhosis in all rats from the AAF/CCl 4 group. PCR analysis (Figure 3C) showed significantly increased hepatic mRNA expression of collagen type 1 and 3, by comparison with that was noted in animals treated only by CCl 4 (ANOVA p<0.05). There was also an induction of hepatic collagen type 4, similar to that observed in group AAF.

An increase of the hydroxyproline content in AAF/CCl 4 group (Figure 3D) up to 7 fold over basal level at week 6, and significantly higher than in CCl 4 and AAF groups (ANOVA p<0.001), was in accordance with increased fibrosis demonstrated by picrosirius red-staining (Figure 3A). Thus, in group AAF/CCl 4 , active deposition of basement membrane components during LPC expansion also takes a part in the fibrogenic process, indicating that AAF, while stimulating an expansion of LPC, exacerbates CCl 4induced liver fibrosis.

Chobert MN et al

Liver inflammation

CCl 4 administration induces an inflammatory response, and macrophages play a critical role in myofibroblastic transformation of HSC and hepatic fibrogenesis. [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF] The amount of liver macrophages, including Kupffer cells and recruited monocytes, was assessed by detection of the pan-macrophage CD68 antigen (Figure 4A). Normal liver displayed a homogenous lobular distribution of CD68-positive cells in accordance with Kupffer cell localization within sinusoids, and CD68 staining covered 2.1 ± 0.5 % of the liver section as estimated by computer-assisted spot detection method (data not shown). In group CCl 4 after 4 weeks, small foci of CD68-positive cells accumulated in centro-central septa (Figure 4A), and increased in number with the duration of CCl 4 treatment, covering up to 6.3% of the liver section at week 6 (Figure 4B). In animals treated with AAF alone, the increase in CD68-labelled cells did not significantly differ from that noticed in group CCl 4 (Figure 4B), but most positive cells were regularly distributed all over liver lobules (Figure 4A), as noted for CD68-stained Kupffer cells in normal liver. In group AAF/CCl 4 , a majority of CD68-positive cells accumulated in the bridging septa above described (Figure 2A). Although extensive fibrosis was observed in AAF/CCl 4 group, the area of the CD68-stained liver tissue was not significantly different from that measured in the group CCl 4 (Figure 4B).

During liver injury, activated macrophages secrete numerous cytokines, including TNF, which are mitogenic for hepatocytes [START_REF] Fausto | Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration[END_REF] as well as for LPC. [START_REF] Knight | TNF/LTalpha double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury[END_REF][START_REF] Knight | Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice[END_REF] In CCl 4 -treated rats, real-time PCR revealed a gradual but limited (less than 4 fold) induction of tumor necrosis factor- (TNF mRNA (Figure 4C) to a level which was not statistically different in AAF and AAF/CCl 4 groups. Even more, chemokine C-C motif receptor 2 (CCR2) mRNA, which is expressed only in recruited monocytes and some T lymphocytes, 37 was not different in the 3 groups (Figure 4D). The low and similar grade of inflammation in the three groups contrasts with the strong difference in fibrosis extension, suggesting that, in our experimental conditions, inflammation had not a major role in the fibrogenic process.

