Exercise and The Nitric Oxide Vasodilator System

LEADING ARTICLE
Sports Med 2003; 33 (14): 1013-1035 0112-1642/03/0014-1013/$30.00/0 Adis Data Information BV 2003. All rights reserved.
Exercise and the Nitric Oxide Vasodilator System

Andrew Maiorana,1,2,3 Gerard ODriscoll,3,4 Roger Taylor2,4 and Daniel Green1,3,4
1 2 3 4 Department of Human Movement and Exercise Science, The University of Western Australia, Crawley, Western Australia, Australia Department of Medicine, The University of Western Australia, Crawley, Western Australia, Australia Cardiac Transplant Unit, Royal Perth Hospital and West Australian Heart Research Institute, Perth, Western Australia, Australia Department of Cardiology, Royal Perth Hospital and West Australian Heart Research Institute, Perth, Western Australia, Australia
Abstract
In the past two decades, normal endothelial function has been identified as integral to vascular health. The endothelium produces numerous vasodilator and vasoconstrictor compounds that regulate vascular tone; the vasodilator, nitric oxide (NO), has additional antiatherogenic properties, is probably the most important and best characterised mediator, and its intrinsic vasodilator function is commonly used as a surrogate index of endothelial function. Many conditions, including atherosclerosis, diabetes mellitus and even vascular risk factors, are associated with endothelial dysfunction, which, in turn, correlates with cardiovascular mortality. Furthermore, clinical benefit and improved endothelial function tend to be associated in response to interventions. Shear stress on endothelial cells is a potent stimulus for NO production. Although the role of endothelium-derived NO in acute exercise has not been fully resolved, exercise training involving repetitive bouts of exercise over weeks or months up-regulates endothelial NO bioactivity. Animal studies have found improved endothelium-dependent vasodilation after as few as 7 days of exercise. Consequent changes in vasodilator function appear to persist for several weeks but may regress with long-term training, perhaps reflecting progression to structural adaptation which may, however, have been partly endothelium-dependent. The increase in blood flow, and change in haemodynamics that occur during acute exercise may, therefore, provide a stimulus for both acute and chronic changes in vascular function. Substantial differences within species and within the vasculature appear to exist. In humans, exercise training improves endothelium-dependent vasodilator function, not only as a localised phenomenon in the active muscle group, but also as a systemic response when a relatively large mass of muscle is activated regularly during an exercise training programme. Individuals with initially impaired endothelial function at baseline appear to be more responsive to exercise training than healthy individuals; that is, it is more difficult to improve already normal vascular function. While improvement is reflected in increased NO bioactivity, the detail of mechanisms, for example the relative
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importance of up-regulation of mediators and antioxidant effects, is unclear. Optimum training schedules, possible sequential changes and the duration of benefit under various conditions also remain largely unresolved. In summary, epidemiological evidence strongly suggests that regular exercise confers beneficial effects on cardiovascular health. Shear stress-mediated improvement in endothelial function provides one plausible explanation for the cardioprotective benefits of exercise training.
Repeated physical activity induces physiological adaptations to the cardiovascular system that improve physical fitness and reduce primary[1-3] and secondary cardiovascular events.[4] The mechanisms involved are multifactorial and probably include modification of established risk factors, such as improved weight control,[5] blood lipid profile[6,7] and glucose tolerance,[8] and blood pressure.[9,10] Furthermore, the positive effects of regular exercise on autonomic tone,[11] blood coagulation[12] and inflammation[13] are likely to contribute to improved vascular health. A growing body of research has identified the vascular endothelium as another therapeutic target for cardiovascular risk reduction. Strategically located at the interface of the circulating blood and the vascular wall, the endothelium is ideally positioned to respond to changes in haemodynamics and to maintain circulatory homeostasis. Originally considered a passive layer of inert cells, the endothelium is now regarded as an important endocrine organ capable of producing and responding to an array of chemical and physical stimuli. Normal endothelial function is not only critical to regulating vascular tone but also to mediating antiatherogenic properties including inhibition of cell growth and proliferation,[14] reduced leucocyte and platelet adhesion to the vessel wall[15] and antithrombotic and fibrinolytic properties.[16] The loss of these protective attributes due to endothelial dysfunction may be an integral part of, and may contribute to, the pathophysiology of atherosclerosis. This link between endothelial dysfunction and clinical vascular disease highlights the therapeutic potential of interventions that improve endothelial function and provides a plausible explanation for a reduction in coronary events associated with these interventions.
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This review will briefly consider the importance of the endothelium in the maintenance of normal vascular function, identify conditions associated with impaired endothelial function, and discuss the effect of exercise training on endothelium-dependent vasodilator capacity in healthy individuals, those with risk factors for cardiovascular disease and those with overt manifestations of cardiovascular disease. 1. How Does the Vascular Endothelium Influence Vasodilator Function? The vascular endothelium produces an array of molecules that influence vascular tone, blood flow, viscosity and cell wall interactions. Some of these, including prostacyclin,[17] putative endothelium-derived hyperpolarising factor,[18] bradykinin[19] and nitric oxide (NO)[20] possess vasodilator properties while others, such as angiotensin II[21] and endothelin,[22] are vasoconstrictor agents. Of the vasoactive agents produced by the endothelium, NO has been the most comprehensively studied and is the focus of this review. An endothelium-derived relaxing factor was first identified in a landmark study by Furchgott and Zawadski[23] who established that the vasodilator effect of acetylcholine (ACh) was only evident in blood vessels with an intact endothelial lining. The compound was subsequently established to be NO, synthesised in endothelial cells from the amino-acid L-arginine through the action of endothelial nitric oxide synthase (eNOS).[24] NO is lipid soluble and rapidly diffuses into the vascular smooth muscle of the tunica media where it binds to the enzyme guanylate cyclase.[25] The resulting increase in cyclic guanosine monoSports Med 2003; 33 (14)
