Effects of Fish Oil-derived N-3 Polyunsaturated Fatty Acids on the Generation and Functional Activities of Extracellular Vesicles
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 42
- 试验地点
- 2
- 主要终点
- Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM
研究概览
简要总结
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.
详细描述
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.
研究设计
- 研究类型
- Interventional
- 分配方式
- Randomized
- 干预模型
- Crossover
- 主要目的
- Prevention
- 盲法
- Triple (Participant, Investigator, Outcomes Assessor)
入排标准
- 年龄范围
- 40 Years 至 70 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •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
排除标准
- •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
结局指标
主要结局
Characterisation of Circulating EVs Subpopulation in PFP Detected by Fluorescence FCM
时间窗: Change in the numbers of circulating EVs subpopulation in PFP by fluorescence FCM after intake period of 12 weeks
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.
Numbers of Circulating Total EVs in Platelet-free Plasma (PFP) Detected by Nanoparticle Tracking Analysis (NTA)
时间窗: Change of circulating total EV numbers in PFP detected by NTA after intake period of 12 weeks
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)
时间窗: Change of total PS+EV numbers in PFP detected by FCM after intake period of 12 weeks
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.
次要结局
- Pro-thrombotic Activities of Circulating EVs in PFP (Lag Time for Thrombin Generation)(Change of pro-thrombotic activities (lag time for thrombin generation)of circulating EVs in PFP after intake period of 12 weeks)
- Concentrations of TC/HDL-C Ratio in Plasma(Change in concentrations of plasma TC/HDL-C ratio after intake period of 12 weeks)
- Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay (CRP-XL Log EC50)(Change in ex vivo platelet activation after intake period of 12 weeks)
- Pro-thrombotic Activities of Circulating EVs in PFP (Peak Thrombin Concentration)(Change of pro-thrombotic activities (peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks)
- Pro-thrombotic Activities of Circulating EVs in PFP (Time to Peak Thrombin Concentration)(Change of pro-thrombotic activities (time to peak thrombin concentration) of circulating EVs in PFP after intake period of 12 weeks)
- Pro-thrombotic Activities of Circulating EVs in PFP (Endogenous Thrombin Potential)(Change of pro-thrombotic activities (endogenous thrombin potential) of circulating EVs in PFP after intake period of 12 weeks)
- Pro-thrombotic Activities of Circulating EVs in PFP (Velocity Index)(Change of pro-thrombotic activities (velocity index) of circulating EVs in PFP after intake period of 12 weeks)
- Ex Vivo Agonist-stimulated Platelet Activation Detected by Plate-based Platelet Aggregation Assay(Change in ex vivo platelet activation after intake period of 12 weeks)
- Pro-thrombotic Activities of Platelet-derived Extracellular Vesicles (PDEVs) Prepared From the Supernatants of Stimulated Platelets (Endpoint and Maximum of Thrombus Formation)(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)(Change in pro-thrombotic activities (area under curve) of PEVs prepared from the supernatants of stimulated platelets after intake period of 12 weeks)
- Circulating EV Total Lipids Analysis(Change in total lipids of EVs after intake period of 12 weeks)
- Plasma Total Phospholipids Analysis(Change of plasma total phospholipids after intake period of 12 weeks)
- Concentrations of Lipid Profile in Plasma(Change in concentrations of plasma lipid profile after intake period of 12 weeks)
- Blood Pressure(Change in blood pressure after intake period of 12 weeks)
研究者
Professor Parveen Yaqoob, MA, DPhil, RNutr, FAfN
Professor Parveen Yaqoob
University of Reading