Mark A. Black

Assessment of flow-mediated dilation in humans: a methodological and physiological guideline

Endothelial dysfunction is now considered an important early event in the development of atherosclerosis, which precedes gross morphological signs and clinical symptoms. The assessment of flow-mediated dilation (FMD) was introduced almost 20 years ago as a noninvasive approach to examine vasodilator function in vivo. FMD is widely believed to reflect endothelium-dependent and largely nitric oxide-mediated arterial function and has been used as a surrogate marker of vascular health. This noninvasive technique has been used to compare groups of subjects and to evaluate the impact of interventions within individuals. Despite its widespread adoption, there is considerable variability between studies with respect to the protocols applied, methods of analysis, and interpretation of results. Moreover, differences in methodological approaches have important impacts on the response magnitude, can result in spurious data interpretation, and limit the comparability of outcomes between studies. This review results from a collegial discussion between physiologists with the purpose of developing considered guidelines. The contributors represent several distinct research groups that have independently worked to advance the evidence base for improvement of the technical approaches to FMD measurement and analysis. The outcome is a series of recommendations on the basis of review and critical appraisal of recent physiological studies, pertaining to the most appropriate methods to assess FMD in humans.

Importance of measuring the time course of flow-mediated dilatation in humans.

Flow-mediated dialation (FMD) is widely used to describe conduit artery endothelial function. The traditional approaches to FMD calculation assess diameter change at arbitrary time points after occluding cuff deflation. The aim of this study was to examine the time course of brachial artery FMD after a 5-minute period of forearm ischemia in 12 young, 12 fitness matched older and 12 older untrained subjects. Edge-detection and wall tracking of high resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate brachial artery diameter, blood flow, and shear rate continuously across the cardiac cycle after forearm ischemia. FMD was significantly higher in young healthy subjects (7.8±3.2%) compared with sedentary older subjects (5.2±2.8%, P<0.05) but not trained older subjects (6.4±2.3%). Time to peak diameter differed between young (50±11 seconds) and both older groups (trained; 80±21, P<0.001; sedentary: 83±36 seconds, P<0.001). A large proportion (>42%) of true peak diameters fell outside the time frames typically used to assess FMD in the literature. When calculated according to the commonly used approach, ie, 60 secs after cuff deflation, FMD was significantly lower compared with true peak FMD in all groups (P<0.001), and no differences were evident between the groups. The time course of FMD differs significantly between young and older subjects. Studies assuming that peak dilation occurs at an arbitrary time point, or within limited time windows, may draw misleading conclusions regarding differences between groups. More sophisticated approaches to measurement of FMD are required if it is to be considered a valid biomarker of vascular disease.

Impact of Shear Rate Modulation on Vascular Function in Humans

Shear stress is an important stimulus to arterial adaptation in response to exercise and training in humans. We recently observed significant reverse arterial flow and shear during exercise and different antegrade/retrograde patterns of shear and flow in response to different types of exercise. The purpose of this study was to simultaneously examine flow-mediated dilation, a largely NO-mediated vasodilator response, in both brachial arteries of healthy young men before and after 30-minute interventions consisting of bilateral forearm heating, recumbent leg cycling, and bilateral handgrip exercise. During each intervention, a cuff inflated to 60 mm Hg was placed on 1 arm to unilaterally manipulate the shear rate stimulus. In the noncuffed arm, antegrade flow and shear increased similarly in response to each intervention (ANOVA; P<0.001, no interaction between interventions; P=0.71). Baseline flow-mediated dilation (4.6%, 6.9%, and 6.7%) increased similarly in response to heating, handgrip, and cycling (8.1%, 10.4%, and 8.9%, ANOVA; P<0.001, no interaction; P=0.89). In contrast, cuffed arm antegrade shear rate was lower than in the noncuffed arm for all of the conditions (P<0.05), and the increase in flow-mediated dilation was abolished in this arm (4.7%, 6.7%, and 6.1%; 2-way ANOVA: all conditions interacted P<0.05). These results suggest that differences in the magnitude of antegrade shear rate transduce differences in endothelial vasodilator function in humans, a finding that may have relevance for the impact of different exercise interventions on vascular adaptation in humans.

