Flow-mediated dilation does reflect nitric oxide-mediated endothelial ...

J Appl Physiol 99: 1233–1238, 2005; doi:10.1152/japplphysiol.00601.2005.

Point:Counterpoint

Point: Flow-mediated dilation does reflect nitric oxide-mediated endothelial function Before you embark on this debate with my redoubtable Canadian friends, you may find it advisable to take something to minimize your cardiovascular risk. I recommend the sensible precaution of a bottle of excellent Australian Shiraz. Pour yourself a generous measure and take a few sips. Now, if your glass appears half full, you might agree that the abundance of flow-mediated dilatation (FMD) studies in the cardiovascular literature in the past decade is attributable to the seductive idea that it provides a functional bioassay for in vivo endothelium-derived nitric oxide (NO) bioactivity in humans. Impaired NO-mediated endothelial function has gained acceptance as a sentinel atherogenic event (4, 11, 21), so a simple and cheap noninvasive marker of NO “dys”-function may provide a “barometer” of cardiovascular disease risk, which could be used to predict individuals at highest risk of future cardiovascular events (2, 27, 30) or those with less stable advanced lesions (4). It might also be possible to optimize medication and risk factor advice to target directly measured vascular health, rather than surrogates such as blood pressure, lipid levels, or inflammatory markers. Risk reduction is, after all, ultimately dependent on changes in the artery wall and a direct measure of arterial “fitness” would provide a powerful tool. Of course, if your glass seems half empty, it is possible that every physician and scientist with access to an ultrasound machine and a delusion of grandeur is riding the latest research bandwagon. So is the mercury rising or falling on the FMD technique and, crucially, is it NO dependent? It is true that when Celermajer and colleagues (6) introduced the idea of using cuff occlusion to examine endothelial function by inducing arterial shear stress in 1992, important assumptions were made. It was known at the time that human conduit arteries dilated in response to increased blood flow (1, 18, 23, 24), that in animals this response was dependent on an intact endothelium (20, 25), that shear stress-sensitive ion channels existed in endothelial cells (7, 14, 19), that the physiological stimulus to NO [endothelium-derived relaxing factor (EDRF) at that time] production in animals was shear stress (22) and that infusion of NO antagonists [e.g., NGmonomethyl-L-arginine (L-NMMA)] decreased FMD in situ (8, 12). Vallance et al. (26) had also established that NO was released basally in humans and in response to pharmacological stimulation. From these atmospherics, it was inferred that the dilator response after cuff deflation was likely to be endothelium dependent and probably NO (EDRF) mediated. Thus, although physiological studies demonstrating NO dependency of the FMD technique had not been performed in humans before the introduction and adoption of the technique, it was a clever idea predicated on sound indicative evidence. Joannides and colleagues (13) published the first study involving L-NMMA infusion to block NO production after cuff occlusion in humans. They imaged the radial artery for diameter and flow at rest and after 3 min of ischemia induced by a wrist cuff placed distal to the ultrasound probe. L-NMMA, infused into the brachial artery upstream, converted the radial artery FMD response (3.6%) to a constriction (⫺2.8%). This abolition of FMD by NO blockade occurred in the absence of http://www. jap.org

