Verena Poinsot
email: verena.poinsot@inserm.fr

Extracellular vesicles of MSCs and cardiomyoblasts are vehicles for lipid mediators

Keywords: Extracellular vesicles, lipid mediator, inflammation, heart, PUFA 1

In many pathophysiological situations, inflammation is an ubiquitous process which can either be positive, or, on the contrary, represent a deleterious mechanism for the organ or tissue. Indeed, the inflammatory response is a prerequisite to initiate the reparatory processes, but intense and persistent inflammation causes tissue damages. The resolution of inflammation is considered to be an active process, influenced by factors released by the matrix and local cells. Considering the heart, myocardial damages involve acute (myocardial infarction) and/or chronic (heart failure) inflammatory phases which most of the time need to be resolved. Different cell types from the stromal and parenchymal compartments can play a role in these processes through the secretion of paracrine factors that modulate inflammation.

Concerning the stromal compartment, mesenchymal stem cells (also known as mesenchymal stromal cells or mesenchymal progenitor cells) are stromal cells that can be isolated from different adult tissues including bone marrow and adipose tissue. Belonging to the family of progenitor cells, they are implied in the renewal of different cell types (chondrocytes, osteocytes, adipocytes, fibroblasts, smooth muscle cells, endothelial cells). In addition, MSCs secrete a variety of paracrine factors, regulating angiogenesis, cell survival and inflammation. The role of the MSCs secretome in regulation of the immune response is particularly relevant. Indeed, MSC have been shown to regulate the activation and differentiation of many cells of the immune system, including T-cells, B-cells, NK cells, monocytes,

dendritic cells, and neutrophils [START_REF] Van Den Akker | Cardiac stem cell therapy to modulate inflammation upon myocardial infarction[END_REF]. The first evidence supporting the role of MSCs in regulation of cardiac homeostasis and repair has been supplied by the studies on cell therapy of heart failure. These studies showed that administration of Bone-Marrow Mesenchymal Stem Cell (BM-MSC) in failing hearts of animal models and humans promoted angiogenesis, decreased fibrosis and improved cardiac function. More recently, several studies showed the existence of cardiac MSC-like cells (cMSCs) playing a critical role in regulation of cardiac function through the cell renewal and the secretion of a variety of paracrine factors [START_REF] D'amario | Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies[END_REF] and in the chronic low grade inflammation associated with aging [START_REF] Martini | Aging induces cardiac mesenchymal stromal cell senescence and promotes endothelial cell fate of the CD90 + subset[END_REF].

Extracellular vesicles are small vesicles (30 to 150 nm), carried by Multi-Vesicular bodies (MVB) and secreted outside of the cell in the stroma after the membrane fusion of the MVBs [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF][START_REF] Blanc | New insights into the function of RabGTPases in the context of vesicular secretion[END_REF][START_REF] Yu | Role of endosomal RabGTPases in cytokinesis[END_REF]. Once secreted, these micro-vesicles can be internalized within neighbouring cells and deliver their molecular material.

Their envelope composition is specific (eg. enriched in lipid raft and tetraspanins), allowing their addressing and incorporation within the neighboring cells. It enables the parent-cell signals to without direct cell to cell contact [START_REF] Waldenström | Role of Extracellular vesicles in Myocardial Remodeling[END_REF].

The parent-cell information, packed into extracellular vesicles in the form of lipids, proteins and nucleic acids, is called "cargo". Extracellular vesicles derived from MSCs (MSC-Ex) have been used in regenerative medicine. MSC-Ex function is often explained by the constant transfer of miRNAs and cytokines [START_REF] Burrello | Stem cell-derived extracellular vesicles and immune modulation[END_REF]. The anti-inflammatory signals as well as the angiogenic signals carried by the extracellular vesicles remain to be fully identified.

Long chain polyunsaturated fatty acids (PUFAs) derivatives, known as lipid mediators or oxilipins, are among the most efficient factors promoting or resolving inflammation. Released from membrane phospholipids or supplied by diet (-linolenic acid), PUFAs are modified (cyclized, oxygenated) by different classes of enzymes. Cycloxygenases (COX) initiated the transformation of arachidonic acid AA (C20 4-6) into prostaglandins (PG) D, E, F or I or into thromboxane (TX) and Lipoxygenases transform them into leukotrienes (LT) or lipoxins (LX) [START_REF] Romano | Lipoxin and aspirin-triggered lipoxins[END_REF][START_REF] Rinaldo-Matthis | Structures and mechanisms of enzymes in the leukotriene cascade[END_REF][START_REF] Lehmann | Lipoxin and resolvin biosynthesis is dependent on 5-lipoxygenase activating protein[END_REF]. Elongases lengthen their chain to 22 carbon atoms, then the lipoxigenase oxidize them to other compounds such as resolvins (Rv) and maresins (Ms) [START_REF] Rajakariar | COX-2 in inflammation and resolution[END_REF][START_REF] Cn. Serhan | Resolution of inflammation: the beginning programs the end[END_REF] thus limiting the synthesis of pro-inflammatory compounds such as PGs or LT. The switch from pro-inflammatory lipid mediators (LT, PG, TX) to pro-resolvers (LX, Rv, Ms) is regulated by the very long chain fatty acids oxidizing enzymes. The LX produced leads to the arrest of the inflammatory response and the recruitment of resolutive macrophages [START_REF] Uderhardt | 12/15-Lipoxygenase during the regulation of inflammation, immunity, and selftolerance[END_REF]. Finally, the n-3 protectants (PD) derived from endogenous -3 docosapentaenoic (-3 DPA) initiated by the 15-LOXs play a role in the macrophage polarization [START_REF] Cn | Novel lipid mediators and resolution mechanisms in acute inflammation: to resolve or not?[END_REF][START_REF] Me | Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment[END_REF][START_REF] Dalli | The novel 13S,14S-epoxymaresin is converted by human macrophages to maresin 1(MaR1), inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage phenotype[END_REF] whose stromal cells are orchestrators [START_REF] Sk | Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm[END_REF][START_REF] Keating | Mesenchymal stromal cells: new directions[END_REF][START_REF] Blanc | Multipotent mesenchymal stromal cells and the innate immune system[END_REF][START_REF] Dj | Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the centre of early regulators of inflammation[END_REF]. These molecules could contribute to the beneficial effects of MSCs as the increase in the production of prostacyclin by MSCs has been shown to be more effective for facilitating perfusion recovery and to promote host-tissue regeneration than MSCs alone [START_REF] Deng | Prostacyclin-producing human mesenchymal cells target H19 lncRNA to augment endogenous progenitor function in hindlimb ischaemia[END_REF]. Beside, inhibition of the COX-2 mediated prostaglandin E2 synthesis enhanced cardiac reprogramming and subsequent silencing of inflammation and fibroblast program [START_REF] Muraoka | Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac Reprogramming[END_REF].

