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PPARβ/δ Priming Enhances the Anti-Apoptotic and Therapeutic Properties of Mesenchymal Stromal Cells in Myocardial Ischemia-Reperfusion Injury


 Background: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARβ/δ (Peroxisome proliferator-activated receptors β/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARβ/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARβ/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. Objectives: The aim of this study was to investigate the role of PPARβ/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction.Methods and results: Naïve MSC and MSC pharmacologically activated or inhibited for PPARβ/δ were challenged with H202. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARβ/δ agonist GW0742 versus naïve MSC. In addition, PPARβ/δ-priming allowed to reveal the anti-apoptotic effect of MSC on co-cultured cardiomyocytes. When injected during reperfusion in an ex vivo heart model of myocardial infarction, PPARβ/δ-primed MSC at a dose of 3.75x105 MSC/heart provided the same cardioprotective efficiency than 7.5x105 naïve MSC, identified as the optimal dose in our model. These enhanced short-term cardioprotective effects were associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 hour of reperfusion. By contrast, inhibition of PPARβ/δ before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. Conclusion: Altogether these results revealed that PPARβ/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARβ/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARβ/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the injected doses. These results could be of major interest to improve MSC e cacy for the cardioprotection of injured myocardium in AMI patients.

Background
Myocardial reperfusion by revascularization of the culprit artery is the only treatment for AMI patients 1 . However, there is no treatment to speci cally prevent irreversible ischemia-reperfusion (IR) injury, considered as the side effects of reperfusion mainly due to apoptosis and triggered by abrupt oxygen restoration in the ischemic tissue. Apoptosis is a highly regulated energy-dependent form of cell death of potential interest for therapeutic intervention because observed in ischemic disease both in animal models and in patients [2][3][4][5] . This regulated mechanism of cell death is speci c to the reperfusion phase.
Mesenchymal Stromal Cell (MSC)-based therapy has been reported to improve the post-MI functional recovery of the myocardium via a myriad of pathways that include the increase of endogenous cell survival, proliferation and angiogenesis. In addition, MSC potently repress in ammation and apoptosis through their plasticity and their capacity to release bioactive molecules and transfer organelles such as mitochondria and extracellular vesicles (EV) 17,18 . Based on these properties, MSC have been tested in several preclinical studies with promising results. Indeed, in preclinical studies of AMI, MSC injection improves tissue repair and cardiac function 19 , regulates the in ammatory response, decreases cell apoptosis 20 and reduces mortality in animals 21 . In clinical trials, although the safety and e cacy of MSC in phase I and II have been demonstrated, inconsistencies in phase III trials to evidence their cardioprotective properties have been reported [22][23][24] . These inconsistencies have been, in part, attributed to the poor in vivo survival rate and engraftment of MSC but also to the source of MSC used and the deleterious effects of the in vitro ampli cation process of MSC obtained from patients 24 . Therefore, to optimize MSC therapeutic potential and bring them into the routine clinical practice, the enhancement of MSC survival rate and engraftment in the damaged tissue as well as their anti-apoptotic properties on cardiac cells appears as a promising approach.
Cardiomyocytes are one of the main and most abundant cardiac cell types in the heart allowing contractile and pacemaker properties. During IR injury, despite their higher tolerance to ischemic injury in close relation to their anaerobic metabolism, this cell type undergoes death through in ammation, oxidative stress and calcium overload 25,26 . Cardiomyocytes seem to be particularly vulnerable during reperfusion, and undergo apoptosis within few minutes of reperfusion in isolated myocardial tissue 27 . Approaches to improve MSC-based anti-apoptotic effects on cardiomyocytes have shown promising results both in vitro and in vivo [28][29][30][31] . However, these properties need to be further enhanced to optimally protect the heart from the massive apoptotic burst occurring during ischemia-reperfusion injury. Moreover, MSCs do not survive long after transplantation into injured organs such as ischemic hearts. Indeed, within 24 hours after systemic injection, only 3% of MSC are found in the marginal zones of the infarct myocardium, and less than 1% of MSC survive for more than a week 32 . Therefore, to signi cantly improve MSC-based therapy, the enhancement of both MSC anti-apoptotic properties and MSC survival abilities should be considered.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that exist in three different isoforms: PPARα, PPARβ/δ and PPARγ. They heterodimerize with RXR (retinoid X receptor) and, after ligand binding, act as transcriptional regulators. Dependent on the tissue ligands and cofactors, PPAR isoforms exert multiple functions 33 . While PPARα is mainly expressed in brown adipose tissue, intestine, heart, liver, kidney, PPARγ is expressed in immune cells, gut, white and brown adipose tissue. PPARβ/δ, ubiquitously expressed, is a proangiogenic member in vascular cell [34][35][36] , where it plays an anti-apoptotic role through an elevation of cAMP 37 . Later studies showed that the anti-apoptotic properties of PPARβ/δ in endothelial cells were mediated through the upregulation of 14-3-3 mRNA and protein levels 38,39 . In  line with these studies, the selective PPARβ/δ agonist, GW501516, was shown to protect the   cardiomyoblast cell line H9c2 from H 2 O 2 -induced cell death 40 . Although the anti-apoptotic effect of PPARβ/δ has been evidenced in cardiomyocytes, the modulation of PPARβ/δ expression, highly expressed by MSC 41 , on MSC anti-apoptotic and cardioprotective properties has never been investigated.
The present study investigates the role of PPARβ/δ on MSC (i) survival and anti-apoptotic functions on cardiomyocytes in vitro and (ii) engraftment and therapeutic potential in an experimental model of MI.

