nutrients Article High Adherence to the Nordic Diet Is Associated with Lower Levels of Total and Platelet-Derived Circulating Microvesicles in a Norwegian Population Gemma Chiva-Blanch 1,2,3, * , Kristian Laake 1,3 , Peder Myhre 1,3 , Vibeke Bratseth 1,3 , Harald Arnesen 1,3 , Svein Solheim 1 , Lina Badimon 2 and Ingebjørg Seljeflot 1,3 1 Center for Clinical Heart Research, Department of Cardiology, Oslo University Hospital Ullevål, 0450 Oslo, Norway; kristian.laake@medisin.uio.no (K.L.); pedermyhre@gmail.com (P.M.); vbratset@ous-hf.no (V.B.); UXHAAR@ous-hf.no (H.A.); svein.solheim@ulleval.no (S.S.); UXINLJ@ous-hf.no (I.S.) 2 Cardiovascular Program-ICCC, Research Institute- Hospital Santa Creu i Sant Pau, 08025 Barcelona, Spain; LBadimon@santpau.cat 3 Faculty of Medicine, University of Oslo, 0372 Oslo, Norway * Correspondence: GChiva@santpau.cat; Tel.: +34-935565613  Received: 23 April 2019; Accepted: 16 May 2019; Published: 18 May 2019  Abstract: Circulating microvesicles (cMV) are small phospholipid-rich blebs shed from the membrane of activated vascular cells that contribute to vascular disease progression. We aimed to investigate whether the quality of the Nordic diet is associated with the degree of blood and vascular cell activation measured by MV shedding in elderly patients after an acute myocardial infarction (AMI). One-hundred and seventy-four patients aged 70–82 years were included in this cross-sectional study. Fasting blood samples were taken within 2 to 8 weeks after an AMI. Annexin V (AV)+ cMV derived from blood and vascular cells were measured through flow cytometry. A patient’s usual diet was recorded with the SmartDiet®questionnaire. Patients with higher adherence to the Nordic diet (highest diet score) had lower levels of total AV+ and platelet-derived (CD61+ /AV+ and CD31+ /AV+ ) cMV. Dietary habits influence cellular activation. A high adherence to the Nordic diet (assessed by the SmartDiet®score) in elderly post-AMI patients was associated with lower levels of platelet activation, which was reflected by a lesser release of MV carrying platelet-derived epitopes, potentially contributing to an explanation of the cardioprotective effects of the Nordic diet. Keywords: Nordic diet; circulating microvesicles; SmartDiet; cardiovascular disease; platelets 1. Introduction Cardiovascular disease (CVD) is one of the main causes of death worldwide. The relationship between diets and CVD is complex, although it has been shown that some dietary patterns, such as the Mediterranean diet [1] or the Nordic diet [2], elicit cardioprotective effects. The Nordic diet is characterized by a high intake of foods produced locally, such as salmon and herring (fatty fish); cod; tubercles; pulses; wholegrain cereals such as barley, oats, and rye; berries (which are very rich in polyphenols); and other fruits. Therefore, this diet is especially rich in long-chain omega 3 fatty acids, accompanied by a moderate-to-high intake of fiber, minerals, and antioxidants, and has been shown to improve endothelial function and alleviate proinflammatory states [2]. Over recent years, this dietary pattern has been the object of increasing interest because it has been associated with decreased overall and cancer mortality, although controversial results have been observed in terms of CVD mortality [3]. Circulating microvesicles (cMV) are a subtype of extracellular vesicles rich in phospholipids that range from 0.1 to 1 micrometer (µm) diameter and shed from cells when activated, injured, or Nutrients 2019, 11, 1114; doi:10.3390/nu11051114 www.mdpi.com/journal/nutrients Nutrients 2019, 11, 1114 2 of 12 undergoing apoptotic or necrotic processes. Circulating microvesicles act as procoagulant agents because an externalization of the procoagulant anionic phosphatidylserine (PS) takes place during MV formation. In addition, cMV carry antigens from their cell of origin and have also been found to be involved in inflammatory processes, thus contributing to both the initiation and progression of CVD. In fact, elevated platelet-, endothelial-, and leukocyte-derived cMV have been observed in several CVD states and have been associated with several CVD risk factors, as reviewed in Reference [4]. The effects of diet on MV shedding have been scarcely investigated (reviewed in Reference [5]), and the relationship between the Nordic diet and MV generation is largely unknown. Therefore, the aim of our study was to investigate whether the quality of the Nordic diet is associated with MV shedding in elderly patients after an acute myocardial infarction (AMI), which is known to increase the levels of cMV [6,7]. 