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# Dietary modulation of large extracellular vesicles: the good and the bad for human health

Laurent-Emmanuel Monfoulet, M. Carmen Martínez

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## To cite this version:

Laurent-Emmanuel Monfoulet, M. Carmen Martínez. Dietary modulation of large extracellular vesicles: the good and the bad for human health. Nutrition Reviews, 2022, 80 (5), pp.1274-1293. 10.1093/nutrit/nuab106. hal-03531018

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Submitted on 31 Mar 2022

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1 **Special Article**

2

3 **Dietary modulation of large extracellular vesicles: the good and the bad for human health**

4

5 Laurent-Emmanuel Monfoulet¹✉ and Maria Carmen Martinez²

6 ¹ Université Clermont Auvergne, INRAE, UNH, F-63000 Clermont-Ferrand, France

7 ² SOPAM, U1063, INSERM, UNIV Angers, SFR, ICAT, Angers, France

8

9 **Corresponding author:**

10 Laurent-Emmanuel Monfoulet

11 laurent-emmanuel.monfoulet@inrae.fr

12 INRAE, Centre de Recherche Clermont Auvergne – Rhône-Alpes, Human Nutrition Unit (UNH), 63122 Saint

13 Genès Chamanelle, France

14
15 Abstract

16 Extracellular vesicles encompassing nanovesicles derived from the endosome system and generated by
17 plasmatic membrane shedding, become of increasing interest in view of their ability to sustain a cell-to-
18 cell communication and with the possibility to be used as surrogate biomarkers of healthy and unhealthy
19 trajectories. Over the last decades, nutritional strategies have been developed to preserve health and
20 their impact on circulating EVs is arousing a growing interest. Data available from published studies are
21 now sufficient for a first integration in order to better understand the role of EVs in the relation between
22 diet and health. Thus, this review focuses on human intervention studies investigating the impact of diet
23 or its components on circulating EVs. Due to analytical bias, only large EVs have been assessed so far. The
24 analysis highlights that poor-quality diets with elevated fat and sugar content increase levels of circulating
25 large EVs that can be partly counteracted by healthy food or some food micronutrients and bioactive
26 compounds. However, knowledge on the content and the biological functions of these diet-induced EVs
27 are still missing. It is of crucial importance to address these aspects in new research in order to state on
28 EVs as mediators of the effects of diet on health.
29

29

30 Keywords

31 Extracellular vesicles, microparticles, exosomes, diet, dietary constituents, human intervention studies,
32 risk factor, nutritional biomarkers

33

34 Introduction

35 Dietary pattern is an important driver of non-communicable diseases (NCDs) such as obesity,
36 diabetes and metabolic syndrome leading to cardiovascular diseases (CVD). In western societies,
37 individuals are routinely exposed to postprandial lipaemia, hypertriglyceridaemia and hyperglycaemia
38 that have been shown to be risk factors for CVD ¹,² including coronary artery diseases (CAD) ³,⁴, and that
39 contribute to the development of atherosclerosis ⁵, ⁶. These metabolic changes induce particularly the
40 activation of circulating immune cells and endothelial cells, one feature of which is the increased release
of circulating extracellular vesicles (EVs) ⁷, ⁸. EVs, including small EVs (previously called exosomes; S-EVs) 41 and large EVs (known as microparticles; L-EVs), are shed membrane particles of less than 1µm in diameter 42 that emerged as candidate meaningful biomarker for the diagnosis and the prognosis of cardiometabolic 43 disorders ⁹⁻¹⁴. S-EVs are vesicles of endo-lysosomal origin that are released as a consequence of the fusion 44 of the multivesicular endosome (namely MVB) with the plasma membrane, whereas L-EVs are generated 45 by budding and fission of the plasma membrane ⁸. Both EV subtypes convey proteins, nucleic acids and 46 lipids with the possibility to be used as markers and conferring to EVs the ability to sustain a cell-to-cell 47 communication. As an example, circulating endothelial L-EVs and their increased level in plasma assessed 48 at fasting state are both emerging biomarkers and active effectors of endothelial dysfunction in CVD ¹⁵⁻¹⁷. 49 Similarly, Crewe et al. ¹⁸ demonstrated that S-EVs are produced locally by endothelial cells in the adipose 50 tissue in fasting mice, but not after feeding. In addition to demonstrate that EVs are regulated by 51 fasting/refeeding, the authors suggest that EVs participate the tissue response to metabolic changes in 52 the systemic nutrient state. 53

Since the 2000s, the relationship between diet and circulating EVs has been the subject of 54 numerous studies. Dietary interventions in animal models have demonstrated that chronic high-energy 55 diet (e.g. high fat or high carbohydrate diet) results in an increased level of circulating S-¹⁹ and L-EVs ²⁰⁻²². 56 Inversely, plant-based diets are highly protective for health ²³. This is partly attributed to EV-associated 57 plant bioactive compounds, especially polyphenols with a body of evidence in preventing cardiometabolic 58 diseases especially through the reduction of vascular dysfunctions associated with ageing and unbalanced 59 nutrition ²⁴⁻²⁶. In addition to improve endothelial function, diet supplemented with polyphenols from red 60 wine has been shown to lower the level of endothelial EVs circulating at fasting state in a rodent model of 61 hypertension ²⁷. 62

Interestingly, food such as fruits and vegetables and cow milk-based products ²⁸, ²⁹ are dietary 63 sources of exogeneous EVs (mainly S-EVs) namely EV-like nanoparticles. As other EVs, they convey 64 proteins, lipids and nucleic acids in a phospholipid bilayer, but they can also encompass nutrients and/or 65 plant bioactive compounds such as polyphenols ³⁰, ³¹. These S-EVs have been demonstrated to be 66
67 absorbed, detected in host tissues few hours after their intake. The biological effects of EV-like
68 nanoparticles on the physiology of the consumer is a matter of concern.

69 The close relationship between diet and health, and the growing development of EV-based
70 biomarkers in medicine make necessary to consider the modulation of EVs by the diet, both at fasting and
71 postprandial states in humans. The present review focuses on human interventions investigating the
72 impact of a single meal and of a chronic intake on circulating EVs. It also introduces how dietary
73 compounds act to trigger physiological pathways controlling the biogenesis and/or the release of
74 endogenous EVs. Finally, this review provides reports on the biological functions of diet-associated EVs
75 and raises the need for further investigations in this domain, especially in order to provide an accurate
76 phenotyping of diet-induced EVs.

77
78 1. Why human nutritional intervention studies focused on L-EVs?
79
80 Since the 2000s, 29 interventional studies have investigated the impact of diet and their constituents
81 on circulating EVs in Humans. In these nutritional intervention studies, EVs were analysed on platelet-
82 poor or platelet-free plasma (PPP or PFP) obtained by centrifugation of blood samples up to 17,000 g. This
83 centrifugation pellets platelets but not the L-EVs and S-EVs that need longer centrifugation at 20,000 g or
84 100,000 g to be pelleted respectively ³². In these PPP/PFP, EVs were detected by flow cytometry based on
85 different combinations of constitutive and/or inducible surface markers (Table 1) after immunostaining.
86 This strategy of analysis limits the detection and the quantification to specific EV subtypes. First, the
87 conventional flow cytometers used in these studies (LSRII, FACSAria, FACScan, Canto, FC500, FACSCalibur)
88 are not sensitive enough to do detect scattered light or emitted fluorescence from particles smaller than
89 300 nm in diameter, and therefore do not allow the direct detection of S-EVs ³³, ³⁴. In addition, plasma
90 lipids (e.g. chylomicrons) due to their size may interfere with the detection of EVs by flow cytometry ³⁵. To
91 avoid this limit, a fluorescent staining mainly based on the detection of phosphatidylserine specifically
92 expressed at the surface of L-EVs was used. Therefore, this explains why these nutritional intervention
93 studies only report on the effect on L-EVs.
Above methodological aspects, the interest in L-EVs may be explained by a greater susceptibility of L-EVs to changes in response to a stimulus than S-EVs<sup>36, 37</sup>. Moreover, the content in proteins of L-EVs was reported as closer to those of the donor cell than the proteome of S-EVs<sup>36, 38</sup>, thus strengthening the interest for the L-EVs in human intervention studies. However, it is worth noting that the secretion, the content and the bioactivity of S-EVs have been reported in *in vitro* studies as modulated by nutritional factors such as hyperglycaemia<sup>39-41</sup> or polyphenol supplementations<sup>42-44</sup>.

Among the subpopulations of EVs present in plasma, endothelial L-EVs whose proportion is estimated to less than 5% are the most investigated ahead of platelet- and erythrocyte-derived L-EVs which account for 70%-95% and 8-16% of the total plasma L-EVs, respectively<sup>45-49</sup>. This large interest in endothelial L-EVs can be explained by the widely documented contribution of endothelial dysfunction in the development and the complications of cardiometabolic diseases<sup>50</sup>. Whatever the subtype of EVs investigated, human intervention studies were controlled by ethical considerations that limit the number and volume of blood sampling and therefore that constrain studies to few timepoints after a nutritional intervention. By consequence, knowledge on the pharmacokinetic of EVs during the postprandial period is still incomplete. Cross-over and/or a placebo control designs have been used to ascertain the impact of dietary intakes on circulating L-EVs. They allowed to demonstrate first the variation of the circulating L-EVs during the postprandial period induced by an acute diet intake (Table 2) and secondarily the effect of chronic intakes on the basal circulating L-EVs at fasting (Table 3).