changes in peak radial artery flow, although L-NMMA decreased the duration of hyperemia, raising the possibility of a confounding nonspecific vasoconstrictor-mediated decrease in radial artery shear stress. This possibility was subsequently ruled out by Lieberman et al. (15), who studied the effect of upper arm cuff occlusion (5 min) on brachial artery FMD in the presence of L-NMMA infused above the ultrasound probe. L-NMMA decreased brachial artery FMD from 21 to 7%, indicating a potent effect of NO inhibition on FMD. Importantly, these investigators also measured the effect of a NOindependent vasoconstrictor (phenylephrine) on FMD. Despite having an equipotent effect on reactive hyperemic forearm blood flow as L-NMMA, phenylephrine did not alter brachial artery FMD, confirming the NO dependence of the FMD response. This study reported relatively high FMD data (⬃21%), possibly due to scanning of the artery below the antecubital fossa, where it is smaller, and the fact that the scanned artery was within the ischemic territory (vide infra). Nonetheless, the authors provided strong evidence that FMD is an endothelium-dependent process, mediated by NO. In 2001, Doshi et al. (10) specifically investigated the issue of cuff placement on NO dependency of the FMD response. A 5-min cuff occlusion at the wrist, distal to the ultrasound probe placed on the brachial artery, was associated with an ⬃7% FMD response that was abolished (0.14%) by L-NMMA infusion. In response to 5 min of occlusion induced by a cuff placed on the arm above the ultrasound probe, the ⬃12% FMD response was only partially decreased by L-NMMA (7.5%). These data therefore suggested that, whereas NO contributed to FMD under both protocols, placement of the cuff was important; dilation of arteries that have been within the ischemic territory is affected by dilators other than NO and may also be complicated by myogenic responses as a result of the pressure fall inside the artery during occlusion. This important study has resulted in general acceptance of the principle that FMD studies should involve cuff occlusion below the antecubital fossa, with proximal brachial artery imaging. A final study that deserves mention is that by Mullen et al. (17), which used clever experimental approaches to determine whether characteristics of the flow stimulus modified the mechanisms involved in conduit artery dilatation. Brachial artery infusion of L-NMMA decreased the radial artery diameter response to 5 min of distal wrist cuff occlusion from ⬃5.3 to 0.7%. The L-NMMA infusion had no effect on either the peak or prolonged flow response after cuff deflation. Conversely, after 15 min of wrist cuff inflation, FMD was 9.6% but L-NMMA had no impact on radial artery dilation (9.6 vs. 9.5%). It was also demonstrated that gradual and stepwise increases in blood velocity through the radial artery, induced by stimuli such as hand warming, substantially increased proximal radial artery diameter in a manner that was not L-NMMA sensitive. These elegant physiological studies indicated that different shear stress stimuli, including different periods of ischemia, induce conduit artery dilation that is dependent on different vasodilator mechanisms. Importantly though, it provided further evidence that the widely used FMD

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Point:Counterpoint 1234 approach in humans (9), involving response to a 5-min occlusion induced by a cuff placed downstream from the imaged artery, is almost entirely abolished by L-NMMA and this is not due to a nonspecific vasoconstrictor effect of L-NMMA. In 2002, an important collaborative guideline was published aimed at standardizing technical approaches to the increasingly popular FMD method (9). The studies detailed above reinforce these guidelines and the use of FMD in humans because they indicate that in response to a period of ⬃5 min of cuff occlusion in the upper limb, where the cuff is placed below the imaging site, FMD is essentially abolished by NO blockade. Under these circumstances, there can be little argument that FMD does reflect NO-mediated endothelial function in humans. It is therefore entirely consistent that FMD measured in this way is impaired by traditional atherosclerotic risk factors (4, 5, 17, 18), predicts future cardiovascular events (3, 30), and is improved by cardioprotective interventions (16, 28), including, for instance, the consumption of wine (29). Cheers! REFERENCES 1. Anderson EA and Mark AL. Flow-mediated and reflex changes in large peripheral artery tone in humans. Circulation 79: 93–100, 1989. 2. Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Delagrange D, Leiberman EH, Ganz P, Creager MA, and Yeung AC. Close relationship of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol 26: 1235–1241, 1995. 3. Brevetti G, Silvestro A, Schiano V, and Chiariello M. Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease. Circulation 108: 2093–2098, 2003. 4. Celermajer DS. Endothelial dysfunction: does it matter? Is it reversible? J Am Coll Cardiol 30: 325–333, 1997. 5. Celermajer DS, Sorensen KE, Bull C, Robinson J, and Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol 24: 1468 –1474, 1994. 6. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340: 1111–1115, 1992. 7. Cooke JP, Rossitch EJ, Andon NA, Loscalzo L, and Dzau VJ. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest 88: 1663–1671, 1991. 8. Cooke JP, Stamler J, Andon N, Davies PF, McKinley G, and Loscalzo J. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am J Physiol Heart Circ Physiol 259: H804 –H812, 1990. 9. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer DS, Charbonneau F, Creager MA, Deanfield J, Drexer H, Gerhard-Herman M, Herrington D, Vallance P, and Vogel R. Guidelines for the ultrasound assessment of flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol 39: 257–265, 2002. 10. Doshi SN, Naka KK, Payne N, Jones CJH, Ashton M, Lewis MJ, and Goodfellow J. Flow-mediated dilatation following wrist and upper arm occlusion in humans: the contribution of nitric oxide. Clin Sci 101: 629 – 635, 2001. 11. Ganz P and Vita JA. Testing endothelial vasomotor function. Nitric oxide, a multipotent molecule. Circulation 108: 2049 –2053, 2003. 12. Hutcheson IR and Griffith TM. Release of endothelium-derived relaxing factor is modulated both by frequency and amplitude of pulsatile flow. Am J Physiol Heart Circ Physiol 261: H257–H262, 1991.