MSC culture
MSC isolation and ampli cation of murine MSC were carried out in conditions previously described 42 .
MSC were then seeded at a density of 0.5×10 6 cells per cm 2 in minimum essential medium (MEM)-α supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 ng/ml human basic broblast growth factor (bFGF). The phenotypic and differentiation potential of MSC were assessed as previously described 42 . Brie y, for the phenotypic characterization, MSC were incubated during 20 minutes with conjugated monoclonal antibodies from BD Biosciences (Le Pont de Claix, France) in PBS supplemented with 0.1% bovine serum albumin.
Regarding the differentiation of MSCs into adipocytes, osteoblasts and chondrocytes, we relied on the inductive conditions already reported 42 . Adipogenic differentiation of MSC was assessed by the analysis of lipid droplets formation after Oil red O staining and by RT-qPCR. MSC chondrogenic differentiation was evaluated by RT-qPCR and the osteogenic differentiation was measured by RT-qPCR and the mineralization of the extracellular matrix.
MSC pre-treatments with PPARβ/δ agonist and antagonists and labeling with CM-DiI.
MSCs were pre-incubated for 24 hours with either PPARβ/δ antagonist GSK0660 (0.1 and 1 µM) or PPARβ/δ agonist GW0742 (0.1 and 1 µM) before to be washed with PBS and used in co-cultures experiments or injected in the myocardium. When indicated, MSCs were labeled with the uorescent celltracer CM-DiI (Molecular Probes). MSC were collected and suspended in 5 mL PBS containing CM-DiI (10 7 cells/10 µg) prior to be incubated at 37°C for 5 minutes followed by 15 minutes at 4°C, in the dark.
Labeled MSCs were washed 2 times in PBS, resuspended in Tyrode solution and maintained at 4°C prior to be injected in the myocardium.

Cardiomyocyte and MSC co-cultures
H9c2 embryonic rat heart-derived (ventricular) cells (myoblasts) were cultured in Dulbecco's modi ed Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 Units/mL penicillin, and 100 µg/mL streptomycin. To induce apoptosis, H9c2 were cultured in a minimal medium (without FBS) containing 350 µM of H 2 O 2 during 4 hours. In order to evaluate the effect of MSCs on cardiomyocytes, a co-culture assay was designed using the transwell system to avoid cell-cell contact. 50,000 H9c2 cells per well (lower part) were plated on a 12-well dish with or 50,000 MSC (upper part) in presence of 350 µM of H 2 O 2 . H9c2 were exposed to H 2 O 2 in minimal medium during 4 hours and after a wash, cells were cocultured in complete medium for 1 hour with MSC. At the end of the protocol, H9c2 were recovered for apoptosis and RT-qPCR analysis.

DNA fragmentation assay
Speci c DNA fragmentation as a hallmark of apoptosis was quanti ed in vitro on cell lysates with an enzyme-linked immunosorbent assay kit (Cell Death ELISA; Roche Diagnostics) designed to measure histone-complexed DNA fragments (mono-and oligonucleosomes) released in the cytosol of cells induced for apoptosis. Data were normalized to the value by obtained in basal conditions of culture.