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phosphate induces smooth muscle relaxation and vascular dilation.[26] NO importantly influences basal vasomotor tone in the coronary and peripheral circulation.[27-30] The tone of the vasculature is determined by the balance between dilator and constrictor influences. At rest, low levels of NO are continuously released. In humans, this phenomenon was initially identified by Vallance et al. who infused the arginine analogue NG-monomethyl-L-arginine (L-NMMA) into the forearm vascular bed to inhibit eNOS and the synthesis of NO, resulting in vasoconstriction.[27] NO bioactivity can be stimulated by pharmacological and physiological stimuli. During exercise, for example, shear stress is imparted to the vascular wall. The endothelium acts as a mechanotransducer[31,32] and increases vasodilator mechanisms, tending to normalise the high shear stress. Although the signalling cascade linking mechanical stimulation to the release of vasoactive molecules has not been fully clarified, a number of mechanisms have been proposed. Endothelial potassium channel activation by flow,[33] possibly as a result of shear stress-mediated deformation of cytoskeletal elements,[34] may lead to release of NO, as found in rabbit iliac arteries.[35] Other investigators have identified that flow increases calcium influx in endothelial cells,[36-38] an effect that is necessary for the synthesis and release of NO. Flow may also induce the release of bradykinin from endothelial cells[39] and, in turn, stimulate bradykinin receptors on the endothelial cell to increase NO bioactivity.[40] More recently, shear stress has been shown to induce phosphorylation of a serine residue, altering eNOS sensitivity to intracellular calcium levels resulting in increased NO production.[41] In humans, NO bioactivity is used as a practical surrogate for endothelial function while, obviously, it does not constitute a comprehensive evaluation. Inhibitors of eNOS are used to examine ambient NO bioactivity while endothelium-dependent vasodilators, such as ACh, are used to stimulate the release of NO from endothelial cells with resultant vasodilation. These drugs are commonly used to assess the responses of resistance vessels. In conduit vessels,
such as the brachial or femoral artery, a response almost exclusively mediated by NO[42] can be induced by the increased flow associated with the transient hyperaemia following a period of ischaemia, known as flow-mediated dilation (FMD). To investigate the vascular smooth muscle component of the NO-dilator pathway, the responses to endothelium-independent vasodilators, such as sodium nitroprusside (SNP) or glyceryl trinitrate (GTN), which release NO to act directly on the smooth muscle, are determined. Evaluating the responses to endothelium-dependent and -independent vasodilators provides information about the contribution to blood-flow control of different components of the NO-dilator system. 2. Endothelial Dysfunction NO-related endothelial dysfunction is found under diverse conditions and, while it is usually apparent in both conduit and resistance vessels and in various vascular beds, differential effects on the many vascular beds might occur under specific conditions, for example, within the coronary circulation.[43-45] The mechanisms proposed to explain depressed NO activity include a loss of endothelial synthesis of NO and a reduction in NO bioavailability due to quenching by superoxide anions associated with an abnormal redox state.[46-48] However, the mechanisms are not well defined and probably depend upon the cause. Several lines of evidence suggest that endothelial dysfunction, along with depressed NO bioactivity, is an important and integral component of atherogenesis although the exact relationship is debated. Endothelial dysfunction occurs initially at coronary branch points,[49] as do atherosclerotic plaques. The endothelium loses its protective effect on the vascular system, which is likely to facilitate the development and progression of atherosclerosis. However, endothelial dysfunction seems reversible to some extent and numerous interventions that improve cardiovascular risk factors and reduce cardiovascular morbidity and mortality also enhance endothelial function,[50-56] further emphasising the potential role of endothelial function in atherosclerosis.
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2.1 Endothelial Dysfunction and Risk Factors for Atherosclerosis
Impaired responses to endothelium-dependent stimuli have been reported in individuals without obvious atherosclerosis but with conventional cardiovascular risk factors,[57] related to the number of risk factors present.[58]
2.1.1 Aging
control alone;[78] enhanced endothelial function is likely to be a major influence,[79,80] perhaps through reduced superoxide production and NO quenching.[48]
2.1.5 Obesity
Aging has been highlighted as a significant predictor of impaired endothelial function,[59,60] function declining with age.[61] Dysfunction has been identified as early as 30 years.[62]
2.1.2 Smoking
Endothelial dysfunction in the obese may contribute to the increased risk of cardiovascular disease in these individuals.[81] It may be particularly associated with central obesity and insulin resistance[81-83] and has been found in the coronary circulation, in addition to the systemic circulation in individuals with normal or only mildly diseased arteries.[84]
2.1.6 Diabetes Mellitus
Cigarette smoking is a major risk factor for atherosclerosis. Endothelial dysfunction has been identified in systemic vessels[50,63] and variably in coronary vessels[58,64] of smokers. It has even been reported in young smokers, dependent upon the amount smoked,[50] and also associated with passive smoking.[63] It may be at least partially reversible when smoking is ceased.[50]
2.1.3 Hypercholesterolaemia
In hypercholesterolaemia, endothelial dysfunction has been identified in both the coronary[58,65] and peripheral circulations,[66-68] and can appear in childhood.[69] The mechanism responsible should accommodate the fact that NO-related endothelial function is responsive to acute elevation[70] or reduction[71] of plasma cholesterol. NO inactivation by superoxide production may be partially responsible.[46]
2.1.4 Hypertension
Mortality from diabetes is largely attributable to atherosclerotic macrovascular complications, while microvascular dysfunction and consequent retinopathy, neuropathy and nephropathy contribute significantly to morbidity.[85,86] Short of these obvious complications, recent evidence suggests that insulin resistance is inextricably linked to endothelial dysfunction, endothelial dysfunction contributing to insulin resistance and vice versa.[86] Impaired endothelial function has been identified in type 2 diabetes,[55,87-91] although not in all studies;[92] diabetic control was better in the latter study. In type 1 diabetes, while one study suggested that the abnormality in vasodilator function may lie within the smooth muscle,[93] most have implicated endothelial rather than smooth muscle dysfunction.[94-96]
2.2 Endothelial Dysfunction in Established Cardiovascular Disease
2.2.1 Coronary Artery Disease (CAD)
Abnormal endothelial function has also been implicated in the pathophysiology of hypertension. It has been identified in several peripheral vascular beds[72,73] and the coronary circulation,[74-76] and more recently reported to be an independent marker for future cardiovascular morbidity in hypertensive individuals.[77] The nexus between hypertension and endothelial dysfunction is unclear. For example, improved cardiovascular outcomes as a result of angiotensin-converting enzyme-inhibitor therapy seem greater than expected from blood pressure
In patients with advanced coronary stenosis, intracoronary ACh causes constriction rather than the vasodilation of angiographically normal vessels, while arteries of individuals with early atherosclerosis demonstrate a heterogenous response.[97] Furthermore, endothelial dysfunction in systemic vessels correlates with the extent ofobstructive coronary artery disease (CAD) and with coronary endothelial dysfunction[98,99] reflecting the generalised nature of endothelial dysfunction. However,
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there can be regional differences within a vascular bed; basal NO function was impaired in large epicardial arteries but normal in small epicardial and resistance vessels,[45] suggesting that the proximal segment is not only more prone to atherosclerosis but also to endothelial dysfunction. Additionally, patients who have experienced recent cardiac events have worse endothelial dysfunction than clinically stable patients.[43,44] Clinical outcomes in patients submitted to coronary angiography have been related to coronary endothelial dysfunction by several authors.[100-102] Brachial endothelial dysfunction has also been found to predict future cardiac events in patients with chest pain,[103] and short-term events in highrisk patients undergoing surgery for vascular disease.[104]