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13. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali E, Thuillez C, and Lu¨scher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91: 1314 –1319, 1995. 14. Lansman JB, Hallam TJ, and Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature (Lond) 325: 811– 813, 1987. 15. Lieberman EH, Gerhard MD, Uehata A, Selwyn AP, Ganz P, Yeung AC, and Creager MA. Flow-induced vasodilation of the human brachial artery is impaired in patients ⬍ 40 years of age with coronary artery disease. Am J Cardiol 78: 1210 –1214, 1996. 16. Maiorana A, O’Driscoll G, Cheetham C, Dembo L, Stanton K, Goodman C, Taylor RR, and Green D. The effect of combined aerobic and resistance exercise training on vascular function in type 2 diabetes. J Am Coll Cardiol 38: 860 – 866, 2001. 17. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, and MacAllister RJ. Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Circ Res 88: 145–151, 2001. 18. Nabel EG, Selwyn AP, and Ganz P. Large coronary arteries in humans are responsive to changing blood flow: an endothelium-dependent mechanism that fails in patients with atherosclerosis. J Am Coll Cardiol 16: 349 –356, 1990. 19. Oleson SP, Clapham DE, and Davies PF. Haemodynamic shear stress activates a K⫹ current in vascular endothelial cells. Nature (Lond) 331: 168 –170, 1988. 20. Pohl U, Holtz J, Busse R, and Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8: 37– 44, 1986. 21. Ross R. Atherosclerosis—an inflammatory disease. New Engl J Med 340: 115–126, 1999. 22. Rubanyi GM, Romero JC, and Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 250: H1145–H1149, 1986. 23. Schretzenmayr A. Uber kreislaufregulatorische Vorgange an den gro␤en Arterien bei Muskelarbeit. Pflugers Arch Ges Physiol 232: 743–748, 1933. 24. Sinoway LI, Hendrickson C, Davidson WRJ, Prophet S, and Zelis R. Characteristics of flow-mediated brachial artery vasodilation in human subjects. Circ Res 64: 32– 42, 1989. 25. Smiesko V, Kozik J, and Dolezel S. Role of endothelium in the control of arterial diameter by blood flow. Blood Vessels 22: 247–251, 1985. 26. Vallance P, Collier J, and Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 2: 997–1000, 1989. 27. Vita JA and Keaney JF. Endothelial function: a barometer for cardiovascular risk? Circulation 106: 640 – 642, 2002. 28. Watts K, Beye P, Siafarikas A, Davis EA, Jones TW, O’Driscoll G, and Green DJ. Exercise training normalises vascular dysfunction and improves central adiposity in obese adolescents. J Am Coll Cardiol 43: 1823–1827, 2004. 29. Whelan AP, Sutherland WH, McCormick MP, Yeoman DJ, de Jong SA, and Williams MJ. Effects of white and red wine on endothelial function in subjects with coronary artery disease. Intern Med J 34: 224 –228, 2004. 30. Widlansky ME, Gocke N, Keaney JF, and Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 42: 1149 – 1160, 2003.

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Danny Green School of Human Movement and Exercise Science The University of Western Australia