2. Materials and Methods 2.1. Study Population The subjects included in this cross-sectional study belong to a subset of the “OMega-3 fatty acids in Elderly patients with Myocardial Infarction” (OMEMI; NCT01841944, Clinical-Trials.gov) cohort [8] at inclusion in the study. The protocol was approved by the Regional Committee for Medical Research Ethics (ref. number: 2012/1422). Patients 70–82 years old were recruited at Oslo University Hospital Ullevål, Oslo, Norway, after a diagnosis of an AMI, and were included in the study after 2 to 8 weeks of surviving the AMI. Patients were treated per guidelines with standard medication. Demographic data, clinical characteristics, cardiovascular history, classic cardiovascular risk factors, and current pharmacological treatment were obtained from all subjects using a standardized report form at inclusion. A patient’s usual diet was recorded at study entry with the SmartDiet®questionnaire (version 3, from May 2009), which is a fast, simple, and validated tool for the assessment of food intake and diet quality, and results in a diet score [9]. As shown in Table 1, the SmartDiet score is built upon 14 questions relating to food quantity and quality, which are scored, summed, and categorized as unhealthy (≤ 27 points, group I), somewhat unhealthy (28–35 points, group II), or healthy diet (≥ 36 points, group III), according to the Nordic Nutrition Recommendations. The study protocol was approved by the Regional Committee for Medical Research Ethics and followed the principles outlined in the Declaration of Helsinki. All participants signed their informed consent. Table 1. Questions and scores of version 3 of the SmartDiet questionnaire used to assess dietary patterns and adherence to the Nordic diet. Least Healthy (1 point) More Healthy (2 points) Most Healthy (3 points) Milk or yogurt What sort of milk do you use low-fat milk or no milk whole milk skimmed milk more often? at all Cream and sour cream extra-light cream or less What sort of cream and sour whole cream light cream than once weekly cream do you use more often? consumption Cheese cheese with rape and What sort of cheese do you use sunflower oil, extra-light white cheese light cheese more often? cheese or less than once weekly consumption Nutrients 2019, 11, 1114 3 of 12 Table 1. Cont. Least Healthy (1 point) More Healthy (2 points) Most Healthy (3 points) Spreading/cured meat lean meats or less than What sort of spreading meat do fat meats - once weekly you use more often? consumption Meat for dinner lean meats or less than What sort of meat do you lean met with fat rim or Fat or processed meat once weekly consume for dinner more often? skin consumption Spreading fish How often do you spread fish in up to 1 slice of bread per between 2 and 4 slices of about 5 or more slices of sandwiches or in salads for week or never bread per week bread per week lunch? Fish for dinner How many times per week do between once per week you eat fish, fish products, 2 times per week 3 or more times per week and never and/or fish dishes? Mayonnaise, remoulade, and kaviar How often do you use Up to 1 slice of bread per 2–7 slices of bread per mayonnaise products on bread week or never or 8 or - week food? more times per week Butter and margarine on bread food What sort of butter and light margarine or low margarine do you use more dairy butter margarine consumption of butter often? and margarine Fat in cooking What sort of fat do you use more oils or use of fat for often when frying, baking, or in dairy butter margarine cooking sauce, such as dressings? Bread and other grain products What sort of bread or grain white bread or low bread whole-grain bread and - cereals do you use more often? consumption cereals Vegetables, fruits and berries How many portions (150 g, equivalent to approximately 2 fewer than 2 portions (< more than 4 portions (> 2–4 portions (300–600 g) carrots or about a big apple) do 300 g) 600 g) you consume daily? Sweet toppings and sweet drinks How often do you use sweet toppings or drinks with glucose 3 or more times per day 2 times per day 0–1 times per day or fructose? Chocolate, snacks, cakes and biscuits How often do you eat snacks? 3 or more times per week 2 times per week 0–1 times per week The points from each item are summed into a total SmartDiet score classified as < 27 points, unhealthy; 28–35 points, somewhat healthy; and >36 points, healthy diet. 2.2. Blood Sampling Fasting venous blood was withdrawn into 3.8% sodium citrate tubes for MV analysis. Blood cells were removed by centrifuging whole blood 20 min at 2500× g at 4 ºC. Plasma was carefully recollected, leaving about 0.2 cm of an undisturbed layer on top of the cells, and aliquots were immediately frozen and stored at −80 ºC until processing for isolation and quantification of cMV. Additionally, biochemical and hematological parameters were quantified with standardized routine methods. Nutrients 2019, 11, 1114 4 of 12 2.3. Circulating Microvesicle Isolation and Quantification The cMV fraction was isolated from citrated and anticoagulated plasma through a two-step high-speed centrifugation procedure. Five-hundred