111
<table><caption>Table 1: Cell surface markers used for the identification of EV subpopulations by flow cytometry.</caption><thead><tr><th>Cell source of large EVs (namely microparticles)</th><th>Cluster of differentiation staining</th><th>Protein Symbol</th><th>Full name</th></tr></thead><tbody><tr><td rowspan="10">Endothelial MPs</td><td>CD31+</td><td>PECAM1</td><td>platelet and endothelial cell adhesion molecule 1</td></tr><tr><td>CD41-</td><td>ITGA2B</td><td>integrin subunit alpha 2b</td></tr><tr><td>CD42a-</td><td>GP9</td><td>glycoprotein IX platelet</td></tr><tr><td>CD42b-</td><td>Gplb</td><td>glycoprotein Ib platelet</td></tr><tr><td>CD45-</td><td>PTPRC</td><td>protein tyrosine phosphatase receptor type C</td></tr><tr><td>CD62b-</td><td>SELP</td><td>selectin P</td></tr><tr><td>CD62e+</td><td>SELE</td><td>selectin E</td></tr><tr><td>CD105+</td><td>ENG</td><td>endoglin</td></tr><tr><td>CD106</td><td>VCAM1</td><td>vascular cell adhesion molecule 1</td></tr><tr><td>CD144+</td><td>CDH5</td><td>cadherin 5</td></tr><tr><td>CD146+</td><td>MCAM</td><td>melanoma cell adhesion molecule</td></tr><tr><td rowspan="6">Platelet MPs</td><td>CD41+</td><td>ITGA2B</td><td>integrin subunit alpha 2b</td></tr><tr><td>CD42b+</td><td>Gplb</td><td>glycoprotein Ib platelet</td></tr><tr><td>CD61+</td><td>ITGB3</td><td>integrin subunit beta 3</td></tr><tr><td>CD62b+</td><td>SELP</td><td>selectin P</td></tr><tr><td>Fibrogen+</td><td>FGB</td><td>fibrinogen beta chain</td></tr><tr><td>TF(CD142)+</td><td>F3</td><td>coagulation factor III, tissue facto</td></tr><tr><td rowspan="6">Leukocyte MPs</td><td>CD4+</td><td>CD4</td><td>CD4 molecule</td></tr><tr><td>CD8</td><td>CD8A</td><td>CD8a molecule</td></tr><tr><td>CD11a+</td><td>ITGAL</td><td>integrin subunit alpha L</td></tr><tr><td>CD11b+</td><td>ITGAM</td><td>integrin subunit alpha M</td></tr><tr><td>CD41-</td><td>ITGA2B</td><td>integrin subunit alpha 2b</td></tr><tr><td>CD45+</td><td>PTPRC</td><td>protein tyrosine phosphatase receptor type C</td></tr></tbody></table>

112

113
<table><caption>114 Table 2: Clinical trials investigating the impact of acute dietary intakes on EVs during the postprandial period.</caption><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Intervention</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Bulut et al. J Cardiovasc Pharmacol 2013<sup>51</sup></td><td>Randomized Cross-over</td><td>EU (Germany)</td><td>Healthy men (n= 10) Age= 44.2 ± 9.6 BMI= 24.4 ± 2.5</td><td>A single meal of French fries and hot pork sausage with sauce (1,200 kcal: 50 % from Fat; 30 % CHO; 20 % Proteins) ± 200 mL water or 200 mL coca cola or 100 mL red wine/100 mL water, or 36 mL liquor (38 vol %)/164 mL water</td><td>AnnexinV<sup>+</sup> L-EVs: <ul><li>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li><li>CD31<sup>+</sup>/CD42b<sup>+</sup> platelet L-EVs</li></ul>From platelet-poor plasma By flow cytometry (FACScalibur, BD)</td><td><ul><li>↑ postprandial (water; 1h: +15 %, 2 h: +30 %)</li><li>AnnexinV<sup>+</sup>/CD31<sup>+</sup>/CD42b<sup>-</sup> L-EVs</li><li>Red Wine consumption: ↓ meal-induced AnnexinV<sup>+</sup>/CD31<sup>+</sup>/CD42b<sup>-</sup> L-EVs compared to water (-83 %)</li><li>Coca: ↑ meal-induced AnnexinV<sup>+</sup>/CD31<sup>+</sup>/CD42b<sup>-</sup> L-EVs compared to water (+109 %)</li></ul></td></tr><tr><td>Eichner et al. Nutrients 2019<sup>52</sup></td><td>NA (US)</td><td>Obese prediabetic (PD, n= 17) and NGT (n= 8) volunteers Male:Female= 4:13 (PD), 1:7 (NGT) Age= 53.5 ± 4.1 (PD), 50.1 ± 5.1 (NGT) BMI= 32.1 ± 1.6 (PD), 33.2 ± 1.4 (NGT)</td><td>Oral Glucose Tolerance Test (a 75 g glucose intake that induced a 136 mg/dl glycaemia at peak, i.-e. 2 h)</td><td>Annexin V<sup>+</sup> EVs: <ul><li>CD31<sup>+</sup>/CD41<sup>+</sup> platelet EVs</li><li>CD45<sup>+</sup>/CD41<sup>-</sup>, CD45<sup>+</sup> leukocyte EVs</li><li>CD105<sup>+</sup>, CD31<sup>+</sup>/CD41<sup>-</sup> Endothelial EVs</li></ul>from Platelet-free plasma (17,000 g) by Flow cytometry (ImageStream MKII Amnis)</td><td><ul><li>↓ total EVs</li><li>↓ CD31<sup>+</sup>/CD41<sup>+</sup> PEVs</li><li>↓ CD31<sup>+</sup> EVs</li><li>Ø CD45<sup>+</sup> LEVs</li></ul></td><td><ul><li>C= Ø effect of single Low-fat meal on postprandial E L-EV levels</li><li>I= ↑ postprandial (1 h: +39 %, 3 h: +70 %) CD31<sup>+</sup>/CD42b<sup>-</sup> Endothelial L-EVs</li></ul></td></tr><tr><td>Ferreira et al. Circulation 2004<sup>53</sup></td><td>Cross-over</td><td>NA (US)</td><td>Healthy volunteers (n= 18) Male:Female= 10:8 Age= 26 ± 3.8 BMI= 23 ± 2.3</td><td>I= High-fat meal (HF; 900 cal: 19 % from fat) C= Low-fat meal (LF; 900 cal: 0 % from fat)</td><td>Annexin V<sup>+</sup> L-EVs: <ul><li>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li><li>CD31<sup>+</sup>/CD45<sup>+</sup> leukocyte L-EVs</li></ul>From platelet-poor plasma (1,000 g) By flow cytometry (Coulter EPICS XL, Beckman)</td><td><ul><li>Test meal induces ↑ CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs from 2h to 6h after intake (+160 %)</li><li>Ø of prior EXC on diet-induced EVs</li></ul></td></tr><tr><td>Harrison et al. Eur J Appl Physiol 2009<sup>54</sup></td><td>Randomized Controlled</td><td>EU (Ireland)</td><td>Healthy active men (n= 8) Age= 26.9 ± 4.1 BMI= 26.0 ± 3.6</td><td>Oral fat tolerance test meal (1,450 kcal per 2 m<sup>2</sup> body surface: 60 % from fat, 34 % from CHO) preceded by a rested time or a prolonged exercise (EXC)</td><td>Annexin V<sup>+</sup> L-EVs: <ul><li>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li><li>CD31<sup>+</sup>/CD42b<sup>+</sup> endothelial L-EVs</li></ul>From platelet-poor plasma (1,500 g) By flow cytometry (LSRII, BD)</td><td><ul><li>Ø effect of single high-fat meal on postprandial (4 h) endothelial L-EVs</li><li>Prior endurance exercise ↓ CD31<sup>+</sup>/CD42b<sup>-</sup> and CD62E<sup>+</sup> endothelial L-EVs (-50 %)</li></ul></td></tr><tr><td>Jenkins et al. J Physiol 2011<sup>55</sup></td><td>Cross-over</td><td>NA (US)</td><td>Healthy active men (n= 10) Age= 27 ± 0.9 BMI= 24.6 ± 0.7</td><td>A single high-fat meal portioned to 386 g/m<sup>2</sup> body surface area (386 g= 1,362 kcal, 84 % from fat) ± prior endurance exercise</td><td>Annexin V<sup>+</sup> L-EVs: <ul><li>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li><li>CD62E<sup>+</sup> endothelial L-EVs</li></ul>From platelet-poor plasma (1,500 g) By flow cytometry (LSRII, BD)</td><td><ul><li>Ø effect of single high-fat meal on postprandial (4 h) endothelial L-EVs</li><li>Prior endurance exercise ↓ CD31<sup>+</sup>/CD42b<sup>-</sup> and CD62E<sup>+</sup> endothelial L-EVs (-50 %)</li></ul></td></tr></tbody></table>
<table><caption>115 Table 2 (To be continued)</caption><thead><tr><td>Publication</td><td>Study design</td><td>Country</td><td>Participants</td><td>Intervention</td><td>Investigated EVs</td><td>Main results</td></tr></thead></table>