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Point:Counterpoint 1235

Counterpoint: Flow-mediated dilation does not reflect nitric oxide-mediated endothelial function The phenomenon of flow-mediated dilation (FMD) relevant to this debate describes the vasodilation in a conduit vessel in response to elevations in flow-associated shear stress. Nitric oxide (NO) is thought to play a key role in vascular health due to its established vasoprotective characteristics (for review, see Ref. 8). Because NO is one of the substances produced by the vascular endothelium in response to elevations in flow-associated shear stress (2), considerable clinical interest has focused on noninvasive assessment of FMD for evaluating NO-specific endothelial function in humans. Celermajer et. al (4) in 1992 were the first to examine FMD induced by elevated forearm conduit vessel blood flow velocity after ischemia-induced downstream vasodilation [reactive hyperemia (RH)] to assess FMD in groups at risk for atherosclerosis and observed a blunted response. Since then, numerous studies assessing FMD in various pathologies and in response to various therapeutic interventions have been conducted. A blunting of the FMD response relative to healthy controls is taken to represent endothelial dysfunction. With regard to NO, Joannides et al. (17) in 1995 were the first to examine the NO contribution to RH-induced FMD in humans and found that NO blockade completely abolished FMD. Their concluding statement was “. . .the present investigation demonstrates that NO is essential for flow mediated dilatation of human radial arteries in vivo. Thus this test can be used as a reliable noninvasive estimate of the capacity of human endothelial cells to release NO. . .” The common clinical view now, as stated in the recent technique report by the International Brachial Artery Reactivity Task Force in 2002 (6), is that vascular endothelial NO production accounts for FMD, and the primary citation supporting this is the above-mentioned Joannides et al. (17) paper. Unfortunately, the history of FMD research represents a clinically driven desire to evaluate endothelial function that has bypassed careful mechanistic dissection of FMD in vivo in humans. The issue here is whether FMD specifically reflects NO-mediated endothelial function. A careful mechanistic exploration of FMD reveals three critical characteristics that seriously question the current dogma that FMD represents NO-mediated endothelial function: 1) the nature of the shear stimulus affects the vasoregulatory mechanisms evoked, 2) shear stress acts as a stimulus for endothelial release of vasoconstrictors, and 3) elevated sympathetic activation, characteristic of numerous pathologies in which endothelial dysfunction is observed, can blunt the FMD response. Shear stimulus characteristics determine FMD mechanisms. It is well established that there are a number of signaling pathways activated with an increase in shear stress and at least three vasodilators who’s production increases have been identified (NO, prostacyclin, endothelium-derived hypopolarizing factor) (3). Therefore, a common stimulus (shear stress) exists for the simultaneous activation of a number of vasoregulatory mechanisms. Mullen et al. (22) investigated the impact of the duration of the shear stress stimulus on the NO dependence of the FMD J Appl Physiol • VOL

response in the radial artery. They found that only the FMD in response to a brief shear stress stimulus was reduced by L-NMMA blockade of NO. In contrast, the FMD response to more sustained stimuli caused by release of 15 min ischemia, skin warming, or distal acetylcholine infusion was unaffected, indicating an NO-independent mechanism(s). Of note, similar blunting of FMD with L-NMMA infusion reported by Joannides et al. (17) can be explained by the L-NMMA infusioninduced attenuation of the reactive hyperemia in their study in healthy humans. In other words, blunted FMD with L-NMMA infusion could be due to a blunted stimulus, not necessarily a reduction in NO-mediated dilation (25). Both the identity and dynamics (when the role of NO ends and that of another vasodilator(s) begins) of the mechanism(s) that is responsible for the FMD in response to a more prolonged elevation in shear stress are therefore still unknown. The relevance of considering shear stress stimulus characteristics beyond the simple 5-min ischemia-induced transient shear elevation is clear when we consider the relevance of this stimulus for FMD in the coronary arteries. Coronary artery FMD is recognized as a critical response for adequate perfusion of cardiac muscle during increased work of the heart (26). The nature of the shear stress stimulus for these vessels is not reflected by the 5 min RH stimulus. Shiode et al. (26) demonstrated that shear stress-induced coronary artery dilation is not dependent on NO. Zeiher et al. (30) demonstrated that impaired coronary artery vasodilation in exercise is associated with myocardial ischemia. This implies that improvements in coronary artery FMD to a sustained shear stress stimulus may attenuate myocardial ischemia. Therefore, FMD in response to sustained shear stress may constitute a critical component of endothelial function for which NO may not be obligatory. Shear stimulus evokes endothelin release. In addition to release of vasodilators in response to elevations in shear stress, it has been demonstrated that endothelin release can also be stimulated (21, 28, 29). Endothelin is a potent vasoconstrictor. Thus alterations in the responsiveness of endothelin release by the vascular endothelium would be expected to influence FMD. Of particular relevance to this point is the observation by Berger et al. (1) that blockade of endothelin-A receptors improves FMD in patients with chronic heart failure. Indeed, these authors also point out the importance of considering that vascular tone is the net result of interaction between simultaneously active vasoregulatory factors. Sympathetic activation can account for blunted FMD in numerous pathologies. Emerging evidence of sympathetic modulation of FMD highlights the consequence of a lack of careful dissection of multiple mechanisms that interact to determine FMD. It is well established that hyperactivation of the sympathetic nervous system is implicated in cardiovascular outcomes (5, 9, 27). Many of the pathologies associated with endothelial dysfunction as evidenced by blunted FMD also demonstrate sympathetic hyperactivity. For example blunted FMD and elevated sympathetic tone is characteristic of aging (7, 11), obstructive sleep apnea (16, 23), heart failure (14, 18),