2.2.2 Chronic Heart Failure
skeletal muscle creates a muscle-pump, which causes an immediate increase in blood flow to the working muscle.[118] As muscle activity continues, metabolites with vasoactive properties are released from the muscle into the interstitial fluid and act directly on terminal arterioles. Substances proposed to be involved in this metabolic vasodilation include potassium ions,[119] metabolites such as adenosine,[120] and, more recently, NO released from the active muscle.[121] Furthermore, NO transported via the blood bound to haemoglobin,[122,123] and acetylcholine spill-over from motor neurones[124] may induce vasodilation of the terminal arterioles. Blood-flow changes due to metabolic vasodilation alone are insufficient to account for the dramatic increases that occur during exercise.[125] Therefore, it is clear that a coordinated dilation of small and large vessels must occur as metabolic demand increases. The structure of the arterial network facilitates such a response to increased local blood flow because terminal arterioles arise, in series, from progressively larger vessels. The principle of ascending vasodilation has been proposed to explain the phenomenon of vasodilation which begins in the microvessels and is then transmitted proximally to larger feed arteries.[125,126] One explanation for ascending vasodilation proposes that, following the onset of exercise, terminal vessels bathed in interstitial fluid and in contact with by-products of metabolism dilate, increasing blood-flow velocity through the upstream feed arteries.[127] This increase in flow increases shear stress on the endothelium,[128] stimulating NO bioactivity and inducing feed artery vasodilation. This hypothetical paradigm explains the observed magnitude of exercise hyperaemia by linking downstream metabolic dilation with dilution of upstream vessels that are not in contact with the by-products of metabolism, but which respond to changes in localised haemodynamics. Certainly, what is now commonly named FMD, a dilator response of conduit arteries in response to muscle ischaemia, is predominantly dependent upon an increase in NO bioactivity in these larger arteries.[42] However, the coordinated dilation of large and small vessels can occur independently
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While a decrease in cardiac function initiates chronic heart failure (CHF), functional capacity is poorly correlated with central haemodynamics[105] and is closely associated with abnormalities of the peripheral circulation, including impaired vasodilator response.[106] Furthermore, systemic vasoconstriction increases left ventricular impedance and ultimately leads to further deterioration in left ventricular function.[107,108] Endothelial dysfunction has been found in the coronary circulation[109] and in peripheral resistance,[110-112] and conduit[113] vessels in individuals with CHF of various aetiologies. Blood-flow responses to GTN, that is, endotheliumindependent NO-related responses, have also been reported to be impaired perhaps reflecting more severe vascular abnormalities and advanced CHF.[114,115] 3. Endothelium-Derived Nitric Oxide (NO) and Exercise Hyperaemia Exercise training involves recurrent exposure to dramatic changes in cardiovascular haemodynamics that occur with bouts of physical activity. In response to acute exercise, numerous phenomena interact to increase blood flow to active muscles as much as 50- to 100-fold.[116,117] At the onset of exercise, the mechanical action of contracting and relaxing
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of changes in endothelial wall shear stress and NO production,[129,130] suggesting that redundant mechanisms may be involved.
3.1 Animal Studies
bition could be explained by changes in resting flow.[141] However, both of these studies measured blood flow after the cessation of muscular contraction and blood flow during recovery may be under different control to that during exercise.[142] The role of NO may increase with the duration of exercise. While it is unlikely that NO plays a major role in the dilator response to a single muscle contraction,[143] it may be important during prolonged exercise. L-NMMA infusion during intermittent forearm hand-gripping at 15%, 30% and 45% of maximum grip strength induced a small, but significant, reduction in exercise-induced vasodilation.[144] Importantly, the greatest effect of L-NMMA was evident at the highest infusion rate of the drug, indicating that the dose of an antagonist may be critical during an exercise study, when increased blood flow effectively dilutes a constant dose delivery. Indeed, Katz et al. indicated that the effect of LNMMA decreased with increasing intensity of exercise.[106] In another study, L-NMMA infusion attenuated the blood-flow response to prolonged forearm hand-gripping by 2030%.[145] Furthermore, Duffy et al. recently confirmed a role for both NO and vasodilator prostanoids in mediating hyperaemia in response to forearm handgrip exercise.[29,146] However, sequential inhibition of these two vasodilators had no additional effect on reducing blood flow over and above that induced by inhibition of one, highlighting the possibility that redundant mechanisms may contribute to blood-flow control during continuous exercise. In the lower limbs, Hickner et al.,[147] using microdialysis probes, measured a reduction in blood flow of 50% following the infusion of L-NMMA during continuous dynamic exercise of the vastus lateralis. However, this technique requires further validation and these results have not been supported by other studies of lower limb exercise. L-NMMA had no effect on blood flow measured using Doppler ultrasound during steady-state submaximal or peak voluntary knee extension exercise.[148] Similarly, when blood flow to the lower limbs was determined using femoral thermodilution, L-NMMA did not reduce blood flow during supine cycling, despite
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A comprehensive account of blood-flow control during acute exercise in animals is provided in an excellent review by Delp and Laughlin.[131] Animal studies offer equivocal support for a major role of NO in exercise-induced local hyperaemia. NO regulated regional flow in dogs[132] and rats[133] while, in the coronary circulation of dogs, endothelial denudation caused the normal epicardial artery vasodilation during exercise to be replaced by constriction. Restoration of normal epicardial vasodilation correlated with normalisation of the response to ACh and to reactive hyperaemia.[134] However, NO inhibition did not alter the hind-limb skeletal muscle microcirculation responses of rabbits[135] or arterial dilation in response to metabolic stimulation of hamster skeletal muscle.[136] In summary, the majority of animal studies suggest a role for NO in exercise-induced vasodilation,[137] but it is likely that interspecies differences exist and human studies are important.
3.2 Human Studies
3.2.1 Skeletal Muscle
Indirect evidence for a role of NO in exercise hyperaemia in humans is provided by the increased levels of plasma nitrite[138] and urinary nitrate[139] in response to prolonged aerobic exercise; however, these measures do not reliably reflect endotheliumderived NO. Therefore, despite technical difficulties associated with measuring blood flow during exercise, a number of investigators have attempted to determine NO involvement in the hyperaemic response to exercise in the peripheral circulation. In an early study, flow was measured during periods of inactivity immediately following two intensities of wrist flexor exercise and concurrent infusion of LNMMA had no significant effect on blood flow.[140] Furthermore, a small reduction in exercise hyperaemia during static hand-gripping following NO inhi Adis Data Information BV 2003. All rights reserved.