<table><thead><tr><td>Publication</td><td>Study design</td><td>Country</td><td>Participants</td><td>Intervention</td><td>Investigated EVs</td><td>Main results</td></tr></thead><tbody><tr><td>Michelsen et al. Thrombosis Res 2009 <sup>56</sup></td><td>Controlled</td><td>EU (Norway)</td><td>Carotid atherosclerosis patients (n= 40, CAP) and matched healthy volunteers (n= 20, H)<br/>Male:Female= 19:21 (CAP) 12:8(H)<br/>Age= 65.4-72.8 (CAP), 66.6-70.1(H)<br/>BMI= 26.4-28.8 (CAP), 24.8-28.4(H)</td><td>Standardized tolerance fat test consisting in sour cream porridge providing 70 % of calories from fat (66 % saturated, 32 % monosaturated, 2 % PUFAs)</td><td>CD41<sup>+</sup>/CD61<sup>+</sup> platelet L-EVs<br/>From platelet-free plasma (11,000 g)<br/>By flow cytometry (Canto, BD)</td><td>↑ postprandial (4 h) total CD41<sup>+</sup>/CD61<sup>+</sup> L-EVs (+49 %)</td></tr><tr><td>Phang et al. JNB 2012 <sup>57</sup></td><td>Controlled Single blinded</td><td>South Pacific (Australia)</td><td>Healthy volunteers (n= 30)<br/>Male:Female= 15:15<br/>Age= 43.7 ± 1.5<br/>BMI= 26.1 ± 0.6</td><td>A single dose of sunola oil (C), 1 g EPA (EPA:DHA= 5:1) or 1 g of DHA (EPA:DHA= 1:5)</td><td>AnnexinV<sup>+</sup>/CD41a<sup>+</sup> platelet L-EVs<br/>From platelet-free plasma (3,000 g)<br/>by flow cytometry (Canto, BD)</td><td>• At baseline, Ø effect gender on the number of platelet L-EVs<br/>• 24h after a single dose of supplementation: Ø effect on AnnexinV<sup>+</sup>/CD41a<sup>+</sup> MP levels<br/>• Only EPA supplementation ↓ thrombin generation mediated by platelet L-EVs (-20 %)</td></tr><tr><td>Spectre et al. Platelets 2019 <sup>58</sup></td><td>EU (Sweden)</td><td></td><td>T2DM (n= 9) and T1DM (n= 11) patients<br/>Male:female= 8:1 (T2DM), 5:4(T1DM)<br/>Age= 55 (40-72, T1DM), 68 (60-76, T2DM)<br/>BMI= 23.5 (T1DM), 30 (T2DM)</td><td>A standardized meal (620 kcal: 30 % fat, 54 % CHO, 16 % protein)</td><td>Annexin V<sup>+</sup> L-EVs:<br/>• CD41<sup>+</sup> ± CD62P<sup>+</sup> ± CD142<sup>+</sup> platelet L-EVs<br/>• CD144<sup>+</sup> or CD62e<sup>+</sup> endothelial L-EVs<br/>• CD14<sup>+</sup> monocyte L-EVs<br/>• CD142(TF)<sup>+</sup> L-EVs<br/>from Platelet-free plasma (13,000 g)<br/>by Flow cytometry (Gallios, BD)</td><td>• ↑ annexin V<sup>+</sup> L-EVs in T2DM and T1DM patients at 90min after meal (+40 %)<br/>• ↑ thrombin generation by postprandial L-EVs compared to pre-meal L-EVs</td></tr></tbody></table>
<table><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Intervention</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Strohacker et al.<br>Appl Physiol<br>Nutr Metabo<br>2012<sup>59</sup></td><td>Randomized<br>Controlled</td><td>NA (US)</td><td>Healthy volunteers (n=8)<br>Male:Female= 4:4<br>Age= 21 ± 3<br>BMI= 23.1 ± 3.92</td><td>Standardized test meal (1,070 kcal: 58 % from fat, 30 % CHO, 9 % proteins) ± prior endurance exercise or rest</td><td>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs<br>From platelet-poor plasma (1,500 g)<br>By flow cytometry (EasyCyte 6HT-2L, EMD Millipore)</td><td><ul><li>Test meal: ↑ postprandial (1h: +50 %) CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li><li>Prior exercise blocks the postprandial ↑ of CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs</li></ul></td></tr></tbody></table>

116

<table><caption>117 Table 2 (To be continued)</caption><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Intervention</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Sustar et al.<br>Lipids in<br>Health &<br>Disease 2011<sup>46</sup></td><td>Cross-over</td><td>EU<br>(Slovenia)</td><td>Healthy volunteers (n=21)<br>Male:Female= 11:10<br>Age= 31 ± 10 (M), 29 ± 13 (F)<br>BMI=not reported</td><td>2 days of food rich in cholesterol, fat and carbohydrates. On day 2, a breakfast consisting of two eggs, bread and a dairy product (7 a.m.) and small meal consisting of a dairy product (10 a.m.)</td><td>CD31<sup>+</sup> and CD42b<sup>-</sup> endothelial L-EVs<br>CD31<sup>+</sup> and CD42b<sup>+</sup> platelet L-EVs<br>CD235<sup>+</sup> erythrocyte L-EVs<br>From plasma L-EVs (17,570 g pellet)<br>By flow cytometry (Altra, Beckman)</td><td><ul><li>postprandial total L-EVs (2 h: +52 %)</li><li>Ø postprandial redistribution of L-EVs with respect to origin</li></ul></td></tr><tr><td>Sutherland et<br>al. Metabol<br>Clin Exp 2010<sup>60</sup></td><td>Randomized<br>Cross-over<br>Single<br>blinded</td><td>South<br>Pacific (New<br>Zealand)</td><td>Healthy volunteers (n=22)<br>Male:Female= 13:9<br>Age= 40 ± 13<br>BMI= 25.3 ± 3.7</td><td>n-6 PUFAs-rich meals containing unheat sunflower oil (USO) or heated SO (HSO) (510 Kcal: 72 % from Fat (mainly PUFAs), 25 % CHO, 2 % Proteins);<br>C= saturated fat-rich meal containing cream (CR) (527 Kcal: 70 % from Fat (mainly SAFAs and MUFAs), 27 % CHO, 4 % Proteins)</td><td>CD144<sup>+</sup> CD62b<sup>-</sup> endothelial L-EVs<br>From platelet-poor plasma (6,000 g)<br>By flow cytometry (FACSCalibur, BD)</td><td>↑ postprandial (3 h: +20 %) CD144<sup>+</sup> endothelial L-EVs after sunflower oil enriched meals but not after CR meal</td></tr><tr><td>Tamburrelli et<br>al.<br>Thrombosis &<br>Haemostasis<br>2012<sup>61</sup></td><td>Not reported</td><td>EU (Italia)</td><td>Subjects at different degree of CV risk (n= 61)<br>Male:Female= 40:21<br>Age= 48.6 ± 13.1<br>BMI= 26.7 ± 3.94</td><td>A standardized fatty meal (675 kcal: 69 % from fat, 15 % CHO, 15 % proteins)</td><td>CD42b<sup>+</sup> L-EVs<br>By flow cytometry (EPICS LX Flow, Beckman)</td><td>↑postprandial (2 h: +150 %) CD42b<sup>+</sup> L-EVs in men, not in women</td></tr><tr><td>Tushuizen et<br>al.<br>Diabetes Care<br>2007<sup>47</sup></td><td>Not reported</td><td>EU (The<br>Netherlands )</td><td>Caucasian men with T2DM (n= 15, T2DM) or healthy (n= 12, H; aged-matched)<br>Age= 55 ± 2 (T2DM)<br>BMI=not reported</td><td>Three consecutive isocaloric meals (breakfast, lunch and dinner; 900 kcal: 50 % from fat, 33 % CHO, 15 % protein)</td><td>AnnexinV<sup>+</sup> CD144<sup>+</sup> endothelial L-EVs<br>From plasma<br>By flow cytometry (FACScan, BD)</td><td><ul><li>At baseline, ↑ CD144+ endothelial L-EVs in T2DM (+130 %)</li><li>4h after the last meal, ↑ CD144+ endothelial L-EVs in T2DM</li></ul></td></tr></tbody></table>
<table><caption>118 Table 2 (To be continued)</caption><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Intervention</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Tushuizen et al.<br>J Thrombosis and Haemostasis<br>2006<br>48</td><td>Randomized Cross-over</td><td>EU (The Netherlands )</td><td>Healthy men (n=17) Age= 25.4 ± 3 BMI= 23.6 ± 1.8</td><td>I= Two consecutive standardized meals (900 kcal: 56 % from fat, 25 % CHO, 13 % protein) as breakfast and 4h later as lunch<br>C= fasting</td><td>■ CD61 for platelet L-EVs<br>■ CD62E, CD106 and CD144 for endothelial L-EVs<br>■ CD4, CD8 and CD14 for leukocyte L-EVs<br>From plasma<br>By flow cytometry (FACScan, BD)</td><td>I= ↑ total L-EVs compared to C (+100 % over the 8 h of the study)<br>I= ↓ FMD<br>compared to healthy volunteers (+260 %)</td></tr></tbody></table>

<table><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Intervention</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Tushuizen et al.<br>Thrombosis Res 2012<br>62</td><td>Randomized Cross-over</td><td>EU (The Netherlands )</td><td>Healthy men (n=12) Age= 26.0 ± 0.9 BMI= 23.9 ± 0.4</td><td>I=Two consecutive standardized meals (900 kcal: 56 % from fat, 25 % CHO, 13 % protein) as breakfast and 4h later as lunch<br>C= fasting</td><td>AnnexinV+ associated with<br>■ CD61 for Platelet L-EVs<br>■ CD62E, CD106 and CD144 for endothelial L-EVs<br>■ CD4, CD8 and CD14 for leukocyte L-EVs<br>from plasma L-EVs (17,590 g pellet)<br>By flow cytometry (FACScan, BD)</td><td>• Over the postprandial period, ↑ erythrocyte-derived L-EVs (+220 %)<br>After meals, ↑ phosphatidylcholine but not of phosphatidylserine in L-EVs compared to fasting</td></tr><tr><td>119</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>120</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>121</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>122</td><td>Abbreviations</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>123</td><td>C: control diet, I: nutritional intervention, BMI: Body Mass Index, CV risk: Cardiovascular risk, T1/2DM: Type 1/2 Diabetes Mellitus, NA: North America, EU:</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>124</td><td>Europe, US: United States, CHO: carbohydrate, PUFAs: Poly Unsaturated Fatty Acid, EPA: EicosaPentaenoic Acid, DHA: DocosaHexaenoic Acid, NGT: Normo</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>125</td><td>Glucose</td><td></td><td></td><td></td><td></td><td>Tolerance</td></tr></tbody></table>
126

