High fat challenges and detection of early perturbations in endothelial health : the use of a comprehensive phenotyping approach

Background:Cardiovascular disease (CVD) is one of the leading causes of morbidity and mortality worldwide. One of the pathophysiology’s that play a pivotal role in the development and progression of CVD is a dysfunctional endothelium. An important lifestyle risk factor for endothelial dysfunction is the diet and several nutrients have been classified to be either beneficial or harmful for the endothelium. Although CVD usually affects middle-aged or older adults, the onset of endothelial dysfunction begins in early life, emphasising the need for primary prevention. We therefore aimed to identify markers of early perturbations in endothelial health by using dietary stressors, e.g. high fat (HF) challenge test. Thereafter we aimed to evaluate if the potential early markers are reversible and can be improved after an intervention with a dietary anti-stressor. Methods:First we validated the HF challenge test as a tool to trigger the endothelial response capacity. For that purpose, we compared the postprandial response after a HF shake with an average breakfast shake in young healthy men by assessing several plasma markers and functional measures of endothelial function. To identify new markers for early perturbations in endothelial health and to optimized the HF challenge test we applied three HF challenges differing in fatty acid type in two populations of middle-aged men, i.e. one at high- and one at low risk for developing CVD and characterized the postprandial response by applying high-throughput metabolomic and transcriptomic tools next to an extensive phenotyping of vascular function and vascular health parameters. Lastly, we evaluated if the, in the studies above, identified potential early biomarker profile is reversible and can be improved after an intervention with a dietary anti-stressor by means of a high flavanol chocolate intervention. Results:In young men, we observed that a HF challenge decreased flow mediated dilation (FMD), but this decrease was also found after the consumption of an average breakfast shake. IL-8 concentrations were more pronouncedly increased after HF shake consumption compared to an average breakfast control shake. In middle-aged men, a HF challenge decreased the augmentation index (AIX) and elicited an activated state of cellular adherence in the circulation as determined by increased plasma soluble adhesion molecules, increased leukocyte cell surface integrin and selectin expression and increased number of leukocytes. A challenge high in mono-unsaturated fatty acids (MUFAs) elicited the highest postprandial triglyceride (TG) concentrations and the most pronounced effects on AIX. By applying high-throughput metabolomic tools, we observed that oxylipin profiles were affected by the HF challenge and that these changes were depended on dietary fatty acid composition. Application of transcriptome profiling revealed that changes in peripheral blood mononuclear cell (PBMC) gene expression profiles after a HF challenge test were different between lean and obese subjects, with the most deviating effect after MUFA intake. The saturated fatty acid (SFA) shake decreased the expression of genes involved in cholesterol uptake and cholesterol biosynthesis and increased expression of genes involved in cholesterol efflux. MUFA increased expression of inflammatory genes and of peroxisome proliferator-activated receptor α (PPARα) target genes involved in β-oxidation. 4-week daily intake of a dietary anti-stressor, e.g. dark chocolate, increased fasting FMD and decreased AIX, and elicited a less activated state of cellular adherence, as determined by a decrease in plasma soluble adhesion molecules, a decrease in leukocyte cell surface integrin and selectin expression and a decrease in the number of leukocytes. Conclusions:In this thesis we extensively characterized the postprandial response to a HF challenge in human subjects with different disease risk profiles and optimized the HF challenge test. We identified MUFAs as most potent fatty acids to trigger the vascular and cellular response capacity, which makes it the optimal fatty acid type to use in a HF challenge test. We demonstrated that besides functional measures of vascular function, also plasma and cellular factors involved in leukocyte adhesion to the endothelium are adversely affected by dietary stressors and are beneficially affected by a dietary anti-stressor. Therefore, we conclude that endothelial health can be more comprehensively measured by means of a biomarker profile consisting not only of the vascular function measures FMD and AIX, but also of a subset of soluble adhesion molecules in the plasma, leukocyte counts and cell surface integrin and selectin expression. To identify potential new leads for biomarkers, we applied whole genome gene expression profiling, combined with the HF challenge test which enabled us to detect small differences in health status. Furthermore, we identified metabolic and inflammatory pathways that are specifically affected by either MUFAs or SFAs. These findings increased our understanding on how a SFA or MUFA challenge exert their distinct effects on stress related and metabolic compensatory cellular processes and provided us with new potential leads to detect early perturbations in endothelial health.