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Point:Counterpoint 1236 and hypertension (13, 20) to name a few. In addition, nonpathological states associated with elevated sympathetic activation also demonstrate blunted FMD. These include diurnal variations in FMD (24) and mental stress, particularly in persons characterized by high levels of hostility (12). Recently, Hijmering et al. (15) examined the effect of lower body negative pressure-induced sympathetic elevation in healthy humans. Their results clearly demonstrate that elevations in sympathetic activation result in considerable blunting of FMD. In addition, interventions that have been demonstrated to improve FMD in sleep apnea (continuous positive airway pressure) (16), aging (10), cardiovascular disease (ACE inhibitors) (19) also either reduce sympathetic activity or have sympathicolytic effects. Taken together, these observations demonstrate that the common assumption that FMD represents NO-mediated endothelial function is particularly misleading in understanding endothelial function and interpreting the impact of therapeutic interventions in conditions where elevated sympathetic activation also exists. In summary, FMD as an “assay” of NO-mediated endothelial function relies on a shear stress stimulus that has a highly complex signal transduction cascade evoking multiple vasoactive mechanisms. FMD in response to many shear stress profiles is not sensitive to NO blockade. In addition, there is unquestionable evidence that sympathetic activation influences the FMD response, and this activation is characteristic of many of the pathologies and altered states in which blunted FMD is observed. Finally, treatment of these pathologies often involves sympathicolytic or sympathetic activation-reducing agents/interventions. Thus we believe the current dogma that FMD reflects NO-mediated endothelial function is in error. REFERENCES 1. Berger R, Stanek B, Hulsmann M, Frey B, Heher S, Pacher R, and Neunteufl T. Effects of endothelin a receptor blockade on endothelial function in patients with chronic heart failure. Circulation 103: 981–986, 2001. 2. Buga GM, Gold ME, Fukuto JM, and Ignarro LJ. Shear stress-induced release of nitric oxide from endothelial cells grown on beads. Hypertension 17: 187–193, 1991. 3. Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, and Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci 23: 374 –380, 2002. 4. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340: 1111–1115, 1992. 5. Cohn JN. Plasma norepinephrine and mortality. Clin Cardiol 18: I9 –I12, 1995. 6. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, and Vogel R. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 39: 257–265, 2002. 7. Dinenno FA, Jones PP, Seals DR, and Tanaka H. Age-associated arterial wall thickening is related to elevations in sympathetic activity in healthy humans. Am J Physiol Heart Circ Physiol 278: H1205–H1210, 2000. 8. Drexler H and Hornig B. Endothelial dysfunction in human disease. J Mol Cell Cardiol 31: 51– 60, 1999. 9. Dupuis J, Tardif JC, Cernacek P, and Theroux P. Cholesterol reduction rapidly improves endothelial function after acute coronary syndromes: the REduction of Cholesterol in Ischemia and Function of the Endothelium (RECIFE) trial. Circulation 99: 3227–3233, 1999. J Appl Physiol • VOL

