Niels Rothermel

Tobias Röther

Tuğçe Ayvali

Luis Miguel Martinez-Pietro

Karine Philippot

Hans-Heinrich Limbach

Bruno Chaudret
email: chaudret@insa-toulouse.fr

Torsten Gutmann
email: gutmann@chemie.tu-darmstadt.de

Gerd Buntkowsky
email: gerd.buntkowsky@chemie.tu-darmstadt.de

Tugce Ayvali

Luis M Martínez-Prieto

Reactions of D 2 with 1,4-Bis(diphenylphosphino) butane-Stabilized Metal Nanoparticles-A Combined Gas-phase NMR, GC-MS and Solid-state NMR Study

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Introduction

Nanoparticles of transition metals, stabilized by organic ligands, are an interesting class of catalysts, since they represent a hybrid between a heterogeneous and a homogeneous catalyst. The combination of both fields is important for hydrogenations of arenes under mild conditions. [START_REF] Debouttière | PTA-Stabilized Ruthenium and Platinum Nanoparticles: Characterization and Investigation in Aqueous Biphasic Hydrogenation Catalysis[END_REF][START_REF] Widegren | A review of soluble transition-metal nanoclusters as arene hydrogenation catalysts[END_REF] In comparison to classical heterogeneous catalysts, such as zeolite-supported noble metals used for aromatic hydrogenations, [START_REF] Stanislaus | Aromatic Hydrogenation Catalysis: A Review[END_REF] metal nanoparticles offer many possibilities for fine tuning with respect to activity and selectivity due to their stabilizing ligand system. This makes it possible to modify nanoparticles not only for operations in different solvents such as aromatics, alcohols and even water, but also for stereoselective operations. [START_REF] Jansat | Synthesis, characterization and catalytic reactivity of ruthenium nanoparticles stabilized by chiral N-donor ligands[END_REF] Particles tuned in this way have been recently used for the regioselective and stereo specific deuteration of a wide range of bioactive aza compounds. [START_REF] Pieters | Regioselective and stereospecific deuteration of bioactive aza compounds by the use of ruthenium nanoparticles[END_REF][START_REF] Taglang | Enantiospecific CH Activation Using Ruthenium Nanocatalysts[END_REF] To design metal nanoparticle systems that fulfill the high demands on activity and selectivity of modern catalysis, it is crucial to gain a deeper understanding of the processes that take place on the nanoparticle surface during the catalytic reaction. For the characterization of surface species and the observation of surface processes on metal nanoparticles (MNPs), solid state and gas phase NMR have proven to be valuable spectroscopic methods. [START_REF] Gutmann | From Molecular Complexes to Complex Metallic Nanostructures-(2) H Solid-State NMR Studies of Ruthenium-Containing Hydrogenation Catalysts[END_REF][START_REF] Gutmann | From Molecular Complexes to Complex Metallic Nanostructures-(2) H Solid-State NMR Studies of Ruthenium-Containing Hydrogenation Catalysts[END_REF][START_REF] Novio | Location and Dynamics of CO Coordination on Ru Nanoparticles: A Solid State NMR Study[END_REF][START_REF] Kinayyigit | Probing the surface of platinum nanoparticles with 13CO by solid-state NMR and IR spectroscopies[END_REF][START_REF] Pan | Ligand-stabilized ruthenium nanoparticles: synthesis, organization, and dynamics[END_REF] For MNPs formed by hydrogenation of organometallic precursors it has been shown by a combination of 1 H gas phase and solid state [START_REF] Widegren | A review of soluble transition-metal nanoclusters as arene hydrogenation catalysts[END_REF] H NMR that hydrides are present on the surface of the particles and that they are mobile and exchangeable by deuterium. [START_REF] Pery | Direct NMR evidence for the presence of mobile surface hydrides on ruthenium nanoparticles[END_REF] Recently, some of us have explored the kinetics of gas-solid hydrogen isotope exchange of ruthenium metal nanoparticles (RuMNPs) in more detail. [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] It was found that when D2 is applied to the latter, a direct reaction with surface hydrides occurs without D2 dissociation leading to the formation of HD in the initial reaction stages.

Whereas we have studied previously RuMNPs containing different stabilizing ligand systems, namely Ru/PVP (PVP = polyvinylpyrrolidone) and Ru/HDA (HDA = hexadecylamine), we have investigated in the present work the more reactive Ru/dppb system (1) (dppb = 1,4-bis(diphenylphosphino)butane) at 1 and 2 bar D2 pressures. As we also wanted to explore the role of the metal, we studied as well the bimetallic system of ruthenium and platinum RuPt/dppb (2) and the monometallic system of platinum Pt/dppb (3). In the course of our studies we observed a hydrogenation of the phenyl groups of dppb and a partial decomposition of the ligand system that leaves into the gas phase. Therefore, the composition of the gas phase has been further analyzed in detail by GC-MS. Finally, multinuclear solidstate NMR experiments have been used to evaluate the impact of hydrogen gas on the structure of the particles' ligand shell.

Experimental Section

General

All reactions were carried out using Schlenk or Fisher-Porter bottle techniques under an inert and dry atmosphere. THF was distilled over CaH2 and pentane over sodium. The solvents were degassed by means of three freeze-pump cycles. 1,4-bis(diphenylphosphino)butane (dppb) was purchased from Sigma-Aldrich -and (Cyclo-octadiene)(cyclo-octatriene)ruthenium(0) (Ru(COD)(COT)) for the synthesis of Ru/dppb (1) from Umicore. For RuPt/dppb (2) the Ru(COD)(COT) precursor was purchased from Nanomeps (Toulouse), dibenzylideneacetone (dba) from Alfa-Aesar and Pt(CH3)2(COD) from Strem and used without further purification. D2 (99.82%) gas for the exchange reactions was purchased from Eurisotop.

Synthesis

The synthesis of all three nanoparticle systems followed the organometallic approach described in the literature, [START_REF] Novio | Location and Dynamics of CO Coordination on Ru Nanoparticles: A Solid State NMR Study[END_REF][START_REF] Kinayyigit | Probing the surface of platinum nanoparticles with 13CO by solid-state NMR and IR spectroscopies[END_REF][START_REF] Lara | On the influence of diphosphine ligands on the chemical order in small RuPt nanoparticles: combined structural and surface reactivity studies[END_REF] where transition metal complexes containing unsaturated ligands are hydrogenated using gaseous H2 in dilute solutions of THF at room temperature, in the presence of a substoichiometric amount of dppb.

Ru/dppb (1)

200 mg of Ru(COD)(COT) (0.634 mmol) were introduced in a Fisher-Porter bottle and left in vacuum for 30 min. 120 mL of freshly distilled THF was then added and the resulting yellow solution was cooled to 193 K. Under rigorous stirring, a solution of 27 mg (0.0633 mmol, 0.1 eq.) dppb in 80 mL THF was slowly added to the precursor solution. The Fisher-Porter bottle was pressurized with 3 bar of H2 gas and the solution was left to slowly reach room temperature (r.t.) under continuous stirring. After 18 h of reaction a black colloidal solution was obtained. The solvent was removed in vacuum to reach a volume of approximately 10 mL. To this solution 80 mL of pentane were added. After 1 h a black precipitate was formed and the solvent was removed by filtration. The black precipitate was further washed with 2 x 35 mL pentane and dried at room temperature under vacuum over night to yield Ru/dppb (1) particles as a black powder.

RuPt/dppb (2)

142 mg of Ru(COD)(COT) (0.451 mmol) and 150 mg of Pt(CH3)2(COD) (0.451 mmol) were introduced into a Fisher-Porter bottle and dissolved in 100 mL THF. The so-obtained yellow solution was cooled to 213 K and a solution of 93.2 mg (0.226 mmol, 0.25 eq.) dppb in 80 mL THF was added under rigorous stirring. After pressurization with 3 bar H2 the solution was left to reach r.t. under continuous stirring. After 18 h the black colloidal solution was reduced to a volume of approximately 10 mL under vacuum. Afterwards 40 mL of cold pentane (243 K) were added to precipitate the particles. The solvent was removed by filtration and the resulting black solid powder was washed with 2 x 40 mL of pentane. The product was dried under vacuum overnight, yielding RuPt/dppb (2) particles.

Pt/dppb (3)

300 mg of Pt(dpa)2 (0.452 mmol) were dissolved in 170 mL THF in a Fisher-Porter bottle. The obtained purple black solution was cooled to 243 K and a solution of 38.6 mg (0.0905 mmol, 0.2 eq.) dppb in 10 mL THF was added under stirring. The mixture was then pressurized with 3 bar of H2 gas and left to slowly reach r.t. under continuous stirring. After 24 h of reaction, the black colloidal solution was reduced to a volume of approximately 20 ml in vacuum and 30 mL of cold pentane were added for a quick precipitation of a black solid. The solvent was removed by filtration and the product was further washed with 6 x 30 mL of cold pentane. The product was dried under vacuum overnight, yielding Pt/dppb (3) particles.

Characterization techniques

Gas Phase NMR

1 H gas-phase NMR spectra were recorded on a Bruker Avance III HD 500 spectrometer at 11.7 T corresponding to a frequency of 500.26 MHz for 1 H. This system is equipped with a Bruker DUL 500 MHz S2 5 mm Probe. 10 mg of the corresponding nanoparticles (1-3) were filled into a sealable pressure resistant NMR sample tube (Wilmad Lab-Glass Quick Pressure Valve NMR Sample Tube, 2.51 mL inner volume). The sample tube was evacuated for at least 30 min before connection to a D2 gas bottle via PFA tubing. The D2 pressure was adjusted to 1 bar (1a-3a) or 2 bar (1b-3b), respectively, and the Teflon seal of the evacuated sample tube was opened quickly to apply a D2 atmosphere to the particles. The tube was sealed again and directly put into the spectrometer to record 1 H NMR spectra of the gas phase containing an assumed mixture of H2, HD, D2 and volatile solvent molecules.

Gas Chromatography and Mass Spectrometry

For RuPt/dppb nanoparticles an analysis of the gas-phase was performed by GC-MS. Experiments were performed on a Fisions MD 800 mass spectrometer equipped with a quadrupole detector and a DB-5 column. For the measurement a sample of 12.6 mg RuPt/dppb was treated with 3 bar D2 at r.t. (2c) and stored for 7 days. The injector was heated to 240°C and a volume of 200 µL of the gas-phase above the RuPt/dppb was injected. A temperature ramp (40°C for 10 min, heating 25°C/min to 150°C, keeping 150°C for 2 min) and He as carrier gas was chosen to separate the different side products that were analyzed by MS.

Solid State NMR

13 C and 31 P CP MAS experiments were recorded on a Bruker Avance III spectrometer at 7 T corresponding to a frequency of 75.47 MHz for [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C and 121.49 MHz for 31 P respectively. This system is equipped with a 4 mm H/X probe. All experiments were performed at spinning rates of 8 kHz and repetition delays of 4 s. The spectra were recorded utilizing ramped CP MAS sequences. [START_REF] Metz | Ramped-Amplitude Cross-Polarization in Magic-Angle-Spinning Nmr[END_REF] For 13 C and 31 P, contact times were set to 3 and 3.2 ms, respectively. Two sets of experiments were performed. One set was performed on the Ru/dppb particles (1) directly after they were synthesized. The other set was performed after treating the same sample with hydrogen gas (RuH/dppb (1c)). The sample preparation constituted of three overnight treatments with 1.5 bar H2 and two intraday treatments with 1.5 bar H2 for 2 h. After each treatment, the sample was exposed to vacuum for at least 30 min to remove decomposition products of the ligands.

