Fish Oil-derived N-3 Polyunsaturated Fatty Acids and Extracellular Vesicles (HI-FIVE)

Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles

N-3 polyunsaturated fatty acids (n-3 PUFA), which are abundant in oily fish and fish oils, have been suggested to play a role in reducing the risk of cardiovascular diseases (CVDs) by modifying a wide range of risk factors, such as blood fats, blood clotting, blood vessel function and inflammation. Extracellular vesicles (EVs) are small particles released from various cells when they are activated or damaged. High numbers of EVs in the blood have been associated with a higher risk of CVDs, and it is thought that this is because they carry 'bioactive' components which can affect many processes involved in CVDs. However, very few clinical trials have investigated the relationships between the consumption of n-3 PUFA and circulating EVs. This study aims to investigate the effects of dietary n-3 PUFA on the generation and functional activities of EVs, which would provide new insight into the benefits of n-3 PUFA on cardiovascular health.

The proposed study will be a randomised, double-blind, placebo-controlled crossover intervention. Subjects (40-70y) at moderate CVDs risk will be supplemented with either fish oil (1.8 g/d n-3 PUFA) or placebo (high-oleic safflower oil) for 12 weeks. After a 12-week washout and then cross-over to the other intervention for another 12 weeks. Blood samples will be collected before and after each intervention. A food frequency questionnaire will be administered to assess the subject's habitual intake of n-3 PUFA. Subjects will also be expected to maintain a low consumption of n-3 fatty acids, refrain from the use of all supplements, and maintain their body weight during the study. The dose is based on our previous work, which demonstrated a reduction in numbers of endothelial-derived EVs (EEVs) and a trend for reduced numbers of platelet-derived EVs (PEVs), and a dose at which beneficial effects of n-3 PUFA on plaque stability are reported. The experimental work will follow two main strands. The first strand will examine the influence of n-3 PUFA supplementation on the characteristics and functional activities of total EVs from plasma. The second strand will examine the influence of n-3 PUFA on the generation of PEVs from platelets taken from subjects and stimulated in vitro; the PEVs generated will subsequently be assessed for their composition and functional activity. This experimental design will allow simultaneous investigation of both the composition and activity of total EVs taken directly from blood, and the generation and activity of PEVs. Based on our previous work, 27 subjects are required to detect a 10% reduction in numbers of EVs following fish oil supplementation with a two-sided significance level of 5% and a power of 90%, and 34 subjects are required for a power of 95%. Also based on previous data, 22 subjects would give 95% power to detect 10% differences in thrombus formation and 30 subjects are required to detect a significant effect of n-3 PUFA on platelet aggregation and phosphatidylserine (PS) exposure. Allowing for a 15% dropout rate, and aiming for 95% power based on a 10% reduction in EVs numbers, we will therefore recruit 40 subjects in total.

Each serving contains 360mg eicosapentaenoic acid (EPA), 270mg docosahexaenoic acid (DHA) and total supplement is 1.8 g per day n-3 PUFA for 12 weeks

Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA)

Circulating EVs were first isolated to obtain fractions 7~9 by size exclusion chromatography (SEC) using Izon qEV columns (Izon Science Ltd, Oxford, United Kingdom). Fractions were then diluted with PBS to maintain the recommended concentration range of particles (1~10*10^8 vesicles/ml) before being analysed on NanoSight 300 (Malvern, Amesbury, United Kingdom). For each analysis, five videos, each of 60 seconds duration, were captured with the camera level at 13. Data were analysed using the instrument software NTA 3.20, which can identify individual particles and estimate their sizes based on the Stokes-Einstein Equation. Finally, a threshold of 70nm was set for NTA to ensure minimal interference by small lipoproteins.

Numbers of Total Phosphatidylserine Positive EVs (PS+EVs) in Platelet-free Plasma (PFP) Detected by Flow Cytometry (FCM)

A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. PS+EVs were identified as Annexin V+EVs when triggering on APC fluorescence.