Fibrogenic liver cells

Chobert MN et al

In chronic liver diseases, portal fibroblasts and activated HSC exhibit phenotypic features of myofibroblasts, namely expression of SMA and extracellular matrix (ECM) proteins. [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF] After 2 and 4 weeks of CCl 4 treatment, immunohistochemical staining of SMA revealed only a vascular labeling in group CCl 4 (Figure 5A), also detected in control livers (data not shown). At week 6, SMA-positive cells were observed in active fibrotic areas of only 3 treated animals out of 6 analyzed (Figure 5A). In contrast, in the AAF/CCl 4 -treated animals, numerous SMA-positive cells were seen around portal areas as early as 2 weeks after starting the treatment (Figure 5A and5B). The SMA labeling was also observed in fibrous septa around centrilobular areas at week 4 and in perinodular fibrosis from cirrhotic rats at week 6 (Figure 5A and5B). In the AAF group, whatever the time of treatment, SMA-staining was restricted to vessels as in normal liver and did not reveal any activated HSC in the areas of ductular reaction (Figure 5A). Immunofluorescence detection of SMA in frozen liver sections which provides a higher sensitivity as compared to immunodetection on paraffin sections, confirmed the lack of SMA-positive cells in the ductular reaction from group AAF and the presence of few activated HSC in fibrous septa in liver from group CCl 4 after 6 weeks of treatment (Figure 6A). Immunofluorescence detection of desmin, a hallmark of quiescent and activated HSC, showed an accumulation of positive cells in group AAF, closely associated to LPC expansion extending from periportal areas (Figure 6A). In group CCl 4 , desmin staining was detected in centrilobular injured areas and in centro-central fibrous septa. Thus, under our experimental setting (1ml/kg of CCl 4 ), the paucity of SMA staining reflected minor myofibroblastic activation in agreement with moderate fibrosis. In summary, HSC transactivation was clearly shown only in liver from AAF/CCl 4 -treated animals, first in close proximity of LPC arising from portal areas, and then in bridging fibrosis, demonstrating that combination of CCl 4 and AAF was necessary to obtain SMA-positive myofibroblasts, which could account for the high degree of fibrosis in this group.

Transforming growth factor- (TGFβ) is the major fibrogenic cytokine mainly produced by SMApositive myofibroblasts, [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF][START_REF] Friedman | Mechanisms of hepatic fibrogenesis[END_REF] and its expression coincides with LPC expansion during liver regeneration after AAF/partial hepatectomy in rats. [START_REF] Pi | Role of connective tissue growth factor in oval cell response during liver regeneration after 2-AAF/PHx in rats[END_REF] Real-time PCR analysis demonstrated that hepatic mRNA expression of this profibrogenic factor was significantly higher in AAF/CCl 4 group (p< 0.05) than in the group which received CCl 4 alone (Figure 6B), in accordance with fibrosis extension. Unexpectedly, Chobert MN et al TGF mRNA induction was higher in AAF than in CCl 4 group (p< 0.001), notably after 2 and 4 weeks of treatment corresponding to the early stages of LPC activation. An increased TGF expression in both groups treated with AAF suggests that LPC are themselves a source of TGF, a result that has already been reported. [START_REF] Knight | Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell[END_REF][START_REF] Braun | Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation[END_REF] In situ hybridization studies were performed to identify more precisely the TGFproducing cells (Figure 6C). In rats treated with AAF alone for 6 weeks, numerous positive-cells for TGF mRNA expression were arranged as duct-like structures that deeply invaded the liver from portal areas. In this group without SMA-positive myofibroblasts (Figure 6A), TGF expression was noted in cells previously identified as LPC by CK19 staining (Figure 2A) and AFP expression (Figure 2C). In cirrhotic liver of AAF/CCl 4 -treated animals, TGF mRNA expression was detected in perinodular septa, within cells of the ductular reaction (Figure 6C); however, it cannot be excluded that part of the signal could also be attributed to SMA-positive myofibroblasts in close contact with LPC (Figure 5 and6A).

These results fit with the induction of TGF mRNA expression (Figure 6B) and the number of activated HSC and/or LPC that emerge in the liver after each treatment. Thus, the 5-fold induction of TGF mRNA after 6 weeks of treatment in the CCl 4 and AAF groups can be accounted by expression in HSC and LPC, respectively. The further induction up to 13 times in group AAF/CCl 4 can be related to TGF expression in both LPC and HSC, as a consequence of AAF-induced LPC expansion and CCl 4 -induced myofibroblastic activation; all these data reveal contribution of LPC to the development of liver fibrosis.