RT-qPCR
Isolation of total RNA for each sample was performed using the RNeasy Mini Kit (Qiagen, Courtaboeuf). The quantity and quality of the RNA extracted were de ned by using a NanoDrop ND-1000 spectrophotometer (NanoDrop ND, Thermo Fisher Scienti c). Then, using the SensiFAST cDNA synthesis kit (Bioline), cDNA was synthesized by reverse transcribing 500 ng RNA into cDNA. Using the SensiFAST™ SYBR (Bioline) and a LightCycler® 480 Detection system Quantitative following manufacturer's recommendations, PCR were performed. Primers speci c for bcl2 were designed using the Primer3 software. Data were normalized to the housekeeping gene ribosomal protein S9 (RPS9) and the values were expressed as relative mRNA level of speci c gene expression as calculated using the 2 −ΔCt method.

Animal experiments
All experiments were carried out on C57BL/6J mice (Charles River laboratories) in accordance with the European Communities Council directive of November 1986 and conformed to the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication 8th Edition, 2011).

Langendorff ex vivo studies
C57Bl6 male mice were anesthetized with a rst intramuscular injection of an anesthetic cocktail comprising ketamine (14 mg/kg, Imalgène®; Merial) and xylazine (14 mg/kg, Rompun®; Bayer), and by a second injection of pentobarbital (76.6 mg/kg; Sano -Aventis). After sternotomy, the heart was excised, quickly cannulated through the aorta and mounted on a homemade Langendorff system. It was retrogradely perfused at pressure-and temperature-constant (37°C) Tyrode solutions.

Ischemia-reperfusion protocols
On the Langendorff system, the heart was subjected to ischemia-reperfusion (IR) protocol comprising, after a 15 min-stabilization period of Tyrode perfusion, a 30 min-period of no-ow to induce global ischemia. Reperfusion was achieved by restoring the perfusion during 60 min with the Tyrode solution alone (IR control) or with MSC cell in Tyrode solution (MSC) administrated during reperfusion. Ischemic postconditioning was induced at the end of global ischemia by the application of a conditioning stimulus comprising 3 cycles of 1 min-reperfusion / 1 min-ischemia, followed by 54 min of reperfusion. For the ex vivo evaluation of MSC-based cell therapy, MSC cells prepared in the Tyrode solution at various concentrations (2,500; 5,000 and 10,000 cells/mL) were perfused in isolated hearts during the 60 minreperfusion phase (perfusion of ≈120 mL per heart).
Infarct size assessment LV were sliced transversally into 1-mm-thick sections and incubated in a 1% solution of 2,3,5triphenyltetrazolium chloride (TTC, Sigma-Aldrich) for 15 min at 37°C. After xation in a 4% paraformaldehyde-PBS solution, the slices were weighted and each side was photographed (Olympus camera). Infarcted area was measured by planimetry with ImageJ software (U. S. National Institutes of Health) and expressed as a percentage of the left ventricle.

Statistical analysis
Statistical analysis was performed only for n ≥ 5 independent experiments. Data (mean ± SD) were analyzed with ANOVA parametric test after con rmation of the normality of the population (Anderson-Darling Test for normal distribution). In case of non-conformity, nonparametric Kruskal-Wallis followed by Dunn's post hoc test for multiple comparison or Mann-Whitney for two groups were used. Data were analyzed with GraphPad Prism (version 8.3.0 for MacOs, GraphPad Software, San Diego, California USA, www.graphpad.com). Statistical signi cance was noted as ns for p> 0.05, * for p<0.05, ** for p<0.01, *** for p<0.001 and **** for p<0.0001.