Recent studies supported the role of lipid-mediators in the heart failure [eg. [START_REF] Muraoka | Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac Reprogramming[END_REF][START_REF] Ozen | Inhibition of microsomal PGE synthase-1 reduces human vascular tone by increasing PGI2 : a safer alternative to COX-2 inhibition[END_REF][START_REF] Bosviel | DHA-derived oxilipins, neuroprostanes and protectins, differentially and dose-dependently modulate the inflammatory response in human macrophages: Putative mechanisms through PPAR activation[END_REF][START_REF] Roy | Nonenzymatic lipid mediators, neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid[END_REF]. Obesity induced heart failure starts with the accumulation of arachidonic acid (C20:4ω-6 or AA) [START_REF] Mourmoura | Body adiposity dictates different mechanisms of increased coronary reactivity related to improve in vivo cardiac function[END_REF] and promotes progression of heart failure [START_REF] Habbout | Postnatal overfeeding in rodents by litter size reduction induces major short-and long-term pathophysiological consequences[END_REF]. Thus partial substitution of ω6 PUFA by ω3 PUFA (C18:3ω-3 ALA), (C20:5ω-3 EPA) and (C22:6ω-3 DHA) in the diet reduces the proportion of AA in cardiac phospholipids [START_REF] Sergiel | Individual effects of dietary EPA and DHA on the functioning of the isolated working rat heart[END_REF] and protect the heart against several diseases [START_REF] Kr | Omega-3 polyunsaturated fatty acids improve endothelial function in humans at risk for atherosclerosis: a review[END_REF][START_REF] Demaison | Influence of the phospholipid n-6/n-3 polyunsaturated fatty acid ratio on the mitochondrial oxidative metabolism before and after myocardial ischemia[END_REF][START_REF] Pl | Dietary fish oil preserves cardiac function in the hypertrophied rat heart[END_REF].

To date, the presence of such lipid cargo in the extracellular vesicles and their role in organs/tissues function is almost unknown. This is probably due to the difficulty to analyse these extremely wellregulated signals within the tenuous extracellular vesicles. One single work has been reported very recently concerning the analysis of Resolvins and prostaglandins in MSC-extracellular vesicles [START_REF] Holopainen | Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells[END_REF].

Combined together, it appeared us interesting to know if extracellular vesicles of BM-MSCs and the cardiomyoblast cell line H9c2are putative vehicles for the inflammation lipid mediator's signalization.

H9c2, are particularly interesting as they present characteristics of proliferative neonatal cardiomyocytes [START_REF] Sj | The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro, In vitro Cell[END_REF]. In addition, these cells seem to be involved in the generation of the inflammation through the release of inflammatory cytokines (interleukin 1 and tumor necrosis factor ) in proinflammatory model [START_REF] Liang | A novel damage mechanism: Contribution of the interaction between necroptosis and ROS to high glucose-induced injury and inflammation in H9c2 cardiac cells[END_REF]. These cells may also modulate inflammation by releasing microRNAs (such as miR-146a) in extracellular vesicles which could attenuate inflammation through an inhibition of the expression of Il-1 and TNF-receptors [START_REF] Beg | Inflammation-associated microRNA changes in circulating extracellular vesicles of heart failure patients[END_REF]. Therefore, we studied and compared the lipid-mediators content of the extracellular vesicles produced by BM-MSCsand H9c2 cells supplemented with ω3-PUFA to investigate if the positive effect of fish oil diet on the heart function can be explained by such extracellular vectorization of lipid mediators.

MATERIAL AND METHODS:

Biological material

The experiments were carried out on three cell types: A commercial H9c2 cardiomyoblast line (ATCCR CRL-1446 ™, Sigma Aldrich), mesenchymal stem cells from adult Lewis rat bone marrow (BM-MSC) previously characterized by our group [START_REF] Mias | Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction[END_REF] and human BM-MSCs obtained from donors (Etablissement Français du sang, EFS).

