Effects of n-3 polyunsaturated fatty acids on the generation and functional activities of microparticles

Microparticles (MPs) are small (50-1000nm), cell-derived particles released from the surfaces of various cell types during activation, damage or cell death, are present in large numbers in the circulation and are emerging as potentially useful indicators of cardiovascular disease. N-3 polyunsaturated fatty acids (PUFA) have well-characterised effects on some aspects of cardiovascular (CV) risk, such as blood triacylglycerols, but there is considerable controversy regarding other endpoints, particularly those associated with thrombosis, inflammation and endothelial dysfunction. Furthermore, there is very little mechanistic data providing a firm basis for effects of n-3 PUFA on these parameters. The uniqueness of MPs and the reason that they hold such promise with respect to modulation by dietary fatty acids is that they are cell-derived and they therefore carry with them not only bioactive cell components, but also cell membranes whose fatty acid composition could be modified by dietary fatty acids. MPs are therefore more than just markers; they carry bioactive cargo, which can initiate thrombosis, potentially influencing a wide range of processes involved in cardiovascular disease. We have recently demonstrated, for the first time, that supplementation with n-3 polyunsaturated fatty acids (PUFA) dramatically reduces numbers of circulating MPs in subjects with mild CV risk, and this may be one of the mechanisms for the protective effects of these fatty acids in CV disease. There is very little other data relating to the effects of diet on MP numbers and almost no information about how diet might influence the activities of MPs. This project will examine the effects of dietary n-3 PUFA on the generation and activities of circulating MPs using newly emerging technology (nanoparticle tracking analysis, NTA, and fluorescence-NTA, fl-NTA) in two main strands with the following overarching hypotheses: (i) MPs derived from subjects supplemented with n-3 PUFA will have altered composition and are less likely to stimulate blood clotting (Strand 1); (ii) platelets derived from subjects supplemented with n-3 PUFA will generate fewer MPs when stimulated (Strand 2); (iii) MPs generated in vitro from n-3 PUFA-enriched platelets will have reduced ability to promote blood clotting (Strand 2). PMPs make up the largest proportion of total circulating MPs; thus an intervention which modulates platelet membrane composition will potentially impact on the largest population of MPs.
Subjects with mild CVD risk will be given fish oil supplements or a placebo (dummy capsule) containing 1.8 g/d n-3 PUFA from fish oil for 12 weeks in a double-blind randomised crossover trial with a 12-week washout. Blood samples will be obtained before and after intervention. The first strand of the project will examine the influence of n-3 PUFA supplementation on the characteristics and functional activities of total MPs from blood. The second strand of the project will examine the influence of n-3 PUFA on the generation of MPs specifically from platelets taken from subjects and stimulated in vitro. This experimental design will allow simultaneous investigation of both the composition and activity of total MPs taken directly from blood, and the generation and activity of MPs derived specifically from platelets under experimental conditions.
The project will advance fundamental knowledge regarding the mechanisms by which n-3 PUFA influence vascular homeostasis. Furthermore, NTA is readily applicable to samples from other dietary interventions, and we propose to collaborate with other DRINC-funded projects to provide added value and new insight into the effects of dietary components on novel biomarkers in other physiological settings/conditions.

N-3 polyunsaturated fatty acids (PUFA) have well-characterised effects on some aspects of cardiovascular (CV) risk, such as blood triacylglycerols, but there is considerable controversy regarding other endpoints, particularly those associated with thrombosis, inflammation and endothelial dysfunction. Microparticles (MPs) are small, cell-derived particles, which carry with them not only bioactive cell components, but also cell membranes whose fatty acid composition could be modified by dietary fatty acids. MPs are therefore more than just markers; they carry bioactive cargo, which can initiate thrombosis, potentially influencing a wide range of atherothrombogenic processes. We have recently demonstrated, for the first time, that supplementation with n-3 polyunsaturated fatty acids (PUFA) dramatically reduces numbers of circulating MPs in subjects with mild CV risk, and this may be one of the mechanisms for the protective effects of these fatty acids in CV disease. There is very little other data relating to the effects of diet on MP numbers and almost no information about how diet might influence the activities of MPs. This project will examine the effects of dietary n-3 PUFA on the generation and functional activities of total circulating MPs and on the mechanisms underlying these effects using both established methods and newly emerging technology (nanoparticle tracking analysis; NTA) in two main strands with the following overarching hypotheses: (i) MPs derived from subjects supplemented with n-3 PUFA will have altered fatty acid composition and reduced pro-coagulant activities (Strand 1); (ii) platelets derived from subjects supplemented with n-3 PUFA will generate fewer platelet-derived MPs (PMPs) upon stimulation due to alterations in PS exposure and intracellular calcium release (Strand 2); (iii) PMPs generated in vitro from n-3 PUFA-enriched platelets will have reduced pro-coagulant activities (Strand 2).

This project will examine the effects of dietary n-3 polyunsaturated fatty acids (PUFA) on the generation and functional activity of circulating microparticles (MPs) using newly emerging technology (nanoparticle tracking analysis; NTA) for characterisation of MPs. Fundamental knowledge on the biological mechanisms by which n-3 PUFA affect atherothrombosis will advance the case for the physiological effects of n-3 PUFA in CVD and enhance the portfolio of scientific evidence underpinning these effects. The food industry will directly benefit from the advance in knowledge because MPs are novel emerging biomarkers with applications across a range of nutritional questions. By working with other DRINC-funded projects (including the Norwich-led project on isothiocyanates in arthritis and the Reading-led project on flavonoids and cognition) we will provide significant added value and new insight into the effects of dietary components on MPs in other physiological settings/conditions and expand the capability and interactions within the DRINC community. A workshop bringing these projects together and engaging the food industry would provide comparative data on the effects of different bioactive ingredients on vascular resilience. The project also brings new researchers into the DRINC forum; Professor Jon Gibbins, Dr Chris Jones and Dr Jim Crawley are internationally renowned experts in platelet biology and thrombosis, whose experience and knowledge will be extremely useful to the DRINC community, and Dr Dionne Tannetta brings 5 years of hands-on experience with NTA and fl-NTA.
There are potential beneficiaries in many areas of research, both academic and commercial. Basic scientists involved in platelet biology, thrombosis, haematology, immunology and inflammation will benefit through the application of new technology and advancement in knowledge and clinicians, cardiologists and epidemiologists will benefit through greater understanding of microparticles as markers of cardiovascular risk. Malvern Instruments (the company which is developing methods to advance sizing, counting and phenotyping of MPs) holds up to 6 workshops and networking events per year, where researchers discuss best practice, hold private and open sessions with real data and explore novel applications of the technology. We intend to work closely with the company during the course of the project to ensure the widest possible engagement with potential beneficiaries and to explore further potential benefits of the technology that may be directly relevant to the food industry.