Although in developing countries an apolipoprotein E4 (apoE4) genotype may offer an evolutionary advantage, as it has been shown to offer protection against certain infectious disease, in Westernised societies it is associated with increased morbidity and mortality, and represents a significant risk factor for cardiovascular disease, late-onset Alzheimer's disease and other chronic disorders. ApoE is an important modulator of many stages of lipoprotein metabolism and traditionally the increased risk was attributed to higher lipid levels in E4 carriers. However, more recent evidence demonstrates the multifunctional nature of the apoE protein and the fact that the impact of genotype on disease risk may be in large part due to an impact on oxidative status or the immunomodulatory/anti-inflammatory properties of apoE. An increasing number of studies in cell lines, targeted replacement rodents and human volunteers indicate higher oxidative stress and a more pro-inflammatory state associated with the epsilon4 allele. The impact of genotype on the antioxidant and immunomodulatory/anti-inflammatory properties of apoE is the focus of the current review. Furthermore, current information on the impact of environment (diet, exercise, smoking status, alcohol) on apoE genotype-phenotype associations are discussed with a view to identifying particular lifestyle strategies that could be adapted to counteract the 'at-risk' E4 genotype.

Cardiovascular risk is determined by the complex interactions between genetic and environmental factors. The apoE genotype represents the most-widely-studied single nucleotide polymorphism in relation to CVD risk, with > 3600 publications cited in PubMed. Although originally described as a mediator of lipoprotein metabolism, the lipoprotein-independent functions of apoE are being increasingly recognised, with limited data available on the potential impact of genotype on these metabolic processes. Furthermore, although meta-analyses suggest that apoE4 carriers may have a 40-50% increased CVD risk, the associations reported in individual studies are highly heterogeneous and it is recognised that environmental factors such as smoking status and dietary fat composition influence genotype-phenotype associations. However, information is often derived from observational studies or small intervention trials in which retrospective genotyping of the cohort results in small group sizes in the rarer E2 and E4 subgroups. Either larger well-standardised intervention trials or smaller trials with prospective recruitment according to apoE genotype are needed to fully establish the impact of diet on genotype-CVD associations and to establish the potential of dietary strategies such as reduced total fat, saturated fat, or increased antioxidant intakes to counteract the increased CVD burden in apoE4 carriers. ApoE genotype: CVD: Dietary fat: Oxidative status: InflammationThe impact of single nucleotide polymorphisms on risk of chronic diseases such as CVD, and the ability of dietary factors to manipulate genotype-phenotype associations, is being increasingly recognised. Undoubtedly, the mostwidely-studied gene variant in relation to CVD is the apoE e (e2, e3, e4) genotype. Since its discovery in 1973 the central role of the apoE protein in lipoprotein metabolism has been comprehensively investigated and reported. The 40-50 % higher risk of CVD in apoE4 carriers (Song et al. 2004) has been traditionally attributed to moderately higher circulating cholesterol and TAG levels. However, it is becoming increasingly recognised that an effect on lipoprotein metabolism alone cannot explain the disease differential and that the impact of an apoE4 genotype is largely lipoprotein independent. Roles of macrophagederived apoE protein on vascular health and atherogenesis are being identified, with apoE thought to impact on oxidative status and in an autocrine and paracrine manner affect macrophage, vascular smooth muscle cell, endothelial cell and platelet function. An impact of genotype on these localised functions of apoE could in part explain the impact of genotype on CVD pathology, as will be discussed.Additionally, apoE genotype has been shown to affect the responsiveness to the total fat content and fatty acid composition of the diet. Manipulation of dietary fat content may serve as a means of reducing the increased CVD burden associated with an apoE4 genotype.

In order to gain a more comprehensive understanding of the aetiology of apolipoprotein E4 genotype-cardiovascular disease (CVD) associations, the impact of the apoE genotype on the macrophage inflammatory response was examined. The murine monocyte–macrophage cell line (RAW 264.7) stably transfected to produce equal amounts of human apoE3 or apoE4 was used. Following LPS stimulation, apoE4-macrophages showed higher and lower concentrations of tumour necrosis factor alpha (pro-inflammatory) and interleukin 10 (anti-inflammatory), respectively, both at mRNA and protein levels. In addition, increased expression of heme oxygenase-1 (a stress-induced anti-inflammatory protein) was observed in the apoE4-cells. Furthermore, in apoE4-macrophages, an enhanced transactivation of the key redox sensitive transcription factor NF-κB was shown. Current data indicate that apoE4 macrophages have an altered inflammatory response, which may contribute to the higher CVD risk observed in apoE4 carriers.

ApoE is secreted by macrophages at the lesion site of the atherosclerotic plaque, where it is thought to play a protective role against atherosclerosis independently of its effects on lipid metabolism. Of the three common isoforms for apoE, apoE4 is associated with higher risk of cardiovascular disease (CVD). In vitro studies have shown that recombinant apoE may act as an antioxidant in an isoform-dependent manner (E2 . E3 . E4). The oxidative status of the macrophages plays a key role in the process of atherosclerosis. In the present study the possible differential actions of apoE3 and apoE4 on several parameters of oxidative status were determined in stably transfected murine macrophages (RAW 264·7-apoE3 andapoE4). No differences between genotypes were observed after peroxide challenge in either protection against cytotoxicity or in cell membrane oxidation, and modest differences were observed in the non-enzymatic antioxidants (glutathione and a-tocopherol) in apoE3 v. apoE4 macrophages. Importantly, cells secreting apoE4 showed increased membrane oxidation under basal conditions, and produced more NO and superoxide anion radicals than the apoE3 macrophages after stimulation. The present data suggest that apoE genotype influences the oxidative status of macrophages, and this could partly contribute to the higher CVD risk observed in apoE4 carriers.

SummaryApolipoprotein E (apoE) is a major constituent of lipoproteins mediating peripheral uptake of lipids including the lipid-soluble vitamin alpha-tocopherol (a-toc). In a recent study, we observed significant lower a-toc concentrations in the lung of apoE4 compared with apoE3 transgenic mice. In this study, we determined the mRNA levels of genes encoding for proteins centrally involved in the uptake, export, and degradation of vitamin E. Receptors of a-toc uptake including scavenger receptor B1 (SR-B1), LDL receptor (LDLrec), and LDLrec-related protein 1 (LRP1) were lower in apoE4 when compared with apoE3 mice with statistical significance for SR-B1 and LRP1. Lung mRNA levels of the ATP-binding cassette A1 and the multidrug resistance transporter 1, surfactant proteins mediating the export of a-toc, were lower in apoE4 than in apoE3 mice. In addition, the mRNA levels of cytochrome P450 3A, a microsomal enzyme family involved in the degradation of a-toc, tended to be higher in the apoE4 when compared with the apoE3 genotype. Current data indicate that genes encoding for proteins involved in peripheral a-toc transport and degradation are affected by the apoE genotype probably accounting for the lower a-toc tissue concentration as observed in apoE4 mice.