To get better insight into TGF expression by LPC, we performed double-immunofluorescent detection of CK19 (red) and TGF (green) in liver sections from AAF/CCl 4 -treated rats. By confocal analysis, there was a yellow signal in merged analysis, revealing TGF staining in CK19-positive cells forming ductular structures (Figure 6D). This result was also confirmed on consecutive liver frozen sections labelled with fluorescent CK19 and in situ hybridization for TGF mRNA (Supplementary Figure S2). Indeed, we noted a clear superimposition of TGF and CK19 signals in serial sections, predominantly in the portal ductular reaction and in septa expanding into the lobules, both areas containing a large population of LPC in AAF-treated animals. DAPI labeling also revealed that TGF mRNA was largely confined to small

Chobert MN et al cells with ovoid nuclei, characteristic of LPC. Altogether, these results might strongly indicate that TGF was expressed by LPC.

Epithelial-mesenchymal transition of LPC

To determine whether LPC undergo an epithelial-to-mesenchymal transition (EMT) in cirrhotic liver, we studied these CK19-positive cells for the expression of mesenchymal markers in AAF/CCl 4 group after 6 weeks of treatment. Double immunofluorescent experiments did not show any expression of SMA in CK19-positive LPC (no merging signals), neither in the ductular reaction arising in portal areas nor in fibrous septa (Figure 7A). Thus, the SMA-positive signal found in close contact of LPC corresponded to labeling of myofibroblastic HSC already described in the ductular reaction. [START_REF] Paku | 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies[END_REF][START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF][START_REF] Knight | Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice[END_REF][START_REF] Van | Expression of matrix metalloproteinase-2 and -9 and of tissue inhibitor of matrix metalloproteinase-1 in liver regeneration from oval cells in rat[END_REF] Desmin staining, which revealed HSC in contact with LPC, extended beyond SMA-labeled cells (Figure 7A), indicating that HSC detected in the ductular reaction are not fully activated into myofibroblastic cells. These results were confirmed by confocal analysis, which clearly showed the absence of SMA and desmin localization in CK19-labeled cells (Figure 7B). Additionally, expression of the fibroblast specific protein FSP1, which was also revealed around CK19-positive LPC (Figure 7A), did not co-stain in the same cells. Lack of colocalization of CK19 and collagen 1 (Figure 7A) suggests that the primary cells responsible for liver fibrogenesis are not LPC but activated HSC surrounding these cells. Therefore, we did not obtain any evidence showing that LPC might undergo EMT in AAF/CCl 4 -treated rats.