Cell cultures

The cell cultures were carried out in a CO2 incubator thermostatically controlled at 37 °C with 5% CO2.Cardiomyoblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM, ATCCR) culture medium and MSCs in Alpha Modified Eagle's Medium (MEM-α no nucleosides). All media were supplemented with 10% foetal calf serum (FCS) and a mixture of antibiotics (100 U / ml of streptomycin and 100 U / ml of penicillin).

The number of apoptotic and necrotic BM-MSCs and H9C2 present in cell cultures after serum starvation and before ultracentrifugation was determined by flow cytometry with Annexin V labelling Kit (BD pharmingen). The number of apoptotic or necrotic cells was always below 5% (Supplementary figure 1). For collecting the vesicular fraction, the dishes were rinsed with 10 mL of PBS and the medium replaced with the same medium but deprived at 1% of ultra-centrifuged foetal serum out of 24 hours. The supernatant is collected over an additional 24 hours period after renewal of the medium.

Dynamic Light Scattering

The extracellular vesicles' size distributions were determined by Dynamic Light Scattering (DLS) using a Malvern Instruments Nano ZS, with a He-Ne laser (633 nm), at a scattering angle of 173 °. The analyses were performed at 25.0 ± 0.1°C. The deconvolution of the measured intensity autocorrelation function of the sample was realized with the CONTIN algorithm on Malvern Instruments software to obtain the distribution of diffusion coefficients (D). The apparent equivalent hydrodynamic diameter (dh) was then obtained using the Stokes-Einstein equation. Hydrodynamic diameter values were obtained from three different runs.

Cryo-SEM

Extracellular vesicles were observed through Scanning Electron Microscopy with freeze-fracture (CryoSEM) using an ESEM Quanta FEG250 (FEI), operating at 5 kV and about 10 -4 Pa. The samples were mounted on pin specimen holders and frozen in liquid nitrogen. They were fractured in a vacuum chamber at -140°C, then a 10 min sublimation at -90 °C was performed. It was followed by a platinum coating using an Ion-sputter JFC-1100 (JEOL). Finally, the specimen holder was transferred to the observation chamber kept at -140 °C under a flow of nitrogen gas.

RNA extraction and RT-PCR

The RNAs were extracted from H9C2 and rat BM-MSC cells using the RNA extraction kit (PromegaRelia prep RNA Cell Miniprep System) and quantified by spectrophotometry (NanoDrop2000). They were then reverse transcribed using the Reverse Transcription Kit (Applied Biosystems). A volume of RNA containing 1 μg of total RNA and 1 volume of RT Master Mix (RT buffer, dNTP (100mM), RT random primers, RNase inhibitor and MultiScribe RT were mixed and then quickly incubated on an appropriate thermal cycler according to manufacturer. Gene mRNA expression was normalized with respect to the housekeeping gene Rplp0.

Table 1 2.6. Sample preparation

The supernatants of the cell culture are centrifuged for 20 minutes at 10,000 g in order to remove the apoptotic bodies. The supernatants were then subjected to precipitation by ultracentrifugation (100,000g; 1h30, 4°C.) (Beckman and Coulter).

Cells were collected using a scraper in 1 mL PBS and transferred to haemolysis tubes. An aliquot of 50 μL was taken in order to carry out an assay of the cellular proteins.

Protein assay and immunodetection

Protein extraction of the samples was carried out using the RIPA lysis buffer containing phosphatase and protease inhibitors (composition: TrisHCl 50 mM; NaCl 150 mM; NP40 1%; Desoxycholic Acid 1%; SDS 0, 1%; EDTA 5 mM; NaF 20 mM). The lysis was carried out in ice, 5 μL of buffer were added to the samples, shaken with a vortex and finally incubated for 30 minutes at 4°C. The determination of total proteins was carried out by the BCA method (BiCinchoninic acid Assay) in a 96-well plate. A standard range was produced from a protein solution of BSA (bovine serum albumin) at 1 mg / ml (0; 62.5; 125; 250; 500; 1000 μg / ml). The samples were diluted 1/10 in PBS and then 10 μL were deposited in the wells in the presence of 200 μL of the BCA reagent. After incubation at 37 ° C for 30 minutes, the absorbance was measured at 570 nm using a spectrophotometer (TECAN). Immunodetection was realized using JESS Separation Capillary Cartridge 12-230 kDa with 1.2 µg of purified extracellular vesicles fraction. Alix protein detected with the rabbit anti Alix antibody (cell signalling 1/50) according to the manufacturer and total protein content was evaluated by protein normalization buffer. The signals generated were detected and analyzed by Compass software (Protein Simple).

Free-lipid extraction

Deuterium-labeled eicosanoids (LxA4-d5, LTB4-d4 and 5-HETE-d8) were mixed at a concentration of 400 ng/mL in MeOH and used at internal standard (IS) solution. The 1 mL cell and extracellular vesicles suspensions were placed in the presence of 10 μL of IS and the lipid extraction was carried out in glass haemolysis tubes with twice 500 μL of CHCl3. The samples were vortexed and then left to settle. The organic phases were collected, mixed and evaporated to dryness under nitrogen flow at 45 ° C and the samples stored in pill boxes with glass inserts in 20 μL of MeOH at -80 ° C, then subjected to spectrometric analysis by LC / MS-MS.