Increased resistance of PPARβ/δ-primed MSC to H 2 O 2 -induced stress
Our previous study demonstrated in an ex vivo model of myocardial IR injury that MSC-induced cardioprotection was lost when PPARβ/δ expression in MSC was knocked out 43 . To determine the role of PPARβ/δ in MSC administered at reperfusion in an infarcted heart, we designed an in vitro protocol mimicking the stress received by MSC during transplantation and evaluated the properties of MSC pharmacologically activated or inhibited for PPARβ/δ. First, we phenotypically and functionally characterized MSC used for the study as previously described 42 . We showed that MSC were negative for CD11b and CD45 and positive for markers expressed on MSC such as CD44 and Sca-1 (Supplementary Figure 1A). After induction of MSC differentiation towards the three main mesenchymal lineages, MSC gave rise to: 1) chondrocytes as shown by the expression of collagen type II (Col2), 2) adipocytes characterized by the expression of pparγ and lpl and the formation of lipid droplets, and 3) osteoblasts characterized by the expression of osteocalcin (OC) and alkaline phosphatase (ALP) and Alizarin Red S staining (Supplementary Figure 1B).
Then, to determine the role of PPARβ/δ on MSC challenged by IR injury, cultured MSC were submitted to an IR-simulated stress in vitro using various concentrations of H 2 O 2 (100, 250, 350, 500 and 750 µM) for 4 hours (see the protocol on Figure 1A). Apoptosis quanti cation was assessed by the mean of speci c DNA fragmentation measurement for the different groups of treatment. We found that speci c DNA fragmentation was induced by H 2 O 2 as low as 100 µM with an equivalent rate for all concentrations from MSC pre-treatment with the selective PPARβ/δ agonist GW0742 improved signi cantly the capacity of MSC to resist against H 2 O 2 -induced stress at 0.1 µM (p****) and 1 µM (p****). By contrast, the pretreatment with the PPARβ/δ antagonist GSK0660 slightly protected the MSC against apoptosis at the concentration 0.1 µM (p*) but not at 1 µM (p ns vs H 2 O 2 group, Figure 1C). The enhanced resistance to apoptosis induced by PPARβ/δ agonist pre-treatment on MSC was con rmed by a signi cant increase in bcl2 gene expression level, in particular after a pre-treatment with GW0742 at 0.1 µM (p**). Conversely, the pre-treatment of MSC with 1 µM antagonist (GSK0660) signi cantly reduced bcl2 gene expression level compared to H 2 O 2 -treated MSC (p**; Figure 1D). In addition, we validated the effect of GW0742 PPARβ/δ agonist pre-treatment on cultured MSC and found that the agonistic effect of GW0742 was associated with increased expression levels of angpl4 target gene allowing to demonstrate the agonist functionality (Supplementary Figure 2). Alltogether, these results revealed that the activation of PPARβ/δ protects MSC from apoptosis induced by H 2 O 2 stress.
PPARβ/δ-priming increases the anti-apoptotic effect of MSC on cardiomyocytes exposed to an IRsimulated stress To determine the role of PPARβ/δ on the anti-apoptotic properties of MSC on stressed cardiomyocytes, we co-cultured in transwell sytems naïve MSC and MSC activated or inhibited for PPARβ/δ with the H9c2 cardiomyoblast cell line, considered as a suitable model to study cardiac ischemia-reperfusion injury 44 . The protocol consisted on exposing H9c2 cardiomyoblasts to 350 µM of H 2 O 2 for 4 hours prior to their co-culture for 1 hour with naive MSC or MSC modulated for PPARβ/δ (Figure 2A). We showed that MSC pre-treatment with 0.1 µM of PPARβ/δ agonist GW0742 allowed to reveal a signi cant protective effect in H9c2 cells against H 2 O 2-induced stress (MSC/0.1 µM GSK0742 vs untreated H9c2, n=8 per group; p**** <0.0001) that was not seen with naïve MSC (MSC vs untreated H9c2; n=8 per group; p ns =0.999 and MSC/0.1 µM GSK0742 vs MSC, n=8 per group; p**=0.0058; Figure 2B). This anti-apoptotic effect was also observed in MSC pre-treated with 1 µM GW0742 (MSC/1 µM GSK0742 vs untreated H9c2, n=8 per group; p**=0.0079). Pre-treatment of MSC with the GSK0660 did not change MSC protective properties compared to naïve MSC (MSC/0.1 µM and GSK0660 MSC/1 µM GSK0660 vs MSC, n=8 per group; p ns >0.999). These results show that PPARβ/δ activation revealed and enhanced MSC anti-apoptotic properties on cardiomyocytes exposed to an IR-simulated stress in vitro.
The cardioprotective effect of MSC administered during reperfusion in an ex vivo model of myocardial IR injury depends on PPARβ/δ In order to evaluate the protective effect of MSC in an ex vivo model of myocardial IR injury, MSC were administered during the reperfusion phase at various concentrations (2,500; 5,000 et 10,000 cells/mL in a Tyrode solution) in isolated hearts subjected to global ischemia (see protocol, Figure 3A). MSC showed a U-shaped dose-response curve with an optimal e cacy at 5,000 cells/mL which was similar to that provided by the PostC (ischemic postconditioning) stimulus, which was considered as the positive control in our experiment (30.27 ± 5.89, for MSC 5,000, n=12 vs 46.51 ± 6.28, n=11 for IR; p*** = 0.0004 and vs 28.13 ± 4.69, n=7 for PostC; p ns >0.999) ( Figure 3B). In addition, neither the 2,500 cells/mL dose (38.64 ± 5.36, n=11 vs 46.51 ± 6.28, n=11 for IR; p ns = 0.80) nor the largest one tested, 10,000 cells/mL (50.28 ± 11.09, n=6 for MSC 10,000 vs 46.51 ± 6.28, n=11 for IR; p ns >0.999) produced a signi cant decrease in infarct size compared to IR.
Since, we demonstrated in previous studies the pivotal role of PPARβ/δ expression on MSC functions including their immunoregulatory properties, we addressed here its role on MSC cardioprotective potential [45][46][47] . To that end, MSC were pre-treated with either PPARβ/δ agonist or antagonist prior to their administration in the ex vivo model of IR injury. According to this result, we investigated whether the use of GW0742 PPARβ/δ agonist at various doses (0.1 and 1 µM) was associated with an improvement of the cardioprotection afforded by MSC administered at the lower dose (2,500 cells/mL) that was not protective by itself ( Figure 3C). We showed that pre-treating MSC with 0.1 and 1 µM PPARβ/δ agonist induced their cardioprotective effect (46.51 ± 6.28, n=11 for IR vs 34.65± 5.36, n=8 for MSC/0.1 µM GW0742; p*=0.0309 and vs 25.77 ± 4.68, n=7 for MSC/1 µM GW0742; p****<0.0001). Pre-treating the cells with GW0472 (1 µM) before administration at 2,500 cells/mL allowed to induce the same cardioprotective effect as the one observed using 5,000 cells/mL naïve MSC (p ns >0.999; Figure 3D). Figure 3E shows that the cardioprotection was abolished when MSC (5,000 cells/mL) were pre-treated during 24 hours with GSK0660 (1 µM) prior to their administration during the reperfusion phase ( This improvement of the therapeutic effect of PPARβ/δ-primed MSC as compared to their naïve counterpart, was associated with an increased rate of MSC engraftment in the infarcted myocardial tissue as assessed by immunohistology experiments using DiI-labeled MSC ( Figure 3F).