Results and Discussion

Gas Phase Measurements

In Figure 1 are depicted typical 1 H gas-phase NMR spectra obtained for all three particle systems after 16 h exposure to 1 bar (1a-3a) or 2 bar (1b-3b) D2 at room temperature. All spectra show a signal at 4.5 ppm arising from gaseous dihydrogen. By line shape analysis (1d, see ESI Figure S1) a line width of about 180 Hz is obtained which corresponds to the one of HD, i.e. the amount of H2 is negligible as it exhibits a much broader line width (see ESI Figure S2 and ref. 13). This result is corroborated by the Raman spectrum (1e, ESI Figure S3) recorded for the gas-phase upon reaction of D2 with the particles, where H2 is barely observable, in contrast to the vibrations of HD and D2.

In addition to HD as main reaction product, the spectra in Figure 1 reveal the formation of side products giving rise to signals at high field between 0 and 1.5 ppm. This region is typical for alkanes which must have been formed by reaction of D2 with the dppb ligands. That reaction is corroborated for Pt/dppb sometimes by the observation of a thin film of a liquid, condensing at the inner wall of the NMR tube when the D2 gas is applied for sample preparation.

We assign these alkane signals as follows. The signal at 1.5 ppm is typical for cyclohexane [START_REF] Ross | NMR spectroscopy of cyclohexane. Gas-phase conformational kinetics[END_REF] and those at 1.35 ppm and 0.95 ppm for the methylene and methyl groups of butane. [START_REF] Tynkkynen | 1H NMR spectral analysis and conformational behavior of n-alkanes in different chemical environments[END_REF] Furthermore, in contrast to the Pt-containing NPs, the spectra of the Ru-containing NPs present an additional signal around 0.2 ppm. That is the chemical shift of methane. [START_REF] Fulmer | NMR Chemical Shifts of Trace Impurities: Common Laboratory Solvents, Organics, and Gases in Deuterated Solvents Relevant to the Organometallic Chemist[END_REF] From Fig. 1 it is evident that the Pt and Ru containing NPs exhibit a different pressure dependence. In case of the Pt/dppb NPs, the alkane signals grow with pressure and in case of the Ru-containing NPs, the alkane signals are weakened with growing pressure. This is a clear indication that the H/D exchange in the alkane moieties of the ligand is more efficient in the Ru/dppb NPs. As the alkanes formed from Ru/NPs will be partially deuterated, one might expect that part of the corresponding alkane signals experience small high field shifts. Indeed, we observe in the 2 bar D2 experiments with Ru-containing NPs that signals of the alkanes released into the gas phase contain high-field shoulders. Therefore, we checked whether these shoulders could arise from H/D isotope effects which lead to high-field shifts of the remaining carbon bound H nuclei. In the case of methane in organic solvents a high-field shift of about 0.045 ppm was observed for CH4/CHD3, which depended on the type of solvent and on temperature. [START_REF] Anet | ON THE PROTON CHEMICAL-SHIFTS IN PARTIALLY DEUTERIATED METHANES[END_REF] However, the shoulders which we observe seem to arise from somewhat larger shifts, their exact value being difficult to obtain. Another explanation would be that some of the alkanes at high pressure are not located in the gas phase but interacting with the particles, either adsorbed or in rapid gas-surface exchange, or as tiny droplets. Finally, we note that in the case of RuPt/dppb the amount of obtained butane is reduced as compared to Ru/dppb, but the amount of methane increased, as expected for more efficient C-C bond breaking processes.

For the above mentioned MNPs, the alkane side products must be formed by the reaction of the dppb ligands at particularly reactive sites. The following reaction scheme is conceivable. Scheme 1 suggests the formation of metal bound PH2 surface species, but the latter might easily oxidize. In order to know the details of these reactions extended kinetic and computational studies are required which were beyond the scope of this work.

GC-MS Measurements

For the detailed characterization of the reaction products, gas-chromatography coupled to mass spectrometry (GC-MS) was applied. As example, RuPt/dppb was treated with 3 bar D2 gas pressure (2c) to produce a higher amount of side products for analysis. RuPt/dppb (2) was chosen because it does not tend to quench the reaction compared to Pt/dppb (3) but it produces larger amounts of side product than Ru/dppb (1). Scheme 1. Hydrogenation and alkane release of a metal dppb ligand by dihydrogen. The phosphine species formed may not be stable but subject to further reactions, in particular with oxygen.

Two main fractions were separated (Figure 2) that are clearly attributed to cyclohexane and butane according to their spectra pattern. This is in excellent agreement with the 1 H gas phase NMR of 1a and 1b (Figure 1), which show the signals of these compounds in the aliphatic region. The appearance of butane and cyclohexane illustrates that the particles facilitate the cleavage of the C-P bond upon decomposition of the dppb ligand, similar to related phosphido bridged transition metal cluster complexes. [START_REF] Garrou | Transition-Metal-Mediated Phosphorus-Carbon Bond Cleavage and Its Relevance to Homogeneous Catalyst Deactivation[END_REF][START_REF] Maclaughlin | Hydrogenation of phosphido bridged ruthenium clusters. A view of P-C bond activation and cleavage in the coordinatively unsaturated molecule : conversion of p2-PPh2 to p3-PPh[END_REF][START_REF] Watson | Diphosphine Isomerization and C-H and P-C Bond Cleavage Reactivity[END_REF] The spectrum of the cyclohexane fraction in Figure 2 shows a "broadening" of the mass distribution compared to the spectrum of pure cyclohexane. The location of the molecular peak at m/z = 84 is shifted to higher m/z values between 84 and 96. This is a clear hint that deuterium is incorporated into the aliphatic parts of the ligand. Similar results are also observed for the fraction of butane (Figure 2). This observation is in excellent agreement with the deuteration of alkanes on Ru/dppb MNPs as demonstrated in our previous work. [START_REF] Rothermel | Surprising Differences of Alkane C-H Activation catalyzed by Ruthenium Nanoparticles: Complex Surface-Substrate Recognition?[END_REF] Comparing the mole peak of the mass spectrum of cyclohexane and the one from butane (m/z value of 58+), one notices a narrower distribution of masses in the case of cyclohexane and a more extensive deuteration. This phenomenon can be explained by different deuteration mechanisms. Due to the aromatic nature of the dppb ligand, some deuterons are incorporated by direct hydrogenation of the aromatic rings or already cleaved benzene. [START_REF] Gutmann | Investigation of the surface chemistry of phosphine-stabilized ruthenium nanoparticles--an advanced solid-state NMR study[END_REF][START_REF] Lara | Organometallic Ruthenium Nanoparticles: A Comparative Study of the Influence of the Stabilizer on their Characteristics and Reactivity[END_REF][START_REF] Rafter | Secondary phosphine oxides as pre-ligands for nanoparticle stabilization[END_REF] This leads to the formation of cyclohexane or partly hydrogenated respectively deuterated C6 analogues. Furthermore, fully deuteration of the cyclohexane and C6 analogues is feasible as shown by the isotopomer patterns for the deuteration of benzene or toluene (ESI Figure S4). This would require a C-H bond activation on the surface of the particles.

Deuteration of the butane fragment on the other hand, is only feasible through C-H-bond activation on the metal surface and not by direct reaction. Such C-H-bond activation is favored as long as the butane moiety is in close proximity to the surface which is highly probable when it is linked to phosphorus that coordinates to the surface. Based on liquid state NMR studies on alkylaminestabilized Ru NPs, it was previously suggested that the presence of such an agostic interaction may lead to the deuteration of alkylic species as in the present case. 11

Solid State NMR

The fate of the dppb ligands of Ru/dppb, RuPt/dppb and Pt/dppb was indirectly elucidated by GC-MS analysis of the side products formed when the particles are exposed to D2 gas. The next step was to study the change of the NP surface structure, when the dppb ligand is transformed. Through the observation of cyclohexane and butane in the side product fraction, the phosphorus moieties are likely transformed into metal bound phosphanes (-PHxC3-x). To shed more light on the structural changes, [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C and 31 P CP MAS NMR measurements were performed before and after hydrogen treatment (Ru/dppb (1) and RuH/dppb (1c)).

The [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C CP MAS spectra of Ru/dppb recorded before and after treatment with H2 gas (Figure 3a,b) both feature signals of aliphatic carbons located around 25 ppm. Before the treatment, a small signal at 130 ppm is visible that is attributed to phenyl groups of non-hydrogenated dppb ligands. This weak aromatic signal in dppb based particles varies in intensity between different particle samples. The intensity can for example be affected by storage time and also by small experimental deviations. In addition to that, the measurement shows that not all ligands on the metal surface undergo decomposition but all aromatic species are hydrogenated. Ligands that undergo decomposition could therefore be located at very reactive sites on the metal surface. Based on previous surface studies with CO adsorbed on Ru/dppb it is assumed that ligands are located on apexes of the particle. [START_REF] Novio | Location and Dynamics of CO Coordination on Ru Nanoparticles: A Solid State NMR Study[END_REF] The observed decomposition products most probably stem from ligands which are not located on apex positions and are therefore labile and readily react with hydrides in their vicinity. The 31 P CP MAS spectra of Ru/dppb recorded before and after treatment with H2 gas (Figure 4a,b), show clear differences in their line shape and an improved resolution and S/N ratio is obtained after H2 treatment. The better resolution and S/N ratio seem to be the consequence of the saturation of the metal surface with hydrogen upon H2 treatment, which improves the cross-polarization efficiency. Nevertheless, no additional signals appeared after treatment with H2 gas, which indicates that no significant changes of the structure occurred during the H2 treatment. An assignment of individual signals has to be done with care. Following previous 31 P solid state NMR studies [START_REF] Gutmann | Investigation of the surface chemistry of phosphine-stabilized ruthenium nanoparticles--an advanced solid-state NMR study[END_REF] the major signal located at 56 ppm is most probably attributed to phosphine species coordinated to the metal surface. Based on the characterization of the dppb chemistry towards decomposition and hydrogenation, a range of different phosphine species is expected to be present on the surface, i.e. alkyl-P(Ph/Cy)2, alkyl-P(Ph/Cy)H, alkyl-PH2, HnP(Ph/Cy)3-n and arene hydrogenation products from all these species, which are not fully hydrogenated to cyclohexyl. Based on the [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C spectra it can be assumed that arene phosphines are the least dominant surface species. The signal located at 29 ppm seems to correspond to co-adsorbed phosphine oxides.

Reactions on dppb stabilized nanoparticles

The results of the previous sections reveal a clearer picture on the surface reactions of dppb stabilized MNPs. For the details of the formal reactions of the dppb ligands with H2 or D2 (Scheme 1) we discuss some possible mechanisms depicted in Figure 5. Gaseous H2 or D2 are consumed and as main products butane and cyclohexane are formed as proven by NMR and GC-MS analysis of the gas phase. On the other hand, some dppb ligands remain as partially hydrogenated/deuterated ligands on the surface as evidenced by [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C and 31 P solid-state NMR.

Together with the results from our former paper on alkane deuteration [START_REF] Rothermel | Surprising Differences of Alkane C-H Activation catalyzed by Ruthenium Nanoparticles: Complex Surface-Substrate Recognition?[END_REF] a variety of side reactions can be identified as illustrated in Figure 5. The reaction steps A1 and A2 describe the C-P bond cleavage catalyzed by the particle that leads to the formation of benzene and butane. The details of these complex reaction steps are illustrated in Figure S5 in the ESI. In step B, benzene reacts further to form cyclohexane. In step C1 and C2, cyclohexane or butane are deuterated by C-H bond activation on the particle surface. In addition to the reactions that involve the dppb ligand, D2 is adsorbed on the surface and is transformed into surface deuterides in step D. Surface deuterides scramble with surface hydrides and form HD that can leave the particle due to the surface/gas phase equilibrium in step E. Finally, in a step F the hydrogenation of the dppb is feasible which leads to 1,4-Bis(dicyclohexylphosphino)butane in case of total hydrogenation.