LC / MS-MS analysis

Mass spectrometric analyzes were performed by Metatoul facilities, according to LeFaouder et al. [START_REF] Faouder | LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites[END_REF].

Briefly, High performance liquid chromatography was performed using an Agilent 1290 Infinity. The analytical column was a ZorBAX SB-C18 column (2.1 mm, 50 mm, 1.8 μm) (Agilent Technologies) maintained at 40 °C. The mobile phases consisted of water, ACN and FA (75:25:0.1; v/v/v) (A) and ACN, FA (100:0.1, v/v) (B). The linear gradient was as follows: 0% B at 0 min, 85% B at 8.5 min, 100% B at 9.5 min, 100% B at 10.5 min and 0% B at 12 min. The flow rate was 0.35 mL/min the injection volume was 5 μL. The HPLC system was coupled on-line to an Agilent 6460 triple quadrupole MS (Agilent Technologies). Electrospray ionization (ESI) was performed in negative ion mode. Analyses were performed in Selected Reaction Monitoring detection mode (SRM). Peak detection, integration and quantitative analysis were done using Mass Hunter Quantitative analysis software (Agilent Technologies) by correlation with the IS response.

RESULTS

Characterization of secreted micro-and nano-vesicles.

The two cell types (BM-MSC and H9c2) were deprived each over 24h to induce the same stress than the ones described in the literature. Then, the supernatants were collected for 24 hours more. The apoptotic bodies (AB) were removed by a 10 000g centrifugation. The extracellular vesicles were collected after ultra-centrifugation at 100000g (1:30 hour), suspended in PBS and submitted to differential light scattering analyzes (DLS).

Figure 1 :

The two cell-supernatants produced very similar DLS signals, therefore we chose to present only the H9c2 results in figure 1 and 2 to gain in clarity. DLS measurements indicated the presence of three populations (figure 1). The first one around 500-1000 nm corresponds to non-spherical objects, probably residual ABs. The second one close to 100 nm correspond the signal of the targeted extracellular vesicles. Finally, particles around 30 nm could be a second population of smaller extracellular vesicles or aggregates of residual proteins. Without information on the refraction index of the nano-vesicles, the DLS-signal is given in intensity, not in in number of particles. It is well establish that Exosomes are impossible to isolate in pure grade, even if the purification and characterization guidelines are in constant amelioration [START_REF] Mathieu | Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication[END_REF][START_REF] Dk Jeppesen | Reassessment of Exosome Composition[END_REF] In order to confirm that the signal around 100 nm could be extracellular vesicles, and to discriminate between both hypotheses for the smallest population, Cryo-SEM experiments were performed. Again three populations of particles could be detected around 1000 nm, 100-200 nm and a smaller one around 50-70 nm. The biggest population (figure 2Aa), which corresponds to non-spherical objects, is most likely residual ABs which confirms our hypothesis. On the contrary, both smaller populations are composed of spherical vesicles, ranging for the biggest one from 100 to 300 nm (figure 2Ab), which could be attributed to micro-vesicles and the smallest population of nano-vesicles (figure 2Ac), around 60 nm, are most probably the expected extracellular vesicles.

To confirm the significant presence or exosomes within these nanometric spherical objects, Immunodetection of Alix, an exosome specific marker, was performed on these purified extracellular vesicles fractions (human and rat BM MSCs or H9C2 (Figure 2B).

Put together, the 100 000 g precipitated fraction is enriched exosomes, even if important residual proteins are clearly visible.

BM-MSC and H9c2 cells grownunder standard culture conditions secrete a wide oxilipins profile

To evaluate the implication of lipid mediators as paracrine signals, we first analyzed the lipid content of BM-MSC and H9c2. The two cell types were cultivated, scraped after 1 to 3 passages, and the lipids were extracted using Chloroform. We tested several solvents (hexane, ethyl acetate, methyl formate, dichloromethane), but chloroform was the most reproducible and the more efficient over the whole profile (Supplementary figure 2). The free-lipids were analyzed using SRM LC-MS/MS. The relative quantification was calculated by comparison with internal perdeuterated standards.

Bone marrow-MSC and cardiomyoblasts exhibit very different oxilipin profiles. The BM-MSCs produce high amount of prostaglandins derived from arachidonic acid indicating a strong COX activity. 13-and 9-HODE as well as 15-HETE were also detected as major compounds. In contrast, only a minor prostaglandins distribution has been observed in H9c2 cells. The oxilipins produced by them under normal culture conditions are almost HETEs and HDoHE. 5-HETE is the precursor of LTB4, but this strong pro-inflammatory compound could not be detected. Resolvins and maresins are reported as the most efficient pro-resolving mediators, but they were not detectable in the cells lipid extracts.

As the biological variation is important under the used culture conditions, around 10 repeats were necessary to obtain acceptable variations around the mean values. The global production of oxilipins was in the 4.5 ng/mg of cell protein range for the BM-MSC and close to 3ng/mg cell protein range for the H9c2 cells.

Figure 3

Extracellular vesicles are oxilipins transporters.

