Brachial Artery Flow-Mediated Dilation in Humans: Establishing Age- and Sex-Related Reference Values with a Focus on the Role of Shear Stress

Abstract

Cardiovascular disease (CVD) is the world-leading cause of mortality. The pathophysiological process underlying CVD is atherosclerosis, often preceded by dysfunction of the endothelium, the inner layer of the artery wall. The endothelium is sensitive to hemodynamic stimuli, including shear stress (i.e. friction force of flowing blood). Endothelial function, assessed using brachial artery flow-mediated dilation (FMD), is a predictor of future CVD risk. In brief, the FMD test consists of ultrasonic assessment of the relative change in brachial artery diameter before and after a 5-minute period of distal limb (forearm) ischaemia, induced by cuff occlusion. Releasing the cuff leads to a transient increase in shear stress through the brachial artery (i.e. shear rate area-underthe-curve; SRAUC), which in turn stimulates vasodilation. Since the introduction of the FMD technique in 1992, efforts have been made to standardise the methodology for the performance of the FMD and expert-consensus protocol guidelines were established. However, large variability in FMD data is present within the literature, likely due to poor adherence to these guidelines. Despite this variation, research has consistently reported age- and sex-specific differences in FMD, where FMD is lower in males compared to females, and declines with age, even in healthy individuals. Reproductive hormones may explain sex-specific differences in FMD, with animal work suggesting that oestrogen improves the relationship between shear stress and vasodilation. Furthermore, exercise/heating protocols have demonstrated the potency of shear stress as a stimulus for improved FMD. The pattern of shear stress is also relevant, given that antegrade and retrograde shear stress impose opposite effects on FMD. However, these studies have manipulated mean shear stress, whilst it could be argued that fluctuations in blood flow and shear stress are more applicable to daily life. Therefore, the aims of this thesis were to: (i) update the expert-consensus guidelines for FMD, (ii) estimate age- and sexspecific reference values for brachial artery FMD in healthy individuals and explore the relation with CVD risk factors, (iii) examine age- and sex differences in the relation between FMD and its eliciting shear stress stimulus, and (iv) assess the acute effect of fluctuations in shear rate on FMD. Chapter 3 aimed to update the expert-consensus methodological guidelines for the performance and analysis of FMD. Importantly, standardised performance of the FMD technique facilitates better between-study comparability, therefore reducing variability. This effort also importantly contributes to the construction of reference values (Chapter 4). Brachial artery FMD data (acquired according to protocol guidelines) and participant characteristics/medical history from 5,362 individuals (4-84yrs; 2,076 females) were pooled into a single database. Healthy individuals (n=1,403 [582 females]) were used to generate age-/sex-specific percentile curves from fractional polynomial regression. Subsequently, individuals with CVD risk factors, but without overt disease were included (un-medicated n=3,167 [1,247 females], and medicated n=792 [247 females]), and multiple linear regression tested the relation of CVD risk factors with FMD. Healthy males showed a negative, curvilinear relation between FMD and age, whilst females revealed a negative linear relation that started higher, but declined at a faster rate than males. Age- and sex-specific differences in FMD relate, at least partly, to baseline artery diameter. FMD was related to CVD risk factors in un-medicated (e.g. systolic-/diastolic blood pressure) and medicated individuals (e.g. diabetes/dyslipidaemia). Sex mediated 12 some of these effects (P40 years, 260 men, 139 women). Secondly, women were grouped based on hormonal status (pre- [n=173] and postmenopausal [n=110]). There was evidence of a weak correlation between SRAUC and FMD in all groups but older men, although there was variation in strength of outcomes. Further exploration using interaction terms (age-sex*SRAUC) in linear regression revealed differential relationships with FMD (young women versus young men (β=-5.8- 4, P=0.017) and older women (β=-5.9-4, P=0.049)). The correlation between SRAUC and FMD in pre-menopausal women (r2=0.097) was not statistically different to postmenopausal women (r2=0.025; Fisher: P=0.30). Subgroup analysis using stringent inclusion criteria for health markers (n=505) confirmed a stronger FMD-SRAUC correlation in young women compared to young men and older women. Chapter 6 aimed to examine the impact of fluctuations in shear, whilst maintaining mean shear levels around baseline. Fifteen healthy males (27.3±5.0 years) underwent bilateral brachial artery FMD assessment before and after unilateral exposure to 30 minutes of intermittent negative pressure (10seconds -40mmHg, 7seconds 0mmHg) to induce fluctuation in shear rate, whilst the contra-lateral arm was exposed to a resting period. Negative pressure significantly increased shear rate, followed by a decrease in shear rate upon pressure release (both P