Conclusions

Three 1,4-Bis(diphenylphosphino)butane stabilized nanoparticle systems (Ru/dppb, Pt/dppb and RuPt/dppb) were investigated in the reaction with D2 gas. Next to HD exchange with gaseous dihydrogen isotopomers, these particle systems were found to be capable of catalyzing the deuteration of aliphatic substrates. The latter was confirmed by GC-MS analysis of the gas phase after reaction with D2 gas, which allowed a detailed characterization of the side products that were observed in the 1 H gas phase NMR spectra. The formation of deuterated butane and cyclohexane isotopomers revealed that all three particle systems not only catalyze an isotope exchange but also catalyze P-C bond cleavage reactions of their dppb ligands in very mild reaction conditions. Exemplary, [START_REF] Limbach | Gas phase 1H NMR studies and kinetic modeling of dihydrogen isotope equilibration catalyzed by Runanoparticles under normal conditions: dissociative vs. associative exchange[END_REF] C and 31 P solid state NMR experiments on Ru/dppb show that even after multiple hydrogen treatments, which trigger the decomposition of the dppb ligand, the latter remains on the particle surface. These results demonstrate that the surface composition is not drastically influenced by this decomposition reaction. For deeper studies of HD exchange kinetics, it is however necessary to pretreat the particles with H2 gas to gain reproducible initial conditions for the surface reaction.

Figure 1 :

 1 Figure1: Typical 1 H gas-phase NMR spectra after exposing solid MNPs at room temperature to 1 bar (1a-3a) or 2 bar (1b-3b) D2 gas for 16 h. The chemical shift of the HD signal is set to 4.5 ppm. For comparison of the signal intensities of the different particle systems, all spectra were processed using the same parameters.

Figure 2 :

 2 Figure 2: Recorded mass spectra of the cyclohexane and the butane fraction of the H/D exchange reaction with RuPt/dppb performed with 3 bar initial D2 gas pressure (2c). The red signals represent the mass spectrum of the individual species with natural isotope distribution as found in a mass database.

Figure 3 :

 3 Figure 3: 13 C CP MAS spectra measured at 8 kHz spinning of Ru/dppb measured before (a) and after (b) treatment with H2 gas.

Figure 4 :

 4 Figure 4: 31 P CP MAS spectra measured at 8 kHz spinning of Ru/dppb measured before (a) and after (b) treatment with H2 gas. Note that signals marked with asterisks are spinning sidebands of the signal at ca. 56 ppm.

Figure 5 :

 5 Figure 5: Reactions taking place on dppb stabilized nanoparticles when exposed to D2.

Acknowledgements

This research was supported by the Deutsche Forschungsgemeinschaft under contract Bu-911-19-1/2 (DFG-ANR project MOCA-NANO): The authors thank Christiane Rudolph, Gül Sahinalp and Alexander Schießer of the mass spectrometry department of TU Darmstadt for their technical support.


Dr Patricia Lara
email: patricia@iiq.csic.es

Dr Karine Philippot

Andres Suarez

Dr Andrés Suárez

Prof Ernesto Carmona

Phosphane-decorated Platinum Nanoparticles as Efficient Catalysts for H 2 Generation from Ammonia Borane and Methanol

Keywords: nanoparticles, platinum, phosphane ligands, dehydrogenation, ammonia borane

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.

Introduction

Chemical energy release from the hydrogen molecule is considered as a practical alternative to replace fossil fuels, and the implementation of a global economy based on hydrogen as an energy vector ("Hydrogen Economy") has been envisaged. [1] At this regard, H2-storage in a chemical reagent by formation of covalent bonds has attracted considerable attention. [2] Among the different possibilities explored, [3] the use of ammonia borane (H3N•BH3) is particularly appealing for the controlled storage and supply of H2 due to its high hydrogen gravimetric content (19.6 %wt) and favorable physical properties including high thermal stability, non-flammability and low toxicity. [4] Hydrogen generation from H3N•BH3 can be accomplished by different methods, [3,5] including thermal and catalytic decomposition. [6] These methods present disadvantages such as the uncomplete release of the maximum available H2 content (3 equiv) and formation of different by-products from which it is difficult to regenerate the H3N•BH3 molecule. [7] As another method, solvolysis of H3N•BH3 by a protic solvent such as water [8,9] or an alcohol [9] usually allow the release of the maximum H2 content and the concomitant formation of a sole H2-depleted by-product. Hydrogen generation from ammonia borane using methanol is particularly interesting given the high solubility of H3N•BH3 in this solvent (23 %wt at 23 o C) and the long-term stability of the solutions. Moreover, methanolysis of H3N•BH3 yields ammonium tetramethoxyborate (NH4B(OMe)4), which unlike the product formed by the hydrolysis of ammonia borane (ammonium metaborate, NH4BO2), can be easily reconverted to H3N•BH3 by reaction with LiAlH4 and NH4Cl. [9] Metal nanoparticles (NPs) are appealing catalytic systems due to their high proportion of surface atoms providing numerous active sites. [START_REF]Nanotechnology in Catalysis[END_REF] The design of well-controlled nanoparticles in terms of size, dispersion and surface state is crucial to achieve good levels of activity and selectivity in a catalytic process. In this context, the organometallic approach for the synthesis of metal nanoparticles has proven to be a powerful method to obtain small and well-controlled nanoparticles displaying hydrides and coordinated ligands at their surface. [START_REF] Philippot | Chaudret in Comprehensive Organometallic Chemistry III[END_REF] Transition metal nanoparticles have been shown to catalyze H3N•BH3 decomposition, [12] including the methanolysis of ammonia borane. [13] Most of the described systems of NPs are stabilized using porous materials such as oxide supports and resins that increase the weight of the resulting H2-storage/delivery system.

Herein, we report the organometallic synthesis of Pt NPs stabilized by sterically demanding phosphanes containing terphenyl groups, and their use as catalysts in H2 generation from ammonia borane and methanol. [START_REF] Cheng | For selected examples of Pt-based catalysts for ammonia borane dehydrogenation[END_REF] A proper adjustment of the phosphane ligand and the phosphane/Pt ratio allowed to obtain small nanoparticles of 1.5-2.2 nm in size that display high catalytic activities. Furthermore, the feasibility of tandem H3N•BH3 dehydrogenation and N-heterocycle hydrogenations catalyzed by these catalysts is demonstrated.

Results and Discussion

Synthesis and characterization of Pt•L nanoparticles

A series of platinum nanoparticles were easily synthesized by decomposing a THF solution of bis(dibenzylideneacetone)platinum, [Pt(dba)2], at room temperature under 3 bar of H2 and in the presence of a phosphane containing a sterically demanding terphenyl group as stabilizing ligand. Three different phosphanes were tested: dimethyl-2,6-bis(2´,6´-dimethylphenyl)phenylphosphane, L1; diethyl-2,6-bis(2´,6´-dimethylphenyl)phenylphosphane, L2; and dimethyl-2,6-bis(2´,6´-di(isopropyl)phenyl)phenylphosphane, L3 (Figure 1, Table 1). A color change from violet to dark brown was FULL PAPER observed after 3 h of reaction, but the reaction mixture was kept under vigorous stirring for 14 h to ensure the total decomposition of the precursor. Then, the dihydrogen pressure was released, and the NPs were isolated by precipitation with cold pentane, giving rise to black powders that were further used for catalytic studies. In order to obtain the best conditions in terms of dispersion and homogeneity, different ligand to metal ratios (L/Pt = 0.2 or 0.5) were examined. The metal content of the purified nanoparticles was obtained by Inductive Coupled Plasma (ICP) with values of 52-69%wt Pt (Table 1). The morphology and size of the nanoparticles were determined by TEM (transmission electron microscopy). Mean sizes in the range 1.5-2.2 nm depending on the ligand used were measured (Table 1, Figure 2). The first Pt/phosphane sample (Pt•L1 0.2 ) was prepared employing 0.2 molar equiv of ligand L1 as stabilizer with respect to platinum. In these synthesis conditions, nanoparticles of 2.2 (0.5) nm were formed (Figure 2). When the reaction was performed using 0.5 equiv of ligand L1, nanoparticles Pt•L1 0.5 displaying a better dispersion and presenting a slightly smaller size (2.0 (0.3) nm) were obtained (Figure 2). This trend (smaller size with more ligand at the surface) has already been observed for Ru and Pt NPs stabilized with different ligands. [15,16] The third Pt/phosphane sample (Pt•L2 0.2 ) was synthesized with 0.2 equiv of ligand L2, leading to a homogeneous population of NPs having a mean size of 1.5 (0.2) nm. Finally, a fourth colloid was prepared in the presence of 0.2 equiv of ligand L3 (Pt•L3 0.2 ). In this case, nanoparticles of mean size 2.0 (0.5) nm were observed by TEM.

Pt NPs

L/Pt ratio %wt Pt [a] Mean size [nm] [b] Pt High-resolution transmission electron microscopy (HRTEM) analysis performed on THF colloidal solution of Pt•L1 0.2 showed the high crystalline character of the nanoparticles and their facecentered cubic structure. Fast Fourier transformation (FFT) evidenced reflections that correspond to the (002), ( 220) and ( 204) atomic planes. The composition of the particles was confirmed by High angle annular dark field imaging (HAADF) coupled with energydispersive X-ray spectrometry (EDX) mapping using scanning transmission electron microscopy (STEM). The EDX spectrum recorded in a selected area of the grid revealed some peaks that can be attributed to Pt (at ca. 1.6, 2.1, 2.8, 8.2 and 9.4 keV) despite an overlapping with peak for phosphorous at ca. 2.0 keV. (see SI).

•L1 0.2 0.2 64 2.2 (0.5) Pt•L1 0.5 0.5 52 2.0 (0.3) Pt•L2 0.2 0.2 67 1.5 (0.2) Pt•L3 0.2 0.2 69

H2 generation by methanolysis of ammonia borane

The catalytic activity of the terphenylphosphane stabilized platinum nanoparticles Pt•L was evaluated in the generation of H2 from methanol solutions of H3N•BH3. Reactions were performed at 30 o C using 0.14-0.19 mol% Pt loadings of Pt•L NPs (Figure 4, Table 2). The monitoring of the catalytic performance was done by measuring the increase of the pressure of gas evolved. Using Pt•L1 0.2 as catalyst, the reaction started with no induction period and led to complete conversion in less than 8 min with the evolution of 3 mol H2 per mol of H3N•BH3. A significant turnover frequency (TOF; calculated from mmol of H2/(mmol Pt x time)) of 191 min -1 was found (Table 2, entry 1). [START_REF] Philippot | Chaudret in Comprehensive Organometallic Chemistry III[END_REF] B NMR spectroscopy analysis of the hydrogen depleted solution showed the presence of only a singlet at  7.5 ppm, supporting the quantitative formation of ammonium tetramethoxyborate, NH4B(OMe)4 (Figure S2). [9] The NPs synthesized with a L1/Pt ratio of 0.5 appeared significantly less active than their Pt•L1 0.2 counterparts (entries 1 and 2) showing a TOF value of 107 min -1 against 191 min -1 , respectively. This decrease of activity can be attributed to a higher coverage of the nanoparticle metal surface by the phosphane ligands that prevents substrates from accessing the reactive sites, as previously observed in other catalytic processes mediated by ligand-stabilized NPs. [16] Hence, the catalytic behavior of the terphenylphosphane stabilized Pt nanoparticles with L/metal ratios of 0.2 was compared, showing that the steric properties of the phosphane ligand have a marked influence on the catalytic activity. Nanoparticles Pt•L2 0.2 , which differ from Pt•L1 0.2 in the alkyl substituent of the phosphane ligand (methyl and ethyl groups, respectively), were found to be a slower catalyst (entry 3). Alternatively, nanoparticles based on L3, having a more sterically demanding terphenyl group, provided a ca. 1.5-fold increase of the catalytic activity with respect to Pt•L1 0.2 , giving rise to a notable TOF of 284 min -1 (entry 4). This high catalytic performance compares favorably with activities provided by other metal catalysts in the methanolysis of H3N•BH3 (see SI, Table S1). It is also worth mentioning that with Pt•L3 0.2 , the catalyst loading could be further decreased to 0.08 mol% without a significant impact on the catalytic activity (entry 5; Figure S3). Furthermore, TEM analysis of the colloid Pt•L3 0.2 after the catalytic reaction showed a very slight increase in the nanoparticles size to 2.3 (0.4) nm (Figure S4).