microliters (µL) of frozen plasma aliquots were thawed on melting ice and centrifuged again at 2500× g for 15 min at room temperature (RT) [10]. Then, 250 µL of plasma were collected and centrifuged at 20000× g for 30 min at RT to pellet cMV. The supernatants were discarded, and the cMV-enriched pellet was washed once with citrate-phosphate buffered saline (PBS) solution (citrate-PBS; 1.4 mM phosphate, 154 mM NaCl, 10.9 mM trisodium citrate, pH 7.4) before a second equal centrifugation step was made. Finally, the remaining cMV pellets were resuspended in 100 µL of citrate-PBS. A triple-label flow cytometry analysis was performed as described before [7,11]. Briefly, 5 µL of washed cMV suspensions were diluted in 30 µL of PBS buffer containing 2.5 mM of CaCl2 (Annexin Binding Buffer, ABB). Thereafter, combinations of 5 µL of allophycocyanin (APC)-conjugated Annexin V (AV, BD-horizon) with two specific monoclonal antibodies (mAb, 5 µL each, Table 2) labeled with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) (or the isotype-matched control antibodies) were added. Table 2. Cell molecules for circulating microvesicle (cMV) identification and characterization. mAb Alternative Name Expression Conjugation Clone Company Widely AV PS-binding protein expressed APC BD Biosciences Widely Sekisui CD142 Tissue Factor expressed FITC VD8 diagnostics Platelet-Related BD CD61 β3 -integrin Platelets PE VI-PL2 Pharmingen CD31 PECAM-1 Activated Cells FITC AC128 Miltenyi Biotec CD62P P-Selectin Platelets PE HI62P Immunotools Glycoprotein Ib alpha Platelets and CD42b chain megakaryocytes PE REA185 MACS Miltenyi Endothelial Cell-Related Melanoma Cell Endothelial BD CD146 Adhesion Molecule Cells FITC P1H12 Pharmingen Endothelial CD62E E-Selectin Cells PE 68-5H11 BD Biosciences HSCs, EPCs, CD309 VEGFR-2 and ECs FITC ES8-20E6 MACS Miltenyi Erythrocyte-Related CD235ab Glycophorin A and B Erythrocytes FITC HIR2 Immunotools Leukocyte-Related Leukocyte Common CD45 Antigen Leukocytes PE MEM-28 Immunotools CD3 T-cell co-receptor T-Lymphocytes FITC HIT3b Immunotools Macrophages, BD CD14 LPS-receptor monocytes PE M5E2 Pharmingen CD62L L-Selectin Leukocytes FITC LT-TD180 Immunotools Macrophage-1 Antigen Neutrophils, CD11b (Mac-1) leukocytes FITC M1/70.15.11.5 MACS Miltenyi Smooth Muscle Cell-Related Smooth Muscle Actin Smooth muscle SMA-α α cells PE 1A4 R&D Systems APC denotes allophycocyanin; AV, Annexin V; CD, cluster of differentiation; ECs, endothelial cells; EPCs, endothelial progenitor cells; FITC, fluorescein isothiocyanate; HSCs, hematopoietic stem cells; LPS, lipopolysaccharide; mAb indicates monoclonal antibody; PE, phycoerythrin; PECAM-1, platelet endothelial cell adhesion molecule; PS, phosphatidylserine; and VEGFR-2, vascular endothelial growth factor receptor-2. Samples were incubated for 20 min at RT in the dark and diluted with ABB before being immediately analyzed with the “Auto Collect” mode in 96-well plates on an AccuriC6 flow cytometer (BD, Accuri®Cytometers, Inc., San Diego, CA, US), except for MV from smooth muscle cells (SMCs), which were quantified as previously described [6,7]. Specifically, for the detection and quantification of cMV from SMC origin, 5 µL of the cMV suspension were incubated for 20 min at RT in the dark with 5 µL of AV-APC and 5 µL of CD142-FITC (tissue factor, TF) in a final volume of 50 µL ABB. cMV were fixed with 250 µL of ABB/paraformaldehyde 2% for 30 min and centrifuged at 20000× g for 30 min to immediately analyzed with the “Auto Collect” mode in 96-well plates on an AccuriC6 flow cytometer (BD, Accuri® Cytometers, Inc., San Diego, CA, US), except for MV from smooth muscle cells (SMCs), which were quantified as previously described [6,7]. Specifically, for the detection and quantification of cMV from SMC origin, 5 µ L of the cMV suspension were incubated for 20 min at RT in the dark with 5 µ L of AV-APC and 5 µ L of CD142-FITC (tissue factor, TF) in a final volume of Nutrients 2019, 11, 1114 5 of 12 50 µ L ABB. cMV were fixed with 250 µ L of ABB/paraformaldehyde 2% for 30 min and centrifuged at 20000 × g for 30 min to pellet cMV. After eliminating the supernatant, cMV were permeabilized with 45 µ LcMV. pellet of ABB/saponin After eliminating0.1% the for supernatant, 20 min at RTcMV in the weredark. After permeabilizing, permeabilized with 45 µL of 5 µABB/saponin L of smooth muscle 0.1% for actin 20 min(SMA)-α-PE at RT in thewere dark.added After to the cMV suspension, permeabilizing, incubated 5 µL of smooth for 20 muscle min(SMA)-α-PE actin at RT in the were dark, and finally diluted with ABB prior to flow cytometer analyses. added to the cMV suspension, incubated for 20 min at RT in the dark, and finally diluted with ABB prior For all cytometer to flow cMV characterization analyses. and quantification, acquisition was performed at 2 min per sample. For allThecMV flow rate was setand characterization