Advances in the understanding of the vascular biology of atherosclerosishave revolutionized the clinical approach to its management. It is now clear that vascular disease begins in childhood and progresses silently for many years until its late clinical manifestations, which include myocardial infarction and stroke, occur. Dynamic changes in vascular biology characterize both the early pre-clinical phase and established atherosclerosis, and the vascular endothelium plays a key role in this process.1 The latter is optimally situated to act as a signal transducer between the circulation and the vessel wall and produces a wide range of factors that regulate vessel tone, cell adhesiveness, vascular growth, and coagulation. 2 Alterations in endothelial function precede the development of morphological changes and contribute to atherosclerotic lesion development and progression.3 – 6 These disturbances in endothelial function also participate in the inflammatory changes in the atherosclerotic artery, which destabilize established plaques to increase the risk of clinical events.1 Appreciation of the role of the vascular endothelium throughout the atherosclerotic disease process has led to the development of a range of invasive and non-invasive techniques which permit evaluation of different aspects of its function.7 These methods have provided insights into the pathophysiology of atherosclerosis. They have also provided opportunities to study the impact of interventions on endothelial function and add greatly to objective assessment of response to treatments in clinical trials. The present review examines the role of flow-mediated dilatation (FMD), which is the most widely used non-invasive ultrasound method to assess endothelial function. The evolution of the technique to provide accurate and reproducible data is described together with its value and limitations in clinical practice and its role in drug development programmes.
Clinical study of endothelial-dependent dilatation
The importance of the vascular endothelium in regulating vascular homoeostasis was first recognized by its effect on vascular tone.Pioneering experimental work, in the 1980s, using isolated blood vessels, demonstrated that stimulation of rabbit aorta with acetylcholine resulted in relaxation that was dependent on the presence of an intact endothelium.8 This endothelium-dependent relaxation was shown to be mediated by nitric oxide (NO).8
Nitric oxide has numerous functions in the maintenance of arterial wall homoeostasis, which are lost when the endothelium becomes dysfunctional. In clinical practice, the impact of NO bioavailability on vasomotion is most commonly studied and evidence suggests that it represents a barometer of other key functions of the endothelium.
In 1986, the experimental observations of Furchgott were adapted for the study of endothelial function in patients undergoing cardiac catheterization.9 Infusion of acetylcholine in patients with coronary artery disease resulted in paradoxical vasoconstriction,
whereas dose-dependent dilatation was observed in patients with ‘smooth’ coronary arteries. Other pharmacological agents can produce similar effects, as can an increase in flow induced by distal infusion of vasodilators such as adenosine and papaverine. In 1992, we reported a new method to perform the same experiment of FMD non-invasively in conduit arteries in the peripheral circulation.10 An increase in flow was induced by inflation and subsequent release of a sphygmomanometer cuff on the distal forearm and the impact of this ‘physiological’ stimulus on artery diameter above the elbow was assessed by high resolution ultrasound. The FMD in the brachial and radial arteries is almost completely abolished by inhibitors of eNOS demonstrating its dependence on local NO bioavailability.11 Many studies have subsequently shown impairment of FMD in response to a range of cardiovascular (CV) risk factors and its recovery after treatment. Anderson et al.12 were the first to report a relationship between endothelial-dependent vasodilatation in the brachial and the coronary circulation using pharmacological agents in the coronary experiments and flow as a stimulus in the peripheral circulation. The coexistence of endothelial dysfunction in the coronary and brachial arteries was confirmed by Takase et al.13 using flow as a stimulus in both vascular beds with a high correlation. This suggests that endothelial function varies throughout the vasculature and, in conjunction with local haemodynamics and other factors, determines the development and progression of focal atherosclerosis.
The measurement of endothelial function in the conduit arteries of the peripheral circulation by FMD enables study of the key
aspects of vascular biology in pre-clinical subjects in whom invasive testing is not feasible.7
Flow-mediated dilatation methodology
While the concept of FMD is simple, measurement is challenging and involves systematic training to reach the plateau of the learning curve for both image acquisition and image analysis.14 Over the last 20 years, technical modifications and the development of analysis software have greatly improved image acquisition and reduced method variability.