In order to obtain a better insight of the potential of our catalytic system, a solution of 15.2 mmol of H3N•BH3 in MeOH was added to a colloidal suspension of 2.5 mol Pt•L3 0.2 (0.016 mol% Pt) in MeOH at 25 o C, and the volume of gas evolved was measured in a gas burette (Figure 5). A steady H2 production was observed until approximately 90% conversion of H3N•BH3, with an average TOF of 122 min -1 . Overall 18240 mmol H2 per mmol Pt were produced in the reaction. 2 for experimental details.

Table 2. Methanolysis of H3N•BH3 catalyzed by Pt•L NPs. [a] Entry Pt•L Cat. loading [b] [mol%]

Conv.

[%] (time, [min]) 5 [b] Pt•L3 0.2 0.08 100 ( 15) 253 Ammonium tetramethoxyborate (NH4B(OMe)4) can be easily sublimated under reduced pressure, and its reconversion to H3N•BH3 has been demonstrated. [9] Since clean transformation of H3N•BH3 to ammonium tetramethoxyborate was observed in the methanolysis of H3N•BH3 catalyzed by the Pt•L nanoparticles (see [START_REF] Philippot | Chaudret in Comprehensive Organometallic Chemistry III[END_REF] B NMR spectroscopy analysis), a reusable H2 delivery system based on catalyst reutilization after vacuum distillation removal of the H2-depleted by-product and MeOH was tested (Figure 6). Over three reuse cycles, complete conversion of H3N•BH3 was observed in the reactions mediated with Pt•L1 0.2 , Pt•L2 0.2 and Pt•L3 0.2 nanoparticles, retaining the catalysts 55-66% of their initial activities after the third reutilization.

Tandem H3N•BH3 dehydrogenation/N-heterocycle hydrogenation

In addition to its use as energy carrier, ammonia borane has found applications as a reductant in synthetic organic chemistry, [4e,17] and the development of metal-catalyzed reductions using H3N•BH3 as hydrogen source has been explored. [18] Interestingly, hydrogen generation by ammonia borane methanolysis allows for an easy separation of the reaction products from the dehydrogenated by-products. Therefore, tandem H3N•BH3 dehydrogenation/hydrogenation of N-heterocycles catalyzed by Pt•L nanoparticles was examined to probe the potential of the catalysts in such a tandem process (Table 3). Hydrogenation of quinoline was cleanly carried out with 3 equivalents of H3N•BH3 in 91 and 58% yields using 1.0 mol% of Pt•L1 0.2 and Pt•L3 0.2 , respectively (entries 1 and 2). Moreover, nanoparticles Pt•L1 0.2 were also effective in the reduction of other N-heterocycles such as phenanthridine, 2-methylquinoxaline and acridine providing the corresponding reduction products in >90% yield (entries 3-5).

Conclusions

Narrowly dispersed Pt nanoparticles stabilized with sterically demanding terphenylphosphane ligands have been synthesized. These nanoparticles displayed high activities in the generation of H2 by methanolysis of H3N•BH3. Thus, by a simple adjustment of the structure of the phosphane ligand and the phosphane/Pt ratio, high turnover frequencies of up to 283 min -1 were observed at 30 o C and low catalyst loadings. Also, reusability studies of the catalysts have shown it is feasible although an erosion of the catalytic activity was observed after three catalytic runs. Finally, the possibility to use these catalysts for tandem H3N•BH3 dehydrogenation/N-heterocycle hydrogenation reactions has been demonstrated. It is worth mentioning that with the colloidal catalysts here described, additional weight and effects produced by supports as used with other nanocatalysts for the target reaction are avoided.

Experimental Section

General procedures and characterization techniques. All the reactions have been carried out in a glovebox or under nitrogen or argon atmosphere using Schlenk-type techniques. Solvents have been dried by already known procedures and distilled under argon or nitrogen atmosphere before their use. Methanol was dried with sodium methoxide (NaOMe) and distilled under argon. Ammonia borane was purchased from Aldrich, and used without any further purification. Terphenylphosphane ligands L1, [19] L2 [19] and L3 [20] were prepared according to already published procedures.

[Pt(dba)2] was obtained by a previously reported method, [21] and its %wt of Pt was determined by ICP. Pt NPs were analyzed by TEM and HRTEM after deposition of a drop of the crude THF colloidal solution or a drop of a solution of the isolated nanoparticles after dispersion in THF on a covered holey copper grid, respectively. TEM analyses were performed at the Centro de Investigación, Tecnología ) was added into the reactor. The Fisher-Porter bottle was pressurized with 3 bar of H2 and the solution was left to reach slowly the room temperature under vigorous stirring. The homogenous solution, which turns black after 30 minutes of reaction, was kept under stirring overnight at room temperature. After this period of time, excess of H2 was eliminated and the volume of solvent was reduced to 10 mL under vacuum. Pentane (40 mL) was added to the colloidal suspension which was cooled down to -30 ºC to precipitate the particles. After filtration under argon with a cannula, the black solid powder was washed twice with pentane (2 x 40 mL) and dried under vacuum. For Pt content determination by ICP and TEM analysis of the nanoparticles, see Table 1.

Catalytic reactions

Experimental setup for small scale H2 production. H2 generation was followed up using a Fisher-Porter vessel (25 mL) connected to a vacuum line and coupled to a ESI pressure gauge model GS4200-USB (0-6 bar) plugged to a computer.

Representative procedure for hydrogen generation. A freshly prepared solution of ammonia borane (1.9 M) in MeOH (1.0 mL, 1.9 mmol H3N•BH3) was added to a colloidal suspension of the nanoparticles Pt•L1 0.2 (1.0 mg, 3.3 mol Pt) in MeOH (0.5 mL) thermostated to 30 ºC and stirred at 750 rpm. H2 generation was monitored by registering the increase of pressure in the system. Quantitative formation of ammonium tetramethoxyborate (NH4B(OMe)4) was determined by 11 B NMR spectroscopy.

Large scale hydrogen generation. A freshly prepared solution of ammonia borane (1.9 M) in MeOH (8.0 mL, 15.2 mmol H3N•BH3) was added over 16 min to a colloidal suspension of nanoparticles Pt•L3 0.2 (0.7 mg, 2.5 mol Pt) in MeOH (0.25 mL) thermostated to 25 ºC and stirred at 750 rpm. H2 generation was monitored by measuring the gas evolved in a gas burette. Quantitative formation of ammonium tetramethoxyborate was determined by 11 B NMR spectroscopy.

Representative procedure for H2 generation with catalyst recycling.

A solution of ammonia borane (1.9 M) in MeOH (1.0 mL, 1.9 mmol H3N•BH3) was added to a colloidal suspension of the nanoparticles Pt•L1 0.2 (1.0 mg, 3.3 mol Pt) in MeOH (0.5 mL) thermostated to 25 ºC and stirred at 750 rpm. H2 generation was monitored by registering the increase of pressure in the system. After the reaction proceeded to completion, solvent and ammonium tetramethoxyborate were distilled off under vacuum. MeOH (0.5 mL) and a solution 1.9 M of H3N•BH3 in MeOH (1.0 mL, 1.9 mmol) were successively added to the nanoparticles and H2 generation was again monitored. The process was repeated over three times.

General procedure for tandem H3N•BH3 methanolysis/N-heterocycle hydrogenation. A 10 mL Fisher-Porter vessel was charged with a suspension of Pt•L1 0.2 (1.0 mg, 3.8 mol Pt) and the corresponding Nheterocycle (0.38 mmol) in MeOH (0.5 mL), and 0.75 mL (1.1 mmol of H3N•BH3) of a freshly prepared solution of H3N•BH3 in MeOH was added.

The reaction mixture was heated to 60 o C for 21 h. CAUTION: hydrogen pressure is developed. Product yield was determined by 1 H NMR spectroscopy using mesitylene as an internal standard.

Figure 1 .

 1 Figure 1. Terphenylphosphanes L used to stabilize Pt nanoparticles.

  %wt Pt content as determined by ICP. [b] Measured from TEM images. Standard deviations in parentheses.

Figure 2 .

 2 Figure 2. TEM images and size histograms of Pt•L1 0.2 (top, left) , Pt•L1 0.5 (bottom, left), Pt•L2 0.2 (top, right) and Pt•L3 0.2 (bottom, right).

Figure 3 .

 3 Figure 3. HRTEM (left) and FFT (right) images obtained for Pt•L1 0.2 .

Figure 4 .

 4 Figure 4. Hydrogen evolution from the methanolysis of H3N•BH3 catalyzed by Pt•L nanoparticles. See Table2for experimental details.

  [a] Reactions conditions, unless otherwise noted: 30 o C, [H3N•BH3] = 1.2 M. TOF values as calculated from (mmol of H2/mmol Pt) x min -1 . [b] [H3N•BH3] = 1.7 M.

Figure 5 .

 5 Figure 5. Large scale methanolysis of H3N•BH3 catalyzed by Pt•L3 0.2 nanoparticles. Reactions conditions: 0.016 mol% Pt, 25 o C, [H3N•BH3] = 1.8 M.

Figure 6 .

 6 Figure 6. Recycling experiments of Pt•L nanoparticles. Reaction conditions: 0.2 mol% Pt, 25 o C, MeOH, [H3N•BH3] = 1.3 M. TOF values as calculated from (mmol of H2/mmol Pt ) x min -1 .

Table 1 .

 1 Terphenylphosphane stabilized Pt NPs.

Table 3 .

 3 Tandem H3N•BH3 methanolysis/N-heterocycle hydrogenation with Pt•L nanoparticles.[a]

	Entry	Pt•L	Substrate	Product	yield [%]
	1	Pt•L1 0.2			91
	2	Pt•L3 0.2			58
	3	Pt•L1 0.2			92
	4	Pt•L1 0.2			>99
	5	Pt•L1 0.2			91
	[a] Reaction conditions: 3 equiv H3N•BH3, 1.0 mol% Pt, 60 o C, MeOH, [S]
	= 0.3 M, 21 h.

Acknowledgements

Financial support (FEDER contribution) from the Spanish MINECO (CTQ2016-80814-R and CTQ2016-81797-REDC) is gratefully acknowledged. CNRS and Université Fédérale de Toulouse are also acknowledged for financial supports. Prof.

thanked for HRTEM -EDX measurements.

