Examining endothelial function in humans in vivo: improving guidelines and exploring novel measures

Chapter 1 introduced the old and new measurement of endothelial function, and vascular health. The first part of this thesis focussed on the “old” flow-mediated dilation measurement, introduced in 1992, which relies on brachial artery vasodilation after a hyperaemic stimulus (e.g. after occlusion of a pneumatic cuff), and the susceptibility of the FMD measurement to variability. In Chapter 2 we sought for factors that might help to improve the reproducibility of the FMD measurement. We performed an analysis including 672 participants with repeated FMD. Overall we found an acceptable reproducibility, with 33% of the FMD measurements showing an excellent-to-moderate reproducibility. We identified several factors that independently increased the variation of the FMD, including the presence of hypertension, a lower resting FMD%, a larger baseline artery diameter, a longer time between subsequent measurements, and less laboratory experience with the measurement. Future studies should take these factors into consideration, as certain measures may lower variability of the FMD or more subjects should be included in individual studies when variation relates to non-modifiable factors. Unfortunately, we also found that a large proportion of our study population demonstrated a moderate-to-poor reproducibility, despite all included studies adhered to expert-consensus guidelines. In Chapter 3, we described the relation between adherence to the expertconsensus guidelines and reproducibility of the FMD. In this meta-analysis, we combined data from twenty-seven studies, comprising 48 study groups, with a total of 1537 subjects. Adherence to expert guidelines was inversely related to the measurement error and adopting the guidelines (with specific notification of the use of a stereotactic probe-holder, continuous diameter recording and the use of automated wall-detection and analysis software) was crucial for improving the reproducibility of the FMD. The second part of this thesis introduced the “new” carotid artery reactivity (CAR) measurement, which depends on carotid artery vasomotor responses following sympathetic stimulation (e.g. after a cold pressor test, CPT). The carotid artery appears to mirror coronary responses to CPT, as it shows dilation in healthy participants, whilst those at risk demonstrate a constriction. In this thesis, we sought to understand the relation of CAR with risk factors, and the similarity with the coronary responses. An attempt was made to unravel the underlying physiological mechanism of CAR. Finally, the prognostic value of CAR was examined in peripheral arterial disease patients, to further investigate to clinical potential of the CAR test. In Chapter 4 we explored the relation of CAR with cardiovascular risk, followed by assessing the similarity in response to sympathetic stimulation between the carotid artery and coronary arteries. We first compared CAR between 50 young and 44 older participants to assess relationships between CAR and traditional cardiovascular risk factors. We found that CAR was lower in participants with ≥2 risk factors, compared to those with lesser risk factors. Secondly, we compared left anterior descending (LAD) artery velocity with carotid artery diameter in a subgroup of 33 participants, to assess similarity between coronary and carotid artery responses. We found that CAR correlated well with coronary artery velocity. This implies that CAR is related to increased CV risk and may represent a surrogate measure for coronary vascular health. In Chapter 5, the physiological mechanism underlying the CAR was further explored, by examining carotid artery responses to different sympathetic stimuli (e.g. the cold pressor test [CPT] and the lower body negative pressure test [LBNP]), exploring the role of α1-receptors, (nor) epinephrine receptors contributing to vasoconstriction, and assessing similarity between carotid and coronary arteries. First, 10 participants underwent both sympathetic tests in randomized order, whilst concurrently measuring CAR and coronary artery velocity. We found distinct carotid artery responses to different tests of sympathetic stimulation (e.g. dilation in response to CPT, and constriction following LBNP). Second, when measurements were repeated following α1-receptor blockade by Prazosin, we found α1-receptors partly contributed to CPT-induced responses. Finally, we found agreement between carotid and coronary artery responses, during both types of sympathetic nerve stimulation as well as during α1-receptor blockade. These data indicate strong similarity between carotid and coronary responses to sympathetic tests and the role of α1-receptors. Since the CAR test was newly introduced, the prognostic value of the test remained unknown and this question is highly relevant to understand its clinical utility. Therefore, in Chapter 6, we examined whether CAR predicts (cardiovascular) events in patients with peripheral arterial disease. A total of 172 PAD patients were included, and we recorded cardiac and cerebrovascular events, mortality and clinical progression to percutaneous transluminal angioplasty or loss of patency during a 12-months follow-up. We found that patients with carotid constriction showed a four-fold higher risk for cardiovascular events and two-fold increased risk for clinical deterioration, even after adjustment for other risk factors. This indicates that CAR provides a simple, novel strategy to predict CV events and progression in PAD patients, which has stronger prognostic value than current techniques. Chapter 7 summarizes, discusses, and explains the findings of these studies, and aims to provide recommendations and implications. We discuss future prospects, and provide perspective for future vascular health assessment