## 2. The postprandial variation of circulating L-EVs

The response of L-EVs to the acute intake of different test meals (providing high dose of glucose, of saturated fat or enriched in polyunsaturated fatty acids) has been investigated during the postprandial period (Table 2)⁴⁶⁻⁴⁸, ⁵¹⁻⁶². The studies reported on the level of L-EVs assessed mainly 2-3 hours after the intake of the test meal in comparison to fasting conditions or to the consumption of a control meal. In healthy young volunteers, the consumption of a meal supplying 19-70 % energy from fat (mainly saturated fat) increased significantly the level of circulating L-EVs at 2 hours postprandially from 20 to 160 % ⁴⁸, ⁵¹, ⁵³, ⁵⁴, ⁵⁹, ⁶⁰, ⁶². Several subtypes of circulating L-EVs increased in response to fat intake: the endothelial L-EVs identified as CD31⁺-CD42b⁻ (+50-160 %) ⁵³, ⁵⁴, ⁵⁹, annexin V⁺-CD31⁺-CD42b⁻ (+30%) ⁵¹, CD144⁺ (+ 20 %) ⁶⁰ and CD144⁺-CD62b⁻ (+ 100-130 %) ⁴⁷, ⁴⁸ EVs, and the platelet-derived L-EVs characterized as annexin V⁺-CD31⁺-CD42b⁺ ⁵¹ and CD41⁺-CD61⁺ ⁵⁶ EVs. Studies from Tushuizen et al. ⁶², Sustar et al. ⁴⁶ and Spectre et al. ⁵⁸ also showed that all annexin V⁺ L-EVs increased postprandially after a high fat meal. One study did not observe a variation of L-EV levels after a high fat meal while they used CD31-CD42b-CD62 markers to identify the endothelial L-EVs ⁵⁵. An acute increase of L-EV levels during the postprandial period was also observed in diabetic patients ⁵⁸, in individuals with cardiovascular risk ⁶¹ and in atherosclerotic patients ⁵⁶, all of them presenting already a higher fasting level of L-EVs when compared to healthy volunteers. It is noteworthy that the postprandial increase of L-EV levels had similar magnitude in individuals at CVD risk than in healthy volunteers ⁵⁶. Furthermore, based on the studies from Table 2, no correlation was found between the percentage of dietary fat and the magnitude of L-EV variation. Other nutrients provided by food such as carbohydrates (CHO) or proteins may affect the postprandial level of L-EVs. To our knowledge, no study has so far investigated the impact of protein intake on the postprandial EVs. Regarding CHO, in vitro studies showed that a high concentration of glucose increased the secretion of L-EVs from endothelial cells ⁴¹, ⁶³, leucocytes ⁶⁴, ⁶⁵, trophoblasts ⁴⁰ or mesangial cells ⁶⁶. In obese volunteers, Eichner et al. ⁵² reported that an oral glucose tolerance test, providing 75 g of glucose and resulting in a 136 mg/dL glycaemia at 2 hours, reduced the level of circulating L-EVs characterized in PFP. In addition, the postprandial level of CD31⁺-CD42b⁻ endothelial L-EVs increased from 30 to 160 %, 2

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hours after the intake of a test meal providing 50-60 % of fat and 30-34 % of CHO (mainly simple sugars) 152
51, 54. Moreover, the study performed by Ferreira et al. 53 has demonstrated that a fat-free meal was not 153
associated to an increased level of postprandial L-EVs compared to an isocaloric meal providing 19 % fat. 154
Altogether, these data suggest that the percentage of CHO in the meal is not a determinant in the 155
postprandial variation of L-EVs contrary to the fat content. 156

Diet may also provide some micronutrients (n-3 PUFAs) or microconstituents (polyphenols) that seem 157
able to counteract the increase of postprandial EVs. The consumption of red wine, providing high amount 158
of polyphenols, alleviated by 83 % the high-fat diet (HFD)-induced L-EVs 51. Phang et al. 57 reported that 24 159
hours after an acute intake of 1 g of n-3 PUFAs, the level of plasma annexin V⁺-CD41a⁺ EVs was unchanged 160
compared to a control meal. However, the intake of n-3 PUFAs reduced the capacity of these platelet- 161
derived L-EVs to induce thrombin formation. Spectre et al. 58 reported that the postprandial annexin V⁺ L- 162
EVs induced a higher thrombin generation that pre-meal annexin V⁺ L-EVs. Studies aiming at deciphering 163
the biological function of postprandial EVs are scarce. Their functional activities could be predicted in 164
silico by an enrichment-based bioinformatics analysis of the proteome and transcriptome of EVs, however 165
the use of this approach is limited by the lack of data on protein content and nucleic acid content of the 166
postprandial EVs. 167