at quantification, 14 µ L/min. Forward scatter acquisition was (FSC), performedsideatscatter (SSC), 2 min per and sample. fluorescence The flow rate was dataset were at 14obtained with the scatter µL/min. Forward settings on the (FSC), sidelogarithmic scale. scatter (SSC), andIn addition, cMV fluorescence were data were identifiedwith obtained andthe quantified based settings on on their binding the logarithmic scale. to In AV and their addition, cMVreactivity to cell-specific were identified mAbs and quantified (Figure based on1). To identify their binding to positive AV and marked events, thresholds their reactivity of fluorescence to cell-specific mAbs (Figure were 1).set Tobased on positive identify samples incubated marked withthresholds events, the same final concentration of fluorescence wereofset isotype-matched based on samples control antibodies incubated after with the sametitration final experiments. of concentration The AV binding level isotype-matched wasantibodies control corrected after for autofluorescence usingThe titration experiments. fluorescence AV binding signals level obtained was withfor corrected MV labeled with AV autofluorescence in fluorescence using a calcium-free buffer signals (PBS). with obtained To reduce background MV labeled with AVnoise, in a buffers werebuffer calcium-free prepared on To (PBS). thereduce same day and filtered background through noise, buffers0.2-µ m pore were size filters prepared on theunder same vacuum. day and filtered through 0.2-µm pore size filters under vacuum. Figure 1. Representative plots for MV identification and characterization. (A) P1 was set according Figure to cMV 1. Representative size plots and granularity for MVasidentification (defined <1 µm). (B) and characterization. Annexin V-APC+ cMV (A) P1 was (M1) wereset according selected to from cMV size and granularity (defined as <1 µ m). (B) Annexin V-APC + cMV (M1) were selected from P1. P1. (C) AV+ cMV binding PE+ (M2)- or (D) FITC+ (M3)-labeled antibodies were selected from P1 and (C) AV+ cMV quantified. binding Double PE+ (M2)- staining or (D)and with FITC- FITC + (M3)-labeled antibodies were selected from P1 and PE-labeled antibodies from M1 (Annexin V+ cMV) was also quantified. Here, cMV indicates circulating microvesicles, APC denotes allophycocyanin, FITC indicates fluorescein isothiocyanate, and PE is phycoerythrin. Data were analyzed with BD CSampler software (version 1.0.264.21, Accuri®Cytometers, Inc.). The cMV concentration (number of cMV per µL of plasma) was determined according to Nieuwland’s formula [12]. Nutrients 2019, 11, 1114 6 of 12 2.4. Statistical Analysis Statistical analyses were performed using the SPSS Statistical Analysis System (version 23.0, IBM Corp. Armonk, NY). Descriptive statistics (mean ± SD or n (%)) were used to describe the characteristics of the patients and the outcome variables. The normality of the variables was assessed with the Shapiro–Wilk test. All variables with a skewed distribution were transformed into their natural logarithms for regression analyses. ANOVA with the Bonferroni post hoc test or t-test analyses were conducted to examine the differences in MV concentrations related to cardiovascular risk factors and potential confounding factors and related to groups of the SmartDiet score. A linear regression was performed to investigate the specific influence of covariates in the relationship between diet and cMV. According to the results, we performed Spearman’s correlations and multivariate linear regressions adjusted for age, sex, use of fish oil supplements, and smoking history to analyze the main associations between the SmartDiet score and cMV. A two-tail p-value of <0.05 was considered statistically significant. 3. Results 3.1. Patients’ Characteristics Patients were divided into three categories according to the SmartDiet score, which was based on the usual intake of the different food groups included in the SmartDiet questionnaire. Group I was defined as ≤ 27 points (n = 39, 22.4%), group II as 28–35 points (n = 106, 60.9%), and group III as ≥ 36 points (n = 29, 16.7%) in the questionnaire, where the higher the score, the healthier the diet, according to the Nordic Nutrition Recommendations [9]. Table 3 shows the characteristics of the 174 patients included in the study according to groups of score. Although cMV concentrations from endothelial cells, erythrocytes, lymphocytes, and monocytes increased over time after an AMI (not shown), the distribution of patients according to the weeks after an AMI was homogeneous within tertiles of adherence to the Nordic diet. Therefore, no statistical interaction was observed between adherence to the Nordic diet and weeks after the AMI at inclusion in the study, for the levels of cMV in the patients studied. The mean age was 75 years, and approximately 60% were hypertensive. Almost all patients were under statin