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decreasing glucose flux across the limb.[149] Leg exercise is associated with a high perfusion-tomuscle mass ratio, and flow may be increased over 10-fold. Possibly, this hyperaemia dilutes L-NMMA concentration to an inadequate blocking level. Using systemic intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME), a potent inhibitor of NO production, leg blood flow during submaximal leg extensor exercise was not reduced, leading the authors to conclude that NO is not essential to exercise hyperaemia. However, this interpretation may be considered controversial, since mean arterial pressure increased and vascular conductance was lower during L-NAME infusion. Furthermore, an increase in arterial pressure is likely to reduce sympathetic outflow during exercise,[150] and this may mask a potential blood-flow reduction due to eNOS inhibition.[151] In summary, of the above results in the peripheral circulation, the evidence for a role of NO in the hyperaemic response to prolonged exercise in the forearms, having a relatively small muscle mass, is reasonably convincing. Because the increased blood flow in these studies was modest (36 times resting levels), the dilution of constant dose infusion of LNMMA would be less than with leg exercise. Evidence is less compelling from studies involving the lower limbs, only one out of four published studies being positive. While this disparity may reflect regional differences between vascular beds, it may depend upon greater dilution of L-NMMA during leg exercise. Further carefully designed studies will be required to clarify this issue. There is also evidence that redundancy of mechanisms of vasodilation exist so that inhibition of one pathway may result in up-regulation of another. A final problem concerns blood-flow measurement techniques. For example, as summarised by Radegran and Saltin,[152] studies indicating an effect of L-NMMA have mostly been undertaken using plethysmography, necessitating measurement of flow during brief rest periods between contractions or exercise bouts. Flows measured using ultrasound/Doppler actually during exercise have, to date, not supported much role for NO
during muscular contraction but have indicated an effect at rest and during recovery. Contrary to this essentially negative conclusion for an additional role of NO during exercise in the active muscle bed, a recent study examined brachial artery blood flow during lower limb exercise,[153] and found that brachial artery infusion of L-NMMA decreased forearm blood flow during cycling at 60, 100, and 160W, compared with the resting state. This finding suggests that systemic production of NO increases during exercise, even in vascular beds feeding metabolically inactive tissue. A subsequent study found that changes in heart rate alone in the absence of exercise were not associated with this phenomenon and, hence, it may be due to pulse pressure dynamics stimulating the endothelium throughout the circulation during exercise.[154]
3.2.2 Cardiac Muscle
Experimentally, an increase in cardiac metabolism is commonly achieved by increasing ventricular rate using cardiac pacing. Indirect evidence for a role of NO in metabolic vasodilation comes from a study showing an increase in by-products of NO in coronary sinus blood following pacing in individuals without CAD risk factors.[155] No increase was evident in individuals with CAD risk factors but coronary sinus adenosine was elevated,[155] raising the possibility that adenosine production may be a compensatory mechanism maintaining myocardial blood flow in the presence of endothelial dysfunction. L-NMMA abolished pacing-induced epicardial vessel dilation, indicating that NO contributes to vasodilation in these conduit vessels in patients free of CAD risk factors[156] and in those with angiographically normal coronary arteries.[157] In another study, L-NMMA reduced pacing-induced dilation of large epicardial arteries, but not of microvessels.[158] However, there have been negative studies in those with angiographically normal arteries, perhaps related to the shortcomings of angiography.[45,159] In yet another study, NO was involved in pacing-induced hyperaemia in patients with risk factors for CAD, including some with mildly irregular arteries[29] despite risk factors leading to a reduction in vasodilation,[156] and an impaired response to
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stimulated NO release.[160] In patients with CAD disease, NO production increased by pacing was abolished at the sites of stenoses.[157] In summary, there is strong evidence for a role of endothelium-derived NO in the metabolic vasodilation of healthy coronary epicardial vessels, but NOdependent vasodilation decreases in the presence of risk factors and is absent or much reduced in grossly atherosclerotic vessels. 4. Does Exercise Training Improve Endothelial Function? If the endothelium is activated during exercise, it can be postulated that repetitive exposure to haemodynamic and shear stress changes may result in an adaptation that up-regulates the NO dilator system. Regular exercise training is associated with increased vasodilator capacity[161,162] and longitudinal studies provide convincing evidence for the antiatherogenic effect of regular physical activity.[2,3,163] These phenomena may be due, in part, to up-regulation of endothelial function.
4.1 Exercise Training and NO Function in Healthy Animals
days of endurance training in pigs.[167] Similarly, 4 weeks of training enhanced ACh-induced vasodilation of the rat aorta and increased eNOS protein levels in aortic tissue.[168,169] In rabbits, 8 weeks of treadmill running increased ACh reactivity in the aorta and pulmonary arteries but not in the carotid artery, a vessel which is partly protected from dramatic changes in flow and shear stress.[170] In contrast to short to moderate periods of exercise training, studies over a longer duration have not consistently shown improvement in NO-related endothelial function. NO-dependent vasodilation was unaltered after 1620 weeks of training in pigs,[171] and 16 weeks in rats despite improved arterial compliance.[172] This seems to indicate that improvement in endothelium-dependent vasodilator responses in the periphery may be a transient phenomenon that is lost with long-term training. Altered expression of eNOS has been implicated in this time dependence. Daily exercise for 1 week resulted in an increased expression of eNOS protein in porcine pulmonary arteries and enhanced ACh-mediated relaxation,[173] changes not present after 16 weeks of exercise training.[174] In various animal models, prolonged exercise training enlarges the diameter of arteries;[175-178] this vascular remodelling appears to be an endothelium and partly NO-dependent phenomenon,[179-184] but one which partly supplants the acutely responsive vasodilator mechanisms. In the coronary circulation, early studies reported exercise training to increase sensitivity to vasodilator agents in dogs[185] and increase transport capacity in miniature swine.[186] Evidence that exercise training enhances NO-mediated dilation was found in a study of short-term (710 days) treadmill exercise training in dogs.[187] This intervention increased dilation of the circumflex artery following both ACh infusion and reactive hyperaemia. Infusion of nitroL-arginine, an NO-synthase inhibitor, eliminated these responses. The training did not change the response to SNP. Evidence that eNOS gene expression is enhanced in the coronary vasculature by exercise training was first published by Sessa et al.,[188] who found that nitrite and NO production, and eNOS gene expression, were increased in coroSports Med 2003; 33 (14)