To conclude, in so far as a meal provides saturated fats, its acute consumption is associated to a 168
rapid rise of plasma L-EVs. In the absence of detailed pharmacokinetic data all over the postprandial 169
phase, the transient character of the postprandial phase cannot be asserted to the EV rise. However, one 170
can speculate that postprandial EVs temporary rise up as the clearance of endothelial- and platelet- 171
derived L-EVs in the human circulation has been demonstrated to vary from 1 to 6 hours 67, 68. Lastly, 172
some dietary constituents such as dietary polyphenols and n-3 PUFAs modulate positively the biology of 173
EVs during the postprandial phase, that is, either by preventing the increase of EV levels or attenuating 174
their bioactivity. 175
<table><caption>Table 3: Human intervention studies reporting the effects of (sub)chronic dietary intake on circulating EVs. C: control diet; I: nutritional intervention.</caption><thead><tr><td>Publication</td><td>Study design</td><td>Country</td><td>Participants</td><td>Dietary intervention</td><td>Duration (weeks)</td><td>Investigated EVs</td><td>Main results</td></tr></thead><tbody><tr><td>Del Turco et al. Haematologica 2008<sup>45</sup></td><td>Randomized Controlled</td><td>EU (Denmarks)</td><td>Post-myocardial infraction patients (n= 21, I; n= 21, C)<br/>Male:Female= 18:3<br/>Age= 65 ± 5 (I), 62 ± 8 (C)<br/>BMI= not reported</td><td>I= 5.2g total n-3 FA (4.3 g EPA+DHA)<br/>C= Olive Oil</td><td>12</td><td>All AnnexinV<sup>+</sup> / TF<sup>+</sup> L-EVs<br/>■ CD61<sup>+</sup> platelet L-EVs<br/>■ CD14<sup>+</sup> monocyte L-EVs<br/>■ CD62E<sup>+</sup> endothelial L-EVs<br/>from 17,500 g pelleted plasma L-EVs</td><td>I=↓ CD61<sup>+</sup> L-EVs, ↓ CD14<sup>+</sup> L-EVs, Ø CD62E<sup>+</sup> endothelial L-EVs, Ø TF<sup>+</sup> L-EVs<br/>I= ↓ thrombin generation by L-EVs<br/>C=Ø L-EVs<br/>At baseline, ↑ total L-EVs in patients compared to healthy matched volunteers (n= 10)</td></tr><tr><td>Durrer et al. Nutrients 2019<sup>69</sup></td><td>Randomized Short-term Controller Cross-over</td><td>EU (UK)</td><td>Healthy men (n= 9)<br/>Age= 21 ± 3<br/>BMI= 23.2 ± 2</td><td>o A 7-day intervention on low-carbohydrate/high-fat diet (2417 ± 267 kcal: 71 % fat, 11 % CHO, 18 % protein) after 2 weeks of typical diet (2584 ± 456 kcal: 37 % fat, 46 % CHO, 17 % protein)<br/>o 75 g oral glucose tolerance test (oGTT) performed before and after nutritional intervention</td><td>1</td><td>■ CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs<br/>■ CD62E<sup>+</sup> endothelial L-EVs<br/>From platelet-poor plasma (1,500 g)<br/>By flow cytometry (LSRII, BD)</td><td>At fasting, no difference in L-EVs levels from LCHFD group compared to typical diet group<br/>↑ post oGTT CD31<sup>+</sup>/CD42b<sup>-</sup> and CD62E<sup>+</sup> endothelial L-EVs (1 h) in LCHFD group<br/>LCHFD: ↓ FMD</td></tr><tr><td>Eitan et al. Aging Cell 2017<sup>70</sup></td><td>Randomized Controlled</td><td>NA (US)</td><td>Prostate cancer patients (n= 38)<br/>Age= 59.26 ± 7.5<br/>BMI= 30.45 ± 5.8</td><td>I= protein restriction diet (2856 ± 198 kcal: 31 % fat, 59 % CHO, 8 % protein)<br/>C= regular diet (2367 ± 444 Kcal: 37 % fat, 45 % CHO, 17 % protein)</td><td>4</td><td>L1CAM immunoprecipitated EVs from Precipitated total plasma EVs by NTA</td><td>I: Ø EV size distribution, Ø total EV concentration<br/>I: ↑ Leptin Receptor<sup>+</sup> /L1CAM<sup>+</sup> neuronal EVs, ↑ pSer312 IRS1<sup>+</sup>/L1CAM<sup>+</sup> neuronal EVs</td></tr><tr><td>Francois et al. Am J Physiol Heart Circ Physiol 2018<sup>71</sup></td><td>Randomized Cross-over Controlled</td><td>NA (Canada)</td><td>T2DM volunteers (n= 16)<br/>Male:Female= 8:8<br/>Age= 64 ± 8<br/>BMI= 34.0 ± 8</td><td>2 isocoloric meals (500 kcal)<br/>o Low-fat control diet (CON: 20 % fat, 55 % CHO, 25 % protein)<br/>o low-carbohydrate high-fat diet (LC: 65 % Fat, 10 % CHO, 25 % protein)<br/>o LC with 15-min post-meal walks (LC-EX)</td><td>4 days</td><td>■ CD62E<sup>+</sup> endothelial L-EVs<br/>■ CD62P<sup>+</sup> platelet L-EVs<br/>From platelet-free plasma (13,000 g)<br/>By flow cytometry (FACSAria III, BD)</td><td>LC: ↓ CD62E<sup>+</sup> endothelial L-EVs (- 50 %); Ø CD62P<sup>+</sup> L-EVs<br/>LC-EX: Ø CD62E<sup>+</sup> endothelial L-EVs; ↓ CD62P<sup>+</sup> L-EVs (- 44 %)<br/>CON: ↑ CD62E<sup>+</sup> endothelial L-EVs (+ 56 %); Ø CD62P<sup>+</sup> L-EVs</td></tr></tbody></table>
<table><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Dietary intervention</th><th>Duration (weeks)</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Gröne et al. JAFC 2019<sup>72</sup></td><td>Randomized Double-blinded Controlled</td><td>EU (Germany)</td><td>Healthy men (n= 39) Age= 26 ± 3 (Young Group, n= 20), 60 ± 8 (Elderly Group, n= 19) BMI= 24.1 ± 2.3 (Young Group), 26.5 ± 3.2 (Elderly Group)</td><td>I= cocoa flavanol drink (CF, providing 450 mg of total flavanol, twice daily) C= nutrients-matched CF free drink</td><td>2</td><td>CD62e<sup>+</sup> endothelial L-EVs CD144<sup>+</sup> endothelial L-EVs CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs from Platelet-free plasma (10,000 g) by Flow cytometry (VERSE, BD)</td><td>I: ↓CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs (Y: -52 %; E: -57 %), ↓CD144<sup>+</sup> endothelial L-EVs (Y: -23 %; E: -21 %), ↓CD62e<sup>+</sup> endothelial L-EVs (Y: -35 %; E: -24 %) in Young and Elderly C: ↑CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs (+52 %), ↑CD144<sup>+</sup> endothelial L-EVs (+35 %), ↑CD62e<sup>+</sup> endothelial L-EVs (+57 %) in elderly vs young</td></tr><tr><td>Horn et al. BJN 2014<sup>73</sup></td><td>Randomized Double-blinded Cross-over</td><td>EU (Germany)</td><td>CAD patients (n= 16) Male:Female= 13:3 Age= 64 ± 3 BMI= 28.8 ± 1.8</td><td>I= high-cocoa flavanol drink (providing 375 mg of total flavanol) C= Low cocoa-flavanol drink (9 mg)</td><td>4</td><td>CD41<sup>+</sup> platelet-derived L-EVs CD144<sup>+</sup> endothelial L-EVs CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs from Platelet-free plasma (10,000 g) by Flow cytometry (Canto II, BD)</td><td>I: ↓CD144<sup>+</sup> endothelial L-EVs (-22 %), ↓CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs (-39 %), Ø CD41<sup>+</sup> PL-EVs in CAD patients C: ↑ CD144<sup>+</sup> (+ 490 %) and CD31<sup>+</sup>/CD41<sup>-</sup> (+ 30 %) endothelial L-EVs in CAD patients vs age-matched healthy volunteers</td></tr><tr><td>Marin et al. AJCN 2011<sup>74</sup></td><td>Randomised Cross-over</td><td>EU (Spain)</td><td>Healthy elderly volunteers (n= 20) Male:female=10:10 Age= 67.1 ± 4.52 BMI= 31.9 ± 5.50</td><td>3 diets providing 1910 Kcal/day - Mediterranean Diet (Met Diet): 38 % fat (24 % MUFAs, <10 % SFA), 47 % CHO, 15 % protein - SFA-rich diet: 38 % fat (12 % MUFAs, 22 % SFAs, 4 % PUFAs), 47 % CHO, 15 % protein - Low-fat high-carbohydrate diet enriched in n-3 PUFAs (CHO-ALA): <30 % fat (<12 % SFAs, 12 % MUFAs, 8 % PUFAs), 55 % CHO, 15 % protein</td><td>4</td><td>AnnexinV<sup>+</sup> CD31<sup>+</sup> endothelial L-EVs CD144<sup>+</sup>/CD62E<sup>+</sup> activated endothelial L-EVs from Platelet-free plasma (13,000 g) by Flow cytometry (FC500, Beckman)</td><td>I: Met Diet and CHO-ALA diet ↓ total (-60 %; -33 %), CD144<sup>+</sup>/CD62e<sup>+</sup> (-76 %; -34 %) and annexinV<sup>+</sup>/CD31<sup>+</sup> (-70 %; -25 %) endothelial L-EVs vs SFA diet I: Met Diet ↓ total (-40 %), CD144<sup>+</sup>/CD62e<sup>+</sup> (-63 %) and annexinV<sup>+</sup>/CD31<sup>+</sup> (-60 %) endothelial L-EVs vs CHO-ALA diet</td></tr></tbody></table>
<table><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Dietary intervention</th><th>Duration (weeks)</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>McFarlin et al. JNB 2015<sup>75</sup></td><td>Randomized Controlled Cross-over</td><td>NA (US)</td><td>Healthy volunteers with normal weight (n= 10, N), over-weight (n= 7, O) or obesity (n=7, Ob)<br>Age= 21 ± 2 (N), 22 ± 3 (O), 22 ± 3 (Ob)<br>BMI= 21.6 ± 1.9 (N), 27.0 ± 1.4 (O), 34.9 ± 3.0 (Ob)</td><td>I= natural cocoa bars (supplying 13.6 mg catechin, 48 mg epicatechin, 640 mg total proanthocyanidins)<br>C= placebo bars</td><td>4</td><td>CD144<sup>+</sup>/CD42a<sup>-</sup>/CD45<sup>-</sup> endothelial L-EVs<br>from Platelet-free plasma (15,000 g)<br>by Flow cytometry (FC500, Beckman)</td><td>I: ↓CD144<sup>+</sup>/CD42a<sup>-</sup>/CD45<sup>-</sup> L-EVs concentration in O (-13 %) and Ob (-31 %).<br>C: ↑baseline CD144<sup>+</sup>/CD42a<sup>-</sup>/CD45<sup>-</sup> L-EVs concentration with ↑BMI (+21 % compared to O; +53 % compared to N)</td></tr><tr><td>Morel et al. Int J Obesity 2011<sup>76</sup></td><td>Cross-over</td><td>EU (France)</td><td>Obese women (n= 24)<br>Age= 39 ± 10<br>BMI= 35 ± 4</td><td>I= diet restriction (600 kcal/day: 15 % Fat, 30 % CHO, 55 % Protein) for 1 month followed by a regular diet (1200 kcal/day: 10 % Fat, 60 % CHO, 30 % protein) for 1 month and a progressive normal intake (1 month)</td><td>12</td><td>AnnexinV<sup>+</sup> L-EVs<br>CD11a<sup>+</sup> leukocyte L-EVs<br>CD4<sup>+</sup>lymphocyte L-EVs<br>CD31<sup>+</sup>CD41<sup>-</sup> endothelial L-EVs<br>CD41<sup>+</sup> platelet L-EVs<br>CD235<sup>+</sup> erythrocyte L-EVs<br>from Platelet-free plasma (13,000 g)<br>by Flow cytometry (Gallios, BD)</td><td>1 month-diet restriction ↑ total AnnexinV<sup>+</sup> L-EVs (including CD41<sup>+</sup> L-EVs, CD235<sup>+</sup> L-EVs)<br>I= ↓ CD4<sup>+</sup> (-20 %), CD11a<sup>+</sup> (-28 %) and procoagulant-platelet derived L-EVs (-43 %)</td></tr><tr><td>Murakami et al. Thrombosis Research 2007<sup>77</sup></td><td>Randomized Controlled</td><td>Asia (Japan)</td><td>Obese (n= 49, O) and healthy non-obese (N= 37, N) volunteers<br>Male:Female= 21:16 (N), 24/25 (O)<br>Age= 49.2 ± 1.8 (N), 50.6 ± 1.4 (O)<br>BMI= 22.8 ± 0.2 (N), 27.4 ± 0.3 (O)</td><td>I= energy restricted diet (1200 kcal/day for women; 1680 kcal/day for men) ± aerobic exercise 3 days/weeks (60 min/session)</td><td>12</td><td>CD41<sup>+</sup> platelet L-EVs<br>From platelet-rich plasma (200 g)<br>By flow cytometry (FACScan, BD)</td><td>At baseline: ↑ CD41<sup>+</sup> L-EVs in O (+97 %)<br>I: energy restricted diet ↓ CD41<sup>+</sup> L-EVs (-30 %)</td></tr><tr><td>Myette-Côté et al. Physiol 2018<sup>78</sup></td><td>Randomized Short-term Controller Cross-over</td><td>EU (UK)</td><td>T2DM volunteers (n= 11)<br>Male:Female= 4:7<br>Age= 64 ± 8<br>BMI= 34.0 ± 8.0</td><td>o Low-fat low-glycemic index diet (GL: 20 % fat, 55 % CHO, 25 % protein)<br>o Low-carbohydrate high-fat diet (LC: 65 % Fat, 10 % CHO, 25 % protein)<br>o LC with 15-min post-meal walks (LC-EX)</td><td>4days</td><td>CD14<sup>+</sup> monocytes L-EVs<br>CD45<sup>+</sup> leukocyte L-EVs<br>From platelet-free plasma (13,000 g)<br>By flow cytometry (FACSAria III, BD)</td><td>GL and LC ↓ both plasma CD14<sup>+</sup> L-EVs (-70 %) and CD45<sup>+</sup> LL-EVs (-63 %; 16 %; tendency)</td></tr></tbody></table>