and dual antiplatelet therapy (98% and 95.5%, respectively), 24% were diabetics, 10% were current smokers, and 50% of the study population used fish oil supplements prior to inclusion. Table 3. Characteristics of the total study population (n = 174) and according to the grouping by diet score. All Patients Group I Group II Group III Mean ± SD or n (%) p (n = 174) (n = 39) (n = 106) (n = 29) Weeks after an AMI at 0.994 inclusion 2 up to 4 weeks 90 (52) 21 (54) 53 (50) 16 (55) 4 up to 6 weeks 41 (24) 9 (23) 25 (24) 7 (24) 6–8 weeks 43 (25) 9 (23) 28 (26) 6 (21) Males 124 (71.2) 28 (71.8) 78 (73.6) 18 (62.1) 0.371 Age (years) 75 ± 4 75 ± 4 74 ± 4 73 ± 3 0.403 BMI (kg/m2 ) 26.11 ± 4.04 25.77 ± 3.68 26.33 ± 4.46 26.16 ± 3.54 0.803 3.68 3.54 Systolic blood pressure (mm 0.092 139 ± 9 138 ± 23 139 ± 18 130 ± 14 Hg) Diastolic blood pressure 0.651 75 ± 10 74 ± 11 76 ± 8 74 ± 10 (mm Hg) Nutrients 2019, 11, 1114 0.410 7 of 12 Total cholesterol (mmol/L) 3.88 ± 0.82 3.74 ± 0.90 3.87 ± 0.83 3.93 ± 0.74 0.973 HDL cholesterol (mmol/L) 1.38 ± 0.44 1.40 ± 0.52 1.37 ± 0.38 1.42 ± 0.45 Table 3. Cont. 0.867 Total cholesterol/HDL ratio 2.97 ± 0.85 2.79 ± 0.57 2.95 ± 0.82 2.95 ± 0.90 All Patients Group I Group II Group III Mean ± SD or n (%) p (n = 174) (n = 39) (n = 106) (n = 29) 0.525 LDL cholesterol (mmol/L) 2.16 ± 0.64 2.05 ± 0.59 2.17 ± 0.64 2.2 ± 0.64 Systolic blood pressure 139 ± 9 138 ± 23 139 ± 18 130 ± 14 0.092 (mm Hg) 0.580 Triglycerides (mmol/L) 1.30 ± 0.84 1.20 ± 0.33 1.24 ± 0.57 1.40 ± 1.69 Diastolic blood pressure 75 ± 10 74 ± 11 76 ± 8 74 ± 10 0.651 (mm Hg) 0.372 Glucose Total (mmol/L) cholesterol (mmol/L) 6.23 3.88 ± ± 1.97 0.82 5.93 3.74±±1.66 0.90 6.22 3.87±±1.89 0.83 5.76 ± 0.98 3.93 ± 0.74 0.410 HDLHbA1C cholesterol (%) (mmol/L) 6.14 ± 1.01 1.38 ± 0.44 1.40±±0.78 6.08 0.52 1.37±±1.12 6.19 0.38 1.42 ± 5.83 0.45 0.246 ± 0.71 0.973 Total cholesterol/HDL 2.97 ± 0.85 2.79 ± 0.57 2.95 ± 0.82 2.95 ± 0.90 0.867 ratio Previous hypertension LDL cholesterol (mmol/L) 103 (59.2) 2.16 ± 0.64 26 (66.7) 2.05 ± 0.59 56 (52.8) 2.17 ± 0.64 21 (72.4) 2.2 ± 0.64 0.4000.525 Previous dyslipidemia Triglycerides (mmol/L) 78 (44.8) 1.30 ± 0.84 15 (38.5) 1.20 ± 0.33 51 (48.1) 1.24 ± 0.57 12 (41.4) 1.40 ± 1.69 0.3460.580 Previous Glucose diabetes(mmol/L) 41 (23.6) 6.23 ± 1.97 10 (25.6) 5.93 ± 1.66 25 (23.6) 6.22 ± 1.89 6 (20.7) 5.76 ± 0.98 0.8640.372 HbA1C (%) Current smokers 6.14 ± 1.01 17 (9.8) 76.08 ± 0.78 (17.9) 6.19 ± 1.12 7 (6.6) 5.83 ± 0.71 3 (10.3) 0.1510.246 Previous hypertension 103 (59.2) 26 (66.7) 56 (52.8) 21 (72.4) 0.400 Medication after AMI Previous dyslipidemia 78 (44.8) 15 (38.5) 51 (48.1) 12 (41.4) 0.346 Acetylsalicylic acid Previous diabetes 166 (95.4) 41 (23.6) 3510(89.7) (25.6) 102 25 (96.2) (23.6) 29 (100) 6 (20.7) 0.2280.864 ADPCurrent receptor inhibitors smokers 163 17 (94.7) (9.8) 367(92.3) (17.9) 1007 (94.3) (6.6) 27 (93.1) 3 (10.3) 0.8850.151 Statins Medication after AMI 170 (97.7) 38 (97.4) 103 (97.2) 29 (100) 0.692 BetaAcetylsalicylic blockers acid 166 (87.9) 153 (95.4) 3435(87.1) (89.7) 102(85.8) 91 (96.2) 29 (96.5) 28 (100) 0.1740.228 ADP receptor inhibitors 163 (94.7) 36 (92.3) 100 (94.3) 27 (93.1) 0.885 Calcium channel Statins 31 170(17.8) (97.7) 738 (17.9) (97.4) 22 103(20.7) (97.2) 29 2 (6.9) (100) 0.2800.692 blockers Beta blockers 153 (87.9) 34 (87.1) 91 (85.8) 28 (96.5) 0.174 ACE inhibitors Calcium channel blockers 58 31 (33.3) (17.8) 137(33.3) (17.9) 36 22(34.0) (20.7) 92(31.0) (6.9) 0.9310.280 ARB ACE inhibitors 44 58 (25.3) (33.3) 813 (20.5) (33.3) 30 36(28.3) (34.0) 69 (20.7) (31.0) 0.5220.931 Nitrates ARB 44 (12.1) 21 (25.3) 38(7.7) (20.5) 30(15.1) 16 (28.3) 62 (20.7) (6.9) 0.2830.522 DiureticsNitrates 21 (12.1) 41 (23.6) 3 (7.7) 8 (20.5) 16 (15.1) 29 (27.3) 42(13.8) (6.9) 0.1320.283 Diuretics 41 (23.6) 8 (20.5) 29 (27.3) 4 (13.8) 0.132 Anticoagulants 23 (13.2) 7 (17.9) 12 (11.3) 4 (13.8) 0.503 Anticoagulants 23 (13.2) 7 (17.9) 12 (11.3) 4 (13.8) 0.503 Omega Omega3 3supplements supplements 88 88 (50.6) (50.6) 2020(51.3) (51.3) 52 52(49.1) (49.1) 16 16 (55.2) (55.2) 0.4560.456 p values refer to comparisons between groups of score from the ANOVA test for quantitative p values refer to comparisons between groups of score from the ANOVA test for quantitative variables or the variables or the chi-squared chi-squared test forvariables. test for categorical categorical variables. Group Group Ias I was defined was defined as ≤ 27points, ≤ 27points, group group II as 28–35 II and points, as 28–35 III as ≥and grouppoints, 36 points groupinIII theasquestionnaire, ≥ 36 points inwhere the the higher the score, questionnaire, wherethethe healthier higherthethe diet. ADPthe score, indicates adenosine diphosphate; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blockers; AMI, acute healthier the diet. myocardial ADP BMI, infarction; indicates adenosine body mass diphosphate; index; HbA1C, glycatedACE, angiotensin-converting