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The synthesis of a series of narrowly dispersed phosphanedecorated Pt nanoparticles is reported. These nanoparticles are highly efficient catalysts for hydrogen generation by methanolysis of ammonia borane (H3N•BH3). Reusability studies of the catalysts have been carried out, as well as the use of these nanoparticles for tandem H3N•BH3 dehydrogenation/Nheterocycle hydrogenation processes.


Liliana Perdomo

Xavier Vidal-Gomez

Raffaella Soleti

Luisa Vergori

Lucie Duluc

Maggy Chwastyniak

Malik Bisserier

Soazig Le Lay

Alexandre Villard

Gilles Simard

Liliana Rap1

Perdomo

Olivier Meilhac

Frank Lezoualc'h

Ilya Khantalin

Reuben Veerapen

Séverine Dubois

Jérôme Boursier

Samir Henni

Frédéric Gagnadoux

Florence Pinet

Ramaroson Andriantsitohaina

PhD M Carmen Martinez
email: carmen.martinez@univ-angers.fr

Large Extracellular Vesicle-Associated Rap1 Accumulates in Atherosclerotic Plaques, Correlates with Vascular Risks and Is Involved in Atherosclerosis

cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis.

INTRODUCTION

Metabolic syndrome (MetS) patients display an approximate two-fold increased risk of developing atherosclerotic cardiovascular diseases. This syndrome is described as a cluster of abdominal obesity, insulin resistance, elevated blood pressure (BP), and obesity-related dyslipidemia consisting in low levels of HDL-cholesterol and high levels of triglycerides. [START_REF] Harris | The metabolic syndrome[END_REF][START_REF] Alberti | Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity[END_REF] In the early stages of atherosclerosis, typical human atherosclerotic lesions contain different cell types, including vascular smooth muscle cells (SMC), endothelial cells, fibroblasts, macrophages, and foam cells involved in inflammatory responses and development of plaque. [START_REF] Ross | Atherosclerosis-an inflammatory disease[END_REF] Proliferation and migration of vascular SMC from the tunica media to the subendothelial region, also known as neointima formation play a crucial role in vascular remodeling, a key step for blood vessel thickening and occlusion. [START_REF] Bentzon | Mechanisms of plaque formation and rupture[END_REF] Over the last years, extracellular vesicles (EVs) have been associated with early processes involved in the development of atherosclerotic lesions, including endothelial dysfunction, inflammation, reactive oxygen species production, and monocyte/macrophage adhesion (for review see). [START_REF] Malloci | Extracellular vesicles: Mechanisms in human health and disease[END_REF] Two main EV subtypes have been described: large EVs (lEVs) released from plasma blebbing and small EVs (sEVs) generated within endosomes forming multivesicular endosomal compartments. Foam cell-derived EVs from atherosclerotic patients were shown to promote SMC migration and adhesion by regulating actin cytoskeleton and focal adhesion pathways after the endocytosis of EVs into SMCs and the subsequent downstream activation of ERK and Akt. [START_REF] Niu | Macrophage foam cell-derived extracellular vesicles promote vascular smooth muscle cell migration and adhesion[END_REF] Regarding MetS, we have reported that circulating lEVs from MetS patients (MetS-lEVs) carrying Fas ligand induce endothelial dysfunction by interacting with Fas receptor on endothelial cells; resulting in a temporal cross-talk between endoplasmic reticulum and mitochondria that increased reactive oxygen species production from cytoplasm and mitochondria via the activation of neutral sphingomyelinase and reduced nitric oxide (NO) release. [START_REF] Agouni | Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome[END_REF][START_REF] Safiedeen | Temporal cross talk between endoplasmic reticulum and mitochondria regulates oxidative stress and mediates microparticle-induced endothelial dysfunction[END_REF] In addition, circulating MetS-lEVs induced hyporeactivity associated with oxidative stress and overexpression of pro-inflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1) and inducible NO synthase. [START_REF] Agouni | Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice[END_REF] However, the effects of circulating MetS-lEVs, on SMC specifically, in the early stage of atherosclerosis have not been fully elucidated.

In the present study, proteomic analysis showed that Rap1 was >1.4-fold increased in MetS-lEVs and that lEVs carrying Rap1 induce aortic SMC migration and proliferation as well as pro-inflammatory cytokine secretion in vitro. All of these effects were abolished when Rap1 carried by lEVs was neutralized or pharmacologically inhibited. Levels of lEVs carrying Rap1 were correlated with waist and hip circumferences, body mass index (BMI), triglyceridemia, insulinemia, high sensitivity C-reactive protein (hs-CRP) and increased stenosis risk of MetS patients. These results suggested that Rap1 carried by lEVs was critical for the development of atherosclerosis associated with MetS. This was further reinforced by the observation that high fat diet (HFD)-fed ApoE -/-mice displayed an increased expression of Rap1 in circulating lEVs that also enhanced SMC proliferation. Also, lEVs from mouse and human atherosclerotic plaques were enriched in Rap1. In conclusion, we propose Rap1 + -lEVs produced in excess as a cause for vascular remodeling and inflammation leading to the progression of atherosclerosis in the setting of MetS.

METHODS

Detailed methods are available in the Online Data Supplement. The data, analytic methods, and study materials for the purposes of reproducing the results or replicating procedures can be made available on request to the corresponding author who manages the information.

MetS patients

This study was approved by the ethics committee of the University Hospital of Angers (France).

Non-MetS donors (nMetS) and MetS patients were recruited in the NUMEVOX cohort (NCT: 00997165). A total of 90 subjects were identified as MetS patients and 72 subjects who displayed two or less MetS criteria were identified as nMetS subjects. Baseline characteristics and clinical data of nMetS and MetS subjects are summarized in Online Table I.

Ultrasonography of carotid arteries

Velocities and resistance indexes of carotid arteries were determined by duplex ultrasonography following the most recent recommendations of the European Society of Cardiology.

lEV isolation from blood

Peripheral blood (20 mL) from nMetS and MetS patients was collected to isolate circulating lEVs and used at the circulating concentration detected for each individual. [START_REF] Agouni | Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome[END_REF][START_REF] Safiedeen | Temporal cross talk between endoplasmic reticulum and mitochondria regulates oxidative stress and mediates microparticle-induced endothelial dysfunction[END_REF]

Proteomic study by 2D-DIGE electrophoresis

Two gels of each were run with a total of 10 μg of mixed fluorescent-labelled proteins per gel containing 5 μg of proteins from lEVs (5 nMetS and 5 MetS patients) randomly labelled with 400 pmol saturation CyDye DIGE fluors (Cy3 or Cy5). After dye-swap, Cy3-and Cy5-labelled samples were mixed and used to rehydrate the immobilized pH gradient (IPG) strip before perform the isoelectric focusing (IEF). After IEF, the IPG strips were incubated in equilibration buffer and transferred for the second dimension electrophoresis (SDS-PAGE) onto 12% PAGE gels and sealed with low-melting agarose. Following electrophoresis, gels were scanned with an Ettan DIGE Imager (GE Healthcare) at excitation/emission wavelengths of 532/580 nm for Cy3 and 633/670 nm for Cy5 to yield images with a pixel size of 100 μm. Differentially regulated spots were excised manually from a preparative gel performed with 500 µg of total proteins. Identifications were performed by MALDI-TOF.

Rap activation assay

Rap1 pull-down experiments were performed using a GST fusion protein containing the Rap1 binding domain of Ral-GDS.

Characterization of lEV phenotype

The counting and phenotyping of the MetS-lEVs were performed by flow cytometry from platelet-free plasma according to the expression of membrane-specific antigens. [START_REF] Agouni | Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome[END_REF][START_REF] Safiedeen | Temporal cross talk between endoplasmic reticulum and mitochondria regulates oxidative stress and mediates microparticle-induced endothelial dysfunction[END_REF] Validation of isolated lEVs was performed by detection of positive and negative markers (Online Figure IA).

Cellular models

Primary human aortic endothelial cells (HAoECs, Promocell, Heidelber, Germany) were grown in endothelial basal medium supplemented with 10% of fetal bovine serum. Primary human aortic smooth muscle cells (HASMC, Gibco) were cultured in Medium 231 (Gibco) supplemented with SMGS (Gibco). Primary vascular SMC were obtained from thoracic aorta arteries from WT C57BL/6 male mice, according to number S380 ethic local protocol.

Trans-epithelial electric resistance (TEER) measurement of HAoECs

HAoECs were seeded in 8 μm pore size Transwells (Corning, Corning, NY) in the upper chamber until 100% confluence. TEER measurement was performed before pre-incubation with the Rap1 inhibitor and after 24 hours, using a Millicell® ERS-2 Voltohmmeter (Merck, Darmstadt, Germany) to assess the integrity of the barrier.

Migration and proliferation assays

For wound healing assay, HASMC were cultured in 231-SMGS medium until they achieve 100% confluence. Confluent monolayer was scratched with a sterile pipette tip. Wound healing closure was followed by phase-contrast microscopy at 0 and 24 hours. Also, migration was analyzed by Transwell assay. Cell Proliferation Assay Kit CyQUANT® (Invitrogen, Carlsbad, CA) and Ki67 staining were used to analyze proliferation.

Western Blotting

After treatment, HASMC were homogenized. Equal amounts of proteins were separated in 4-15% TGX-midi gels by electrophoresis and then transferred to nitrocellulose sheets. Densitometric analyses of Western blots were performed using ImageJ software.

Quantitative RT-PCR analysis

After treatments, frozen cell pellets were used to analyze the genetic expression of human MCP-1 and IL-6 mRNA by RT-PCR.

Animal model

All animal studies were carried out using approved institutional protocols (n°APAFIS320).

Male ApoE -/-were allowed free access to standard diet (STD) or HFD. lEVs were isolated from blood and from atherosclerotic plaque. The effects of circulating lEVs isolated from the ApoE - /-mice on the proliferation of primary SMC isolated from WT mice was evaluated.

Staining and imaging of mouse aorta wall by confocal microscopy

Frozen mouse aortic ring sections were prepared and treated with primary antibodies. Following three washes with PBS, secondary antibodies were applied to detect F4/80 and Rap1. Nuclei were counterstained with DAPI. Confocal microscopy and digital image recording were used.

lEVs from human atherosclerotic plaque

Human atherosclerotic plaques from 5 patients undergoing carotid endarterectomy who gave their informed consent were used for lEV isolation.

Statistical Analysis

Data normality was assessed using the Shapiro-Wilk test. Comparisons were made between all conditions for each panel. Statistical analysis was performed by using ANOVA followed by Tukey post-hoc test for multiple comparisons. In cases where normality was not confirmed, statistical analysis from two groups and more than two groups were analyzed by Mann-Whitney

RESULTS

MetS patients showed a significant increase in BMI, waist circumference indicating greater visceral obesity, BP, insulinemia, glycaemia, HbA1c, triglycerides, and hs-CRP levels, and decreased plasma HDL levels (Online Table I). In addition, the total number of circulating lEVs was significantly increased in patients with MetS compared to nMetS subjects (Online Table I). As we have previously shown, [START_REF] Agouni | Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome[END_REF][START_REF] Safiedeen | Temporal cross talk between endoplasmic reticulum and mitochondria regulates oxidative stress and mediates microparticle-induced endothelial dysfunction[END_REF][START_REF] Agouni | Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice[END_REF] MetS patients displayed an increase of ~1.7-fold platelet (CD41 + )-derived lEVs (Online Table I).