To explore the participation of lipid mediators in extracellular vesicles mediated cell communications, we measured their cargo. The oxilipin profiles of the vesicles deriving from the two types of parent cells are different from those of the cells. For example, even if the prostaglandin PGF2 was abundantly present in BM-MSCs, it was not detected in their extracellular vesicles. In contrast LXB4, weakly observable in cells, was largely represented in extracellular vesicles. Actually, LXB4 and 9-and 13-hydroxy-octadecadienoic acid (9-/13-HODE) principally derived from the dietary linoleic acid were the major products of BM-MSCs and H9C2 extracellular vesicles. In addition, we also analysed the presence of eicosanoids in the culture media depleted from extracellular vesicles. The oxilipin quantified in the medium were at least 100 times less abundant than in the vesicular-fractions. This points out that the analysed lipid mediators are specifically included in the extracellular vesicles.

The cells express genes encoding for lipoxygenase and CYP450

The principal routes of synthesis of lipoxins involve the action of a series of enzymeson arachidonic acid which then gets converted into 15-HpETE. Therefore, we examined the expression of lipoxygenase. Both cell types expressed Alox15, whereas Alox5 was not detected. Alox12 expression could be found only in BM-MSCs.

Lipid profiles of human BM-MSC cells resemble the rat BM-MSC profiles

We next investigated the lipid mediator content of human BM-MSC, cells extensively used for therapy purpose. Actually, the human BM-MSC had a comparable profile with rat BM-MSC, they also exhibited large cellular content of prostaglandins suggesting that tissue origin of cells determined the lipid profile independently of the animal species. Even if the extracellular vesicles derived from the human cells carry important amounts of PGF, again, a LXB4 enrichment is observed in the human cell-derived EVs. The free fatty acid concentration reported in the literature ranges from 450µM to 100 µM (eg. for humans [START_REF] Burla | MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines[END_REF]; for mice [START_REF] Awada | n-3 PUFA added to high-fat diets affect differently adiposity and inflammation when carried by phospholipids or triacylglycerols[END_REF]). The -3 fatty acids are considered as representing between 5 and 10% of the total fatty acids [START_REF] Awada | n-3 PUFA added to high-fat diets affect differently adiposity and inflammation when carried by phospholipids or triacylglycerols[END_REF][START_REF] Jk | Serum n-6 polyunsaturated fatty acids and risk of death: the KuopioIschaemic Heart Disease Risk Factor Study[END_REF]. Therefore, under normal diet, the circulating PUFA concentration is estimated to be in the 10-40 µM. Therefore, we decided to feed the cells with a PUFA concentration of 10µM. As the so called free fatty acids are circulating as complexes with lipid binding proteins, we combined the PUFA with bovine albumin (BSA).

Independently from the cell type, the supplementation of the culture media with 10µM BSA associated-PUFAs, increased the oxilipin production by a factor 10 to 100.

According to the lipid mediator biosynthetic cascade, EPA was mostly converted, in cell, into 18-HEPE.

More surprisingly, it activated also the COX pathway as PGE2 and PGD2 levels increased significantly.

DHA was converted as attempted in 17-and 14-HDoHE and, more surprisingly, also activated the production of 18-HEPE, but not the COX-pathway. Neither EPA nor DHA was further converted into E resolvins and D resolvins respectively. As expected, the supplementation with AA, an-6 fatty acid, supplied the production of prostaglandins PGE2 and PGD2 but also the formation of 15-HETE, an AA metabolite produced by the 15-LOX and /or the CYP450. Again, even if it was in a less measure compared to DHA, AA seems also to activate the production of 18-HEPE.

We never were able to observe robust amounts of Resolvins in all the cells types.

Even after AA supplementation, and a huge cellular increase, the extracellular vesicle cargo was poorly loaded with PGs. After AA or EPA addition, the extracellular vesicle contained an important load of 18-HEPE and the amount of LXB4 was maintained at the normal level (around 0.1 ng/mg cell proteins). Extracellular vesicles -as observed under standard nutrition-are efficient hydroxylated-lipid (HDoHE and HEPE) transporter, as the HEPE content in extracellular vesicles is higher compared to the one of the parent cells.

The PUFA response is dose dependent

The cells were grown in culture media supplemented with different concentrations of AA or DHA. The oxilipins profiles were measured in the extracellular vesicles fraction produced over 24h. For the DHA, the profiles are constant in composition, but differed in abundancy. At 1µM, the PUFAs demonstrated no activity and increase linearly between 10 and 50 µM. The situation differs for AA, as the prostaglandins are efficiently produced at 1µM. In contrast, the HETE evolve in the same manner than the ones produced after DHA supplementation. The lipid mediators observed at 1µg of PUFAs were similar in composition and concentration to the ones observed without PUFA supplementation.

Table 2

DISCUSSION

Omega-3 fatty acids are associated to the prevention of cardiovascular risks. Our knowledge of pathways and cells targeted by these molecules are limited. In this study, we provide an assessment of cellular and vesicular contents of oxilipins in basal conditions and after n-3 PUFA supplementation.

We observed that each cells models analyzed exhibited efficient transport of lipid mediators in their extracellular vesicles that may constitute the principal vehicles for inflammatory signalization mediated by oxylipin. BM-MSCs and cardiomyoblasts, two cell types associated with beneficial effect during myocardial injuries, showed in standard conditions, similar vesicular oxilipin profiles despite different cellular contents. Human and rat BM-MSCs had abundant cellular level of the prostaglandin PGE2, PGF2a and PGD2 derived from the arachidonic cascade. However, neither thromboxane nor lipoxins appear in quantity at beside prostaglandins while TXB is expressed in these cells.