hemoglobin; enzyme; LDL, HDL, high-density lipoprotein; ARB,low-density angiotensin II receptor blockers; AMI, acute myocardial infarction; BMI, body mass index; lipoprotein. HbA1C, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Figure 2 depicts the main cell origin of cMV. No significant differences were observed in the Figure 2 depicts the main cell origin of cMV. No significant differences were observed in the percentage of cMV from each cell origin or in clinical and biochemical characteristics of patients percentage of cMV from each cell origin or in clinical and biochemical characteristics of patients according to groups of diet score. according to groups of diet score. Figure 2. Distribution of cell origins of circulating microvesicles in the 174 patients studied. The pie charts show the distribution of cMV from the patients studied (n = 174) by major cell origins, indicated by percentages of each marker relative to cell lineage. Only cMV that were Annexin V-positive were considered. The used markers were CD61 for platelets, CD146 for endothelial cells, CD235ab for erythrocytes, CD45 for total leukocytes, CD3 for lymphocytes, CD14 for monocytes, and SMA-α for smooth muscle cell origins. Other leukocyte-derived cMV were positive for CD45 but negative for CD3 or CD14 staining. The markers used for MV characterization are defined in Table 2. Here, cMV indicates circulating microvesicles. Nutrients 2019, 11, 1114 8 of 12 3.2. Food Items and SmartDiet Score In general terms, the mean score for each item (Table 4) was higher in group III compared to group II and group I, and also was higher in group II compared to group I for nearly each food item, except for the “mayonnaise, remoulade, and kaviar” and “sweet toppings and sweet drinks” food items, in which no differences were observed between the three groups of the SmartDiet score. Hence, patients in group III had a higher score for almost every food item (12 out of 14) of the questionnaire compared to patients allocated to group II or group I. Table 4. Mean of score for each food item according to groups of the SmartDiet score. N (%) Group I Group II (Somewhat Group III p (Unhealthy Diet) Unhealthy Diet) (Healthy Diet) Milk or yogurt 2.0 ± 0.5 a 2.2 ± 0.5 a,b 2.7 ± 0.5 b 0.001 Cream and sour cream 1.7 ± 0.8 a 2.5 ± 0.6 b 2.8 ± 0.4 b <0.0001 Cheese 1.1 ± 0.3 a 1.6 ± 0.8 b 2.7 ± 0.6 c <0.0001 Spreading/cured meat 1.7 ± 1.0 c 2.6 ± 0.8 b 3.0 ± 0.0 b <0.0001 Meat for dinner 1.7 ± 0.9 a 2.6 ± 0.7 b 3.0 ± 0.0 b <0.0001 Spreading fish 1.8 ± 0.7 a 2.0 ± 0.7 a 2.5 ± 0.7 b 0.027 Fish for dinner 1.8 ± 0.7 a 2.2 ± 0.7 b 2.7 ± 0.4 c <0.0001 Mayonnaise, remoulade, 1.4 ± 0.5 1.4 ± 0.5 1.4 ± 0.5 0.950 and kaviar Butter and margarine on 1.7 ± 1.0 a 2.5 ± 0.8 b 2.9 ± 0.3 b <0.0001 bread food Fat in cooking 1.4 ± 0.8 a 2.3 ± 0.9 b 2.8 ± 0.4 c <0.0001 Bread and other grain products 1.5 ± 0.9 a 2.5 ± 0.9 b 2.7 ± 0.8 b <0.0001 Vegetables, fruits, and berries 1.5 ± 0.5 a 1.8 ± 0.6 b 2.2 ± 0.4 c 0.003 Sweet toppings and sweet drinks 2.6 ± 0.5 2.7 ± 0.6 2.9 ± 0.3 0.259 Chocolate, snacks, cakes, 2.1 ± 0.8 a 2.5 ± 0.7 b 3.0 ± 0.0 c 0.001 and biscuits The results are expressed as mean ± SD of the score (ranging from 1 to 3) for each food item within each group of diet quality classification. p is from the ANOVA with the Bonferroni post hoc test comparing the mean scores for each food item between groups of total SmartDiet score. Values in rows with different superscript letters are significantly different. Group I was defined as ≤ 27 points (n = 39, 22.4%), group II as 28–35 points (n = 106, 60.9%), and group III as ≥ 36 points (n = 29, 16.7%) as a product of the sum of the 14 items of the questionnaire, where the higher the score, the healthier the diet, according to the Nordic Nutrition Recommendations. 3.3. Associations between Circulating Microvesicles and SmartDiet Score The Spearman’s analyses showed an inverse correlation between the SmartDiet score and total AV+ , and CD61+ /AV+ and CD31+ /AV+ cMV, reflecting platelet activation (R = −0.162, −0.176, and −0.159, and p = 0.045, 0.029, and 0.049, respectively). Thus, the association between SmartDiet score and these cMV was further analyzed by linear regression, adjusting for age, sex, use of fish oil supplements, and smoking history. Multivariable adjustments did not significantly modify the observed results (Table 5). As depicted in Figure 3, patients allocated at the highest score range (healthy diet) had significantly lower total (AV+ ) and platelet (CD61+ /AV+ and CD31+ /AV+ ) cMV (p = 0.028, 0.017, and 0.038, respectively, one-way ANOVA with the Bonferroni post hoc test) compared to patients allocated at the first and second tertiles of score. cMV R p β p Total cMV (AV ) + Unadjusted −0.179 0.027 - - Multivariable adjusted* 0.275 0.031 −0.190 0.024 Nutrients 2019, 11, 1114 Platelet-Derived cMV 9 of 12 CD61+/AV+ Table 5. Multivariable