Discussion

Hepatic fibrosis is the liver response to chronic liver injury. It is characterized by excessive matrix deposition and accumulation of LPC, which are usually initiated in periportal areas, whatever is the location of the damage in the lobule. [START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF][START_REF] Clouston | Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis[END_REF]14 Classical models of fibrosis in rodents are based on chronic CCl 4 intoxication leading to hepatocyte necrosis and matrix accumulation, which appeared initially around the central veins, without any LPC proliferation. [START_REF] Zhao | Newly proposed fibrosis staging criterion for assessing carbon tetrachloride-and albumin complex-induced liver fibrosis in rodents[END_REF] AAF administration associated with single CCl 4 injection or partial hepatectomy is a classical experimental model to study LPC-dependent regeneration in rat. [START_REF] Petersen | Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats[END_REF] In this study, we developed a new experimental model, which recapitulates the Chobert MN et al events commonly observed in human fibrosis and is suitable to investigate the role of LPC in fibrogenesis. Indeed, the severity of liver fibrosis has been correlated to LPC expansion in human [START_REF] Lowes | Oval cell numbers in human chronic liver diseases are directly related to disease severity[END_REF][START_REF] Clouston | Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis[END_REF][14][START_REF] Roskams | Oxidative stress and oval cell accumulation in mice and humans with alcoholic and nonalcoholic fatty liver disease[END_REF] and recently, unexpected activation of LPC compartment in Egr1 -/-mice enhanced hepatic fibrosis after chronic CCl 4 exposure. [START_REF] Pritchard | Hepatic fibrosis is enhanced and accompanied by robust oval cell activation after chronic carbon tetrachloride administration to Egr-1-deficient mice[END_REF] To achieve that goal, we combined an AAF treatment, known to induce regeneration from LPC, [START_REF] Paku | Origin and structural evolution of the early proliferating oval cells in rat liver[END_REF][START_REF] Petersen | Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats[END_REF] with repeated CCl 4 injections, known to induce liver fibrosis in rodents. Activation of LPC is a complex process that is not elucidated yet. It involves secretion of cytokines such as TNF that activates the entry of quiescent stem cell in the cell cycle in a context where liver regeneration from hepatocytes is severely impaired as reported in AAF/CCl 4 treated rats. [START_REF] Knight | Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice[END_REF] Other cytokines produced by immune cells in response to liver injury such as interferon- have mitogenic effects on LPC and anti-proliferative effects on hepatocytes. [START_REF] Viebahn | What fires prometheus? The link between inflammation and regeneration following chronic liver injury[END_REF] Interestingly, hedgehog (Hh) ligands released from dying hepatocytes can promote LPC expansion and the resistance of LPC to the antiproliferative response of TGF They are probably other players of the LPC response in injured liver and by favouring activation of HSC or portal fibroblasts into myofibroblasts, [START_REF] Nguyen | Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation[END_REF][START_REF] Omenetti | Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation[END_REF][START_REF] Jung | Signals from dying hepatocytes trigger growth of liver progenitors[END_REF] may also promote cirrhosis in AAF/CCl 4 group. Numerous studies have reported paracrine interactions between LPC and HSC or portal fibroblasts located in close proximity. [START_REF] Paku | Origin and structural evolution of the early proliferating oval cells in rat liver[END_REF][START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF]14,[START_REF] Knight | Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice[END_REF][START_REF] Evarts | Cellular and molecular changes in the early stages of chemical hepatocarcinogenesis in the rat[END_REF][START_REF] Yovchev | Identification of adult hepatic progenitor cells capable of repopulating injured rat liver[END_REF] Activated HSC [START_REF] Akhurst | Differential lymphotoxin-beta and interferon gamma signaling during mouse liver regeneration induced by chronic and acute injury[END_REF]49 and portal fibroblasts [START_REF] Omenetti | Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation[END_REF] are a major source of growth factors for LPC and recent data established that myofibroblasts are required for LPC response [START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF][START_REF] Pintilie | Hepatic stellate cells' involvement in progenitor-mediated liver regeneration[END_REF] and are a component of the LPC niche. [START_REF] Lorenzini | Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver[END_REF] Conversely, it has not been clearly shown whether LPC could cause HSC or portal fibroblast activation. The SMA staining, the most widely used technique that reveals myofibroblasts, could not distinguish in our study between both types of cells. In the present study, we demonstrated that LPC express TGF, the most potent activator of quiescent HSC [START_REF] Friedman | Mechanisms of hepatic fibrogenesis[END_REF] and portal fibroblasts [START_REF] Li | Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture[END_REF] into myofibroblasts, therefore contributing to hepatic fibrogenesis. The fact that TGF was also detected in LPC from rats treated with AAF alone in the absence of SMA-positive cells close to LPC, points out that LPC and TGF alone are not sufficient to induce activation of HSC or of portal fibroblasts. In the group AAF, we observed an enhanced hepatic expression of collagen 4 and an accumulation of laminin (data not shown), two components of the Chobert MN et al basement membrane secreted by quiescent HSC in contact with LPC, [START_REF] Paku | 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies[END_REF][START_REF] Lorenzini | Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver[END_REF] which may account for the faint picrosirius red labeling around portal areas. In summary, these data suggest that LPC alone are not fibrogenic when there is no hepatocellular damage, and they emphasize the need of CCl 4 -induced injury to promote HSC activation and subsequent fibrosis. In other words, in addition to TGF secreted by LPC, the release of numerous factors from dying hepatocytes seems necessary to trigger the further myofibroblastic activation of HSC and the subsequent fibrogenic response.