PPARβ/δ-priming increases the cardioprotective effects of MSC ex vivo by decreasing caspase 3 activation in IR heart
The potent anti-apoptotic effect of the PPARβ/δ-primed MSC on cardiomyocytes was further con rmed on whole hearts. Western blot analysis in hearts subjected ex vivo to IR injury allowed to show that pretreating MSC during 24 hours with the GW0742 PPARβ/δ agonist (1 µM) allowed to reveal a signi cant inhibition of caspase 3 cleavage compared to the IR condition (p* = 0.0303 versus IR) that was not observed with naïve MSC (p ns = 0.4986 vs IR; Figure 4B). By contrast, the administration of MSC inhibited for PPARβ/δ in the ex vivo IR heart at reperfusion, did not reduce caspase 3 activation as compared to the IR control condition.
In addition, evaluation of the phosphorylation patterns of ERK1/2 and AKT pro-survival kinases showed a signi cantly increased phosphorylation ratio pAkt/Akt in MSC-treated versus non-treated IR hearts ( Figures 4C, D) when MSC were pre-treated with GW0742 PPARβ/δ agonist (1 µM) prior to their administration during reperfusion. By contrast, there was no change in the pERK1/2 /ERK1/2 ratio when naive MSC were administered in the post-ischemic phase of the IR protocol.