Rap1 activation is increased in lEVs from patients with MetS

To identify proteins carried by MetS-lEVs that could play a role in vascular remodeling, we performed a differential proteomic analysis by 2D-DIGE comparing the replicates (Cy3-and Cy-5 labeling) prepared from nMetS and MetS. Among the 608 spots detected (Online Figure IB), we found 10 spots differentially expressed (P<0.05) between nMetS-and MetS-lEVs (Figure 1A). Identification of proteins present in each spot by MALDI-TOF allowed us to identify 8 proteins differentially expressed in MetS subjects compared to lEVs isolated from nMetS subjects (Online Table II). Interestingly, spot 534 corresponds to the Ras-related protein Rap1 which has emerged as a central player in cardiovascular pathophysiology. [START_REF] Métrich | Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology[END_REF] Detailed 2Dspots corresponding to Rap1 are presented for each sample of lEVs from nMetS and MetS patients analyzed (Online Figure IC). We confirmed by immunoblot that MetS-lEVs displayed 50% increase of Rap1 expression compared to nMetS-lEVs (Figure 1B). We also analyzed Rap1 activation by pull down assay using the Ral-GDS fusion protein in lEVs. Interestingly, we found that MetS-lEVs had higher levels of activated Rap1 (Figure 1C). To determine the location of this protein within the lEVs, Rap1 was analyzed on native or permeabilized MetS-lEVs. As shown in Figure 1D, Rap1 labeling was similar in non-permeabilized and permeabilized lEVs suggesting that Rap1 is essentially exposed to the surface of lEVs. Thus, further experiments were performed in non-permeabilized lEVs. Under these conditions, the concentration of circulating lEVs bearing Rap1 were significantly higher in MetS compared to nMetS subjects (Figure 1E). The majority of Rap1 + -lEVs were also CD41 + suggesting that they originated from platelets (Figure 1F). We have also detected low levels of Rap1 + -EVs from endothelial cells (CD146 + ), erythrocytes (CD235a + ), leukocytes (CD45 + ) and granulocytes (CD11b + ). It should be noted that all of these subpopulations of Rap1 + -lEVs were increased in MetS patients versus nMetS patients. Altogether, these results show that lEVs from MetS patients had higher levels of activated Rap1 at their surface.

Expression level of Rap1 in lEVs is closely correlated with a cluster of cardiometabolic risk factors in MetS patients

We analyzed the relationship between circulating levels of lEVs and a cluster of anthropometric parameters and cardiometabolic risk factors (Figure 2). Neither total circulating lEVs, nor platelet-and endothelial-derived lEVs were correlated with any of these parameters (not shown). Interestingly, circulating lEVs carrying Rap1 were positively correlated with waist and hip circumferences, BMI (r= 0.1936, p= 0.0219), the mean heart rate, insulinemia, plasma triglyceride concentration and hs-CRP concentration (Figure 2A-F). Consistently, the triglyceride-glucose index (TyG index) was also positively correlated with the circulating level of Rap1-lEVs in MetS (Figure 2G), reflecting insulin resistance in these patients.

Vascular parameters such as aorta diameter or pulse wave velocity did not correlate with the number of MetS criteria neither with lEVs carrying Rap1 (not shown). Interestingly, the right common carotid end diastolic velocity (EDV) was positively correlated with the levels of Rap1-lEVs (Figure 2H). Finally, the analysis performed on 162 subjects displaying 0 to 5 of MetS criteria showed that circulating levels of lEVs carrying Rap1 increased depending on the number of the components of MetS, thus its severity (Figure 2I). These data suggest that circulating levels of Rap1 carried by MetS-lEVs are associated with risk factors of cardiovascular events in MetS patients, identifying Rap1 carried by MetS-lEVs as a potential molecular link leading to the development of vascular diseases, such as atherosclerosis.

lEVs from MetS patients increase endothelial cell permeability sensitive to the Rap1 inhibitor, GGTi-298

Incubation for 24 hours with lEVs from MetS patients decreased transendothelial electrical resistance (TEER) whereas those from nMetS patients had not significant effects (Figure 3A).

Interestingly, the pharmacological Rap1 inhibitor, GGTi-298, improved MetS-lEVs-induced reduction of TEER values (Figure 3A). In addition, MetS-lEVs increased monocyte transcytosis through the endothelial cell monolayer and this effect was prevented by GGTi-298 (Figure 3B).

Altogether, these data indicate that lEVs from MetS patients, but not those from nMetS patients, enhance endothelial permeability by a mechanism sensitive to GGTi-298. These results also suggest that MetS-lEVs can reach SMC into the vessel through this pathway.

Rap1 carried by circulating lEVs induces migration and proliferation of human aortic smooth muscle cells (HASMC)

To decipher the interaction between lEVs and vascular SMC, we studied lEV internalization in HASMC by confocal microscopy and showed that lEVs from nMetS and MetS patients labeled MetS-lEVs, but not nMetS-lEVs, significantly increased the number of HASMCs and this effect was abolished by neutralization of Rap1 (Figure 3E). In line with these findings, the increase of cell number induced by MetS-lEVs was abolished in the presence of the Rap1 pharmacological inhibitor, GGTi-298 (Figure 3F). Similar results were observed when HASMC proliferation was measured by Ki67 staining assay (Online Figure IIE). Altogether, these results suggest that Rap1 carried by lEVs from MetS plays a critical role in migration and proliferation of HASMC, as general features of vascular remodeling.

Rap1 carried by lEVs activates MEK5-ERK5 and p38 pathways to induce proliferation of human aortic smooth muscle cells

Since the mechanisms underlying the crosstalk between small GTPase proteins and MAPK signaling have also been intensively investigated, [START_REF] Romano | Differential involvement of the Ras and Rap1 small GTPases in vasoactive intestinal and pituitary adenylyl cyclase activating polypeptides control of the prolactin gene[END_REF] we analyzed the effects of MetS-lEVs on MAPKs signaling pathway in HASMC, with a particular focus on ERK1/2, ERK5 and p38 MAPK. Neither PDGF, 8-CPT, nMetS-lEVs nor MetS-lEVs increased phosphorylation of ERK1/2 (Online Figure IVA-IVB). Interestingly, PDGF, 8-CPT, MetS-lEVs, but not nMetS-lEVs, significantly increased ERK5 phosphorylation (Figure 4A-B) which was significantly reduced after neutralization of Rap1 (Figure 4A-B). Moreover, the ability of MetS-lEVs to enhance the number of HASMC was abrogated in the presence of ERK5 inhibitor, BIX02189 (Figure 4C). These data suggest that ERK5 pathway triggers the ability of lEVs from MetS carrying Rap1 to increase HASMC proliferation. Finally, MetS-lEVs, but not PDGF, 8-CPT or nMetS-lEVs, increased p38 MAPK phosphorylation in HASMC and this effect was inhibited after neutralization of Rap1 carried by MetS-lEVs (Figure 4A-B). Altogether, these results showed that ERK5 and p38 MAPK, but not ERK1/2, were activated by MetS-lEVs in HASMC.

MetS-lEVs increase MCP-1 and IL-6 expression in a Rap1-dependent manner

Rap1 is an important regulator of the vascular inflammatory response. [START_REF] Métrich | Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology[END_REF] To confirm the contribution of Rap1-lEVs on the inflammatory response observed during MetS, the effects of nMetS-lEVs and MetS-lEVs were tested on mRNA expression of different pro-inflammatory cytokines in HASMC. We found that MetS-lEVs, but not nMetS-lEVs, significantly increased mRNA expression of MCP-1 and IL-6 (Figure 5A-5B). Neutralizing Rap1 carried by MetS-lEVs completely abrogated their ability to increase MCP-1 and IL-6 mRNA expressions in HASMC (Figure 5A-5B). In contrast, neither MetS-lEVs nor nMetS-lEVs modified VCAM-1 and ICAM-1 protein expressions and NF-κB pathway activation (Online Figure IVC). These data suggest that MetS-lEVs increase the expression of inflammatory cytokines, MCP-1 and IL-6, which have been previously described to be overexpressed in patients with increased cardiovascular risk factors.

Pathophysiological relevance of Rap1 in the atherosclerotic process

To study the pathophysiological relevance of Rap1 in the atherosclerotic process, we used the HFD-fed ApoE -/-mouse model, exhibiting an exacerbation of the formation of atherosclerotic lesions. Macrophage infiltration was analysed by confocal microscopy by staining of F4/80 together with the expression of Rap1 in aortic roots from STD-and HFD-fed ApoE -/-mice. We observed an enhanced macrophage infiltration in aorta of HFD-fed ApoE -/-mice when compared to STD-fed ApoE -/-mice (Figure 6A-6B). Rap1 staining was increased in the medial layer of the aorta from HFD-fed ApoE -/-compared to that from STD-fed ApoE -/-mice (Figure 6A-6B). Circulating lEVs from HFD-fed ApoE -/-displayed greater expression of Rap1 compared to those from STD-fed ApoE -/-mice (Figure 6C). Interestingly, circulating lEVs from HFD-fed ApoE -/-mice, but not those from STD-fed ApoE -/-, enhanced the number of primary murine aortic SMC (Figure 6D).

We tested whether lEVs expressing Rap1 were localized in atherosclerotic plaques in ApoE -/- mice. For this, lEVs were isolated from atherosclerotic plaques and characterized by flow cytometry. lEVs essentially were from SMC (74%), leukocytes (11%), macrophages (5%) and platelets (6%) (Online Figure V). In addition, atherosclerotic aortic plaques from HFD-fed ApoE -/-mice contained higher levels of lEVs carrying Rap1 than arterial wall of aorta from STD-fed ApoE -/-mice (Figure 6E). Interestingly, the content of Rap1 + -lEVs increased depending on the stage of plaque development, being similar at 8, 12 or 16 weeks in STD-fed mice, but increasing between 12 and 16 weeks in HFD-fed mice (Figure 6E). These results indicate that accumulation of Rap1 + -lEVs in atheroma plaques is associated with the progression of the atherosclerotic plaque in ApoE -/-mice and strengthened our hypothesis that Rap1 carried by circulating lEVs plays a role in the development of atherosclerosis in HFD-fed ApoE -/-mice.

Finally, we analyzed the presence of Rap1 + -lEVs on human atherosclerotic plaque samples.

Rap1 + -lEVs were more abundant in human atherosclerotic plaques than in the arterial wall adjacent to the lesion (Figure 7). The analysis of the content of lEVs from human atherosclerotic plaque showed that they were mainly originated from SMC (42%), leukocytes (19%), platelets (16%) and endothelial cells (14%) (Online Figure V). These results corroborate that lEVs expressing Rap1 accumulate in atherosclerotic lesions.

DISCUSSION

The present study highlights the role of circulating lEVs carrying Rap1 as a crucial driver of the early steps of atherosclerosis such as SMC hyperplasia and inflammation in MetS.

Circulating MetS-lEVs, but not nMetS-lEVs, increased endothelial cell permeability and monocyte transcytosis suggesting that, in vivo, they can reach smooth muscle layer into the vessels. In addition, we demonstrated that MetS-lEVs overexpressed the small protein GTPase Rap1 on the surface of lEVs in its active GTP-bound form. Moreover, circulating levels of Rap1-lEVs were not only positively correlated with the severity of MetS and obesity indicators, such as waist and hip circumferences and BMI, but also with risk factors to develop cardiovascular diseases, such as, increased heart rate, hyperinsulinemia, hypertriglyceridemia, TyG index, hs-CRP and right common carotid EDV. At cellular and molecular level, lEVs carrying Rap1 induced SMC migration and proliferation by a mechanism involving Rap1/ERK5 axis and pro-inflammatory profile. Finally, by using a mouse model of atherosclerosis and plaques from patients undergoing carotid endarterectomy, we showed that lEVs carrying Rap1 accumulated in atherosclerotic lesions strongly supporting the critical role of Rap1 carried by lEVs in vascular remodeling leading to atherosclerosis. Since SMC hyperplasia is a critical mechanism involved in the early steps of atherosclerosis, Rap1 carried by lEVs could represent a promising therapeutic target to limit the development of atherosclerosis through the reduction of both SMC proliferation and SMC pro-inflammatory profile.