Prostaglandins exert various effects on cardiovascular system through the activation of specific G protein coupled receptors. The PGE2-EP3 interaction triggers acute inflammation (vascular permeability, oedema, leukocyte infiltration) [START_REF] Donnini | Nitric Oxide and PGE-2 Cross-Talk in EGFR-Driven Epithelial Tumor Cells[END_REF][START_REF] Morimoto | Prostaglandin E2-EP3 signaling induces inflammatory swelling by mast cell activation[END_REF], whereas the PGE2-EP4 mediated cardioprotection during myocardial ischemia. Moreover, although all 3 prostaglandins were found in rat mesenchymal cells, their extracellular vesicles presented only PGE2 indicating a selective secretion.

Similarly, the extracellular vesicles produced by different cell-types share important amounts of LXB4 (as compare to the cells). Unfortunately, only few is published concerning the biological effect of LXB4.

H9C2 and MSCs contained also large amount of 9-and 13-HODE, these oxygenated polyunsaturated fatty acids are metabolite of linoleic acid formed by enzymatic or free radical-mediated oxygenation and mediate acute nociception and/or inflammatory hyperalgesia [START_REF] Shapiro | Beyond the classic eicosanoids: Peripherally-acting oxygenated metabolites of polyunsaturated fatty acids mediate pain associated with tissue injury and inflammation[END_REF]. It is noteworthy that 18-HEPE faintly observable in extracellular vesicles was tremendously increased after EPA and DHA supplementation. 18-HEPE has been shown to inhibit macrophage-mediated pro-inflammatory activation of cardiac fibroblasts and prevent cardiac pathology [START_REF] Endo | 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive cardiac remodeling[END_REF].

The incubation of MSCs with arachidonic acid enhanced the cell production in prostaglandins but no minor thromboxanes or leukotrienes amounts could be observed. Surprisingly, although these compounds were over-produced by the cell and efficiently transported by the extracellular vesicles, the eicosanoid profile was not deeply modified by AA supplementation. Indeed, PGD2 and PGE2 were increased by 1.5 fold and 3.2 fold respectively when arachidonic acid was introduced at 50 µM as compared to 1µM suggesting a saturation in transport and/or packaging processes.

In MSCs incubated with media enriched in EPA or DHA, the profile of the cells and the extracellular vesicles are in good accordance. Interestingly, resolvins could not be detected in significant amounts in cells, but few were found in extracellular vesicles following DHA addition. It is also note full that the EPA cascade mainly ended with the 18-HEPE and the DHA cascade with 17-or 14-HDoHE. This point out, that the 5-LOX (encoded by alox5) is not active and was confirmed by absence of alox 5 expression measured RT-PCR in both H9C2 and BM-MSCs. The resolvins obtained with high concentration of DHA may result from residual non enzymatic oxygenation. In the cardiovascular system, leukocytes represent the main source of 5-LOX.

The low abundance of all lipid mediators in the ultra-centrifugated culture media demonstrated their selective enrichment in extracellular vesicles. Indeed, the oxilipins characterized in the extracellular vesicle-fraction are two magnitude more concentrated than the ones analysed in the medium.

Moreover, it cannot be excluded that even in the medium, the few amounts of mediators reflect residual unprecipitated or collapsed extracellular vesicles. This points out that the lipid mediators are specifically included in the extracellular nanovesicles, making extracellular vesicles specific shuttles of cell communications.

Excepted from the very recent publication [START_REF] Holopainen | Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells[END_REF], no publication had reported on the composition of free fatty acids transported by extracellular vesicles (EV). Actually, several works have focused on one or the other of the mediators and in particular on prostaglandins (and prostacyclins) [START_REF] Subra | Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[END_REF] or leukotriene [START_REF] Esser | Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration[END_REF]. Only, rare publications consider a large panel of these compounds whom balance is at the origin of the activity.

Hoplainen et al. determined that after supplementation of hMSCs with polyunsaturated fatty acids these were incorporated into the main membrane phospholipid classes with different dynamics, with phosphatidylcholine serving as the first acceptor. They also demonstrated that the PUFA modifications were transferred into hMSC-EVs, which are known to mediate hMSC immunomodulation [eg. 50].

These membrane-incorporated PUFAs increased the production of downstream prostaglandin E2 and Resolvins (E2 and D6). The authors demonstrated that the production of LMs was enhanced by a highly pro-inflammatory stimulus, which an increase number of mediators, especially prostaglandins, while other stimulatory conditions had less a pronounced impact. In this work, we analyzed and compared the whole profile of lipid mediators after PUFA supplementation and compared the cells with their derived extracellular vesicles. Our results are globally in accordance with Hoplainen et al., but revealed that EPA and DHA are even more efficient to increase the production of lipid mediators as huge amounts of the intermediates 18-HEPE and 14-or 17-HDoHE are obtained. We demonstrated that the limited impact of these two PUFA on the resolving profiles was mostly linked to the low expression of the 5-LOX.We also demonstrated that the PUFA modifications were transferred into the EVs, but we demonstrated that this occurs in a specific manner, as the vesicular lipid mediator profile differed from the cell ones. To our Knowledge, it is the first report on lipoxins transport by extracellular vesicles. Moreover, we could measure that even in this class of compounds, a selection occurs as more LXB4 is found in the extracellular vesicles compared to the cells.

