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

微颗粒（MPs）很小（50-1000nm），是在激活、损伤或细胞死亡期间从各种细胞类型表面释放的细胞来源颗粒，在循环中大量存在，并正在成为心血管疾病的潜在有用指标。N-3多不饱和脂肪酸（PUFA）对心血管（CV）风险的某些方面具有明显的影响，例如血液中的甘油三酯，但对于其他终点，特别是与血栓、炎症和内皮功能障碍相关的终点，存在相当大的争议。此外，很少有机制数据为n-3 PUFA对这些参数的影响提供坚实的基础。MPs的独特性和它们在膳食脂肪酸调节方面具有如此前景的原因是，它们是细胞衍生的，因此它们不仅携带生物活性细胞成分，而且携带脂肪酸组成可以被膳食脂肪酸修饰的细胞膜。因此，MPs不仅仅是一个标记；它们携带生物活性货物，可引发血栓形成，潜在地影响心血管疾病的广泛过程。我们最近首次证明，补充n-3多不饱和脂肪酸（PUFA）可显著降低轻度CV风险受试者的循环MPs数量，这可能是这些脂肪酸对CV疾病保护作用的机制之一。关于饮食对MPs数量的影响的其他数据很少，几乎没有关于饮食如何影响MPs活动的信息。该项目将使用新兴技术（纳米颗粒跟踪分析，NTA和荧光-NTA， fl-NTA）在两个主要链中研究饮食中n-3 PUFA对循环MPs的生成和活性的影响，并提出以下主要假设：(i)补充n-3 PUFA的受试者产生的MPs将改变其组成，并且不太可能刺激血液凝固（链1）；（ii）来自补充了n-3 PUFA的受试者的血小板在受到刺激时会产生更少的MPs (Strand 2)；（iii）从富含n-3 pufa的血小板体外产生的MPs促进血液凝固的能力会降低（Strand 2）。PMPs占流通MPs总量的最大比例；因此，调节血小板膜组成的干预措施将潜在地影响最大的MPs群体。在为期12周的双盲随机交叉试验中，有轻度心血管疾病风险的受试者将被给予鱼油补充剂或含有1.8 g/d鱼油n-3 PUFA的安慰剂（假胶囊），并进行12周的洗脱期。干预前后将采集血液样本。该项目的第一部分将研究n-3 PUFA补充对血液中总MPs的特征和功能活性的影响。该项目的第二部分将检查n-3 PUFA对MPs产生的影响，特别是从受试者身上提取的血小板，并在体外刺激。该实验设计将允许同时研究直接从血液中提取的总MPs的组成和活性，以及在实验条件下特异性从血小板中提取的MPs的生成和活性。该项目将推进有关n-3 PUFA影响血管稳态机制的基础知识。此外，NTA很容易适用于其他饮食干预的样本，我们建议与其他drc资助的项目合作，为其他生理环境/条件下饮食成分对新型生物标志物的影响提供附加价值和新见解。

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.