MetS criteria including central obesity, hypertension, hyperglycemia, hypertriglyceridemia, and low HDL levels, are commonly associated with the risk of developing atherosclerosis and cardiovascular events. [START_REF] Barrachina | Application of Extracellular vesicles proteomics to cardiovascular disease: guidelines, data analysis, and future perspectives[END_REF] However, there is no existing valid biomarker to accurately predict the risk of developing cardiovascular diseases since it is difficult to demonstrate the relationship between cardiovascular diseases and each MetS criterion, as they overlap. EVs are important players in intercellular communication and have been described as potential biomarkers in different pathologies, such as diabetes and obesity. It has been recently reported that EVs have an impact on cardiovascular physiology and atherothrombotic events. [START_REF] Barrachina | Application of Extracellular vesicles proteomics to cardiovascular disease: guidelines, data analysis, and future perspectives[END_REF][START_REF] Martínez | Extracellular vesicles in metabolic syndrome[END_REF] Interestingly, lEVs have been highlighted as important actors during the initiation and the progression of atherosclerotic lesions due to their capacity to interact with endothelial cells, SMC and circulating cells such as monocytes. [START_REF] Malloci | Extracellular vesicles: Mechanisms in human health and disease[END_REF] In the present study, we showed that lEVs isolated from MetS patients had higher levels of activated Rap1 at their surface compared to nMetS-lEVs. It has been shown that when Rap1 is localized in the plasma membrane, the GTPase is activated; whereas the intracellular pool of Rap remained GDP-bound and, in consequence, not activated. [START_REF] Bivona | Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion[END_REF][START_REF] Torti | Structure and function of Rap proteins in human platelets[END_REF] Since Rap1 is detected by flow cytometry without permeabilization of lEVs, we hypothesize that activated Rap1 is present at the membrane of lEVs and that probably limits the ability of Rap1 to cycle between the GDPand GTP-bound states. We also demonstrated that circulating levels of lEVs carrying Rap1 increased depending on the severity of MetS, and positively correlated with abdominal obesity and BMI, and with the cardiovascular risk factors, including enhanced heart rate, hyperinsulinemia, hypertriglyceridemia and TyG index reflecting insulin resistance. These findings corroborate previous work of our team showing that obese patients displayed enhanced levels of circulating lEVs, [START_REF] Amosse | Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor[END_REF] and strongly suggest that lEVs expressing Rap1 might be useful to early identify individuals at a high risk of developing cardiovascular events. In this context, Cooney and colleagues [START_REF] Cooney | Elevated resting heart rate is an independent risk factor for cardiovascular disease in healthy men and women[END_REF] have shown that the cardiovascular risk associated with each 15 beats/min increase in resting heart rate was 1.24 in men and 1.32 in women, demonstrating a relationship between heart rate and incident cardiovascular. Also, hyperinsulinemia can predict adverse cardiac events in type 2 diabetes mellitus, [START_REF] Srinivasan | Basal hyperinsulinemia beyond a threshold predicts major adverse cardiac events at 1 year after coronary angiogram in type 2 diabetes mellitus: a retrospective cohort study[END_REF] hypertriglyceridemia is an independent marker of ischemic events risk, [START_REF] Toth | High triglycerides are associated with increased cardiovascular events, medical costs, and resource use: a real-world administrative claims analysis of statin-treated patients with high residual cardiovascular risk[END_REF] while TyG index can identify early individuals at a high risk of developing cardiovascular events. [START_REF] Sánchez-Íñigo | The TyG index may predict the development of cardiovascular events[END_REF] Levels of lEVs carrying Rap1 also correlated with levels of hs-CRP which is a described marker to estimate the risk of plaque instability [START_REF] Garcia | High-sensitivity C-reactive protein in high-grade carotid stenosis: risk marker for unstable carotid plaque[END_REF] and vulnerability of atheromatous lesion. [START_REF] Zhong | Role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism[END_REF] To corroborate this, lEVs carrying Rap1 were also correlated with right common carotid EDV, which is associated with a high probability to develop stenosis. [START_REF] Strosberg | Common carotid artery end-diastolic velocity and acceleration time can predict degree of internal carotid artery stenosis[END_REF] All these clinical data strongly support that circulating levels of lEVs harboring Rap1 could be useful to evaluate the risks of developing cardiovascular events in MetS patients.

Our data show that MetS-lEVs, but not those from nMetS patients, enhanced endothelial permeability and monocyte transcytosis by a mechanism sensitive to GGTi-298 suggesting that MetS-lEVs can reach SMC into the vessel. In line with these results, we have previously reported that lymphocyte-derived lEVs injected in vivo were able to interact directly with SMC as evidenced by an increased CD4 labelling in the media layer of aortas from lEV-treated mice compared to control vessels. [START_REF] Tesse | Upregulation of proinflammatory proteins through NF-CIRCRES/2019/316082R1 kappaB pathway by shed membrane microparticles results in vascular hyporeactivity[END_REF] We also found that iv injection of MetS-lEVs into mice induced vascular hypo-reactivity that is associated with up-regulation of inducible NO-synthase and increased production of NO. [START_REF] Agouni | Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice[END_REF] Activation of Rap1 was associated with the enhancement of vascular SMC migration in vitro and the development of neointimal thickening in fetal rat aortic tissues in organ culture. [START_REF] Yokoyama | Epac1 is upregulated during neointima formation and promotes vascular smooth muscle cell migration[END_REF] To confirm the involvement of Rap1 carried by lEVs in the early stages of atherosclerosis, we analyzed the effects of lEVs on HASMC migration, proliferation and cytokine production. Our findings show that MetS-lEVs significantly increased the migration and the proliferation of HASMC via a mechanism involving Rap1 and the subsequent activation of ERK5. We observed that the effects of Rap1 carried by MetS-lEVs on HASMC proliferation was inhibited by using a selective small-molecule ATP-site inhibitor of MEK5/ERK5 signaling, BIX02189. These results are in accordance with a recent study published by Li and colleagues [START_REF] Li | Rap1 promotes proliferation and migration of vascular smooth muscle cell via the ERK pathway[END_REF] suggesting that transfection with the activated Rap1 protein directly modulates the phosphorylation of ERK pathways and promotes proliferation, differentiation and migration of vascular SMC. Indeed, as other small G proteins, Rap1 is able to directly activate several MAPKs. In this respect, it has been shown that, in cortical neurons, constitutive active Rap1 is sufficient to activate ERK5 via the MEKK2-MEK5 signaling cascade. [START_REF] Wang | Brain-derived neurotrophic factor activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade[END_REF] ERK5 has also been described as a central mediator of TLR2-dependent inflammatory signaling in human endothelial cells and monocytes; and activation of TLR2 led to the up-regulation of inflammatory mediators via NF-κB, JNK, and p38 MAPK, promoting the attachment of human neutrophils to lung microvascular endothelial cells. [START_REF] Wilhelmsen | ERK5 protein promotes, whereas MEK1 protein differentially regulates, the Toll-like receptor 2 protein-dependent activation of human endothelial cells and monocytes[END_REF] We also looked at p38 MAPK activation which has been previously associated with the development and progression of atherosclerosis by up-regulating migration, proliferation, permeability, apoptosis and adhesion molecule expression in endothelial cells. [START_REF] Blanc | Synchronous activation of ERK 1/2, p38mapk and PKB/Akt signaling by H2O2 in vascular smooth muscle cells: potential involvement in vascular disease[END_REF] It has also been described that an increase of Rap1 mediated by p38 MAPK activation enhanced migration and invasion in fibroblasts. [START_REF] Priego | C3G knock-down enhances migration and invasion by increasing Rap1-mediated p38α activation, while it impairs tumor growth through p38α-independent mechanisms[END_REF] Similar to ERK5, we observed that MetS-lEVs increased p38 MAPK phosphorylation, which was prevented upon Rap1 neutralization. Migration and proliferation processes are altered during vascular remodeling as part of the reparative response to injury and can lead to neointimal formation associated with infiltration of inflammatory cells and matrix production. [START_REF] Renna | Pathophysiology of vascular remodeling in hypertension[END_REF] Furthermore, our findings showed that MetS-lEVs, by a Rap1-dependent mechanism, increased mRNA expression of the pro-inflammatory cytokines MCP-1 and IL-6, which were described to be involved in the development of atherosclerosis. [START_REF] Lima | Adipokines, diabetes and atherosclerosis: an inflammatory association[END_REF] Neither MetS-lEVs nor nMetS-lEVs modified VCAM-1 and ICAM-1 expression and NF-κB pathway activation. Several hypotheses can be advanced to explain these findings. Thus, it has been described that whereas VCAM-1 and ICAM-1 expression is mainly regulated by NF-κB pathway, [START_REF] Amrani | Up-regulation of ICAM-1 by cytokines in human tracheal smooth muscle cells involves an NF-kappa B-dependent signaling pathway that is only partially sensitive to dexamethasone[END_REF] IL-6 and MCP-1 release can be regulated by other transcription factors different to NF-κB, such as CREB [START_REF] Song | A novel TLR4mediated signaling pathway leading to IL-6 responses in human bladder epithelial cells[END_REF] or AP-1. [START_REF] Lee | 5, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability[END_REF] Nevertheless, these results and those previously described [START_REF] Agouni | Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice[END_REF] showing an increased MCP-1 expression in the aorta after iv injection of MetS-lEVs into mice suggest that MetS-lEVs could potentially increase the recruitment of pro-inflammatory circulating cells such as leukocytes, under conditions associated with vascular inflammation. Finally, by using an animal model prone to the atherosclerotic lesion, the HFD-fed ApoE -/- mouse model, we confirmed the crucial role of Rap1 in the development of atherosclerosis. We showed that the aorta of HFD-fed ApoE -/-mice displayed enhanced expression of Rap1 and macrophage infiltration. In addition, Rap1 was overexpressed in circulating lEVs from atherosclerotic mice; and that these lEVs induced proliferation of murine aortic SMC.

Moreover, since the content of Rap1 + -lEVs increased depending on the stage of plaque development in HFD-fed mice and Rap1-lEVs accumulated in atherosclerotic lesions from humans undergoing carotid endarterectomy, our results strongly suggest the involvement of Rap1-lEVs in the progression of atherosclerosis.

Limitations: A large number of patients declined to participate in the vascular function analysis.

Accordingly, vascular function measurements were performed only on 20% of nMetS subjects and 50% of MetS patients. Although the number of patients is low, significant correlation between lEVs carrying Rap1 and right common carotid EDV was found suggesting that this vascular parameter is strongly associated with lEVs expressing Rap1. Also, it should be noted that 73% of nMetS patients displayed 1 or 2 criteria of MetS, and this could explain the trend to increase SMC migration and proliferation induced by lEVs from nMetS patients.