Discussion
Our study is the rst demonstration that the priming of murine MSC using an agonist of PPARβ/δ allows to protect them from apoptosis induced by an H 2 O 2 -induced stress and to enhance their anti-apoptotic and cardioprotective properties against myocardial IR injury ex vivo. First, we evidenced in vitro that PPARβ/δ priming allowed to increase MSC resistance against H 2 O 2 -induced oxidative stress as assessed by decreased apoptosis rate and by an increased amount of bcl2 transcripts. Then, we observed that PPARβ/δ-primed MSC had a more potent anti-apoptotic effect on cardiomyocytes than naïve MSC.
Interestingly, the priming of MSC with a PPARβ/δ agonist before their injection in an ex vivo model of myocardial IR injury allowed them to confer signi cant cardioprotective properties not observed with naïve MSC when evaluated in the same model. Importantly, PPARβ/δ-priming reduced by two the number of MSC administered to obtain the optimal e ciency in decreasing infarct size. This enhanced short-term cardioprotection, within 1 hour after reperfusion, was also associated with an increased number of MSC detected in the ventricular wall and also enhanced anti-apoptotic effects as assessed by western blot analysis of caspase 3 activation. Accordingly, the phosphorylation ratio of AKT pro-survival kinase was increased in IR hearts treated by PPARβ/δ-primed versus naive MSC. Of note, the cardioprotective effects of MSC were lost when pre-treated with a PPARβ/δ antagonist prior to their administration, con rming our previous results using MSC derived from PPARβ/δ knockout mice.
Reperfusion, even if considered as a double-edged sword, is the only treatment recommended for patients with acute myocardial infarction 48 . Despite obvious bene cial effects in limiting the ischemic insult, revascularization of the culprit artery leads to an abrupt return of oxygen in the threatened myocardial tissue and generates subsequent deleterious side effects. Lethal reperfusion injury corresponds to the death of cardiac cells viable at the end of the ischemic period occurring at the onset of reperfusion and mainly related to apoptotic death 49,50 . Regarding MSC-based therapy against myocardial IR injury, MSC undergo apoptosis in particular when they are transplanted in vivo in injured tissues, such as damaged hearts after IR injury 51 . Several strategies were developed to inhibit apoptosis of MSC after in vivo administration to help them survive hypoxia-reoxygenation injury, such as heat-shock treatment 52 or a hypoxia-regulated Heme oxygenase-1 (HO-1) vector modi cation 53 of MSC. Among the factors impacting negatively the therapeutic effect of MSC, the poor survival rate of transplanted cells is of major importance. So, MSC resistance to oxidative stress should be increased to improve their therapeutic e cacy against IR injury 54 . Here, we demonstrate for the rst time that PPARβ/δ-priming of MSC enhanced their capacity to be protected against apoptosis when they were subjected to an H 2 O 2 -induced stress revealing that PPARβ/δ-priming is able to increase MSC resistance to oxidative stress in vitro. These results are in line with the anti-apoptotic effects of PPARβ/δ stimulation described, in vitro, on several cell types including endothelial cells, H9C2 cardiomyoblasts, neurons or keratinocytes [38][39][40] . In endothelial cells, PPARβ/δ anti-apoptotic properties were associated to an increased protection against oxidative stress 38,39 . In the present study, RT-qPCR experiments have shown that MSC-increased resistance to H 2 O 2 -induced stress upon PPARβ/δ-priming i.e., stimulation of the receptor by an agonist, was associated with an increased amount of anti-apoptotic bcl2 gene transcripts. Other anti-apoptotic mechanisms have also been highlighted in other cell types following the activation of PPARβ/δ receptors, such as the activation of AKT1 pathway. Gamdzyk's team has shown in the context of neuronal stress that the activation of PPARβ/δ by its agonist GW0742 leads to the inhibition of the pro-apoptotic pathway ASK1/p38 MAPK 55 . In the context of PPARβ/δ MSC priming, the decreased rates of apoptosis after an H 2 O 2 -induced stress could be of major importance to explain the increase of their therapeutic properties. Indeed, we demonstrate for the rst time that PPARβ/δ-priming of MSC was associated with their enhanced anti-apoptotic potential both in vitro and ex vivo. H9C2, described as a suitable model to study cardiac ischemia-reperfusion injury in vitro 44 , were subjected to an H 2 O 2 -induced stressed prior to be co-cultured with MSC. Then, speci c DNA fragmentation was measured as a hallmark of apoptosis in H9C2 co-cultured with PPARβ/δ-primed or naïve MSC. Ex vivo, a strong pro-apoptotic stress was assessed by western blot analysis of cleaved caspase 3 expression in isolated hearts subjected to severe IR injury showing a 46.5%-mean infarct size (expressed as a % of the LV, SHAM, n=6 vs IR, n=10; p**=0.0047; data not shown). MSC-induced cardioprotection was associated with a decreased amount of cleaved (activated) caspase 3 measured in the left ventricle as already reported for MSC injected in the injured myocardium 56 . MSC priming with PPARβ/δ agonist exacerbated the anti-apoptotic properties, allowing in particular to unmask potent anti-apoptotic effects that were absent when using naïve MSC at the same dose. Targeting regulated cell death pathways, in particular apoptosis, after an ischemic event, appears as a main strategy to prevent reperfusion injury and to limit infarct size [7][8][9][10][11][12][13][14][15][16] . Anti-apoptotic strategies allow to inhibit speci cally reperfusion-induced apoptotic burst of cell death, providing longterm cardioprotective effects, a strategy evidenced recently by our group in the mouse heart 57 .
In terms of cardioprotection, MSC administered during the reperfusion phase provided a 35%-decrease in infarct size. Regarding their concentration, we observed a U-shaped curve clearly showing that MSCinduced cardioprotection is dose-dependent as reported in clinical trials. Indeed, in AMI patients, a recent study reported that only doses less than 10 7 MSC administered within 1 week after AMI enhanced the systolic function of left ventricle whereas administration of more than 10 7 cells had the opposite effect 58 . In agreement with this study, a U-shaped curve for MSC cardioprotective effects in the treatment of chronic advanced ischemic heart failure was also reported indicating reduced outcomes at the highest dose of cells 59 . The U-shape curve in our study allowed to identify the optimal dose providing a maximal cardioprotective effect (5,000 cells/mL administered during 60 minutes of reperfusion). The corresponding number of cells that was administered per heart was around 7.5x10 5 cells, which corresponds to a relatively low dose compared to other reports in the literature. The use of a higher dose of MSC induced the loss of cardioprotection. Interestingly, we observed also that MSC primed with a PPARβ/δ agonist allowed to decrease the dose of MSC injected in the heart by half. Indeed, in our study, the cardioprotective effect observed ex vivo using PPARβ/δ-primed MSC injected at a dose of 3.75x10 5 MSC was similar to that obtained with naive MSC administered at the optimal dose (7.5x10 5 MSC).
Inhibition of PPARβ/δ activity prior to their administration ex vivo in IR injured hearts provided the same results obtained in our previous study using MSC derived from PPARβ/δ knockout mice that failed to protect myocardial tissue against IR injury ex vivo 60 . By contrast, MSC priming with GW0742 PPARβ/δ agonist allowed to unmask and potentiate their cardioprotective effects. For the optimal dose of GW0742 agonist used to prime MSC, a shift from 0.1 to 1 µM was observed between in vitro and ex vivo results. It seems reasonable to speculate that this difference may be due to the fact, that the ex vivo system integrates several cell types including broblast, endothelial but also resident immune cells and that effect of MSC might differ depending on the cell type with which they interact.