10. Eskurza I, Monahan KD, Robinson JA, and Seals DR. Effect of acute and chronic ascorbic acid on flow-mediated dilatation with sedentary and physically active human ageing. J Physiol 556: 315–324, 2004. 11. Franzoni F, Ghiadoni L, Galetta F, Plantinga Y, Lubrano V, Huang Y, Salvetti G, Regoli F, Taddei S, Santoro G, and Salvetti A. Physical activity, plasma antioxidant capacity, and endothelium-dependent vasodilation in young and older men. Am J Hypertens 18: 510 –516, 2005. 12. Gottdiener JS, Kop WJ, Hausner E, McCeney MK, Herrington D, and Krantz DS. Effects of mental stress on flow-mediated brachial arterial dilation and influence of behavioral factors and hypercholesterolemia in subjects without cardiovascular disease. Am J Cardiol 92: 687– 691, 2003. 13. Greenwood JP, Stoker JB, and Mary DA. Single-unit sympathetic discharge: quantitative assessment in human hypertensive disease. Circulation 100: 1305–1310, 1999. 14. Hasking GJ, Esler MD, Jennings GL, Burton D, Johns JA, and Korner PI. Norepinephrine spillover to plasma in patients with congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation 73: 615– 621, 1986. 15. Hijmering ML, Stroes ES, Olijhoek J, Hutten BA, Blankestijn PJ, and Rabelink TJ. Sympathetic activation markedly reduces endotheliumdependent, flow-mediated vasodilation. J Am Coll Cardiol 39: 683– 688, 2002. 16. Ip MS, Tse HF, Lam B, Tsang KW, and Lam WK. Endothelial function in obstructive sleep apnea and response to treatment. Am J Respir Crit Care Med 169: 348 –353, 2004. 17. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, and Luscher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91: 1314 –1319, 1995. 18. Katz SD, Biasucci L, and Sabba C. Impaired endothelium-mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol 19: 918 –925, 1992. 19. Langes K and Bleifeld W. [Sympathetic activity in patients with heart failure due to idiopathic dilated cardiomyopathy: effect of ACE inhibitors and other vasodilators]. Herz 15: 164 –170, 1990. 20. Lauer T, Heiss C, Preik M, Balzer J, Hafner D, Strauer BE, and Kelm M. Reduction of peripheral flow reserve impairs endothelial function in conduit arteries of patients with essential hypertension. J Hypertens 23: 563–569, 2005. 21. Moreau P, Takase H, and Luscher TF. Effect of endothelin antagonists on the responses to prostanoid endothelium-derived contracting factor. Br J Pharmacol 118: 1429 –1432, 1996. 22. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, and MacAllister RJ. Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Circ Res 88: 145–151, 2001. 23. Narkiewicz K, Pesek CA, Kato M, Phiollips BG, Davison. DE and Somers VK. Sympathetic activity in obese subjects with and without obstructive sleep apnea. Circulation 98: 772–776, 1998. 24. Otto ME, Svatikova A, Barretto RB, Santos S, Hoffmann M, Khandheria B, and Somers V. Early morning attenuation of endothelial function in healthy humans. Circulation 109: 2507–2510, 2004. 25. Pyke KE, Dwyer EM, and Tschakovsky ME. Impact of controlling shear rate on flow-mediated dilation responses in the brachial artery of humans. J Appl Physiol 97: 499 –508, 2004. 26. Shiode N, Morishima N, Nakayama K, Yamagata T, Matsuura H, and Kajiyama G. Flow-mediated vasodilation of human epicardial coronary arteries: effect of inhibition of nitric oxide synthesis. J Am Coll Cardiol 27: 304 –310, 1996. 27. Wilmink HW, Stroes ES, and Erkelens WD. Influence of folic acid on postprandial endothelial dysfunction. Arterioscler Thromb Vasc Biol 20: 185–188, 2000. 28. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411– 415, 1988. 29. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, and Yazaki Y. Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 161: 859 – 864, 1989.


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Point:Counterpoint 1237 30. Zeiher AM, Krause T, Schachinger V, Minners J, and Moser E. Impaired endothelium-dependent vasodilation of coronary resistance vessels is associated with exercise-induced myocardial ischemia. Circulation 91: 2345–2352, 1995.

Michael E. Tschakovsky Kyra E. Pyke School of Physical and Health Education Queen’s University Kingston, Ontario, Canada E-mail: mt29@post.queensu.ca

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REBUTTAL FROM DR. GREEN

At the risk of being controversial, I will begin by stating that I agree with much of the Tschakovsky and Pyke statement. When the FMD technique was introduced in humans there was indeed no direct evidence it was NO dependent. However, one should not assume that the vision of a cart dragging a horse necessarily infers the horse is lame. Further studies not only proved the FMD approach NO dependent (3, 7–9), but also that the L-NMMA effect was definitively not due to attenuation of reactive hyperemia (8, 9) (e.g., Fig. 2A of Ref. 9). Although FMD can technically be defined as the response to any shear stress stimulus, the widely adopted approach involves quantification of radial or brachial vasodilator response to an ⬃5-min ischemic stimulus where the cuff is distal to the probe (2, 3). The papers reviewed previously (3, 7–9) indicate that, under these circumstances, FMD is NO mediated and Tschakovsky and Pyke do not directly contradict this. Rather they make the case that stimulus-response specificity exists in FMD responses, where FMD is more broadly defined (e.g., different ischemic periods). I agree. Ultimately, however, the point is that we have a noninvasive technique, which, if performed appropriately, provides a valid index of NO function in vivo. The example of coronary FMD being different to that in the periphery misses the point. Although coronary lesions are focal in nature, atherosclerosis is a systemic disease (5) that can be interrogated via peripheral NO bioassay (1). This is why FMD predicts cardiovascular events (10) and why it may reflect the compound effect of risk factors, including elevated sympathetic nervous system tone, on arterial health. Furthermore, shear stress-mediated upregulation of NO synthase expression and phosphorylation occurs in vivo (5). Finally, the Hijmering paper provides an important reminder that controls need to be instituted before comparing FMD responses, especially between groups. However, this study did not infuse L-NMMA and in no way invalidates others that indicate that FMD is NO dependent (3, 7–9), including those that have isolated improvement in endothelial function to enhanced NO bioavailability (4, 6). Tschakovsky and Pyke are to be lauded for bringing the issue of stimulus-response specificity to the forefront of the FMD debate. Their conclusion, that “FMD in response to many shear stress profiles is not sensitive to NO blockade”. . . is qualified (“. . .many. . .”), but true. However, the point remains that when the appropriate FMD approach is adopted, it provides a valid index of NO bioactivity in vivo. REFERENCES 1. Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Delagrange D, Leiberman EH, Ganz P, Creager MA, and Yeung AC. Close J Appl Physiol • VOL