In peripheral vessels, short-term exercise training in rats, of 24 weeks, increased endothelial NO synthesis in skeletal muscle arterioles and increased the vasodilator responses to ACh and L-arginine, but not to SNP, suggesting enhanced endotheliumdependent function but unchanged smooth muscle cell sensitivity to NO.[164] A subsequent study of similar duration also demonstrated augmented dilator response in rat muscle arterioles following training, which was partially abolished by L-NMMA infusion.[165] Vascular structure was not obviously altered. These findings suggest that increased production of endothelial NO constitutes an initial phase in the adaptive response to exercise training. Another study found that 4 weeks of daily exercise in rats improved flow-induced dilation in skeletal muscle arteries, but not in mesenteric vessels, which are not subject to exercise hyperaemia.[166] In large conduit vessels, improved endothelium-dependent vasodilation has been observed after as few as 7
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nary arterioles following 10 days of training in dogs. Similarly, in a porcine model, 16 weeks of training increased eNOS mRNA[189] and increased bradykinin induced vasodilation[190] in coronary resistance vessels, suggesting that enhanced endothelium-dependent dilation persists in such vessels for at least 4 months. However, 1622 weeks of training augmented vasodilator responses to adenosine in the large epicardial arteries of pigs, even after removal of the endothelium. This one piece of evidence suggests that changes in vasomotor response in cor-
onary arteries with longer-term training might be due, at least in part, to adaptations within smooth muscle.[191] In summary, animal studies investigating the peripheral and coronary vasculature suggest the hypothetical adaptation schema diagrammatically represented in figure 1. Short-term exercise training enhances NO production and bioactivity to buffer increased shear. After extended training, increased NO production, and possibly other mediators, induce structural changes to the vessel resulting in an
Fig. 1. Hypothesised response of arteries to increased flow and shear stress following varying duration of exercise training. (a) Untrained vessel; baseline endothelial release of NO, which diffuses to smooth muscle, activates GC leading to production of cyclic GMP. Cyclic GMP leads to calcium channel opening causing smooth muscle relaxation and vasodilation of the vessel (intermediate steps have been overlooked for simplification). Localised fluctuations in release of NO act to homeostatically regulate wall shear. (b) Vessel following medium-term exercise training (several weeks); acute increase in shear stress, associated with repetitive exposure to increased flow during bouts of exercise, stimulates increased endothelial NO production and consequent vasodilation. Up-regulation of the NO-dilator system, including eNOS expression, occurs to buffer increased shear stress. (c) Vessel following long-term exercise training; structural adaptation occurs, possibly partly due to NO-mediated changes in smooth muscle cells, resulting in chronic increase in vessel calibre. Shear stress is normalised and NO function returns towards baseline levels. eNOS = endothelial nitric oxide synthase; GC = guanylate cyclase; GMP = guanosine monophosphate; GTP = guanosine triphosphate; NO = nitric oxide.
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increase in lumen diameter.[184] Shear stress is hence structurally normalised and endothelial NO activity returns towards initial levels. Whilst the evidence upon which this is based is currently limited, these data suggest that functional and structural adaptations of the vasculature to exercise training may alter with training duration. Further research will be required to answer this intriguing hypothesis.
4.2 Exercise Training and NO Function in Animal Models of Pathological States
Hypercholesterolaemia is associated with reduced aerobic capacity in mice and this correlates with impaired endothelium-dependent vascular relaxation.[192] Four weeks of exercise training in hypercholesterolaemic mice partially reverses endothelial dysfunction, and this is correlated with improved aerobic capacity.[193]
4.2.2 Hypertension
failure did not improve aortic vasodilation to agonists of NO.[198] However, treadmill running in dogs during the development of pacing-induced heart failure preserved endothelium-dependent vasodilation of peripheral and coronary arteries[198,199] and vascular gene expression of eNOS.[199] In these studies, exercise training was initiated before overt CHF developed. This is potentially clinically significant because it demonstrates preservation of endothelial function despite the development of a failing central circulation. In summary, there is preliminary evidence that exercise training can improve NO-related endothelial function in animal models of hypercholesterolaemia, hypertension, diabetes and heart failure.
4.3 Exercise Training and NO Function in Healthy Volunteers
In in-vitro preparations of aortic and mesenteric rings from spontaneously hypertensive rats, AChinduced relaxation was significantly greater following exercise training.[194] Furthermore, in the same model, exercise training was associated with increased NO production[195] and an increase in plasma nitrate.[196]
4.2.3 Diabetes
Only one animal study has examined the effect of exercise training on endothelial function in type 2 diabetes.[197] Diabetic rats with impaired aortic endothelial function were randomised to sedentary, exercise-trained and food-restricted groups. Both exercise training and food restriction reduced plasma glucose and insulin, and increased insulin sensitivity, but only exercise training improved endothelium-dependent relaxation to histamine and increased urinary excretion of nitrite. This suggests that exercise training improves endothelial function independent of changes in glucose tolerance in this model of diabetes.
4.2.4 Heart Failure
Animal models of heart failure provide conflicting evidence. Exercise training of rats with heart
Early descriptive studies included the finding that 4 weeks of handgrip training improved forearm maximal blood flow.[162] Due to the relatively small muscle mass involved, the training stimulus was thought insufficient to alter sympathetic tone, suggesting the presence of an alternative local mechanism. Supportive studies found enhanced vasodilator capacity in the dominant limb of tennis players[161] and in the forearm with the resumption of activity following immobilisation by casting.[200] Cross-sectional studies highlight a significantly greater capacity for vasodilation among highlytrained middle-aged distance runners than agedmatched inactive men.[201] Furthermore, exercise training increased plasma nitrite and nitrate, persisting for 4 weeks of detraining but returning to baseline after 8 weeks.[202] An early investigation to elucidate mechanisms associated with blood-flow changes following exercise training was conducted in young males by Green et al.[203] Four weeks of hand-grip training reduced minimum vascular resistance following an ischaemic stimulus in the trained limb but not the untrained contralateral arm, nor in sedentary controls. However, neither the forearm blood-flow response to the endothelium-dependent vasodilator methacholine chloride nor to SNP were altered following training. Similar findings were