adjusted Unadjusted −0.198associations0.014 between the SmartDiet - score and cMV. - Multivariable adjusted* 0.288 SmartDiet0.043 Score −0.229 0.006 CD31+/AV+ cMV R1 p1 β2 p2 +) Unadjusted Total cMV (AV −0.168 0.037 - - Unadjusted 0.328 Multivariable adjusted* −0.179 0.0160.027 -−0.179 - 0.033 Multivariable 1R- and p-values are adjusted* from the ANOVA 0.275 regression in the linear −0.190 0.031 model, and β2- and0.024 p-values are for the Platelet-Derived cMV contribution of the SmartDiet score in the multivariable adjusted model. *Adjusted for age, sex, use CD61+ /AV+ of omega 3 supplements, Unadjusted smoking −0.198 and history. The markers used 0.014 - for microvesicle - (MV) characterization are defined in adjusted* Multivariable Table 2. cMV indicates 0.288 circulating 0.043 microvesicles, −0.229 and AV is Annexin 0.006 V. CD31+ /AV+ Unadjusted −0.168 0.037 - - Multivariable adjusted* 0.328 0.016 −0.179 0.033 As depicted in Figure 3, patients allocated at the highest score range (healthy diet) had 1 R- and p-values are from the ANOVA in the linear regression model, and β2 - and p-values are for the contribution significantly lower total (AV+) and platelet (CD61+/AV+ and CD31+/AV+) cMV (p = 0.028, 0.017, and of the SmartDiet score in the multivariable adjusted model. *Adjusted for age, sex, use of omega 3 supplements, and 0.038,smoking respectively, one-way history. The ANOVA markers used with the for microvesicle Bonferroni (MV) postarehoc characterization test) defined compared in Table to patients 2. cMV indicates circulating allocated at themicrovesicles, and AV is first and second Annexin tertiles ofV.score. Figure 3. Concentration of circulating microvesicles according to groups of score from the SmartDiet Figure 3. Concentration questionnaire. Total cMV of concentration (AV+ ), platelet-derived circulating microvesicles (CD61+of according to groups /AV + ) cMV, score fromand the cMV SmartDiet from questionnaire. Total + cMV +concentration (AV +), platelet-derived (CD61+/AV+) cMV, and cMV from activated cells (CD31 /AV ) concentrations in the three groups of scores. *Significantly different from activated the other cells groups(CD31 +/AV+) concentrations in the three groups of scores. *Significantly different from (one-way ANOVA with the Bonferroni posthoc test). The markers used for cMV the other groupsare characterization (one-way definedANOVA in Table 2.with the IBonferroni Group was defined posthoc test). The as ≤27points (n =markers used 39, 22.4%), for cMV group II as characterization 28–35 points (n =are defined 106, 60.9%),inand Table 2. Group group III as I≥36 waspoints defined (n =as29, ≤27points 16.7%) in (nthe = 39, 22.4%), group questionnaire, II as where 28–35 points the higher the(nscore, = 106,the60.9%), andthe healthier group diet,III as ≥36 points according to the(n = 29, 16.7%) Nordic in the Nutrition questionnaire, where Recommendations. Here, the higher the score, the healthier the diet, according cMV indicates circulating microvesicles, and AV is Annexin V. to the Nordic Nutrition Recommendations. Here, cMV indicates circulating microvesicles, and AV is Annexin V. 4. Discussion 4.1. Previous Knowledge A healthy diet should be the cornerstone of CVD prevention. In this setting, a high adherence to the Nordic diet has been related to reduced blood pressure [13], improved blood lipid profile [14] and insulin sensitivity [15], improved endothelial function [2], and reduced low-grade inflammation [16]. However, this is the first time that high adherence to the Nordic diet, assessed through the validated SmartDiet questionnaire (which was particularly developed to evaluate a subject’s diet regarding risk of CVD in the context of the Nordic diet), has been related to MV shedding. 4.2. Main Findings of the Study The main finding in this cross-sectional study is that patients with higher adherence to the Nordic diet presented around 32% lower levels of total AV+ cMV, 41% lower levels of CD61+ /AV+ cMV, and 30% lower levels of CD31+ /AV+ cMV, compared to patients allocated to the first and second tertiles of score, despite being under dual antiplatelet therapy. No differences in cholesterol levels were observed across tertiles of score, probably because almost all patients were under statin treatment, as Nutrients 2019, 11, 1114 10 of 12 per guidelines after an AMI. Taking this into consideration, this inhibitory effect on platelet activation may contribute to the beneficial effects of the Nordic diet, which is associated with reduced mortality in patients with established CVD [17]. Interestingly, the mean score for each item was higher in group III compared to group II and group I, except for two food items, in which no differences were observed between groups of the SmartDiet score. This observation indicates that the group classification from the SmartDiet questionnaire, at least within the patients included in the study, reflected a dietary pattern rather than high/low consumption of a specific/single food group. Although the SmartDiet questionnaire has been validated by dietary fat and fiber [9], the protective effects of the Nordic diet may be considered to be a product of the sum of healthy consumption of single food items and should not be only attributed to a single food or food compound. 