Finally, the focus of the literature is almost on BM-MSC, but we demonstrated here that the inflammatory signalization is also handled by other stromal cells. Therefore, it appears as very interesting to analyze the cooperative effect of the different members of the pathological microenvironment to increase our understanding on the benefic effect of the cell therapies.

CONCLUSION:

In this study, we analysed the lipid profile of Rat BM-MSC and cardiomyoblasts (H9c2) secretome. We demonstrated that these cells used as models for cell-therapy, have a specific lipid secretome included in their extracellular vesicles. At basal conditions, the EVs of both cell types were enriched in LXB4 and PGE2. After incubation of cells with the -3 and-6 PUFAs, EPA, DHA and AA respectively, the PUFAs derived metabolites were transferred into the EVs, in a specific manner, as the nanovesicles lipid mediator profile differed from the cell ones. Put together, these results indicate that extracellular vesicles are vehicles for lipid mediators and may open novel therapeutic opportunity to improve the benefit of cell therapy on post-infract.

FIGURE AND TABLE CAPTIONS

Figure 2 :

 2 Figure 2:

Figure 4 Figure 5 :

 45 Figure 4

Figure 6

 6 Figure 6

Figure 7 3. 6 .

 76 Figure 7

Figure 8

 8 Figure 8

Figure 1 :

 1 Figure 1: Top: Size distribution obtained from the intensity value with DLS analyzes of the extracellular vesicle fraction ofH9c2 cells. Bottom: Approximated Size distribution in numbers, integrating that small particles are less capable to scatter the light.

Figure 2 :

 2 Figure 2: Top: SEM images obtained after freeze fracture of the sample of extracellular vesicle fraction of H9c2 after 1:30h UC at 100 000g. Bottom: Immunodetection of Alix in purified extracellular vesicles fraction of human and rat BM-MSCs and H9c2 (left panel). Alix, one of the exosome specific marker, is present in important amounts in the exosomes. The right panel indicate the presence of a large protein distribution in the EV fraction.

Figure 3 :

 3 Figure 3: Fatty acid profile measured in BM-BMSCs and H9c2 cells corresponding to rat BM-MSCs (dark) and to H9c2 (light grey). The results are expressed as ng/mg cell protein, n =7 and 12 experimental replicates per group respectively.

Figure 4 :

 4 Figure 4: Comparison of the lipid mediator profiles obtained upon two different cultures of H9c2 cells After extraction of the isolated vesicles (pellets) and the 100 times freeze-dried concentrated medium (supernatant) obtained after ultracentrifugation.

Figure 5 :

 5 Figure 5: Oxilipins profile obtained in pg/mg cellular proteins for rat BM-MSC (dark) and cardiomyoblaste (H9c2) (grey) extracellular vesicles. The extracellular vesicles collected after the two step-centrifugation were directly extracted by chloroform and analyzed using SRM LC-MS/MS. The relative quantification occurs through comparison with internal perdeuterated standards.

Figure 6 :

 6 Figure 6: RT-PCR quantification of enzymes involved in lipoxin biosynthesis pathways without cell activation. mRNA were normalized to the housekeeping Rplp0 expression for each group. Data are presented in mean ± SEM. ND: not detected.

Figure 7 :

 7 Figure 7: Comparison of oxilipins profiles (in pg/mg cellular proteins) from a) rat BM-MSC cells (dark) and human BM-MSC cells (grey) b) in the EV fraction for each cell-type.

ACKNOWLEGMENTS:

We thank the lipidomic platform of the Metatoul network for the mass spectrometric analyses of le lipid mediators, especially P. LeFaouder and J. Bertran-Michel.

FUNDINGS:

The INSERM, CNRS and University of Toulouse 3 financial supports allowed this work to be performed

AUTHOR CONTRIBUTIONS:

Pizzinat N.: collection of data, analysis of data, writing of manuscript and work conception Ong-Meang V.: collection of data Bourgailh-Tortosa F.: collection of data Blanzat M.: collection of data, analysis of data, writing of manuscript Perquis L.: collection of data Cussac D.: writing of manuscript Parini A.: work conception Poinsot V.: work conception, collection of data, analysis of data and writing of manuscript


Mouche Alexis
email: lotfi.aouf@meteo.fr

Perspectives for directional spectra assimilation : Results from a study based on joint assimilation of CFOSAT synthetic wave spectra and observed SAR spectra from Sentinel-1A

Keywords: assimilation, surface ocean wave model, SAR, CFOSAT, forecast, surface ocean wave spectra

INTRODUCTION

The assimilation of satellite wave observations plays a key role in the operational wave forecasting system of Météo-France. The assimilation system corrects efficiently the wave model errors, which are mainly related to the wind forcing. To ensure reliable marine security bulletins the assimilation system is upgraded by wave data provided by new satellite missions. Since 2010 improvements have been obtained for the parametrization of the dissipation by wave breaking [START_REF] Ardhuin | Semi empirical dissipation source functions for windwave models : Part I, definition and calibration and validation at global scales[END_REF] but there is still possible improvement for directional properties of swell. The assimilation of directional wave spectra such as those provided by the Synthetic Aperture Radar (SAR) can be very helpful in order to catch the best initial conditions of swell generated by storms. The wave forecasting system of Météo-France assimilated in operations SAR level 2 directional wave spectra provided by ENVISAT since March 2011 until the end of mission. Lessons from this experience showed that the assimilation of SAR directional wave spectra improves the normalized scatter index of the peak period of long waves larger than 10 seconds by 20% with respect to buoy data [START_REF] Aouf | On the assimilation of ASAR directional wave spectra in the operational wave model MFWAM[END_REF]. Since June 2015 SAR wave spectra from Sentinel-1A (S-1A) are globally provided by the European Space Agency (ESA). The wave mode in S-1A has two incidence angles. The imagettes (size of 20 by 20 km) on the same incidence angle are separated by 200 km.