Time course of change in vasodilator function and capacity in response to exercise training in humans.

Studies of the impact of exercise training on arterial adaptation in healthy subjects have produced disparate results. It is possible that some studies failed to detect changes because functional and structural adaptations follow a different time course and may therefore not be detected at discrete time points. To gain insight into the time course of training‐induced changes in artery function and structure, we examined conduit artery flow mediated dilatation (FMD), an index of nitric oxide (NO)‐mediated artery function, and conduit dilator capacity (DC), a surrogate marker for arterial remodelling, in the brachial and popliteal arteries of 13 healthy male subjects (21.6 ± 0.6 years) and seven non‐active controls (22.8 ± 0.2 years) studied at 2‐week intervals across an 8‐week cycle and treadmill exercise training programme. Brachial and popliteal artery FMD and DC did not change in control subjects at any time point. FMD increased from baseline (5.9 ± 0.5%) at weeks 2 and 4 (9.1 ± 0.6, 8.5 ± 0.6%, respectively, P < 0.01), but returned towards baseline levels again by week 8 (6.9 ± 0.7%). In contrast, brachial artery DC progressively increased from baseline (8.1 ± 0.4%) at weeks 2, 4, 6 and 8 (9.2 ± 0.6, 9.9 ± 0.6, 10.0 ± 0.5, 10.5 ± 0.8%, P < 0.05). Similarly, popliteal artery FMD increased from baseline (6.2 ± 0.7%) at weeks 2, 4 and 6 (9.1 ± 0.6, 9.5 ± 0.6, 7.8 ± 0.5%, respectively, P < 0.05), but decreased again by week 8 (6.5 ± 0.6%), whereas popliteal DC progressively increased from baseline (8.9 ± 0.4%) at week 4 and 8 (10.5 ± 0.7, 12.2 ± 0.6%, respectively, P < 0.05). These data suggest that functional changes in conduit arteries occur rapidly and precede arterial remodelling in vivo. These data suggest that complimentary adaptations occur in arterial function and structure and future studies should adopt multiple time point assessments to comprehensively assess arterial adaptations to interventions such as exercise training in humans.

Impact of age, sex, and exercise on brachial artery flow-mediated dilatation

Flow-mediated dilatation (%FMD), an index of nitric oxide (NO)-mediated vasodilator function, is regarded as a surrogate marker of cardiovascular disease. Aging is associated with endothelial dysfunction, but underlying sex-related differences may exist and the effects of fitness and exercise on endothelial dysfunction in men (M) and women (W) are poorly understood. We compared %FMD of the brachial artery in 18 young [Y, 26 +/- 1 yr; 9 M and 9 W], 12 older fit (OF, 57 +/- 2 yr; 6 M and 6 W), and 16 older sedentary (OS, 59 +/- 2 yr; 8 M and 8 W) subjects. Glyceryl trinitrate (GTN) administration was used to assess endothelium-independent vasodilatation, and the FMD-to-GTN ratio was calculated to characterize NO dilator function in the context of smooth muscle cell sensitivity. Brachial %FMD in Y (7.1 +/- 0.8%) was significantly higher compared with OS (4.8 +/- 0.7%, P < 0.05), but not OF (6.4 +/- 0.7%). Differences between Y and OS subjects were due primarily to lower FMD in the OS women (4.3 +/- 0.6%). OS women exhibited significantly lower FMD-to-GTN ratios compared with Y (P < 0.05) and OF women (P < 0.05), whereas these differences were not apparent in men. Exercise training improved brachial artery NO dilator function (FMD-to-GTN ratio) after 24 wk (P < 0.05) in OS women, but not men. These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents the age-related decline in the brachial artery vasodilator function evident in women. In OS men, who had relatively preserved NO dilator function, no training adaptations were observed. This study has potential implications for the prevention of conduit artery endothelial dysfunction in men and women.

