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

Lead Research Organisation: University of Reading
Department Name: Food and Nutritional Sciences

Abstract

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.

Technical Summary

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.

Publications

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Spencer JP (2012) Neuroinflammation: modulation by flavonoids and mechanisms of action. in Molecular aspects of medicine

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Spencer JP (2010) The impact of fruit flavonoids on memory and cognition. in The British journal of nutrition

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Spencer JP (2009) Flavonoids and cognition: the molecular mechanisms underlying their behavioural effects. in Archives of biochemistry and biophysics

 
Description Mars Symboscience 
Organisation Mars Incorporated UK
Department Mars Symboscience
Country United States 
Sector Academic/University 
PI Contribution R&D of new products based on the research
Collaborator Contribution Help with the synthesis of physiologically relevant metabolites of flavanols, through the use of their facilities.
Impact Manuscript and addtional funding
 
Description The effects of Champagne wine consumption on brain health 
Organisation Comité Interprofessionnel du Vin de Champagne (CIVIC)
Country France 
Sector Charity/Non Profit 
PI Contribution Wrote proposals and hosted PhD students
Collaborator Contribution Two Partly funded PhD studentships. Further expands the flavonoid/brain and vascular research programme.
Impact Partly funded PhD studentship. Further expands the flavonoid/brain research programme.
Start Year 2006
 
Description The effects of citrus flavanones on neuronal function 
Organisation PepsiCo
Department Nutrition Dept PepsiCo
Country United States 
Sector Private 
PI Contribution ethics applications, conduction the research, research discussions
Collaborator Contribution ethics application, interventionism research discussion
Impact three papers in preparation
Start Year 2012
 
Description The effects of flavanols on brain and vascular health 
Organisation Mars Incorporated UK
Country United Kingdom 
Sector Private 
PI Contribution Wrote successful grant application and hosted PhD student
Collaborator Contribution Supply of research intervention diets for human clinical trial (£60,000). Fully funded PhD studenship (stipend + fees + runing costs). Enhances my groups work in the flavonoid vascular area.
Impact Greatly expands the flavonoid brain and vascular research programme.
Start Year 2007
 
Description Sixth Form Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact organisation and running of the laboratory sessions as part of the School of Food Biosciences 'Sixth Form Open Day'. This course gives A-level students a chance to experience the academic research environment. Experiments related to this MRC grant were used in order to show how certain nutrients may be beneficial to human brain health. June 2005, 2006, 2007 and 2008

Increased recruitment of students.
Year(s) Of Engagement Activity
 
Description The Wye Valley School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk to 6th-form students about University level science and research.

Increased recruitment and interest in science in general.
Year(s) Of Engagement Activity 2012