Michael E. Tschakovsky

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.

The relationship between shear stress and flow‐mediated dilatation: implications for the assessment of endothelial function

Endothelium‐dependent flow‐mediated dilatation (FMD) describes the vasodilatory response of a vessel to elevations in blood flow‐associated shear stress. Nitric oxide (NO), one of many vasoactive substances released by the endothelium in response to shear stress, is of particular interest to researchers as it is an antiatherogenic molecule, and a reduction in its bioavailability may play a role in the pathogenesis of vascular disease. The goal of many human studies is to create a shear stress stimulus that produces an NO‐dependent response in order to use the FMD measurements as an assay of NO bioavailability. The most common non‐invasive technique is the ‘reactive hyperaemia test’ which produces a large, transient shear stress profile and a corresponding FMD. Importantly, not all FMD is NO mediated and the stimulus creation technique is a critical determinant of NO dependence. The purpose of this review is to (1) explain that the mechanisms of FMD depend on the nature of the shear stress stimulus (stimulus response specificity), (2) provide an update to the current guidelines for FMD assessment, and (3) summarize the issues that surround the clinical utility of measuring both NO‐ and non‐NO‐mediated FMD. Future research should include (1) the identification and partitioning of mechanisms responsible for FMD in response to various shear stress profiles, (2) investigation of stimulus response specificity in coronary arteries, and (3) investigation of non‐NO FMD mechanisms and their connection to the development of vascular disease and occurrence of cardiovascular events.

Initial orthostatic hypotension: review of a forgotten condition

Several studies have shown that standing up is a frequent (3-10%) trigger of loss of consciousness both in young and old subjects. An exaggerated transient BP (blood pressure) fall upon standing is the underlying cause. IOH (initial orthostatic hypotension) is defined as a transient BP decrease within 15 s after standing, >40 mmHg SBP (systolic BP) and/or >20 mmHg DBP (diastolic BP) with symptoms of cerebral hypoperfusion. It differs distinctly from typical orthostatic hypotension (i.e. BP decrease >20 mmHg SBP and/or >10 mmHg DBP after 3 min of standing) as the BP decrease is transient. Only continuous beat-to-beat BP measurement during an active standing-up manoeuvre can document this condition. As IOH is only associated with active rising, passive tilting is of no diagnostic value. The pathophysiology of IOH is thought to be a temporal mismatch between cardiac output and vascular resistance. The marked decrease of vascular resistance during rising is similar to that observed at the onset of leg exercise and is absent during head-up tilting. It is attributed to vasodilatation in the working muscle through local mechanisms. Standing up causes an initial increase in venous return through the effects of contraction of leg and abdominal muscles. The consequent sudden increase in right atrial pressure may contribute to the fall in systemic vascular resistance through a reflex effect. This review alerts clinicians and clinician scientists to a common, yet often neglected, condition that occurs only upon an active change of posture and discusses its epidemiology, pathophysiology and management.

Is sympathetic neural vasoconstriction blunted in the vascular bed of exercising human muscle

Sympathetic vasoconstriction of muscle vascular beds is important in the regulation of systemic blood pressure. However, vasoconstriction during exercise can also compromise blood flow support of muscle metabolism. This study tested the hypothesis that local factors in exercising muscle blunt vessel responsiveness to sympathetic vasoconstriction. We performed selective infusions of three doses of tyramine into the brachial artery (n= 8) to evoke endogenous release of noradrenaline (norepinephrine) at rest and during moderate and heavy rhythmic handgrip exercise. In separate experiments, tyramine was administered during two doses of adenosine infusion (n= 7) and two doses of sodium nitroprusside (SNP) infusion (n= 8). Vasoconstrictor effectiveness across conditions was assessed as the percentage reduction in forearm vascular conductance (FVC), calculated from invasive blood pressure and non‐invasive Doppler ultrasound blood flow measurements at the brachial artery. Tyramine evoked a similar dose‐dependent vasoconstriction at rest in all three groups, with the highest dose resulting in a 42‐46 % reduction in FVC. This vasoconstriction was blunted with increasing exercise intensity (e.g. tyramine high dose percentage reduction in FVC; rest −43.4 ± 3.7 %, moderate exercise −27.5 ± 2.3 %, heavy exercise −16.7 ± 3.6 %; P < 0.05). In contrast, tyramine infusion resulted in a greater percentage reduction in FVC during both doses of adenosine vs. rest (P < 0.05). Finally, percentage change in FVC was greater during low dose SNP infusion vs. rest (P < 0.05), but not different from rest at the high dose of SNP infusion (P= 0.507). A blunted percentage reduction in FVC during endogenous noradrenaline release in exercise but not vasodilator infusion indicates that sympathetic vasoconstriction is blunted in exercising muscle. This blunting appears to be exercise intensity‐dependent.