Exercise prevents age-related decline in nitric-oxide-mediated vasodilator function in cutaneous microvessels

Ageing is associated with impaired endothelium‐derived nitric oxide (NO) function in human microvessels. We investigated the impact of cardiorespiratory fitness and exercise training on physiological and pharmacological NO‐mediated microvascular responses in older subjects. NO‐mediated vasodilatation was examined in young, older sedentary and older fit subjects who had two microdialysis fibres embedded into the skin on the ventral aspect of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42°C, with Ringer solution infused in one probe and N‐nitro‐l‐arginine methyl ester (l‐NAME) through the second. In another study, three doses of ACh were infused in the presence or absence of l‐NAME in similar subjects. The older sedentary subjects then undertook exercise training, with repeat studies at 12 and 24 weeks. The NO component of the heat‐induced rise in cutaneous vascular conductance (CVC) was diminished in the older sedentary subjects after 30 min of prolonged heating at 42°C (26.9 ± 3.9%CVCmax), compared to older fit (46.2 ± 7.0%CVCmax, P < 0.05) and young subjects (41.2 ± 5.2%CVCmax, P < 0.05), whereas exercise training in the older sedentary group enhanced NO‐vasodilator function in response to incremental heating (P < 0.05). Similarly, the NO contribution to ACh responses was impaired in the older sedentary versus older fit subjects (low dose 3.2 ± 1.3 versus 6.6 ± 1.3%CVCmax; mid dose 11.4 ± 2.4 versus 21.6 ± 4.5%CVCmax; high dose 35.2 ± 6.0 versus 52.6 ± 7.9%CVCmax, P < 0.05) and training reversed this (12 weeks: 13.7 ± 3.6, 28.9 ± 5.3, 56.1 ± 3.9%CVCmax, P < 0.05). These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents age‐related decline in indices of physiological and pharmacological microvascular NO‐mediated vasodilator function. Since higher levels of NO confer anti‐atherogenic benefit, this study has potential implications for the prevention of microvascular dysfunction in humans.

Brachial Artery Blood Flow Responses to Different Modalities of Lower Limb Exercise

INTRODUCTION/PURPOSE Cycling is associated with a reproducible systolic anterograde and diastolic retrograde flow pattern in the brachial artery (BA) of the inactive upper limb, which results in endothelial nitric oxide (NO) release. The purpose of this study was to examine the impact of different types and intensities of lower limb exercise on the BA flow pattern. METHODS We examined BA blood flow and shear rate patterns during cycling, leg kicking, and walking exercise in 12 young subjects (24 +/- 3 yr). BA diameter, blood flow, and shear rate were assessed at baseline (1 min) and at three incremental intensity levels of cycling (60, 80, and 120 W), bilateral leg kicking (5, 7.5, and 10 kg), and walking (3, 4, and 5 km x h(-1)), performed for 3 min each. Edge detection and wall tracking of high-resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate conduit artery diameter and anterograde/retrograde blood flow and shear rate continuously across the cardiac cycle. RESULTS BA mean blood flow and shear rate increased significantly throughout each exercise protocol (P < 0.001), and BA anterograde blood flow and shear rate showed comparable increases throughout each protocol (P < 0.001). Retrograde blood flow and shear rate, however, demonstrated a significant increase during cycling and walking (P < 0.001) but not during leg kicking. CONCLUSION Rhythmic lower limb exercise (cycling and walking) results in an increase in BA systolic anterograde blood flow and shear rate, directly followed by a large retrograde flow and shear rate. This typical pattern, previously linked with endothelial NO release, is not present during a different type of exercise such as leg kicking.

Changes in vascular and cardiac function after prolonged strenuous exercise in humans