Swath alternates incidence angles between near range and far range which are 23° and 36.5°, respectively. The retrieved level 2 directional wave spectra are described in 60 frequencies up to 0.22 Hz and 72 directions by a step of 5°. The goal of this work is firstly to evaluate the impact of the assimilation of SAR spectra from S-1A in the wave model MFWAM. Secondly the impact of using several sources of wave data such as altimeters, SAR spectra and CFOSAT synthetic spectra is investigated in the period of analysis and forecast. We recall that the instrument SWIM of CFOSAT will provide directional wave spectra for waves larger than about 70 m in wavelength, while the SAR has a cut-off at roughly 200 m in wavelength.

METHODOLOGY

The operational wave forecasting system of Météo-France is composed of the wave model MFWAM and the assimilation system using the wave observations from satellites. The model MFWAM is based on the ECWAM code with a dissipation by wave breaking developed by Ardhuin et al. [START_REF] Ardhuin | Semi empirical dissipation source functions for windwave models : Part I, definition and calibration and validation at global scales[END_REF] and a swell damping term induced by the air friction at the sea surface. The wave model MFWAM has been upgraded in November 2014 with major improvements thanks to the My wave European FP7 research project [START_REF] Janssen | [END_REF]. The assimilation technique is based on an optimal interpolation technique with, for spectral information, assimilation of wave main parameters (significant wave height, mean period and direction) of up to 3 partitions of the full two-dimensional spectrum [START_REF] Aouf | Assimilation of directional wave spectra in the wave model WAM : an impact study from synthetic observations in preparation to the SWIMSAT satellite mission[END_REF].

Synthetic directional spectra from CFOSAT are obtained from the simulator FAWASSI developed at the French Space Agency (CNES) in 2012. This simulator uses as input an ocean surface reconstructed from directional wave spectra provided by the wave model MFWAM. As an output the simulator FAWASSI provides synthetic spectra as they are seen by the real aperture radar SWIM of CFOSAT. The run of the wave model MFWAM driven by analysed winds from ECMWF gives at CFOSAT orbit tracks the input directional spectra for FAWASSI.

In this study several runs of the model MFWAM have been performed in order to evaluate the contribution of each wave observations (altimeters, SAR and CFOSAT). To make sure that the synthetic spectra and the first guess of the wave model are uncorrelated, 3-day forecast winds from ECMWF atmospheric model is used to drive the model runs. The assimilation is performed every 6 hours. Also a baseline run of the model without assimilation is performed.

RESULTS AND DISCUSSIONS

In our previous work [START_REF] Aouf | On the assimilation of ASAR directional wave spectra in the operational wave model MFWAM[END_REF] we have demonstrated that only the assimilation of directional wave spectra induces a significant impact on peak wave period of swell dominant sea state, as illustrated in figure 1. This latter showed also when we assimilate conjointly CFOSAT synthetic spectra, Envisat wave spectra and altimeters the scatter index of peak period is improved regarding to the use of Envisat and altimeters.

The assimilation of SAR wave spectra from S-1A shows a significant impact in the forecast period, as illustrated in figure 2. The difference of mean wave period can reach after 1-day forecast about 1.5 seconds as indicated in the eastern part of the pacific ocean. This is related to the intense hurricane season in the east pacific on September 2015. The validation of the results with independent wave data such altimeters and buoys has been performed. Figure 3 shows the variation of the normalized scatter index of significant wave height in the forecast period. The smaller the scatter index is, better the performance of the system is. It is clearly indicated that when we use both altimeters and SAR directional wave spectra the impact is more pronounced. We can also see that the impact stays efficient until 3-day forecast. Results on model runs with both S-1A and CFOSAT spectra will be commented in the final paper. Further comparison with buoys data will be also discussed.

CONCLUSIONS

With a low cut-off for the directional wave spectra CFOSAT will impact both mixed sea and swell. The complementary use of CFOSAT and SAR wave spectra will enhance the impact of the assimilation and therefore induce a more accurate sea state. The CFOSAT mission is on one hand a big challenge for operational wave forecasting system, and on the other hand a good opportunity for wave modellers in order to improve the source and dissipation terms in the wave model.

Figure 1 .Figure 2 .

 12 Figure 1. Normalized scatter index of peak period (in %) for several runs with respect to NDBC buoys. SWIM+SAR, SAR, SWH-only and NOASSI stand for the assimilation of all satellites (CFOSAT and Envisat spectra and altimeters),the assimilation of Envisat spectra and altimeters, the assimilation of altimeters only, and the control run without assimilation.

Figure 3 .

 3 Figure 3. Variation of the normalized scatter index (in %) of significant wave height in the forecast period based on comparison with altimeters (Jason-2 and Saral). Triangles, circles and pluses stand for runs of the model MFWAM with assimilation of altimeters and SAR spectra from S-1A, with assimilation of SAR spectra from S-1A onlyand without assimilation, respectively.