Conclusions
Our study provides evidence for an enhanced MSC-based cell product inducing cardioprotection against IR injury with potent anti-apoptotic effects and an increased number of surviving MSC in the injured myocardium, one hour after the onset of reperfusion.
These results could be of major interest in the clinical setting to improve MSC e cacy for the cardioprotection of injured myocardium in AMI patients. Availability of data and material

Abbreviations
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare no competing interests.  Figure 1 Decreased apoptosis in PPARβ/δ-primed MSC exposed to H202 stress. (A) 48 hours after the seeding step in complete medium, MSC were subjected to H2O2-induced oxidative stress during 4 hours in minimal medium with serum deprivation. Pharmacological preconditioning of MSC using PPARβ/δ agonist or antagonist was performed 24 hours before oxidative stress exposure. At the end of the protocol, cell death or relative gene expression was analyzed measuring speci c DNA fragmentation by  Increased anti-apoptotic effects of PPAR β/δ-primed MSC on cardiomyocytes. (A) After 24 hours of MSC pharmacological preconditioning using PPARβ/δ agonist, cells were co-cultured in classical culture medium (CM) using transwells (upper chamber) with H9c2 (lower chamber) previously exposed to 350 µM of H2O2 in minimal medium (serum deprivation) during 4 hours. At the end of the protocol, speci c DNA fragmentation was quanti ed in H9c2 cells and values were compared with those obtained for co-