Prolonged exercise has been shown to result in an acute depression in cardiac function. However, little is known about the effect of this type of exercise on vascular function. Therefore, the purpose of the present study was to investigate the impact of an acute bout of prolonged strenuous exercise on vascular and cardiac function and the appearance of biomarkers of cardiomyocyte damage in 15 male (32 +/- 10 yr) nonelite runners. The subjects were tested on two occasions, the day before and within an hour of finishing the London marathon (229 +/- 38 min). Function of the brachial and femoral arteries was determined using flow-mediated dilatation (FMD). Echocardiographic assessment of cardiac strain, strain rate, tissue velocities, and flow velocities during diastole and systole were also obtained. Venous blood samples were taken for later assessment of cardiac troponin I (cTnI), a biomarker of cardiomyocyte damage. Completion of the marathon resulted in a depression in femoral (P = 0.04), but not brachial (P = 0.96), artery FMD. There was no change, pre- vs. postmarathon, in vascular shear, indicating that the impaired femoral artery function was not related to hemodynamic changes. The ratio of peak early to atrial radial strain rate, a measure of left ventricular diastolic function, was reduced postmarathon (P = 0.006). Postrace cTnI was elevated in 12 of 13 runners, with levels above the recognized clinical threshold for damage in 7 of these. In conclusion, when taken together, these data suggest a transient depression in cardiac and leg vascular function following prolonged intensive exercise.

Heterogeneity in conduit artery function in humans: impact of arterial size.

To determine whether conduit artery size affects functional responses, we compared the magnitude, time course, and eliciting shear rate stimulus for flow-mediated dilation (FMD) in healthy men (n = 20; 31 +/- 7 yr). Upper limb (brachial and radial) and lower limb (common and superficial femoral) FMD responses were simultaneously assessed, whereas popliteal responses were measured in the same subjects during a separate visit. Glyceryl trinitrate (GTN)-mediated responses were similarly examined. Edge detection and wall tracking of high-resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate conduit artery diameter, blood flow, and shear rate continuously across the cardiac cycle. Baseline artery size correlated inversely with the FMD response (r = -0.57, P < 0.001). Within-artery comparisons revealed a significant inverse correlation between artery size and FMD% for the radial (r = -0.66, P = 0.001), brachial (r = -0.55, P = 0.01), and popliteal artery (r = -0.48, P = 0.03), but not for the superficial and common femoral artery. Normalization of FMD responses for differences in eliciting shear rate did not abolish the between-artery relationship for artery function and size (r = -0.48, P < 0.001), suggesting that differences between artery function responses were not entirely due to size-related differences in shear rate. This was reinforced by a significant between-artery correlation for GTN responses and baseline artery size (r = -0.74, P < 0.001). In summary, systematic differences exist in vascular function responses of conduit arteries that differ in size. This raises the possibility that differences in artery size within or between individuals may influence functional responses.

Does arterial shear explain the magnitude of flow-mediated dilation?: a comparison between young and older humans

Flow-mediated dilatation (FMD) has become a commonly applied approach for the assessment of vascular function and health in humans. Recent studies emphasize the importance of normalizing the magnitude of FMD to its apparent eliciting stimulus, the postdeflation arterial shear. However, the relationship between shear stress and the magnitude of FMD may differ between groups. The aim of this study was to examine the relationship between the brachial FMD and four different indexes of postdeflation shear rate (SR) in healthy children (n = 51, 10 +/- 1 yr) and young (n = 57, 27 +/- 6 yr) and older (n = 27, 58 +/- 4 yr) adults. SR was calculated from deflation (time 0) until 9 s (peak), 30 s (0-30), 60 s (0-60), or until the time-to-peak diameter in each individual (0-ttp). Edge detection and wall tracking of high resolution B-mode arterial ultrasound images were used to calculate the conduit artery diameter. In young adults, the brachial artery FMD demonstrated a significant correlation with the area under the SR curve (SR(AUC)) 0-30 s (r(2) = 0.12, P = 0.009), 0-60 s (r(2) = 0.14, P = 0.005), and 0-ttp (r(2) = 0.14, P = 0.005) but not for the peak SR(AUC) 0-9 s (r(2) = 0.04, P = 0.12). In children and older adults, the magnitude of the brachial artery FMD did not correlate with any of the four SR(AUC) stimuli. These findings suggest that in young subjects, postdeflation SR(AUC) correlates moderately with the magnitude of the FMD response. However, the relationship between FMD and postdeflation shear appears to be age dependent, with less evidence for an association in younger and older subjects. Therefore, we support presenting SR(AUC) stimuli but not normalizing FMD responses for the SR(AUC) when using this technique.