4.3. Review of Previous Data cMV were identified and quantified through AV binding, which has a high affinity for PS and therefore identifies PS externalization. AV+ cMV expose PS on their surfaces, providing a catalytic surface for factor X and prothrombin activation, thus promoting thrombin generation and clot formation [18]. Besides the externalization of PS, which confers procoagulant activity to cMV, cMV were also identified and quantified on the basis of antigens from their cell of origin and on markers of cell activation. In this line, platelet-derived cMV, which contain epitopes and biomolecules from platelets such as CD61 and/or CD31 and account for 74% of cMV AV+ , amplify platelet activity on coagulation cascade and leukocyte (and platelet) adhesion/activation [19], promoting inflammation and increasing platelet deposition on damaged arteries and thrombus formation [18], thus increasing the atherothrombotic burden [7]. In fact, total AV+ and CD31+ /AV+ cMV map and predict coronary atherosclerosis and calcification in familial hypercholesterolemia patients [20], contributing to an increased risk of a major adverse CV event. Therefore, nutritional strategies to decrease total AV+ and platelet-derived cMV release may improve CVD prognosis. Few studies have analyzed the effects of diet on MV release. Cocoa flavonols have been shown to decrease CD31+ cMV concentrations in patients with coronary artery disease after one month of intake [21], as did one month of an intervention with the Mediterranean diet in elderly healthy individuals [22]. Supplementation with omega 3 fatty acids has been shown to reduce CD31+ [23] and CD61+ [24] cMV concentrations, and replacement of saturated fats with monounsaturated and omega 6 fatty acids reduced the number of CD31+ cMV [25]. Taking into consideration that the Nordic diet is rich in polyunsaturated fatty acids and polyphenols, our observations are endorsed by the literature. 4.4. Limitations and Scope of Future Studies Our study is not exempt from limitations. The current study was performed in elderly patients with a relatively recent AMI, and therefore, these findings may not be extrapolated to healthy subjects or to other AMI populations. In addition, females accounted for only 29% of the study population, and thus sex-specific differences were not analyzed. This was an association study, and therefore it was hypothesis-generating. Although flow cytometry is the most widely used method for MV quantification and characterization, its limit of quantification is ranged between 0.1 and 0.3 µm of particle size. Therefore, MP populations below the limit of quantification were not considered and could be influenced by the Nordic diet quality. Considering all of these aspects, future studies should be focused on the evaluation of the effects of the Nordic diet on MV shedding from different sizes and cell origins through randomized trials, in order to confirm a cause–effect relationship and the potential sex-specific differences in the observed effects. 5. Conclusions Dietary habits significantly affect MV shedding, known to directly influence cellular functions. This study showed that the higher the adherence to a healthy Nordic diet was, the lower the levels of Nutrients 2019, 11, 1114 11 of 12 total AV+ and platelet-derived AV+ cMV found in the circulating blood of Norwegian elderly patients with a recent AMI. The lower release of MV may potentially contribute to the beneficial effects of the Nordic diet. Author Contributions: Conceptualization, G.C.-B., L.B., and I.S.; data curation, K.L., P.M., and S.S.; formal analysis, G.C.-B., K.L., P.M., and V.B.; funding acquisition, S.S. and I.S.; investigation, G.C.-B., K.L, P.M., and V.B.; methodology, G.C.-B. and V.B.; resources, H.A., S.S., and I.S.; software, G.C.-B.; supervision, H.A.; validation, K.L., P.M., and V.B.; writing—original draft, G.C.-B.; writing—review and editing, H.A., S.S., L.B., and I.S. Funding: This research was funded by the Stein Erik Hagens Foundation of Clinical Heart Research, Oslo, Norway. Gemma Chiva-Blanch is a Juan de la Cierva-Incorporación Postdoctoral Fellow (IJCI-2015-26358) from the Spanish Ministry of Economy and Competitiveness (MINECO), Spain. Acknowledgments: We are indebted to Mrs. Charlotte Holst for her invaluable assistance with the SmartDiet questionnaire. Conflicts of Interest: The authors declare no conflicts of interest. References 1. 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