A 5μl of PFP was added into nonsticky microcentrifuge tubes (Alpha Laboratories Ltd, Hampshire, United Kingdom), which contained 5μl FcR blocking reagent (Miltenyi Biotec Ltd, Surrey, United Kingdom) and Annexin V buffer and incubated for 15 minutes in the dark at room temperature. Antibodies and isotype-matched controls were then added and samples incubated for another 15 minutes in the dark at room temperature. After incubation, samples were diluted with 200μl Annexin V buffer and transferred into FACS flow tubes (BD Biosciences, Wokingham, United Kingdom), ready to be analysed by FCM. Platelet-derived EVs (PDEVs) were identified as Annexin V+EVs which also stained positive for CD41-PE in APC vs PE quadrant plot, and endothelial-derived EVs (EDEVs) were identified as Annexin V+EVs which also stained positive for CD105- eFluor450 in APC vs PB quadrant plot.

Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks

A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)

Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks

A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)

Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks

A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)

Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks

A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)

Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks

A commercially available, plate-based thrombin generation assay was used to measure thrombin generation in either a standard, pooled vesicle and platelet-free plasma (termed vesicle-free plasma or VFP) or in the same VFP but with added circulating EVs from subjects in the intervention study. This enabled the assessment of TF-dependent thrombin generation specifically attributed to circulating EVs in samples from the intervention study. Results were presented as five variables: (i) lag-phase for initiation of thrombin generation after addition of the trigger (time to 1/6 of the peak height) (min); (ii) peak thrombin concentration (nM); (iii) time to reach the peak (min); (iv) velocity index, defined as = [peak height/(time to peak - lag time)] and (v) area under the curve, defined as endogenous thrombin potential (ETP) (expressed as nM thrombin × min)

Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks

96-well high-throughput aggregometry technique, allowing testing of a wide range of concentrations of different agonists, was used to examine the influence of n-3 PUFA supplementation on platelet function. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from each study visit was used in the platelet aggregation assay using pre-prepared 96-well microplates, containing the agonists (ADP, EPI, TRAP-6 and U46619). Dose-response curves in response to each agonist were obtained and results were represented as a LogEC50 (log concentration of agonist, M, giving a response halfway between maximum and minimum aggregation).

Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50)

96-well high-throughput aggregometry technique, allowing testing of a wide range of concentrations of different agonists, was used to examine the influence of n-3 PUFA supplementation on platelet function. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from each study visit was used in the platelet aggregation assay using pre-prepared 96-well microplates, containing the agonists (CRP-XL). Dose-response curves in response to each agonist were obtained and results were represented as a LogEC50 (log concentration of agonist, mg/ml, giving a response halfway between maximum and minimum aggregation).

Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation)

Ex vivo thrombus formation was measured by the addition of in vitro-generated PDEVs from stimulated platelets into whole blood under flow. Results were presented as three variables: (i) endpoint for ex vivo thrombus formation (FU); (ii) endpoint for ex vivo thrombus formation (FU); (iii) area under curve.

Change in pro-thrombotic activities (endpoint and maximum of thrombus formation)of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks

Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Area Under Curve)

Ex vivo thrombus formation was measured by the addition of in vitro-generated PDEVs from stimulated platelets into whole blood under flow. Results were presented as three variables: (i) endpoint for ex vivo thrombus formation (FU); (ii) endpoint for ex vivo thrombus formation (FU); (iii) area under curve.

Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks

A 500μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and subjected to SEC for the isolation and purification of EVs. The fractions 7~9 were pooled together, and 800μl of pooled fractions was prepared for total lipid extraction and methyl esterification. The EV total lipid methyl esters were then analysed by gas chromatography on a Hewlett-Packard 6890 series GC (Hewlett-Packard, California, United States), with the following protocol: split ratio was set as 30:1 for plasma and EV analysis. The injection volume was 1μl for plasma and 5μl for EVs, respectively. The temperature of both injector and detector were kept at 300°C and the temperature program was initial temperature 115°C for 2 minutes, increased at 10 °C/min to 200°C and hold at this temperature for 16 minutes, and finally increased at 60°C/min to 240°C for 2 minutes (total run time: 29.2 minutes). Samples were analysed by using ChemStation software and Microsoft Excel.