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evident in studies comparing preferred and nonpreferred arms of elite tennis players[204] and recovery from forearm immobilisation from casting.[205] In another study, which employed 4 weeks of handgrip training, exercise-induced vasodilation was increased following training. However, neither ACh nor SNP infusion during exercise increased forearm vascular conductance above the level observed during exercise alone, suggesting that exercise training does not increase the response to these vasodilators, at least during exercise.[206] On the whole, these studies suggest that forearm muscle training (i.e. localised exercise of a small muscle group) does not influence endothelial function in apparently healthy individuals, despite demonstrable increased capacity for peak blood flow. Exercise involving large muscle groups of the legs is likely to produce systemic changes in vascular haemodynamics not apparent with localised exercise of the upper limb. In healthy individuals, Kingwell et al.[207] examined the systemic effect of 4 weeks of cycling on NO production in the forearm. Following training, L-NMMA-induced vasoconstriction was increased, suggesting enhanced basal NO production. Conversely, there was no significant effect of training on the response to ACh or SNP, suggesting that stimulated endothelium-dependent and -independent function were unaltered. However, following a 10-week programme of daily aerobic and anaerobic exercise training in young military recruits, FMD- but not GTN-mediated vasodilation of the brachial artery was significantly improved.[208] This study also involved predominantly lower-limb exercise, indicating that the improvement in endothelial function was likely to be systemic in nature although, despite an intensive training regime, the change in FMD was relatively small, increasing from 2.23.9%. Even after training, these values are lower than commonly reported in healthy individuals,[57,99] the explanation for which is unclear as baseline diameters did not differ substantially from other studies.[99,209-211] Recently, in a randomised, crossover study of combined aerobic and resistance exercise in healthy middle-aged men, training did not affect endothelium-dependent
or -independent function.[212] This finding is in contrast with studies in which an identical mode and intensity of training improved endothelium-dependent and independent function in CHF patients[213] and endothelium-dependent function in type 2 diabetes.[209] NO-related endothelial function is impaired in CHF and type 2 diabetes (discussed earlier in this review). It is therefore probable that depressed endothelial function is more capable of augmentation by moderate exercise training than is the well-preserved function of healthy individuals, which may, however, be improved by more intense training. Cross-sectional studies suggest that regular physical activity may partially reduce the age-related decline in NO-related endothelial function. A comparison of young and elderly athletes, and agedmatched sedentary controls, found that vasodilation to ACh was not different between young trained and sedentary individuals but was significantly reduced in elderly sedentary individuals compared with elderly athletes.[214] Furthermore, L-NMMA reduced vasodilation to ACh in elderly athletes compared with the untrained. No difference was seen in the response to SNP between the trained and control individuals. In the elderly control individuals, ascorbic acid (vitamin C) restored vasoconstriction to L-NMMA, suggesting that the aged-related decline in NO bioavailability may be due partially to oxidative stress. DeSouza et al. agreed with these findings in a study of men aged 2235 and 5076 years who were either sedentary or exercise trained.[53] Among the sedentary men, the forearm blood-flow response to ACh was reduced in the older group by 25%. However, there was no agerelated decline in endothelial function among the older men who were endurance trained. In a subset of the sedentary older group, 3 months of moderate training increased vasodilation to ACh by 30%, to levels similar to those in the young group and older endurance trained men, suggesting that endothelial function can be improved by exercise in previously sedentary older men. Two recent studies raise the possibility that there may be an optimal threshold of training, beyond
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which exercise may decrease endothelial function. Bergholm et al. reported that 3 months of highintensity running reduced endothelium-dependent function but not endothelium-independent function.[215] The degree of endothelial dysfunction following training was significantly correlated with a decrease in serum uric acid and was most severe in individuals with the greatest improvement in maxi 2max). The authors postulatmal oxygen uptake (VO ed that the training-induced decrease in circulating antioxidant levels may adversely affect endothelial function in the highly-trained or overtrained state. However, this finding is also consistent with a training-induced change in vascular structure as proposed earlier and depicted diagrammatically in figure 1. In a study comparing the effects of different intensities of exercise training in healthy young 2max) exercise men, moderate intensity (50% VO training augmented endothelium-dependent vasodi 2max) and highlation, but low-intensity (25% VO intensity (75% VO2max) training did not.[216] These findings suggest that low-intensity training may fall below the threshold for improving endothelial function while the increase in markers of oxidative stress associated with high-intensity training in this study raise the possibility that increased reactive oxygen species may reduce NO bioavailability, abolishing any improvement in vascular function as a result of increased NO production.
4.4 Exercise Training and NO Function in Pathological States in Humans
after 3 months of combined exercise and diet education in individuals with elevated low-density lipoprotein (LDL)-cholesterol, despite increases in 2max and reduction in LDL cholesterol.[218] In a VO randomised, crossover study, 8 weeks of training also failed to alter endothelial function in unmedicated individuals, but improved FMD and ACh responses in individuals taking hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor lipid-lowering therapy. This disparity is intriguing; it was probably due to the longer duration and greater severity of hypercholesterolaemia in the treated individuals and their depression of NO-related vasodilation compared with the untreated and healthy individuals, again consistent with the greater potential for improving depressed compared with normal function. Alternatively, as HMG-CoA reductase inhibitors and exercise training have both been reported to increase eNOS activity,[219,220] and perhaps both affect the oxidant state,[221,222] there is a hypothetical possibility of synergistic benefit from the two interventions. Improvement in basal[217] and stimulated endothelial function with exercise training have been reported in the absence of a decrease in serum lipids, and reductions in lipid levels are not necessarily associated with improved endothelial function.[218] Therefore, exercise-induced improvements in endothelial function in hypercholesterolaemia appear to result from mechanisms independent of the effect of exercise training on serum cholesterol reduction. Another interesting outcome of the study by Walsh et al. is that, in the hypercholesterolaemic patients receiving statin therapy, both endotheliumdependent (FMD) and endothelium-independent (GTN) responses were impaired at the outset of the study compared with matched healthy controls. Exercise training improved endothelium-dependent vasodilation but not the impaired smooth muscle function. This is consistent with findings from several animal studies, which suggest that exercise trainingmediated improvement in endothelial function precedes improvements in structure and function in the medial layer.[167,171-174,176]
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Hypercholesterolaemia is associated with impaired NO-related endothelium-dependent vasodilation[67,68] and is an important cardiovascular risk factor. Several studies have examined the effect of exercise training on endothelial function in this group. Four weeks of cycle training in unmedicated individuals enhanced basal NO bioactivity but did not alter ACh-stimulated vasodilation nor smooth muscle sensitivity to SNP in the forearm.[217] Unchanged endothelium-dependent function was found
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4.4.2 Hypertension
4.4.4 Diabetes
Twelve weeks of daily walking increased forearm reactive hyperaemia[223] and the blood-flow response to ACh[224] in essential hypertension. These effects were abolished by L-NMMA, indicating that the improvement following training was due to augmented bioactivity of NO. There was a reduction in blood pressure as a result of exercise training in this study, but it did not correlate with improved endothelial function.