Regulation of oxygen consumption at the onset of exercise.

HUGHSON, R.L., M.E. TSCHAKOVSKY, and M.E. HOUSTON. Regulation of oxygen consumption at the onset of exercise. Exerc. Sport Sci. Rev., Vol. 29, No. 3, pp 129–133, 2001. Increased aerobic production of ATP at the onset of exercise could be limited by availability of metabolic substrates independent of O2, or interaction between O2 and metabolic substrates. We point out the importance of feedback control to match O2 supply to demand and discuss metabolic control at the onset of exercise.

Nitric oxide is not obligatory for radial artery flow-mediated dilation following release of 5 or 10 min distal occlusion.

This study investigated the nitric oxide (NO) dependence of radial artery (RA) flow-mediated dilation (FMD) in response to three different reactive hyperemia (RH) shear stimulus profiles. Ten healthy males underwent the following three RH trials: 1) 5 min occlusion (5 trial), 2) 10 min occlusion (10 trial), and 3) 10 min occlusion with cuff reinflation at 30 s (10-30 trial). Trials were performed during saline infusion and repeated during N(G)-monomethyl-L-arginine (L-NMMA) infusion in the brachial artery. RA blood flow velocity was measured with Doppler ultrasound, and B-mode RA images were analyzed using automated edge detection software. Shear rate estimation of shear stress was calculated as the blood flow velocity/vessel diameter. L-NMMA decreased baseline vascular conductance by 35%. L-NMMA infusion did not affect the peak shear rate stimulus (P = 0.681) or the area under the curve (AUC) of shear rate to peak FMD (P = 0.088). The AUC was significantly larger in the 10 trial vs. the 10-30 or 5 trial (P < 0.001). Although percent FMD (%change in diameter) in the 10 trial was larger than that in the 5 trial (P = 0.035), there was no significant difference in %FMD between the saline and L-NMMA conditions in any trial: 5 trial, 5.62 +/- 1.48 vs. 5.63 +/- 1.27%; 10 trial, 9.07 +/- 1.16 vs. 11.22 +/- 2.21%; 10-30 trial, 6.52 +/- 1.43 vs. 7.98 +/- 1.51% for saline and L-NMMA, respectively (P = 0.158). We conclude the following: 1) RH following 10 min of occlusion results in an enhanced stimulus and %FMD compared with 5 min of occlusion. 2) When the occlusion cuff is reinflated 30 s postrelease of a 10 min occlusion, it does not result in an enhanced %FMD compared with that which results from RH following 5 min of occlusion. 3) The lack of effect of L-NMMA on FMD suggests that NO may not be obligatory for radial artery FMD in response to either 5 or 10 min of occlusion in healthy volunteers.

Brachial artery flow-mediated dilation during handgrip exercise: evidence for endothelial transduction of the mean shear stimulus.

Exercise elevates shear stress in the supplying conduit artery. Although this is the most relevant physiological stimulus for flow-mediated dilation (FMD), the fluctuating pattern of shear that occurs may influence the shear stress-FMD stimulus response relationship. This study tested the hypothesis that the brachial artery FMD response to a step increase in shear is influenced by the fluctuating characteristics of the stimulus, as evoked by forearm exercise. In 16 healthy subjects, we examined FMD responses to step increases in shear rate in three conditions: stable shear upstream of heat-induced forearm vasodilation (FHStable); fluctuating shear upstream of heat-induced forearm vasodilation and rhythmic forearm cuff inflation/deflation (FHFluctuating); and fluctuating shear upstream of exercise-induced forearm vasodilation (FEStep Increase). The mean increase in shear rate (+/-SD) was the same in all trials (FHFluctuating): 51.69 +/- 15.70 s(-1); FHStable: 52.16 +/- 14.10 s(-1); FEStep Increase: 50.14 +/- 13.03 s(-1) P = 0.131). However, the FHFluctuating and FEStep Increase trials resulted in a fluctuating shear stress stimulus with rhythmic high and low shear periods that were 96.18 +/- 24.54 and 11.80 +/- 7.30 s(-1), respectively. The initial phase of FMD (phase I) was followed by a second, delayed-onset FMD and was not different between conditions (phase I: FHFluctuating: 5.63 +/- 2.15%; FHStable: 5.33 +/- 1.85%; FEStep Increase: 5.30 +/- 2.03%; end-trial: FHFluctuating: 7.76 +/- 3.40%; FHStable: 7.00 +/- 3.03%; FEStep Increase: 6.68 +/- 3.04%; P = 0.196). Phase I speed also did not differ (P = 0.685). In conclusion, the endothelium transduced the mean shear when exposed to shear fluctuations created by a typical handgrip protocol. Muscle activation did not alter the FMD response. Forearm exercise may provide a viable technique to investigate brachial artery FMD in humans.