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10.

relationship of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol 26: 1235–1241, 1995. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer DS, Charbonneau F, Creager MA, Deanfield J, Drexer H, Gerhard-Herman M, Herrington D, Vallance P, and Vogel R. Guidelines for the ultrasound assessment of flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol 39: 257–265, 2002. Doshi SN, Naka KK, Payne N, Jones CJH, Ashton M, Lewis MJ, and Goodfellow J. Flow-mediated dilatation following wrist and upper arm occlusion in humans: the contribution of nitric oxide. Clin Sci (Lond) 101: 629 – 635, 2001. Goto C, Higashi Y, Kimura M, Noma K, Hara K, Nakagawa K, Kawamura M, Chayama K, Yoshizumi M, and Nara I. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans. Role of endothelium-dependent nitric oxide and oxidative stress. Circulation 108: 530 –535, 2003. Hambrecht R, Adams V, Erbs S, Linke a Krankel N, Shu Y, Baither Y, Geilen S, Thiele H, Gummert JF, Mohr FW, and Schuler G. Regular physical activity improves endothelial function in patients with coronary artery disease by increasing phosphorylation of endothelial nitric oxide synthase. Circulation 107: 3152–3158, 2003. Hornig B, Maier V, and Drexler H. Physical training improves endothelial function in patients with chronic heart failure. Circulation 93: 210 –214, 1996. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali E, Thuillez C, and Lu¨scher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91: 1314 –1319, 1995. Lieberman EH, Gerhard MD, Uehata A, Selwyn AP, Ganz P, Yeung AC, and Creager MA. Flow-induced vasodilation of the human brachial artery is impaired in patients ⬍ 40 years of age with coronary artery disease. Am J Cardiol 78: 1210 –1214, 1996. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, and MacAllister RJ. Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Circ Res 88: 145–151, 2001. Widlansky ME, Gocke N, Keaney JF, and Vita JA. The clinical implications of endothelial dysfunction. JACC 42: 1149 –1160, 2003.

REBUTTAL FROM DRS. TSCHAKOVSKY AND PYKE

We have taken the sage advice of our erudite colleague Dr. Green to heart . . . namely indulging in a bottle of robust Australian Shiraz. We, first off, thank him for his suggestion as it has helped us to identify within this debate a classic blunder. Equating FMD with NO-mediated endothelial function repeats the folly of the simple tourist who sees one black sheep and concludes that black is the color of all the sheep in England. The first well-characterized FMD response in humans (2) was found to be NO dependent (4, 11) and this has lead to the assumption that all FMD must share this mechanism. Recent publications do not support this position (12) and it is time to . . . “shear” erroneous assumptions from our concept of FMD. The argument by Dr. Green could be partially correct if the statement to be defended qualified the type of FMD as 1) brachial or radial artery specific (4, 11), 2) specific to 5 min of distal occlusion reactive hyperemia without ischemic handgrip exercise (1, 12), and 3) in healthy subjects (11). This is critical because primarily NO-mediated FMD has been confirmed only under this specific combination of conditions (4, 11). Furthermore, it has been demonstrated that slight deviations from these conditions resulting in small stimulus profile alterations can dramatically alter the NO dependence of the FMD response (12). Readers! Pay attention to the following statement in the very guidelines that Dr. Green cites to support a consensus on FMD assessment of endothelial NO function: “Studies have variably used either upper arm or forearm cuff occlusions and there is no consensus as to which technique


