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

Abstract

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.

Fig. 1.

Schematic representation of steps involved in flow-mediated dilation (FMD) generation from the initiation of the shear-stress stimulus (step 1) to the resultant vessel diameter change (step 6). Blood flow-associated shear stress is sensed by deformation of mechanosensitive structures at the cell membrane. These structures could include the glycocalyx, the primary cilia, and mechanosensitive ion channels (19). Shear-stress mechanotransduction activates a signaling cascade that results in vasodilator production (step 2). The vasodilators produced and predominantly involved in FMD appear to depend on the nature of the shear-stress stimulus (100) and the endothelial phenotype (96). Vasodilators must diffuse from the endothelial cell into the smooth muscle cell (step 3). Nitric oxide (NO) may react with reactive oxygen species (ROS), decreasing its bioavailability (103). In the vascular smooth muscle, vasodilators trigger a signaling cascade that results in a lowering of calcium concentration and vasorelaxation (step 4). Vessel wall structural factors (e.g., relative proportions of collagen and elastin or wall-to-lumen ratio) may influence the diameter change that results from a given degree of smooth muscle relaxation (69) (step 5). FMD is quantified as the change in vessel diameter from baseline dimensions before the application of the shear-stress stimulus (step 6). Given the established vasoprotective properties of NO, FMD is often intended as an index of NO bioavailability (37, 100). Thus efforts have been directed at identifying specific protocols and stimulus profiles that are able to isolate the NO pathway (step 7). Examining vasodilatory responses to exogenous nitroglycerin can isolate and interrogate function downstream of the endothelium. EDHF, endothelial-derived hyperpolarizing factor; PG, vasodilatory prostaglandins; CYP 450, cytochrome P450; eNOS, endothelial NO synthase.

Fig. 2.

Schematic presentation of diameter and shear-stress (or rate) responses following cuff deflation in response to a 5-min ischemic stimulus. The gray area represents the relevant shear rate area-under-the-curve (AUC) that is believed to be the main stimulus for the peak diameter.

Fig. 3.

Mean and individual brachial artery diameter time to peak dilation following a period of 5 min of forearm ischemia in healthy young (n = 12, •), old fit (n = 12, ▪), and old unfit (n = 12, □) subjects. Error bars represent standard error of the mean. [Adapted from Black et al. (10).]

Fig. 4.

Hypothetical data in which no between-subject relationship between shear and FMD is observed despite the presence of (variable) within-subject shear-FMD relationships. Thin solid lines represent within-subject shear-FMD relationships when exposed to 5 different shear-stress stimuli. Circled points represent the shear-stress stimulus and FMD response in each subject during a single reactive hyperemia test. The thick solid line represents the between-subject shear-FMD relationship. Box 1 represents a selection of data in which subjects received the same shear-stress stimulus. In this selection it is straight-forward to conclude that differences in the FMD response represent biological variability in steps 2-4 described Fig. 1. Box 2 represents a selection of data in which subjects have the same FMD response but experienced very distinct shear-stress stimuli. In this data selection the subjects have differences in endothelial function, and only the variable shear-stress stimulus has allowed them to experience a similar FMD response. This highlights the importance of determining an effective method to account for the stimulus magnitude.

Fig. 5.

For any normalization approach to be appropriate, the covariate (i.e., shear) should be at least moderately correlated to the outcome (i.e., FMD) (5). Thin solid lines represent hypothetical within-subject shear-FMD relationships, where the dilation is presented to 5 incremental shear-stress stimuli. The good within-subject shear-FMD relationship suggests that normalization is appropriate when using a within-subject comparison in this data set. The thick solid line represents the between-subject correlation fitted across the mean values for the 5 subjects. A between-subject correlation is present in this data set, which is a necessary assumption for normalization in cross-sectional studies. Interindividual variation is expected in 1) the response of shear to the hyperemic stress, 2) the slope of within-subject relationship, and 3) the intercept of within-subject relationship.