4.4.3 Obesity
The combined effects of exercise training and dietary restriction on vascular endothelial function have been investigated in a non-randomised study in obese adults.[225] Following the weight loss intervention, ACh responses significantly improved and the magnitude of this improvement was significantly correlated with changes in body mass index, waist girth and insulin resistance. No changes were evident in response to SNP. The authors concluded that weight loss may affect endothelial function by improving insulin sensitivity, decreasing oxidative stress and increasing NO bioavailability. The effect of exercise training alone has recently been investigated in obese children (unpublished data) and adolescents.[226] Baseline endothelial function was significantly impaired in both groups relative to age- and sex-matched controls. Adolescents performed combined resistance and aerobic exercise three times a week for 8 weeks and children undertook 8 weeks of physically active game play. Exercise intensity was monitored at 6085% maximum heart rate in both groups. Study participants and their guardians were specifically requested not to alter diet intake across the study period and this was checked using repeated diet diaries. In both groups, the exercise intervention was associated with decreased abdominal fat mass (dual-energy x-ray absorbtiometry assessment) and improved FMD compared with an inactive control period. These findings highlight the importance of regular exercise, even in the young, in controlling risk factors to reduce the risk of cardiovascular disease later in life.
One recently published trial has examined the effects of combined, predominantly lower limb, resistance and aerobic exercise training on vascular function in type 2 diabetes.[209] Despite the avoidance of forearm muscle contractions, exercise training enhanced endothelium-dependent reactivity to ACh and FMD in forearm resistance and conduit vessels, respectively, without a change in endothelium-independent response. This improvement in endothelial function appears, therefore, to be a systemic adaptation. Changes in conduit and resistance vessel endothelial function were not significantly related to changes in fasting blood glucose or glycated haemoglobin, suggesting that improved endothelial function occurs independently of glycaemic control. In keeping with a systemic effect of exercise training, 4 months of cycle training in patients with long-standing type 1 diabetes also increased FMD of the brachial artery, and occular fundus pulsation amplitudes to intravenous L-NMMA, effects which were lost after 8 months of detraining.[227]
4.4.5 CAD
Physical inactivity is an established risk factor for cardiovascular disease[228] and exercise training has been associated with a 2025% reduction in mortality following a primary cardiac event.[4] To date, there appear to be five studies of the effect of exercise training on endothelial function in patients with established CAD. Four weeks of highfrequency exercise training improved endotheliumdependent vasodilation in epicardial coronary and resistance vessels, but did not alter endotheliumindependent function.[229] These benefits can be maintained, albeit to a reduced degree, with homebased exercise of reduced daily exercise duration.[230] Eight weeks of combined resistance and aerobic exercise, that did not involve forearm muscle contraction, improved FMD of the brachial artery in patients with CAD suggesting a systemic effect of training.[210] In minor contrast, 10 weeks of lower extremity training increased FMD significantly in the tibial artery by 2%, but the 1.9% increase in the brachial artery was not statistically signifSports Med 2003; 33 (14)
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icant.[231] The baseline FMD in the former study[210] was substantially lower than in the latter,[231] reflecting greater impairment of endothelial function, a condition that may be more sensitive to exercise training. An insight into the possible mechanisms responsible for improved endothelial function following exercise training has recently been presented by Hambrecht et al.[232] in a study of patients undergoing coronary artery bypass graft surgery. Four weeks of daily exercise, undertaken immediately prior to surgery, improved peak flow velocity in response to ACh and FMD in the left internal mammary artery. No changes were evident in inactive controls. Following surgery, a section of this vessel was harvested and used for in vitro assessment of endothelial function, immunochemistry, and quantification of mRNA and protein expression. eNOS mRNA and vascular protein expression were higher in trained individuals and increased eNOS phosphorylation was significantly correlated with the change in ACh-induced average peak flow velocity following training. These important findings support the hypothesis that an increase in eNOS protein may mediate an improvement in endothelial function through shear stress-induced phosphorylation. In summary, exercise training can improve endothelial function both locally and systemically in patients with existing CAD. This may enhance myocardial perfusion and contribute to the well-documented benefits of exercise training, such as reduction in the incidence and severity of exercise-induced myocardial ischaemia.[233,234]
4.4.6 Chronic Heart Failure
change was evident in the untrained contra-lateral arm[239] and that comparable training protocols have not altered systemic cardiac output, heart rate or plasma norepinephrine or lactate levels[242] support the argument that training adaptations to hand-gripping result from local mechanisms. Because greater blood-flow responses to ACh were noted following combined exercise and supplementation with oral L-arginine compared with either treatment alone,[240] increased substrate availability appears to compliment shear stress-induced up-regulation of eNOS. However, localised exercise training has not universally resulted in improved endothelial function. In one study, training increased endotheliumdependent vasodilation and peak hyperaemic blood flow in control individuals but not in CHF patients;[243] however, the negative results may reflect the low intensity of training undertaken. On balance, the preceding findings provide good support for the efficacy of exercise training, involving relatively small muscle mass (i.e. the forearm), in stimulating a localised improvement in endothelium-dependent vasodilation in patients with CHF, who initially exhibit endothelial dysfunction. The vascular effects of exercise training involving a large muscle mass have been examined in several studies in patients with CHF.[213,244-246] Twelve weeks of low intensity cycling (<50% 2max) significantly increased peak reactive hyVO peraemia locally in the calf, but did not provide a systemic effect assessed in the forearm.[244] A localised training effect on basal and stimulated NOrelated dilation of the femoral artery was also evident following 6 months of cycle training without a change in the smooth muscle response to GTN.[245] Increases in endothelium-dependent vasodilation in vessels supplying the trained musculature were significantly correlated with changes in functional capacity in both of these studies.[244,245] Furthermore, exercise training was also associated with an increase in stroke volume, a small but significant reduction in left ventricular end diastolic diameter and volume, and a reduction in total peripheral resistance at rest and during peak exercise, the latter correlating with ACh-enhanced blood flow in the
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Due to the clinical significance of peripheral abnormalities in CHF, which limit peak oxygen uptake,[235,236] a strong correlate of prognosis,[237,238] the effects of exercise training on endothelial function have been thoroughly investigated. Studies have examined the effect of localised and systemic training protocols in the upper and lower limbs. Handgrip training improved NO-related function in conduit[239,240] and resistance vessels[241] following 4 and 8 weeks, respectively, without altering endothelium-independent function. The observations that no