Abbreviations: oGTT: oral Glucose Tolerance Test, MUFA: Mono Unsaturated Fatty Acid, SFA: saturated fatty acids.
<table><thead><tr><th>Publication</th><th>Study design</th><th>Country</th><th>Participants</th><th>Dietary intervention</th><th>Duration (weeks)</th><th>Investigated EVs</th><th>Main results</th></tr></thead><tbody><tr><td>Phang et al. Nutr Metabo CVD 2016<sup>79</sup></td><td>Randomized Controlled Double-blinded Cross-over</td><td>South Pacific (Australia)</td><td>Healthy volunteers (n=94)<br/>Male:Female= 43:51<br/>Age= 39.6 ± 1.7<br/>BMI= 24.6 ± 3.7</td><td>I= EPA rich oil (500:100 mg EPA:DHA) or DHA rich oil (100:500 mg EPA:DHA)<br/>C= sunola oil</td><td>4</td><td>AnnexinV<sup>+</sup>/CD41<sup>+</sup>/CD62<sup>+</sup> platelet L-EVs<br/>From platelet-free plasma (3,000 g) by flow cytometry (Canto, BD)</td><td>At baseline, Ø effect gender on the number of endothelial L-EVs<br/>I= Ø AnnexinV<sup>+</sup>/CD41<sup>+</sup>/CD62<sup>+</sup> L-EVs</td></tr><tr><td>Wekesa et al. Appl. Physiol Nutr Metabo 2016<sup>80</sup></td><td>Randomized Cross-over</td><td>EU (Ireland)</td><td>Overweight women (n=28)<br/>Age= 48.4 ± 0.6<br/>BMI= 28.1 ± 2.7</td><td>I= low-carbohydrate diet (1228 Kcal: 48 % fat, 29 % CHO, 23 % protein)<br/>C=normal diet (1626 Kcal: 38 % fat, 44 % CHO, 18 % protein)</td><td>24</td><td>CD31<sup>+</sup>/CD41<sup>-</sup> Endothelial L-EVs<br/>CD144<sup>+</sup> or CD146<sup>+</sup> or CD105<sup>+</sup> Endothelial L-EVs (namely endothelial L-EVs)<br/>from Platelet-poor plasma (13,000 g) by Flow cytometry (FC500, Beckman)</td><td>I= ↓ CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs (-30 %), Ø endothelial L-EVs</td></tr><tr><td>Wu et al. AJCN 2014<sup>81</sup></td><td>Randomized Controlled Double-blinded Cross-over</td><td>EU (UK)</td><td>Moderate CVD volunteers (n=84)<br/>Male:Female= 29:55<br/>Age= 45.8-49.3<br/>BMI= 25.6-26.6</td><td>I= fish-oil supplementation providing 1.5g LC n-3 PUFAs<br/>C=corn-oil supplementation</td><td>8</td><td>CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs<br/>CD31<sup>+</sup>/CD42b<sup>+</sup> PL-EVs<br/>from platelet-poor plasma (1,000 g) by flow cytometry (FACSCalibur, BD)</td><td>I= ↓ CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs (-10 %)</td></tr><tr><td>Zhang et al. MNFR 2014<sup>82</sup></td><td>Randomized Cross-over</td><td>EU (UK)</td><td>T2DM patients (n=22)<br/>Mixed gender<br/>Age from 40 to 75 years<br/>BMI= not reported</td><td>o oat-rich habitual diet (131 g/day, OAT)<br/>o standard dietary advice (SDA)<br/>o habitual diet (HAB)<br/>Challenge meal (720 Kcal: 33 % fat, 53 % CHO, 14 % protein)</td><td>8</td><td>C61<sup>+</sup> ± Fibrogen<sup>+</sup> or TF (CD142)<sup>+</sup> or CD62P<sup>+</sup> platelet L-EVs<br/>CD45<sup>+</sup> ± CD14<sup>+</sup> ± CD11b<sup>+</sup> leukocyte L-EVs<br/>from platelet-poor plasma (2,000 g) by flow cytometry (not detailed)</td><td>• OAT: ↓ Fibrogen<sup>+</sup> (-44 %) and TF<sup>+</sup> (-43 %) platelet L-EVs, CD11b<sup>+</sup> (-74 %) ML-EVs compared to HAB<br/>• SDA: ↓ Fibrogen<sup>+</sup> L-EVs compared to HAB (-47 %)<br/>During the postprandial period after challenge meal:<br/>• Transient ↓ of leukocyte-derived L-EVs in all groups<br/>• ↓ of monocyte L-EVs and CD11b<sup>+</sup> L-EVs in OAT and SDA; ↑ in HAB</td></tr><tr><td>Weech et al. An. J. Clin. Nutr. 2018<sup>83</sup></td><td>Randomized, controlled, single-blind</td><td>EU (UK)</td><td>Men and Women with moderate CVD risk (n=190)<br/>Male:Female=82:108<br/>Age=45 ± 1<br/>BMI= 26.3 ± 0.5</td><td>I=replacement of 8% SFAs with MUFAs or n-6 PUFAs for 16 weeks</td><td></td><td>■ CD31<sup>+</sup>CD42b<sup>-</sup> for endothelial L-EVs<br/>■ CD31<sup>+</sup>CD42b<sup>+</sup> for platelets L-EVs<br/>From platelet-poor plasma by flow cytometry (FACSCalibur, BD)</td><td>• Replacement by MUFAs: ↓ endothelial L-EVs (-47.3 %) and platelet L-EVs (-36.8 %)<sup>°</sup><br/>• Replacement by n-6 PUFAs: ↓ endothelial L-EVs (-44.9 %) and platelet L-EVs (-39.1 %)<sup>°</sup><br/>• SFA diet: ↑ endothelial L-EVs (+</td></tr></tbody></table>
3. Chronic dietary interventions modulate plasma L-EVs at fasting

Acute interventions have revealed that dietary intakes can drive the release of L-EVs during the postprandial period. Outside of this period (e.g., at fasting state), there still have detectable L-EVs in the circulation and have been of interest as reliable biomarkers for diagnosis and prognosis since a proportional increased level has been observed in several conditions, including ageing<sup>72, 84</sup>, physical activity<sup>85</sup>, metabolic disorders such as obesity<sup>12, 86</sup>, type 2 diabetes<sup>12, 13</sup> and metabolic syndrome<sup>87</sup>, and CVD<sup>88</sup>. Based on the established relationship between diet and health<sup>23</sup>, some research has been done to decipher the effects of (sub)chronic dietary interventions on plasma remnant EVs in different categories of population (e.g., healthy, young or normo-weighted persons, at risk, patients) (Table 3)<sup>45, 69-83</sup>. The efficiency of these interventions, lasting from 4 days to 24 weeks, has been assessed by comparing the plasma levels of L-EVs between the beginning (baseline) and the end of the study period or by comparing levels between study groups, depending on their design.

Two studies reported that diet restriction aiming at reducing the dietary energy intake of volunteers with a BMI over 25 kg/m² significantly reduced the level of circulating L-EVs. In obese women, a 600 kcal/day diet restriction for one month followed by two months of regular (1200 kcal/day) and normal diets results in lowering plasma levels of lymphocyte-derived (CD4⁺, -20 %), leukocyte-derived (CD11a⁺, -28 %) and procoagulant platelet-derived L-EVs (-43 %) <sup>76</sup>. In healthy middle-age volunteers, the increase of resident platelet-derived L-EVs (CD41⁺) associated to obesity can be attenuated by 30 % after 12 weeks under an energy-restricted diet<sup>77</sup>. Another randomized controlled trial showed that a low protein diet (8%) administrated to prostate cancer patients did not affect the level of circulating cancer-derived L-EVs identified using L1CAM markers, but enriched some of the L-EV surface markers (Leptin Receptor and phospho-Ser-IRSII), supporting the effectiveness of the dietary intervention to affect level of plasma L-EVs<sup>70</sup>.

Dietary CHO, including sugars, starches, celluloses and gums serve as major energy sources of human diet. The physiological effects of dietary CHO are highly dependent on the rate and extent of digestion and absorption in the small intestine and fermentation in the large intestine by the gut
microbiota, these latter interactions being known to promote health. Thereby, the modulation of the CHO
fraction could be useful to reach a healthier quality. Wekesa et al. ⁸⁰ reported that a low-CHO diet
decreases the level of circulating CD31⁺/CD41⁻ endothelial L-EVs (30 %) in middle-aged over-weight
healthy women. However, low-CHO content is often offset by an increased fat supply to maintain the
energy content of the diet, that is why several studies have investigated the impact of a low CHO-high fat
diet. Durrer et al. ⁶⁹ did not observe any variation of CD31⁺/CD42b⁺ and CD62⁺ endothelial L-EVs in fasting
plasma of young healthy volunteers after a 7 days- intervention on a low CHO-high fat diet However,
higher levels of plasma L-EVs were observed after a metabolic challenge consisting in an oral glucose
tolerance test only in volunteers that consumed the low CHO-high fat diet. In type 2 diabetes volunteers,
a low CHO-high fat diet reduced the level of plasma CD62⁺ endothelial L-EVs (-45 %) ⁷¹ and leukocyte-
derived L-EVs (-63 % of CD45⁺ L-EVs, -70 % of CD14⁺ L-EVs) ⁷⁸. In another study, the consumption for 8
weeks of an oat-rich diet (providing complex CHO, extensively metabolized by the gut microbiota) has led
to a lower level of fasting circulating fibrogen⁺, TF⁺-CD61⁺ platelet L-EVs (around -40 %) and of
leukocytes-derived L-EVs after a challenge test meal in type 2 diabetes (T2DM) patients ⁸². Taken
together, these results highlight that dietary CHO content affects the basal level of circulating L-EVs. Most
of the aforementioned studies did not specify the CHO quality used while a relationship between CHO
quality and health have been demonstrated ⁸⁹, especially regarding CHO with a low glycaemic index and
those poorly absorbable but highly metabolized by the gut microbiota. The nutritional interventions
presented in this review provide low amount in total CHO ⁶⁹, ⁷¹, ⁸⁰ or high in low glycaemic index CHO ⁷⁸, ⁸²
and have been associated with a decrease in circulating L-EVs.

An intervention based on a Mediterranean diet, one of those peculiarity is to be rich in n-3 PUFAS
(and in a diversity of plant food bioactives), reduce the level of circulating endothelial L-EVs identified as
annexin V⁺-CD31⁺ and CD144⁺-CD62e⁺ vesicles in elderly volunteers ⁷⁴. In the same study, the authors
have also demonstrated that a low-fat – high CHO diet enriched in n-3 PUFAs (8 %) modulated similarly
the level of circulating endothelial L-EVs. In another randomized controlled trial, some subtypes of
circulating L-EVs (namely CD61⁺ platelet-derived and CD14⁺ monocyte-derived L-EVs) present in high
amounts in post-myocardial infraction patients were reduced by a 12 weeks- supplementation with n-3 PUFA <sup>45</sup>. Likewise, a fish-oil supplementation providing long chain n-3 PUFAs and saturated fatty acids replacement by MUFA or n-6 PUFAs reduced the circulating level of CD31<sup>+</sup>/CD42b<sup>-</sup> endothelial L-EVs and/or platelet L-EVs in volunteers with moderate risk of CVD <sup>81, 83</sup>. In contrast, in healthy middle-age volunteers, the 4-weeks diet enrichment in n-3 PUFAs failed to modulate the level of circulating annexin V<sup>+</sup>-CD41<sup>+</sup>-CD62<sup>+</sup> platelet L-EVs <sup>79</sup>.

Beyond their potential contribution to the observed modulatory effects of a Mediterranean Diet on circulating L-EVs <sup>74</sup>, the efficiency of polyphenols *per se* to modulate circulating L-EVs has also been investigated. Cocoa polyphenols have been of interest due to their well-known protective effects improving CV health <sup>90</sup>. Thus, McFarlin et al. <sup>75</sup> reported that a 4-weeks dietary supplementation with cocoa bars supplying flavanols and proanthocyanins (700 mg/d in total), reduced by 13% the concentration of endothelial L-EVs (identified as CD144<sup>+</sup>/CD42a<sup>-</sup>/CD45<sup>-</sup>) in over-weight young people. Similarly, the 52% higher level of CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs observed in healthy elderly men compared to young men can be reduced by 50% after only 2 weeks of nutritional intervention supplying a cocoa flavanol rich drink twice a day (450 mg/daily dose) <sup>72</sup>. Lastly, the consumption of this cocoa flavanol rich drink for 4 weeks has been demonstrated to reduce significantly the levels of CD144<sup>+</sup> endothelial L-EVs (-22 %), CD31<sup>+</sup>/CD41<sup>-</sup> endothelial L-EVs (-39 %) but not of CD41<sup>+</sup> L-EVs in CAD patients <sup>73</sup>.