The ability of LPC to transform into myofibroblasts through EMT is an interesting issue. [START_REF] Sicklick | Evidence for epithelial-mesenchymal transitions in adult liver cells[END_REF][START_REF] Sackett | Foxl1 is a marker of bipotential hepatic progenitor cells in mice[END_REF][START_REF] Wang | Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?[END_REF] The role of EMT in fibrosis was established in lung and kidney and, in the liver, transition of hepatocytes, biliary cells and HSC has also been already reported. [START_REF] Kaimori | Transforming growth factor-beta1 induces an epithelial-tomesenchymal transition state in mouse hepatocytes in vitro[END_REF][START_REF] Zeisberg | Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition[END_REF][START_REF] Robertson | Biliary epithelial-mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis[END_REF][25][START_REF] Battaglia | Liver cancer-derived hepatitis C virus core proteins shift TGF-Beta responses from tumor suppression to epithelial-mesenchymal transition[END_REF] TGF which we found secreted by LPC, is a wellknown EMT inducer [START_REF] Kaimori | Transforming growth factor-beta1 induces an epithelial-tomesenchymal transition state in mouse hepatocytes in vitro[END_REF][START_REF] Zeisberg | Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition[END_REF]25 that could trigger LPC transition by an autocrine/paracrine pathway and provide a new potential mechanism of fibrosis. However, we could not demonstrate the expression of mesenchymal markers (i.e., SMA, desmin and FSP1) in CK19-positive LPC cells from AAF/CCl 4treated rats in vivo. Transition of LPC organized in ductular structures into isolated myofibroblasts could be difficult to identify by colocalization of epithelial and mesenchymal markers, particularly if the loss of epithelial characteristics occurs early during EMT process. For these reasons, proof of LPC undergoing EMT in vivo needs a genetic fate mapping approach to track the fate of LPC during liver injury.

