Apolipoprotein E genotype and macrophage inflammatory status: Impact of antioxidant/anti-inflammatory dietary components

心血管疾病（CVD），包括冠心病和中风，在欧盟每年约有190万人死亡，占总死亡率的42%。一个人的疾病风险是由环境（运动，吸烟，药物，饮食等）和遗传因素决定的。基因的变异（多态性），包含在我们的DNA中，它携带着制造体内所有蛋白质所必需的信息，可以影响所得蛋白质的结构或产生的水平。这可能随后影响身体代谢和疾病的风险，如心血管疾病。一种这样的多态性是apoE多态性，通常观察到该基因的3个版本，即E2、E3或E4。携带apoE 4基因型（E3/E4或E4/E4，占英国人口的25%）的人患CVD的可能性比常见的E3/E3基因型（60%英国人口）高出约50%。然而，这种风险增加的生理原因知之甚少。我们和其他人主要使用实验室中生长的细胞（细胞培养技术）生成的初步数据表明，apoE蛋白可能在体内充当天然抗氧化剂，E4版本的抗氧化能力显著低于E3或E2版本。已知低抗氧化状态有助于促进CVD发展的氧化应激。此外，我们迄今为止进行的研究表明，E4蛋白的这种低抗氧化能力可能会影响巨噬细胞的行为方式。巨噬细胞是一种白色血细胞，它在我们的动脉壁上积累胆固醇形成斑块，这是动脉粥样硬化的主要特征（动脉的“水垢”）。除了积累胆固醇，巨噬细胞还产生一系列物质，称为炎症化合物，加速动脉粥样硬化过程。由于巨噬细胞在体内产生高达20%的apoE，并且已知对氧化应激敏感，因此假设E4个体中CVD风险增加部分是由于基因型对巨噬细胞功能的影响。这项研究的主要目的是调查这一假设。此外，将研究饮食中存在的一系列物质对抗E4基因型对巨噬细胞的拟议负面影响的能力。简言之，将使用已插入人E3或E4基因的小鼠巨噬细胞进行一系列细胞培养实验。细胞将在模拟体内条件的条件下生长，并研究基因型对巨噬细胞代谢许多方面的影响。人类饮食中含有许多天然抗氧化剂或炎症剂，如维生素C，鱼油脂肪和水果和蔬菜中的类黄酮。将进行第二系列实验以确定这些化合物是否可以抵消E4基因型对巨噬细胞功能的负面影响。最后，将对人类志愿者进行基因分型，并要求10名E3/E3、E3/E4和E4/E4个体提供血液样本，从中分离单核细胞（前巨噬细胞）。将进行最后一系列测试，以确保我们使用方便的小鼠细胞进行的实验中的主要发现在使用人类细胞时也是明显的。从科学的角度来看，目前研究的结果将扩大我们目前关于apoE 4基因型导致CVD风险的机制的知识。从公共卫生的角度来看，该研究将调查天然膳食成分部分抵消apoE 4个体中观察到的疾病风险增加的能力。如果更广泛的人群遗传分析将被采用作为减少或延迟慢性病发作的政策，那么应对“风险”基因型带来的风险增加的策略（饮食或其他生活方式）至关重要。

Cardiovascular disease (CVD), which includes coronary heart disease and stroke, accounts for about 1.9 million deaths per year in the EU, which represents 42% of total mortality. An individual's risk of disease is determined by both environmental (exercise, smoking, medication, diet etc) and genetic factors. Variations in genes (polymorphisms), contained within our DNA, which carry the information necessary for the manufacture of all proteins in the body, can affect both the structure of the resultant protein or the levels produced. This may subsequently impact on body metabolism and risk of diseases such as CVD. One such polymorphism is the apoE polymorphism with 3 versions of the gene i.e. E2, E3 or E4, commonly observed. Carriers of the apoE4 version (E3/E4 or E4/E4, 25% of the UK population) are about 50% more likely to develop CVD compared to the common E3/E3 (60% UK population) genotype. However, the physiological reasons for this increased risk are poorly understood. Preliminary data generated by ourselves and others, mainly using cells grown in the laboratory (cell culture techniques), suggest that the apoE protein may act as a natural antioxidant in the body, with the antioxidant capabilities of the E4 version significantly lower than either the E3 or the E2 version. Low antioxidant status is known to contribute to oxidative stress which promotes the development of CVD. Furthermore our studies conducted thus far are suggestive that this low antioxidant capacity of the E4 protein may affect the way macrophages behave. Macrophages are a type of white blood cell, which accumulate cholesterol in our artery walls to form a plaque, which is a main feature of atherosclerosis ('furring' of the arteries). In addition to accumulating cholesterol macrophages produce a range of substances, called inflammatory compounds, which speed up the atherosclerotic process. As macrophages produce up to 20% of apoE in the body, and are known to be sensitive to oxidative stress it is hypothesised that the increased CVD risk in E4 individuals is in part due to an impact of genotype on macrophage function. The main aim of the study is to investigate this hypothesis. Furthermore the ability of a range of substances present in the diet to counter-act the proposed negative effect of the E4 genotype on macrophages will be investigated. In brief, a range of cell culture experiments will be conducted using mouse macrophages, into which the human E3 or E4 gene has been inserted. The cells will be grown under conditions which simulate conditions in the body, and the effect of genotype on many aspects of macrophage metabolism will be investigated. The human diet contains many components which act as natural antioxidant or inflammatory agents, such as vitamin C, fish oil fats, and flavonoids found in fruit and vegetables. A second series of experiments will be conducted to ascertain if these compounds can counteract the negative effect of the E4 genotype on macrophage function. Finally human volunteers will be genotyped, and ten E3/E3, E3/E4 and E4/E4 individuals will be asked to provide a blood samples from which monocytes (pre-macrohpages) will be isolated. A final series of tests will be conducted to ensure that the main findings in our experiments using the convenient mouse cells, are also evident using human cells. From a scientific point of view the output from the current study will expand our current knowledge regarding the mechanisms by which an apoE4 genotype contributes to CVD risk. From a public health viewpoint the study will investigate the ability of natural dietary components to in part counteract the increased disease risk observed in apoE4 individuals. If more widespread population genetic profiling is going to be adopted as a policy to reduce or delay the onset of chronic diseases, then strategies (dietary or other lifestyle) to counteract the increased risk posed by an 'at-risk' genotype are essential.