In conclusion, the analysis of these (sub)chronic intervention studies shows that dietary habits can affect the level of circulating L-EVs. Interventions aiming at providing a healthier diet, such as at low-glycaemic index or rich in n-3 PUFAs and plant food bioactives (e.g. polyphenols) appear efficient to reduce the level of circulating endothelial L-EVs and platelet-derived L-EVs in healthy volunteers or in patients with cardiometabolic disorders.

258

259

**4. Dietary modulation of EVs: what potential underlying mechanisms of action?**

The release of S-EVs resulting the fusion of the MVB with the plasma membrane and the budding of L-EVs are mechanisms both highly regulated. They involved a wide range of factors which regulate (i)

260

261
262 deformability and bending of the membrane inside MVB or at the plasma membrane (i.e. the endosomal
263 sorting complexes required for transport or ESCRT; the generation of ceramide; the tetraspanins),
264 membrane lipid composition, intracellular trafficking (e.g. Rab GTPases and the soluble N-ethylmaleimide-
265 sensitive factor activating protein receptors or SNAREs) and (ii) organization of the actomyosin
266 cytoskeleton (Rho GTPases) ⁸, ⁹¹⁻⁹⁶. In the perspective that, as suggested by the present review of available
267 data, the diet can impact EVs both in fasting and postprandial conditions, it would be interesting to
268 understand how some macro-/micro-nutrients (glucose, amino acids, triglycerides, and n-3 PUFAs) and
269 microconstituents (such as polyphenols, as main plant food bioactives) act to trigger physiological
270 pathways controlling the biogenesis and/or the release of EVs.

271 The postprandial period is characterized among other metabolic changes by a transient increase
272 in plasma glucose concentration whose magnitude depends on glucose load and on physiological state. In
273 *vitro* studies have demonstrated that a moderate elevation in glucose concentration (from 1g/L (5.5 mM)
274 to 1.9 g/L (11 mM)) or higher (up to 9g/L (50 mM)) increases the release of L-EVs by endothelial cells ³⁹, ⁴¹,
275 ⁶³, monocytes/leukocytes ⁶⁴, ⁶⁵ or renal mesangial cells ⁶⁶. However, Eichner et al. ⁹⁷ observed that the level
276 of plasma L-EVs (platelet-derived CD31⁺-CD41⁺ EVs, endothelial-derived CD31⁺-CD41⁻/CD105⁺ EVs) is
277 lowered in obese adults with prediabetes at 2-h following administration of a single 75-g oral glucose load
278 leading to 1.36 g/L plasma glucose. In contrast, an increased level of basal EVs has been reported in
279 prediabetic individuals compared to individuals with normal glucose tolerance ⁹⁸. Consistently, chronic
280 dietary interventions showed a direct correlation between CHO diet content and the level of plasma L-EVs
281 ⁷¹, ⁷⁸, ⁸⁰. This suggests that dietary glucose can drive L-EV release. The underlying molecular mechanisms
282 supporting the action of plasma glucose on L-EV release have not been properly explored. High glucose
283 can activate several signalling pathways including mitogen-activated protein kinases (MAPK) and Rho-
284 GTPases (as Rho A-ROCK) ⁹⁹⁻¹⁰¹ that have been reported to target the production of EVs ¹⁰², ¹⁰³. The rise in
285 glucose level also coincides with a rapid insulin secretion from pancreatic beta cells that exerts a
286 hypoglycaemic action. To our knowledge, the effect of insulin on EV biogenesis has never been
287 investigated. However, it is known that insulin regulates the intracellular vesicle trafficking machinery to
288 deliver the glucose transporter (GLUT4) to the cell surface and ensure glucose uptake. The insulin
289 pathway engages small GTPases of RAB and ARF families (including the ADP-ribosylation factor 6, ARF6),
290 tethering complexes and the vesicle fusion machinery (including the remodelling of the actin cytoskeleton
291 and SNARE proteins) <sup>67, 104, 105</sup>. Some of these molecular actors are also involved in the biogenesis and the
292 release of L-EVs <sup>8, 91-96</sup> suggesting that hyperinsulinemia may directly contribute to L-EV release.

293 The digestion of dietary proteins leads to a plasma increase of essential and branched-chain
294 amino acids (BCAAs) from 40 to 120 min after protein intake <sup>106-108</sup>. The concomitant increase of
295 aminoacidaemia and plasma EVs sustains a potential role of amino acids in the regulation of S-EV release.
296 The main cellular amino acid sensors are the amino acid-sensitive kinase complex, mechanistic Target of
297 Rapamycin Complex 1 (mTORC1) and the General Control Non-depressible Kinase 2 (GCN2) <sup>109-111</sup>. Briefly,
298 under BCAA deficiency, the phosphorylate GCN2 contributes to the inhibition of mTORC1 and by
299 consequence to the down regulation of protein synthesis. The low amino acids level is compensated by an
300 enhance activity of autophagy or protein degradation mediated by the proteasome system to recycle
301 damaged proteins back into BCAAs <sup>112</sup>. Evidences have disclosed that a crosstalk between proteolytic
302 activities (e.-g. autophagy, proteasome and lysosome) and the release of S-EVs, with a balance to a
303 reduced EV biogenesis under starvation conditions <sup>113, 114</sup>. After a meal, the increase of aminoacidaemia
304 and of insulin will restore the activity of mTORC1 contributing to alleviate the proteolytic activities and
305 stimulate S-EV release <sup>115, 116</sup>. mTORC1 activation can also be driven by the phosphatidic acid of EV layers
306 that has been detected in EV-like nanoparticles<sup>117</sup>. Specifically, in endothelial cells, the activation of
307 mTORC1 contributes through reactive oxygen species (ROS) to impairment of the endothelial function <sup>118</sup>,
308 <sup>119</sup>, which one feature is the increased release of endothelial L-EVs <sup>7</sup>. In addition, the endothelial
309 dysfunction induced by a high-fat diet during the postprandial period can be attenuated by a dietary
310 supply in proteins (mainly proteins rich in arginine and cysteine) <sup>120-122</sup>. Taken together, these
311 observations support that dietary proteins can prevent EV release associated to postprandial cell
312 dysfunctions. However, the lack of scientific evidence sustains the interest to explore the link between
313 dietary BCAAs and the release of EVs.
Postprandial lipaemia is characterized by a rise in circulating free fatty acids (FFAs) and triglyceride-rich proteins (TRPs) that include intestine-derived chylomicrons and their remnants and liver-derived VLDLs <sup>123</sup>. The postprandial lipaemia peak occurs at 2 hours after meal intake, kinetic that depends on dietary factors (e.g. diet composition, lipid quantity, food structure), intrinsic factors (e.g. gender, age, genetic background, health status) and life style factors (e.g. physical activity, smoking) <sup>124</sup>, <sup>125</sup>. The postprandial FFAs can directly drive the release of S- and L-EVs <sup>126, 127</sup> acting on specific receptors such as the death receptor 5 (DR5) <sup>127</sup>. With TRPs, they can also bind to other receptors such as the toll-like receptor 4 (TLR-4), contributing to induce a transient postprandial low-grade inflammation <sup>128-131</sup>. In this way, postprandial lipaemia contributes to the development of a postprandial endothelial dysfunction <sup>128, 132</sup> associated with an enhanced release of EVs. In addition, metabolic changes associated to the postprandial period, mainly postprandial glucose and lipids, participate to generate oxidative stress <sup>133-135</sup> that sustains the postprandial development of low-grade inflammation state contributing to the release of EVs. Dietary n-3 PUFAs, in contrast to palmitic acids, participate lowering level of plasma EVs through a potent multi-level action that include their anti-inflammatory effect and their ability to modulate the mTOR pathway <sup>136, 137</sup>.

Some dietary microconstituents such as polyphenols peak as metabolites in the plasma during the postprandial period <sup>138</sup>. Dietary polyphenols have been reported to prevent the release of EVs but the underlying mechanism of action is unknown. Regarding their effects on endothelial-derived L-EVs, one can speculate that polyphenols act through their capacity at preserving endothelial function <sup>139</sup> and cell dynamics <sup>140</sup> to modulate the release of L-EVs. In addition, polyphenols have been demonstrated to counteract glucose-induced inhibition of autophagy in endothelial cells <sup>141</sup> suggesting that they can restore cellular homeostasis and by consequence L-EV secretion <sup>142</sup>. Polyphenols display potent mTOR modulator through the attenuation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, that can lead the regulation of L-EV synthesis and release as previously shown <sup>143</sup>.

Taken together, these sustain that diet-derived metabolites that circulate during the postprandial period can constitute modulators of EV secretion as summarized in Figure 1. Nevertheless, further
investigations are needed to decipher the underlying mechanisms of action. Furthermore, only the
release of EVs has been considered here, while EV content that support EV bioactivity has never been
investigated. Thus, another important question to address in future is how the diet can modulate the
content of EVs, and by consequence their functions.

### 5. Food-derived EVs: can they affect human health?

EVs from exogenous dietary sources such as milk or vegetables can be also found in bloodstream
and affect homeostasis of the host or improve pathophysiological conditions. This is the particular
interest taking in consideration the ability of EVs to protect some molecules with short half-life from
degradation. For instance, EV-like nanoparticles from grapefruits and milk resist to digestion products of
the host such as gastric enzymes and intestinal pancreatic and bile extract solution. Furthermore, these
nanoparticles do not modify serum levels of interferon gamma (IFN-γ) and the liver enzymes in mice
suggesting the lack of deleterious effects on inflammation<sup>23</sup>.

