Fish Oil-derived N-3 Polyunsaturated Fatty Acids and 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.

Eligibility Criteria:
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