A 400μl aliquot of frozen PFP was defrosted and centrifuged to remove denatured protein. The 400μl of 0.9% NaCl was added to the PFP sample to make up 800μl in total, and 30μg of phosphatidylcholine (PC) and 15μg of phosphatidylethanolamine (PE) internal standards were then added for the quantitative analysis. After lipid extraction, separation of PC and PE, and methyl esterification of plasma phospholipid extracts, samples were analysed by GC.

A 250μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and centrifuged at 500xg for 5 minutes at room temperature (Eppendorf Centrifuge 5415 R, DJBlabcare, United Kingdom). Then the sample was analysed by a RANDOX clinical analyser (RANDOX Daytona+ Analyser, Randox Laboratories Ltd, United Kingdom) for the concentration of TC, TAG, HDL-C, LDL-C and TC/HDL-C ratio.

A 250μl aliquot of frozen PFP was defrosted at room temperature using a roller mixer and centrifuged at 500xg for 5 minutes at room temperature (Eppendorf Centrifuge 5415 R, DJBlabcare, United Kingdom). Then the sample was analysed by a RANDOX clinical analyser (RANDOX Daytona+ Analyser, Randox Laboratories Ltd, United Kingdom) for TC/HDL-C ratio.

Subjects were asked to have a rest for 10 mins before blood pressure detection, and then blood pressure cuff was placed firmly on their upper left arms approximately 2 cm above the elbow with the indicator mark on the cuff over the brachial artery to start measurement. Subjects should put their arms at the level of the heart and should not speak and cross their legs during the measurement. Measurement was performed three times and waited for 2 mins between each reading ((Omron M2 Upper Arm Blood Pressure Monitor, OMRON Healthcare Europe BV, United Kingdom). The average of the three readings was taken to obtain the final result.

Inclusion Criteria: Aged 40-70 years Non-smoker At moderate risk of cardiovascular diseases The risk will be evaluated by an online calculator called "QRISK2". This online calculator (https://qrisk.org/2016/), which use traditional risk factors (age, systolic blood pressure, smoking status and ratio of total serum cholesterol to high-density lipoprotein cholesterol) together with body mass index, ethnicity, measures of deprivation, family history, will provide a percentage of risk of having a heart attack or stroke within the next 10 years. Subjects with 10%-20% will be regarded as being at moderate risk Exclusion Criteria: BMI: <18.5 kg/m2 Anaemia (haemoglobin concentration <12.5 g/L in men and<11.5 g/L in women) Hyperlipidaemia (total cholesterol concentration >8 mmol/L) Diabetes (diagnosed or fasting glucose concentration >7 mmol/L) or other endocrine disorders Angina, stroke, or any vascular disease in the past 12 months Renal, gastrointestinal, respiratory, liver or bowel disease Inflammatory disease Take drug treatment for hypertension, hyperlipidaemia, inflammation, depression or thyropathy. Take aspirin, ibuprofen or other nonsteroidal anti-inflammatory drugs (NSAIDs) > 4 times per month, or once in the week preceding the study Take any other anti-platelet or anti-coagulant drugs, like triflusal, clopidogrel and warfarin. Have allergies Smoking (including e-cigarettes and nicotine products) Alcohol misuse or intakes >21 units/wk for men and >15 units/wk for women or have a history of alcohol misuse Regularly consume oily fish and/or dietary supplements Planning to start or on a weight reducing regimen Intense aerobic exercise (>20 min, three times a week) Females who are pregnant, lactating, or if of reproductive age and not using a reliable form of contraception (including abstinence) Have participated in another clinical trial within the last three months