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Point:Counterpoint 1238 provides more accurate or precise information . . . The change in diameter is similar after 5 and 10 min of occlusion; therefore the more easily tolerated 5-min occlusion is typically used” (3). These statements by experts in the field do not acknowledge the critical importance of the stimulus creation technique in determining the NO dependence of the FMD response. Finally, we reiterate that, even when the methodological constraints that allow for an NO-dependent FMD response are followed, other factors may influence the magnitude of vasodilation. Specifically, the elevated sympathetic activation, common in pathologies also associated with endothelial dysfunction, may blunt the FMD response (10). In these groups, FMD in response to even 5 min of distal occlusion reactive hyperemia is a reflection of combined NO bioavailability and sympathetic activation. Finally, it has been clearly demonstrated that some FMD responses in humans are not NO mediated (12). Under these circumstances, to state that FMD as a whole reflects NOmediated endothelial function is to ignore this or to imply that all other mechanisms of FMD are irrelevant. This is inappropriate on two counts. First, FMD is an important vasoregulatory mechanism in both the coronary and peripheral vasculature systems (6, 9) and thus all mechanisms of FMD should be studied. Second, it has not been clearly established that all coronary FMD is NO dependent (13). In atherosclerotic coronary arteries dilation in response to increases in blood flow (regardless of the mechanism responsible) can help to attenuate myocardial ischemia (5, 7, 8). Therefore from a clinical perspective, all mechanisms of FMD in this vascular bed require focused research. In conclusion, at present FMD can only be said to reflect NO-mediated endothelial function if it is in response to a very narrowly defined stimulus in only the radial or brachial arteries. To imply that FMD in response to other stimulus profiles in other areas of the vasculature is NO dependent might be akin to, dare we say, embracing the only black sheep in the family.

3. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, and Vogel R. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 39: 257–265, 2002. 4. Doshi SN, Naka KK, Payne N, Jones CJ, Ashton M, Lewis MJ, and Goodfellow J. Flow-mediated dilatation following wrist and upper arm occlusion in humans: the contribution of nitric oxide. Clin Sci (Lond) 101: 629 – 635, 2001. 5. Duffy SJ, Castle SF, Harper RW, and Meredith IT. Contribution of vasodilator prostanoids and nitric oxide to resting flow, metabolic vasodilation, and flow-mediated dilation in human coronary circulation. Circulation 100: 1951–1957, 1999. 6. Gaenzer H, Neumayr G, Marschang P, Sturm W, Kirchmair R, and Patsch JR. Flow-mediated vasodilation of the femoral and brachial artery induced by exercise in healthy nonsmoking and smoking men. J Am Coll Cardiol 38: 1313–1319, 2001. 7. Gielen S and Hambrecht R. Effects of exercise training on vascular function and myocardial perfusion. Cardiol Clin 19: 357–368, 2001. 8. Gielen S, Schuler G, and Hambrecht R. Exercise training in coronary artery disease and coronary vasomotion. Circulation 103: E1–E6, 2001. 9. Gordon JB, Ganz P, Nabel EG, Fish RD, Zebede J, Mudge GH, Alexander RW, and Selwyn AP. Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise. J Clin Invest 83: 1946 –1952, 1989. 10. Hijmering ML, Stroes ES, Olijhoek J, Hutten BA, Blankestijn PJ, and Rabelink TJ. Sympathetic activation markedly reduces endotheliumdependent, flow-mediated vasodilation. J Am Coll Cardiol 39: 683– 688, 2002. 11. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, and Luscher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91: 1314 –1319, 1995. 12. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, and MacAllister RJ. Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia. Circ Res 88: 145–151, 2001. 13. Shiode N, Morishima N, Nakayama K, Yamagata T, Matsuura H, and Kajiyama G. Flow-mediated vasodilation of human epicardial coronary arteries: effect of inhibition of nitric oxide synthesis. J Am Coll Cardiol 27: 304 –310, 1996. POINT:COUNTERPOINT CALL FOR COMMENTS

REFERENCES 1. Betik AC, Luckham VB, and Hughson RL. Flow-mediated dilation in human brachial artery after different circulatory occlusion conditions. Am J Physiol Heart Circ Physiol 286: H442–H448, 2004. 2. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340: 1111–1115, 1992.

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