Nevertheless, transition of LPC to myofibroblasts after treatment with TGF has indeed been previously obtained in vitro, using LPC isolated from rats fed a choline-deficient diet supplemented with ethionine. [START_REF] Wang | Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?[END_REF] In a search of the capability of LPC to undergo EMT into HSC, we cultured human HepaRG liver progenitor cells [START_REF] Parent | Origin and characterization of a human bipotent liver progenitor cell line[END_REF] and followed their epithelial and fibroblastic phenotypic characteristics in response to TGF. HepaRG underwent a phenotypic change from epithelial to mesenchymal-like cells in vitro on the basis of an increased expression of snail, SMA and collagens and a paralleled decrease of E-cadherin (unpublished data) as observed by Wang et al on rat LPC. [START_REF] Wang | Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?[END_REF] Demonstration of EMT in vivo is at the heart of a controversy and some investigators consider that EMT observed in vitro could be an artefact of cell culture. [START_REF] Taura | Hepatocytes do not undergo epithelial-mesenchymal transition in liver fibrosis in mice[END_REF][START_REF] Duffield | Epithelial to mesenchymal transition in injury of solid organs: fact or artifact?[END_REF] Although isolated and cultured LPC possess some mesenchymal markers of HSC, such as collagen 1 and 3 [START_REF] Wang | Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?[END_REF] and SMA [START_REF] Yovchev | Identification of adult hepatic progenitor cells capable of repopulating injured rat liver[END_REF][START_REF] Sicklick | Evidence for epithelial-mesenchymal transitions in adult liver cells[END_REF] , an elegant fate-mapping study using the LPC marker Foxl1 demonstrated that all Foxl1-expressing cells and their progeny did not acquire a mesenchymal phenotype Chobert MN et al during LPC expansion in vivo. [START_REF] Sackett | Foxl1 is a marker of bipotential hepatic progenitor cells in mice[END_REF] These Foxl1-positive cells are surrounded by fibroblastic cells expressing elastin, desmin and SMA, which reveals a potential crosstalk between LPC and these fibrogenic cells. In accordance with these data, we show that LPC neither acquire a mesenchymal phenotype through EMT in vivo nor directly participate to excessive ECM deposition. It remains that CK19-negative cells expressing mesenchymal markers were detected in close proximity to LPC, confirming that LPC are accompanied by SMA-positive cells that derive from HSC or periportal fibroblasts. [START_REF] Svegliati-Baroni | Hepatic fibrogenesis in response to chronic liver injury: novel insights on the role of cell-to-cell interaction and transition[END_REF][START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF][START_REF] Knight | Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice[END_REF] Other evidence suggests that HSC could differentiate into LPC with an intermediate phenotype, [START_REF] Sicklick | Evidence for epithelial-mesenchymal transitions in adult liver cells[END_REF][START_REF] Yang | Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers[END_REF] adding further complexity to the mechanisms by which HSC or LPC contribute to liver fibrogenesis. Nevertheless, mesenchymal to epithelial transition of HSC could not be demonstrated in liver fibrosis induced by bile duct ligation or CCl 4 administration. [START_REF] Scholten | Genetic labeling does not detect epithelial-tomesenchymal transition of cholangiocytes in liver fibrosis in mice[END_REF] In summary, we show that LPC expansion aggravates liver fibrosis. This strong fibrogenic response does not appear to be the direct consequence of hepatocellular lesions or inflammatory response, but seems to be due to the accumulation of TGF-producing LPC, thus driving the myofibroblastic transformation of fibroblasts and/or HSC in injured liver. Fibrogenic properties of periportal LPC provide an explanation for the development of predominant periportal fibrosis, whatever is the primary location of hepatocellular damage, leading to porto-central fibrous septa progressing to cirrhosis. Proliferation of these cells with small ovoid nuclei in periportal areas was also observed in AAF-treated rats (n=4). PT denotes portal tracts. Supplementary

  Primary antibodies were mouse monoclonal anti-cytokeratin 19 (CK19) (1:150, Novocastra laboratories), anti-alpha-smooth muscle actin (SMA) (1:1000, Sigma-Aldrich), anti-desmin (1:200, DakoCytomotion), anti-CD68 (1:150, Serotec) and rabbit polyclonal anti-collagen 1 (1:200, Serotec) or anti-TGF (1:50 Santa Cruz). Fluorescent labeling of CK19 and CD68 was achieved on 6 µm frozen sections using secondary goat antimouse IgG Alexa fluor 488 and 555 respectively (1:1000), after incubation with the Image-iT TM FX signal enhancer to reduce the background (Invitrogen). Computer-assisted quantitation of the relative surface Chobert MN et al stained by CD68 on liver section at magnification x100 was performed with ImageJ (NIH).