Nitric oxide and muscle blood flow in exercise.

Despite being the subject of investigation for well over 100 years, the nature of exercising muscle blood flow control remains, in many respects, poorly understood. In this review we focus on the potential role of nitric oxide in vasodilation of muscle resistance vessels during a bout of exercise. Its contribution is explored in the context of whether it contributes to steady-state exercise hyperemia, the dynamic adjustment of muscle blood flow to exercise, or the modulation of sympathetic vasoconstriction in exercising muscle. It appears that the obligatory role of nitric oxide in all three of these categories is modest at best. The elucidation of the integrated nature of exercise hyperemia control in terms of synergy and redundancy of mechanism interaction remains in its infancy, and much more remains to be learned about the role of nitric oxide in this type of integrated control.

Are the dynamic response characteristics of brachial artery flow-mediated dilation sensitive to the magnitude of increase in shear stimulus?

The purpose of this study was to determine the dynamic characteristics of brachial artery dilation in response to step increases in shear stress [flow-mediated dilation (FMD)]. Brachial artery diameter (BAD) and mean blood velocity (MBV) (Doppler ultrasound) were obtained in 15 healthy subjects. Step increases in MBV at two shear stimulus magnitudes were investigated: large (L; maximal MBV attainable), and small (S; MBV at 50% of the large step). Increase in shear rate (estimate of shear stress: MBV/BAD) was 76.8 +/- 15.6 s(-1) for L and 41.4 +/- 8.7 s(-1) for S. The peak %FMD was 14.5 +/- 3.8% for L and 5.7 +/- 2.1% for S (P < 0.001). Both the L (all subjects) and the S step trials (12 of 15 subjects) elicited a biphasic diameter response with a fast initial phase (phase I) followed by a slower final phase. Relative contribution of phase I to total FMD when two phases occurred was not sensitive to shear rate magnitude (r(2) = 0.003, slope P = 0.775). Parameters quantifying the dynamics of the FMD response [time delay (TD), time constant (tau)] were also not sensitive to shear rate magnitude for both phases (phase I: TD r(2) = 0.03, slope P = 0.376, tau r(2) = 0.04, slope P = 0.261; final phase: TD r(2) = 0.07, slope P = 0.169, tau r(2) = 0.07, slope P = 0.996). These data support the existence of two distinct mechanisms, or sets of mechanisms, in the human conduit artery FMD response that are proportionally sensitive to shear stimulus magnitude and whose dynamic response is not sensitive to shear stimulus magnitude.

Heterogenous vasodilator pathways underlie flow-mediated dilation in men and women.

This study investigated the sex differences in the contribution of nitric oxide (NO) and prostaglandins (PGs) to flow-mediated dilation (FMD). Radial artery (RA) FMD, assessed as the dilatory response to 5-min distal cuff occlusion, was repeated after three separate brachial artery infusions of saline (SAL), N(G)-monomethyl-L-arginine (L-NMMA), and ketorolac (KETO) + L-NMMA in healthy younger men (M; n = 8) and women (W; n = 8). In eight subjects (4 M, 4W) RA FMD was reassessed on a separate day with drug order reversed (SAL, KETO, and L-NMMA + KETO). RA FMD was calculated as the peak dilatory response observed relative to baseline (%FMD) and expressed relative to the corresponding area under the curve shear stress (%FMD/AUC SS). L-NMMA reduced %FMD similarly and modestly (P = 0.68 for sex * trial interaction) in M and W (all subjects: 10.0 ± 3.8 to 7.6 ± 4.7%; P = 0.03) with no further effect of KETO (P = 0.68). However, all sex * trial and trial effects on %FMD/AUC SS for l-NMMA and KETO + l-NMMA were insignificant (all P > 0.20). There was also substantial heterogeneity of the magnitude and direction of dilator responses to blockade. After l-NMMA infusion, subjects exhibited both reduced (n = 14; range: 11 to 78% decrease) and augmented (n = 2; range: 1 to 96% increase) %FMD. Following KETO + l-NMMA, seven subjects exhibited reduced dilation (range: 10 to 115% decrease) and nine subjects exhibited augmented dilation (range: 1 to 212% increase). Reversing drug order did not change the nature of the findings. These findings suggest that RA FMD is not fully or uniformly NO dependent in either men or women, and that there is heterogeneity in the pathways underlying the conduit dilatory response to ischemia.