Interestingly, plant-derived EVs can display beneficial effects under pathological conditions due to
the plant bioactive molecules that they carry, as recently reviewed<sup>43, 144, 145</sup>. Indeed, it has been shown
that grapefruit-derived nanovesicles can be used as immune modulators in the digestive tract during
colitis in mice. Thus, through the direct action on intestinal macrophages, these vesicles inhibit the
production of IL-1β and TNF-α in these cells and allow the maintaining of intestinal macrophage
homeostasis<sup>23</sup>. In the same pathology, broccoli-derived nanoparticles decrease the weight loss, the
infiltration of inflammatory cells in the mucosa and the pro-inflammatory cytokine production<sup>30</sup>.
Interestingly, broccoli-derived nanoparticles activate AMPK in dendritic cells leading to a dual effect
associated to the prevention of dendritic cell activation and the induction of tolerant dendritic cells. Also,
lemon-derived EVs display anti-tumor properties in several tumor cell lines by arresting cell proliferation
of tumor cells via the induction of TRAIL-mediated cell death<sup>146</sup> and downregulating proteins implicated
in the lipid metabolism pathway<sup>147</sup>. Dietary plant-derived EVs had also effects on lipid metabolism as
illustrated by the fact that ginger-derived EVs can protect against the accumulation of lipid droplets
during alcohol-induced liver damage<sup>147</sup> and lemon EVs reduce LDL cholesterol in healthy volunteers<sup>148</sup>.
366 Milk is another food source of EV-like nanoparticles that could modulate several functions of
367 human milk-consumers triggering several host tissues ¹⁴⁹. In human food, milk and its derived products
368 can be pasteurized and/or fermented. Milk-derived EVs numbers are not affected by pasteurization
369 compared to other method of sterilization¹⁵⁰, ¹⁵¹. Thus, pasteurized milk is a new source of large and small
370 EVs for human consumers of western diet allowing the transfer of milk-derived microRNAs. These small
371 EVs of pasteurized milk convey oncogenic micro RNAs (e.g., microRNA-21 and microRNA-148a) which
372 both inhibit PTEN (Phosphatase and TENsin homolog) expression and drive mTORC1-mediated
cancerogenesis ¹⁵²⁻¹⁵⁴. In another study, the authors observed that bovine milk-EVs (orally administrated
373 at 25 mg/kg) reduce primary tumor growth but accelerate metastasis in breast and pancreatic cancer
374 mouse models¹⁵⁵. The oral administration of milk-derived S-EVs have been also demonstrated to
375 attenuate the severity of colitis ¹⁵⁶ and of arthritis ¹⁵⁷. These beneficial effects have been partly associated
376 to the ability of milk-derived S-EVs to reduce the expression of inflammatory cytokines (e.g. the monocyte
377 chemo-attractant protein (MCP-1), inteleukin-6 and TNF-α) and to up-regulate the expression of anti-
378 inflammatory mediators such as the transforming growth factor beta (TGF-β). In addition, milk-derived S-
379 EVs convey miRNAs that can act as epigenetic regulators contributing to the effects of these EVs on
380 biological functions ¹⁵⁸. In addition, milk-derived S-EVs can drive some pathways such as the AMPK-
mTORC1 activation¹⁵², ¹⁵⁹, that can result in the release of EVs from the host recipient cells.. Furthermore,
381 due to their small size EV-like nanoparticles from foods can probably cross the epithelial barrier including
382 the blood brain barrier¹⁶⁰. This hypothesis is supported by the loss of spatial learning and memory
383 observed in mice fed a diet with bovine milk depleted in S-EVs for up to 20 weeks compared to mice
384 consuming a complete diet¹⁶¹ and the detection in brain of fluorescent-labelled milk S-EVs administered
to adult mice through oral gavage ¹⁶². 385

388 Together, these data underline the strength of EV-like nanoparticles from foods to impact on
389 biological functions and the interest to consider these EVs as relevant dietary contributors on human
health.
390
391
392

# 6. Diet-induced EVs: friends or foes for human health?

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It is widely demonstrated that EVs are important mediators of cell-to-cell communication. EV-mediated intercellular communication begins by the recognition and the capture of circulating EVs by target cells<sup>92, 163</sup>, that is influenced by both EV surface markers and EV size. Information are transmitted from EVs to cells by acting at the cell membrane, with or without the delivery of their content. The mechanisms of uptake and cargo delivery into recipient cells are still incompletely understood. But evidences support that EVs drive phenotypic changes mostly by spreading the phenotypic features of donor cells to acceptor cells<sup>164</sup>. Thus, the pending question that is raised in the present review focuses on the biological function(s) of diet-induced EVs. Knowledge on this topic is still scarce and is constrained by the heterogenicity of circulating diet-induced EVs. As previously mentioned, diet modulates EV release mainly from endothelial cells, leukocytes and platelets. In addition to this board of cell source, it has been reported that cells release various subpopulations of EVs with distinct size, protein cargo and miRNAs content<sup>36, 165-168</sup>.

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Spectre et al.<sup>58</sup>, who reported an increased level of annexin V<sup>+</sup> L-EV derived from endothelial cells, platelet and monocytes after a standard meal, have demonstrated that these EVs generate more thrombin than pre-meal EVs. In contrast, acute and chronic n-3 PUFA supplementation have been shown to lower plasma annexin V/TF<sup>+</sup> L-EV levels and their thrombin generation capacity<sup>45, 57</sup>. Endothelial-derived L-EVs carrying TF<sup>+</sup> and platelet-derived L-EVs exposing at their surface phosphatidylserine are known to be procoagulant and to increase the risk of venous thrombo-embolism<sup>169</sup>. Circulating L-EVs released in dyslipidaemia induced by a high-energy diet have been shown to contribute to inflammation and vascular damage by increasing the endothelial expression of intercellular adhesion molecule 1 (iCAM-1) and the production of reactive oxygen species and by reducing vascular relaxation of aortic rings<sup>21</sup>. These EVs also exert an antiatherogenic action through the reduction of caveolin 1 endothelial. Similarly, Jansen et al.<sup>170</sup> demonstrated that high glucose-induced L-EVs impair endothelial function as determined by a lower endothelial-dependent relaxation in EV-treated mice and by an increased adhesion of monocytes to L-EV-treated human coronary artery endothelial cells partly due to an up-regulation of
418 iCAM-1 and vCAM-1 in the treated cells. Similarly, L-EVs released from activated endothelial cells, such as
419 those released during the transient diet-induced inflammation and endothelial dysfunction, exert a dual
420 role. Luna et al. ¹⁷¹ reported that endothelial L-EVs induced by inflammation alter the function of
421 endothelial cell progenitors whereas Mahmoud et al. showed that they reduce oxidative stress and
422 prevent lipid-induced endothelial damage ¹⁷². On another note, it has been observed in the Framingham
423 Offspring cohort the association of circulating level of endothelial L-EVs (referred as CD31, CD144⁺ EVs
424 and known to reflect damaged endothelium) with the presence of cardiometabolic risk factors,
425 particularly dyslipidaemia ¹⁷³.

426 Other EVs subtypes (e.g. from adipose tissues, skeletal muscles), not detected so far in human
427 plasma in response to a meal intake or a chronic dietary supplementation, participate to attenuate
428 glucose intolerance and to improve insulin resistance ¹⁷⁴⁻¹⁷⁷. Wu et al. ¹⁷⁸ demonstrated that S-EVs derived
429 from liver and enriched in miR-130a contribute to the recovery of insulin resistance induced by an HFD in
430 mice. A study by Kumar et al. ¹⁷⁹ revealed that an HFD affect intestinal epithelial S-EVs that are enriched in
431 phosphatidylcholine resulting in an inhibition of the insulin response in mice. The authors report the same
432 alteration in lipid composition in S-EVs from patients with T2DM. Together, these results reveal that S-EVs
433 are potential contributors to the development of insulin resistance and that diet can be an efficient
434 strategy to attenuate their impact.

435 In absence of more investigations on the biological function(s) of diet-induced EVs, available data
436 support that unbalanced diet could drive through metabolic changes (glycaemia, lipaemia, oxidative
437 stress and related inflammation) the release of EVs that convey pro-inflammatory, pro-thrombotic and
438 pro-atherogenic messages, these latter representing in case of chronicity an increased risk for health.
439 However, this risk could be attenuated by an improved diet supplying protective micronutrients and plant
440 food bioactives.

441

442 **Conclusion**
This review highlights the importance to consider diet as an important regulator both in postprandial and fasting conditions of EV biology and to develop further investigations to extend knowledge in this promising but still underexplored research field.

EVs modulated by diet have been identified using the same methods (i.e. surface markers) than for the detection of fasting EVs in patients with a diagnosis or prognosis purpose. By consequence, diet-associated EVs share similar phenotype characteristics with disease-associated EVs. Apart the "fast" feature during the postprandial period, the kinetic of release of diet-associated EVs has never been fully described. Consequently, their persistence in the bloodstream till a fasting state cannot be excluded. If so, this phenomenon could skew the use of fasting EVs as a reliable health biomarker and this aspect must be addressed properly. One bias of the methods used to explore human plasma EVs is to exclude S-EVs that may be modulated by diet and could be use as reliable nutritional biomarker as the L-EVs. Further, EV content, that is considered as a fingerprint of their cells of origin, has not been reported for diet-associated EVs. One critical question in this regard is how the cargo (e.g. proteins, miRNAs and lipids) of diet-associated EVs allow to differ them from EV released in health disturbances. In addition, an accurate characterization of diet-associated EV content will contribute to a better understanding of their biological functions. In 2021, Mantilla-Escalante et al.¹⁸⁰ report that the long-term consumption of a Mediterranean diet enriched in extra-virgin olive oil or nuts from participants of the PREDIMED study improves the number and modulates the miRNA profiles of circulating exosomes. This modulation could help to explain how such diet lead to beneficial health effects. Based on the biological functions reported for diseases associated-EVs or *in vitro* induced EVs sharing similar phenotype features, EVs induced after consumption of an unbalanced diet are assumed to constitute a risk factor for health, that could potentially be slow down by supplying diet with plant food bioactives protective for health. Answering these pending questions will contribute to substantiate the interest of EVs and their content as innovative tools to explore the relationship between nutrition and health.

468 Acknowledgements
469 We are grateful to Christine Morand (INRAE, UNH) for her valuable advice during the preparation of the

470 manuscript.

471 This paper was supported and approved by INRAE.

472 Declaration of interest: none.

473

474 Author contribution

475 LEM performed literature search and analysis, and he wrote the manuscript. MCM contributed to the

476 editing and reviewed the manuscript.

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966

967 **Table Legend:**

968 **Table 1:** Cell surface markers used for the identification of EV subpopulations by flow cytometry.

969 **Table 2:** Clinical trials investigating the impact of acute dietary intakes on EVs during the postprandial
970 period.

971 **Table 3:** Human intervention studies reporting the effects of (sub)chronic dietary intake on circulating
972 EVs. C: control diet; I: nutritional intervention.

973 **Legend figures**

974 **Graphical abstract:** Summary of key aims reviewed

975 **Figure 1:** Potential effects of dietary nutrients and metabolites driving the biogenesis and the release of
976 EVs.

977