Combined AAF and CCl 4

 4 treatment led to extensive liver fibrosis and cirrhosis with marked LPC expansion, whereas CCl 4 intoxication alone caused moderate fibrosis without expansion of LPC compartment. Unexpectedly, chronic and discontinuous AAF treatment by itself induced also an LPC expansion in absence of an external mitogenic stimulus. Actually, low AAF doses generated low toxicity as revealed by slight induction of ALAT, but liver injury was accompanied by an increase of apoptosis. This is in line with the proposal that AAF metabolites are able to uncouple mitochondrial respiratory chain and open the permeability pore leading to apoptosis.[START_REF] Neumann | Aromatic amines in experimental cancer research: tissue-specific effects, an old problem and new solutions[END_REF] As a consequence, injured hepatocytes eliminated by apoptosis might provide a sufficient signal to stimulate the LPC compartment while normal hepatocytes cannot proliferate due to AAF toxicity. In all AAF/CCl 4 -treated rats, accumulation of LPC in fibrous septa was associated with enhanced activation of desmin-positive HSC and expression of profibrotic genes such as collagen 1 and 3, leading to cirrhosis. As recently reported,[START_REF] Van Hul | Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury[END_REF] SMA-positive cells were first detected in close association with LPC expansion in the portal region of the lobule at week 2. Activated HSC were observed later in necrotic centrilobular areas and in porto-central bridging fibrosis at week 6. In contrast, very few SMA-positive cells were detected in the liver of animals treated with CCl 4 only, probably because of the low dose of CCl 4 used in our experimental conditions, which represented only 50% of the amount generally used in fibrogenic protocols. Since paracrine and autocrine secretion of TGF is the main factor involved in matrix deposition by activated HSC, 8 reduced fibrosis in liver from CCl 4 -treated rats is in accordance with low level of TGF mRNA. Resident and recruited macrophages also play a critical role in myofibroblastic activation of HSC, notably by releasing factors including TGF.[START_REF] Ramadori | Portal tract fibrogenesis in the liver[END_REF] However, the increased fibrogenic process in AAF/CCl 4 -treated rats did not essentially depend on the inflammatory response, which remained moderate in our experimental setting and similar in both CCl 4 and AAF/CCl 4 groups, as Chobert MN et al assessed by CD68 labeling, TNF and CCR2 mRNA levels. To investigate the underlying role of LPC on fibrosis, it was important to exclude a distinct effect in liver damage of combined AAF/CCl 4 treatment from CCl 4 alone. A similar level of liver injury was achieved in both groups, as shown by similar serum ALT and similar apoptosis in liver extracts at any experimental time point. This probably excludes additional AAF-induced toxicity as a complicating factor in AAF/CCl 4 -treated animals. Thus, defective HSC activation into SMA-positive cells and reduced fibrosis in CCl 4 -treated rats seemed to be related to the lack of LPC compartment expansion.

Figure 1 :

 1 Figure 1 : Liver injury and histopathology. A) Serum ALAT activity was increased to a level which was

Figure 2 :Figure 3 :

 23 Figure 2 : LPC compartment activation. A) Representative immunofluorescent detection of CK19-

Figure 4 :

 4 Figure 4 : Macrophage recruitment and inflammation. A) CD68 immunofluorescent detection (original

Figure 5 :

 5 Figure 5 : HSC or portal fibroblast activation. A) Immunohistochemical detection of SMA-positive

Figure 6 :

 6 Figure 6 : Detection of fibrogenic liver cells. A) Immunofluorescent detection of SMA and desmin after 6 weeks of treatment confirms activation of HSC into SMA-expressing myofibroblasts in cirrhotic liver of AAF/CCl 4 -treated rats. Very few SMA-positive HSC were noted in fibrous septa after CCl 4

Figure 7 :

 7 Figure 7: Immunolocalization of mesenchymal proteins on CK19-positive LPC from AAF/CCl 4 -treated

Figure S1 :

 S1 Figure S1: Representative photomicrographs (original magnification x200) on consecutive frozen liver

Figure S2 :

 S2 Figure S2: Representative photomicrographs (original magnification x200) on consecutive frozen liver

  At week 4, we observed gradual formation of fibrous septa interconnecting centrilobular areas, without disorganization of the lobular architecture. After 6 weeks, multiple centro-central fibrotic septa incompletely compassed and divided hepatic lobules. Unexpectedly, in AAF-treated animals after 4

4 , fibrosis was first demonstrated around centrilobular venules, with deposition of thin collagen fibers in necrotic and steatotic areas (data not Chobert MN et al shown).

Acknowledgement

This work was supported by INSERM, University Paris-Est Creteil and Agence Nationale pour la Recherche (ANR-06-Physio-022-2). We are grateful for X. Decrouy from Platform for cell imaging (IMRB, Creteil) for technical support in confocal microscope.

Conflict of interest

The authors declare no conflict of interest.

Supplementary information is available at Laboratory Investigation's website