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lower limb.[247] This is consistent with exerciseinduced improvement in endothelium-dependent function in the peripheral circulation benefiting afterload and cardiac function. Systemic changes in vascular NO bioactivity have been identified in two lower body exercise training studies in CHF patients.[213,246] In a randomised, crossover study, combined aerobic and resistance exercise, which specifically avoided forearm exercise, improved forearm resistance vessel function in CHF patients,[213] suggesting a systemic effect. Although the response to training was predominantly endothelium-dependent, there was a slight improvement in endothelium-independent dilation and an increase in reactive hyperaemia, which suggests a change in smooth muscle function and/or vascular structure.[201,248] Cycle training has also been associated with a systemic effect on endothelial function in CHF patients after 4 weeks[246] and heart transplant recipients after 6 months.[249] These findings suggest that the endothelial dysfunction evident in CHF may be improved throughout the vasculature with exercise training protocols that activate sufficient muscle mass to increase pulse wave dynamics and shear stress. The minimum training threshold for this effect may lie between 50%[244] 2max.[246] and 70% of VO
4.5 Exercise Cessation and Detraining
5. Conclusions It should be emphasised that NO-related vasodilation is only one of the endothelium-related effects of NO, and that NO is only one of many mediators produced by the endothelium. However, NO plays a key role in endothelial function and its vasodilator function provides a practical index of endothelial function. Acute bouts of exercise produce changes in haemodynamics that act directly on the endothelium. Recurrent exposure to these forces can result in endothelial adaptation, including up-regulation of NO bioactivity and an increased capacity for vasodilation. Exercise training involving relatively small muscle groups, such as hand-gripping, can improve vasodilator function locally;[239,241] whilst exercise involving a large area of active muscle, such as cycling or jogging, causes heart rate and pulse pressure to rise, resulting in increased pulsatile flow and pulse pressure systemically.[153] This increases vascular shear stress and recurrent exposure to this stimulus, by way of exercise training, can enhance endothelial NO-related vasodilator capacity both locally[231,245] and systemically[208-210,213,246] under a range of conditions, although variable results have been found.[203,206,212] Importantly, exercise training effects on the endothelium appear to extend to the coronary circulation, at least in patients with existing CAD.[229] It appears that a greater training load is required to improve normal endothelial function in the young and healthy,[208] for which evidence is equivocal, than in conditions associated with endothelial dysfunction,[250] or to prevent age-related decline in endothelial function.[53] This raises the intriguing possibility of a threshold effect for training on endothelial function which may be determined by age, fitness or pathological state. Endothelial function may improve after as few as 7 days in trained animals.[167,187] In healthy humans, 4 weeks of training improved basal NO-related endothelial function in healthy individuals.[207] However, studies that have identified improved stimulated endothelium-dependent vasodilation have prescribed at least 10 weeks of training in healthy
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Localised and systemic exercise-induced changes in endothelial function are quickly lost following the cessation of relatively short duration training. Six weeks after training was ceased in patients with CHF, radial artery diameters had returned to pre-training size.[239] Similarly, endothelial function returned to baseline after 8 weeks in patients with CHF,[213] hypercholesterolaemia and type 2 diabetes.[209] This is consistent with the time course of deconditioning associated with other physiological adaptations to exercise training. Therefore, these data suggest that an exercise programme, at some level as yet undetermined, would be necessary to maintain vascular benefits. To date, no data exist on the effect of detraining on endothelial function in healthy individuals.
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individuals.[53,208] Pathological states associated with depressed NO bioactivity may respond more rapidly to training, with endothelial function improving after as little as 4 weeks,[229,239] but returning rapidly to baseline following the cessation of training.[239] Conditions in which exercise training can improve NO-dependent endothelial vasodilator function include CHF, CAD, type 2 diabetes, hypercholesterolaemia and hypertension, although the literature remains sparse except in the case of CHF. With extended exposure to the recurrent haemodynamic changes of exercise training, there is evidence from animal[191] and human[201] studies that this adaptation may progress to an increased sensitivity of smooth muscle to NO. There is also some evidence that vascular remodelling with increase in vessel diameter can eventually occur.[175,176,251] The latter two processes, effectively, may constitute longer term mechanisms for reducing shear-stress on a more sustained basis, allowing NO activity to return towards pre-training levels. However, endothelial mechanisms responsible for mediating vessel structural changes remain largely undefined; there is evidence that NO plays an important role,[179-183] while there might also be other mediators. Throughout the course of a lifetime, the vascular endothelium is exposed to physical, chemical and biological stressors that compromise function with increasing age, exposure to risk factors and development of cardiovascular disease. Endothelial dysfunction, characterised by reduced bioactivity of NO, is almost certainly an integral component of clinical atherogenesis, and important in the pathophysiology of the circulation in heart failure, diabetes and in the presence of vascular risk factors. Regular physical activity is associated with improved outcomes in these conditions, which may be due, in part, to the positive effect of exercise on endothelial function. Acknowledgements
The authors wish to thank Paul Ricketts, Multimedia Designer, DUIT Multimedia, for constructing the diagram that appears in this manuscript. No sources of funding were used to assist in the preparation of this manuscript. The
authors have no conflicts of interest that are directly relevant to the content of this manuscript.
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Correspondence and offprints: Dr Andrew Maiorana, Cardiac Transplant Unit, Royal Perth Hospital, Wellington Street, GPO Box X2213, Perth, WA 6847, Australia. E-mail: Andrew.Maiorana@health.wa.gov.au
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Exercise and The Nitric Oxide Vasodilator System

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Exercise and The Nitric Oxide Vasodilator System

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LEADING ARTICLE

Exercise and the Nitric Oxide Vasodilator System

Exercise Training and Endothelial Function

2.1 Endothelial Dysfunction and Risk Factors for Atherosclerosis

Exercise Training and Endothelial Function

Exercise Training and Endothelial Function

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Exercise Training and Endothelial Function

Exercise Training and Endothelial Function

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

Exercise Training and Endothelial Function

Adis Data Information BV 2003. All rights reserved.

Sports Med 2003; 33 (14)

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