A number of patient related and environmental factors have been shown to influence FMD, including mental stress, food, drugs, and temperature.15,16 Current guidelines recommend that subjects should have FMD measurement in a fasting state (8–12 h) and be studied in a quiet, temperature-controlled room.14,17 Tobacco use should be avoided for at least 4–6 h before the study
and, for female individuals, the phase of the menstrual cycle should be reported, since hormonal changes may affect FMD
results.18,19 Recent data, however, from population studies, demonstrate that the contribution of environmental factors to
the variability of FMD is relatively small, and these should not be considered limiting factors for FMD assessment when the ‘ideal’
conditions cannot be achieved.20
Image acquisition and site selection
Before initiating an FMD study, the subject should rest for .10 min to ensure stable conditions during scanning.21 In most cases, the brachial artery is the preferred site of measurement (usual diameter 2.5–5 mm). In pre-pubertal children, the femoral artery can be also studied, but is too large in older subjects. Arteries with smaller diameter are difficult to image accurately and reproducibly and very small changes in absolute diameter are consequently reported as large percentage changes. Longitudinal images of the brachial artery are obtained with a highresolution ultrasound probe (usually 7–12 MHz), while the subject lies in supine position with the arm resting in a comfortable position in a cradle support. The interface between the near and far arterial wall and the vessel lumen has to be clearly defined. Brachial diameter measurements are obtained in end-diastole, identified by the onset of the R-wave.14 This period is preferred since functional characteristics of the artery such as vessel compliance, which influence the extent of systolic expansion, are unlikely to interfere with diameter measurements.14 Finally, anatomical landmarks have to be identified and the use of a stereotactic adjustable probe-holding device is necessary to ensure that image quality and plane are maintained throughout the study (Figure 1). To ensure that the examiner will be able to restudy the original position of the brachial artery when the subject is re-examined it is important that the positions of the arm, hand, and head are noted, along with the distance between the pneumatic cuff and the probe during the first examination. It is also helpful to make a thermal print of the arterial image for matching at subsequent visits.22
Sphygmomanometer probe position
In the original description for brachial FMD measurement, the sphygmomanometer blood pressure cuff was placed on the forearm, and the brachial artery was imaged above the antecubital fossa. This remains the technique of choice, with placement of the cuff 1–2 cm distal to the elbow crease. Placement of the cuff more distally around the wrist results in lower reactive hyperaemia and lower FMD.23 Placement of the occlusion cuff above the imaging probe has also been advocated.24 Proximal cuff positioning may affect the magnitude and peak vasodilatory response and also the time course of the peak response.23 Although this has been advocated to enable the differentiation of subtle differences in an FMD response between different groups, there are major concerns about the validity of this approach. Imaging is challenging as the artery can collapse or distort during cuff inflation and the measured vasodilatation obtained with a proximal cuff placement is confounded by ischaemic mediators other than NO, making this approach inapplicable for studies where NO bioavailability is the focus of interest.24 (Figure 1).
Cuff occlusion time
The original cuff occlusion duration of 5 min has stood the test of time. Subsequent studies have shown that cuff occlusion of
,1.5 min is not followed by significant dilatation, whereas FMDincreases linearly with increasing cuff occlusion times of up to 4.5 min.16 However, no further increase in maximal dilator response is seen when the cuff occlusion time is extended further to 10 min.16 The cuff is inflated to ≥50 mmHg above systolic pressure to occlude arterial inflow and this causes ischaemia and subsequent dilatation of downstream resistance vessels by autoregulatory mechanisms. On cuff deflation, reactive hyperaemia in the brachial artery occurs and results in vasodilatation if endothelial function is intact.25
Flow-mediated dilatation analysis
A number of studies have demonstrated that maximal increase in diameter occurs 45–60 s after the release of the cuff. New semiautomatic measurement software is now available and this permits faster, less subjective and more reproducible measurement of the arterial diameter compared with previously used manual measurement. 22 Additionally, analysis software can characterize the whole profile of change in brachial artery diameter. It has been suggested that measurement of vasoconstriction, which can occur during cuff inflation may provide additional information on endothelial function and can complement FMD.26
Characterization of the flow-mediated dilatation response
Flow-mediated dilatation is commonly reported as percentage change from the baseline diameter. Other parameters such as FMD at 60 s, area under the response curve and time to peak have also been used to describe the dilatory response.22
However, recent data show that per cent maximum FMD is the most reproducible measure and the best discriminator between
health and disease.22 The influence of baseline diameter on FMD is important. Vessel size influences an FMD response, both by an impact on blood flow-related shear stress on the vessel wall, and also by being part of the calculation of percentage FMD. Absolute change in FMD (millimetre) is unrelated to resting vessel size. Percentage FMD is currently recommended, for interventional and longitudinal studies in which baseline vessel diameter remains stable over time.22 However, measurement and reporting of the baseline diameter, absolute change and percentage FMD in diameter are advisable. Equally important is the characterization of the hyperaemic stimulus for serial measurements and comparisons between groups.