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

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.

There is currently a lack of information regarding the molecular-mechanisms underlying the 50% increased risk of cardiovascular disease in individuals with an apoE4 genotype (25% of the UK population). Our recent studies are suggestive, that in contrast to the accepted dogma, the increased CVD risk in E4 carriers may be in large mediated by lipoprotein-independent mechanisms, including an impact of genotype on antioxidant status and macrophage inflammatory gene expression. The current study is a progression of our preliminary findings, with the main study hypothesis being that an apoE4 genotype is a significant determinant of the macrophage inflammatory response, which is in part attributable to an impact of genotype on NF-KB signalling. A series of cell culture experiments will be conducted using (a) human E3- and E4- stably transfected murine monocytes (RAW 264.7), and (b) primary human monocytes isolated from blood samples provided by E3/E3, E3/E4 and E4/E4 healthy volunteers. Gene expression will be determined using targeted inflammatory specific array and real time PCR, whilst the impact of genotype on the protein phenotype will be determined by quantifying a number of proinflammatory mediators in the cell culture medium. The impact of genotype on NF-KB signalling will be established by, determining the gene expression (real time PCR), phosphorylation status (Western Immunoblotting) and activity (specific activity assays) of a number of central components of the signalling cascade, and nuclear translocation (confocal microscopy) of NF-KB. Furthermore the culture of cells in physiological relevant concentration of vitamin C/long chain n-3 PUFA/quercetin and its metabolites, will be conducted to assess the ability of these antioxidant and anti-inflammatory dietary components to counter-act the predicted pro-inflammatory status in E4 macrophages.