Nevertheless, the effects of lEVs from nMetS patient were not significant and never prevented when Rap1 in nMetS-lEVs was neutralized or inhibited suggesting that Rap1 does not account for these effects. In addition, the fact that nMetS-lEVs did not increase endothelial cell permeability strongly suggests that the ability of circulating nMetS-lEVs to reach smooth muscle layer under in vivo conditions is very restricted. The present study shows the crucial role of Rap1 carried by lEVs on the processes involved in SMC remodeling including SMC proliferation, migration and secretion of pro-inflammatory cytokines; however, the molecular link between Rap1 and pro-inflammatory cytokine production needs to be further explored.

Several approaches may be used but none with optimal results. Firstly, although constitutive Rap1 -/-mice are available, their use cannot help us since whole-body knockout implicates the lack of Rap1 also in target SMC. Another possibility is the use of Rap1 plasmids to overexpress Rap1; however, in this case, only one type of cells generating Rap1-enriched lEVs will be transfected, and in the present study, lEVs overexpressing Rap1 are from several cell type origin (Figure 1F). Nonetheless, we demonstrate by using a specific antibody and a pharmacological inhibitor the involvement of Rap1 carried by lEVs from MetS patients in the early steps of atherosclerosis.

In conclusion, circulating lEVs from MetS patients had higher levels of activated Rap1 protein and that correlated with cardiovascular risk factors. MetS-lEVs carrying Rap1 increased mRNA expression of MCP-1 and lL6 as well as the phosphorylation level of ERK5 and p38 MAPK.

By activating ERK-MAPK pathways, MetS-lEVs induced the migration and proliferation of SMC, processes involved in vascular remodeling. All of these effects were reduced or abolished when Rap1 was neutralized or pharmacologically blocked. In vivo, atherosclerotic HFD-fed ApoE -/-also displayed high expression of Rap1 in circulating lEVs which also promoted proliferation of murine aortic SMC. Since we have previously shown that, in endothelial cells, MetS-lEVs induce endoplasmic reticulum stress associated with an increase of oxidative stress through the activation of the Fas/Fas ligand pathway, [START_REF] Safiedeen | Temporal cross talk between endoplasmic reticulum and mitochondria regulates oxidative stress and mediates microparticle-induced endothelial dysfunction[END_REF] the results of the present study highlight the deleterious effects of these lEVs on vascular cells. In this respect, it has been shown that the Fas/Fas ligand pathway triggers activation of Rap1 signaling leading to integrin activation, and then, the induction of pro-inflammatory cell recruitment. [START_REF] Gao | Endothelial cellderived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion[END_REF] Thus, in a more integrative model, MetS-lEVs act first on the endothelial cells and through both Fas/Fas ligand and Rap1 pathway they evoke the decrease in NO production and the increase on endothelial cell permeability; secondly, MetS-lEVs reach SMC and favor early steps of atherosclerosis. Taken together, these results support the hypothesis that Rap1 + -lEVs produced in excess could represent a promising therapeutic target to prevent or limit the progression of atherosclerosis in MetS subjects, as well as a biomarker to predict vascular consequences of MetS.

DISCLOSURE

The authors have declared that no conflict of interest exists.

ONLINE MATERIALS

Expanded Materials & Methods.

Online Tables I-IV. I-V.

Online Figures

U

  test, and Kruskal-Wallis test with Dunn's test post-hoc, respectively. No experiment-wide multiple test correction was applied. Correlations were performed by Spearman correlation test. Values shown in the text and figures represent the mean ± the standard deviation (SD) or the median [IQR] for the clinical data. P values were shown in Figures and Tables. When P>0.05, values of P were not shown. Statistical analysis was performed using GraphPad Prism (version 5.0; GraphPad Software Inc., San Diego, CA).

  with PKH67 were internalized by HASMC after 4 hours of incubation (Online Figure IIA). To study the role of Rap1 carried by lEVs on HASMC and vascular remodeling, we measured HASMC migration induced by nMetS-and MetS-lEVs by wound healing assay. MetS-lEVs, but not nMetS-lEVs, significantly increased HASMC migration (Figure 3C-D). Pre-incubation of MetS-lEVs with Rap1 antibody abolished HASMC migration induced by MetS-lEVs (Figure 3C-D). Similar results were obtained by Transwell assays (Online Figure IIB-IIC). Positive controls using platelet-derived growth factor (PDGF) or 8-(4-Chlorophenylthio) adenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT), (an activator of Rap1-GTP via the exchange factor Epac), are shown in Online Figure III both for migration and proliferation.

Figure 1 .F

 1 Figure 1. Characterization of lEVs from non-metabolic subjects (nMetS) and metabolic syndrome (MetS) patients. (A) Representative 2D-DIGE gel and bioinformatic analysis of polypeptidic spots; circles are corresponding to those differentially abundant in lEVs from nMetS and MetS subjects. The positions of molecular weight (MW) pattern is indicated on the left and the isoelectric point (Pi) on the bottom of the gels (n=5). (B) Representative Westernblot of Rap1 protein expression in lEVs from nMetS and MetS subjects. Rap1 expression was significantly higher in MetS-lEVs (n=21) compared to nMetS-lEVs (n=21). (C) Representative immunoblot of Rap1 pull-down experiments and quantification of Rap1-GTP (n=10). Rap1-GTP levels were significantly higher in MetS-lEVs. (D) Differential Rap1 expression was analyzed on native and permeabilized MetS-lEVs by flow cytometry. Non-significant change was observed in Rap1 labeling after permeabilization (n=8). (E) Circulating lEV levels expressing Rap1 in patients with MetS (n=90) compared to nMetS patients (nMetS n=72). (F) Cellular origins of Rap1 + -lEVs in nMetS (white) and MetS (gray) patients. CD41 (n=55, n=75), CD146 (n=7, n=10), CD235a (n=7, n=12), CD45 (n=7, n=10) and CD11b (n=6, n=7) for nMetS and MetS, respectively. Data are shown as medians and IQRs of numbers of lVs. P values in B, C, E were determined by Mann-Whitney U test; F, Kruskal Wallis test with Dunn's test post-

Figure 2 .Figure 3 .

 23 Figure 2. Correlation between circulating levels of Rap1-lEVs and clinical findings observed in non-metabolic (nMetS) and metabolic syndrome (MetS) subjects. Correlations were performed between levels of Rap1 expression in lEVs and waist circumference (cm) (A), hip circumference (cm) (B), mean heart rate expressed in beats per minute (bpm) (C), insulinemia (µmol/L) (D), plasma triglycerides levels (g/L) (E), high-sensitivity C-reactive protein levels (hs-CRP, mg/L) (F), TyG index (G), right common carotid end diastolic velocity (EDV, cm/s) (H) and number of components of MetS (I). Rap1 levels in circulating lEVs were detected by flow cytometry and expressed in log. Data are shown as medians and IQRs of numbers of lVs. (A-H) Correlations were performed by Spearman correlation test. For panel I, P values were determined by Kruskal Wallis test with Dunn's test post-hoc between all conditions. P values were not indicated when non-significant.

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Rap1 carried by lEVs regulates activation of MEK5-ERK5 and p38 pathways to induce proliferation of human aortic smooth muscle cells (HASMC). (A, B) Representative immunoblot and quantification of ERK5 and p38 activation induced by plateletderived growth factor (PDGF), 8-(4-Chlorophenylthio) adenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT), nMetS-lEVs (n=5) or MetS-lEVs (n=11). lEVs were pre-incubated with anti-Rap1 antibody or its control IgG. (C) Histograms showing fluorescence intensity representing the cell number of HASMC induced by either PDGF or MetS-lEVs in the absence or presence of BIX, a MERK5-ERK5 inhibitor (n=8). Data are shown as mean ± SD. P values were determined by Kruskal Wallis test with Dunn's test post-hoc between all conditions for each panel. P values were not indicated when non-significant. Representative immunoblots were selected to represent the mean value of each condition

Figure 7 .

 7 Figure 7. Rap1 expression in lEVs isolated from human atherosclerotic plaques.Quantification of Rap1 + -lEVs isolated from atherosclerotic plaques (AP) and from the arterial wall adjacent to the lesion from patients undergoing carotid endarterectomy (n=5). Data are shown as interconnected dots between conditions for each related sample. P value was determined by Mann-Whitney U test.

ACKNOWLEDGMENTS

We thank S. Marechal-Girault and P. Vandeputte for analysis of clinical data of Numevox cohort and the staff of SCAHU (Angers) for the care of animals. Also, we thank G. Hilairet, P. Mallegol and C. Besnard for help in confocal microscopy analysis, PCR experiments, and flow cytometry measurements, respectively. Metabol study Group composition: CLINICS (Centre Hospitalo-Universitaire d'Angers (CHU), Angers, France) • Hepatology: Jérôme Boursier, Paul Calès, Frédéric Oberti, Isabelle Fouchard-Hubert, Adrien Lannes • Diabetology: Séverine Dubois, Ingrid Allix, Pierre-Henri Ducluzeau • Pneumology: Frédéric Gagnadoux, Wojciech Trzepizur, Nicole Meslier, Pascaline Priou • Functional Vascular Explorations: Samir Henni, Georges Leftheriotis, Pierre Abraham • Radiology: Christophe Aubé BASIC SCIENCE (INSERM U1063, SOPAM, Angers University, France) Ramaroson Andriantsitohaina, M Carmen Martinez, Soazig Le Lay, Raffaella Soleti, Luisa Vergori STATISTICS: Gilles Hunault BIOLOGICAL RESSOURCE CENTER: Odile Blanchet, Belaid Sekour COORDINATION: Jean-Marie Chrétien, Sandra Girre SOURCES OF FUNDING This work was supported by INSERM, Université d'Angers and CHU Angers. LP was recipient of a post-doctoral fellowship from Angers Loire Metropole and AV a doctoral fellowship from Région Pays de la Loire. XVG was supported by Fondation pour la Recherche Médicale (SPF201809006985).

AUTHOR CONTRIBUTIONS

LP, XVG, RS, LV, LD, MC, MB, AV conducted experiments, acquired data, analyzed data; FP, performed proteomic analysis; FL, performed Rap activation assay; SLL, GS discussed the study; SD, JB, SH, FG, OM, IK, RV provided human samples; RA, MCM conceived and supervised the study, designed the experiments; LP, RA, MCM wrote the manuscript.

Novelty and Significance

What Is Known?

• Circulating levels of large extracellular vesicles (lEVs) from metabolic syndrome (MetS) patients are increased and participate to endothelial dysfunction.

• Proliferation, migration and proinflammatory cytokine secretion from vascular smooth muscle cells play a crucial role in vascular remodeling, a key step for atherosclerosis progression.

What New Information Does This Article Contribute?

• lEVs from MetS patients carry the small GTPase Rap1 and Rap1-lEVs levels correlate with cardiovascular risks including stenosis.

• MetS-lEVs promote migration and proliferation of human aortic smooth muscle cells and increase expression of pro-inflammatory cytokines via a Rap1-dependent mechanism.

• lEVs accumulate in plaque atherosclerotic lesions in both mice and humans.

Although it is known that extracellular vesicles from different origins are involved in atherosclerosis by inducing endothelial dysfunction and proliferation of smooth muscle cells, few data are available concerning lEVs from MetS patients. Here, we perform proteomic analysis of the content of these lEVs and identify overexpression of Rap1 in lEVs from MetS patients. Rap1-expressing lEVs play a key role in the progression of atherosclerosis by enhancing migration, proliferation and proinflammatory cytokine expression of vascular smooth muscle cells. Most interestingly, the analysis of atheroma lEVs indicates that they accumulate in atheroma plaque. These data define Rap1 + -lEVs produced in excess as a promising therapeutic target to prevent/limit the progression of atherosclerosis in MetS subjects, as well as a biomarker to predict vascular consequences of MetS.