The Assessment of Endothelial Function

HomeCirculationVol. 126, No. 6The Assessment of Endothelial Function Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessReview ArticlePDF/EPUBThe Assessment of Endothelial FunctionFrom Research Into Clinical Practice Andreas J. Flammer, MD, Todd Anderson, MD, David S. Celermajer, MD, Mark A. Creager, MD, John Deanfield, MD, Peter Ganz, MD, Naomi M. Hamburg, MD, Thomas F. Lüscher, MD, Michael Shechter, MD, Stefano Taddei, MD, Joseph A. Vita, MD and Amir Lerman, MD Andreas J. FlammerAndreas J. Flammer From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Todd AndersonTodd Anderson From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , David S. CelermajerDavid S. Celermajer From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Mark A. CreagerMark A. Creager From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , John DeanfieldJohn Deanfield From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Peter GanzPeter Ganz From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Naomi M. HamburgNaomi M. Hamburg From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Thomas F. LüscherThomas F. Lüscher From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Michael ShechterMichael Shechter From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Stefano TaddeiStefano Taddei From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author , Joseph A. VitaJoseph A. Vita From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author and Amir LermanAmir Lerman From the Division of Cardiovascular Diseases, Mayo Clinic and College of Medicine, Rochester, MN (A.J.F., A.L.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada (T.A.); Department of Cardiology, Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia (D.S.C.); Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.A.C.); Vascular Physiology Unit, Great Ormond Street Hospital for Children, University College London, London, UK (J.D.); Division of Cardiology and the Center of Excellence in Vascular Research, San Francisco General Hospital, University of California San Francisco, San Francisco (P.G.); Whitaker Cardiovascular Center, Boston University School of Medicine, Boston, MA (N.M.H., J.A.V.); Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zürich, Zürich, Switzerland (A.J.F., T.F.L.); Leviev Heart Center, Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (M.S.); and Department of Internal Medicine, University of Pisa, Pisa, Italy (S.T.). Search for more papers by this author Originally published7 Aug 2012https://doi.org/10.1161/CIRCULATIONAHA.112.093245Circulation. 2012;126:753–767IntroductionThe discovery of nitric oxide (NO) as a crucial endothelium-derived molecule for vascular relaxation and the recognition of the endothelium as more than a passive interface between blood and the vessel wall led to substantial progress in the field of vascular research.1 Endothelial dysfunction is a pathological condition characterized mainly by an imbalance between substances with vasodilating, antimitogenic, and antithrombogenic properties (endothelium-derived relaxing factors)2 and substances with vasoconstricting, prothrombotic, and proliferative characteristics (endothelium-derived contracting factors).3 Among the most important vasodilator molecules, particularly in muscular arteries, is NO, which also inhibits other key events in the development of atherosclerosis such as platelet adhesion and aggregation, leukocyte adhesion and migration, and smooth muscle cell proliferation. Particularly in the microcirculation, prostacyclin and endothelium-derived hyperpolarization factors (an umbrella term for substances and signals hyperpolarizing vascular myocytes by opening voltage channels4) also play an important role.Generally, loss of NO bioavailability indicates a broadly dysfunctional phenotype across many properties of the endothelium. Thus, the assessment of its vasodilator properties resulting from NO and other molecules may provide information on the integrity and function of the endothelium. Interestingly, most, if not all, cardiovascular risk factors are associated with endothelial dysfunction,5 and risk factor modification leads to improvement in vascular function. Endothelial dysfunction has been detected in the coronary epicardial and resistance vasculature and in peripheral arteries, so endothelial dysfunction can be regarded as a systemic condition.6 Importantly, the process of atherosclerosis begins early in life, and endothelial dysfunction contributes to atherogenesis and precedes the development of morphological vascular changes.7Over the past 25 years, many methodological approaches have been developed to measure the (patho)physiological function of the endothelium in humans.8 Although the ability to measure endothelial function has boosted clinical research in this field, its use as a clinical tool in daily practice is not established, nor has any method been recommended in clinical guidelines for planning primary or secondary prevention of vascular disease.The aims of this review are to give a short overview of the most commonly used methods to measure endothelial function in humans, particularly noninvasive techniques (Table 1), and to summarize the clinical implications of endothelial dysfunction in the population and in individual patients. The possible future role of endothelial function measurement for individualized medicine is also considered.Table 1. Advantages and Disadvantages of the Most Commonly Used Techniques to Assess Endothelial FunctionTechniqueVascular BedAdvantagesDisadvantagesStimulus (Examples)Coronary epicardial vasoreactivity (QCA)Epicardial macrovascular Conduit arteriesAssessment directly in the coronary vascular bedInvasiveAchExpensiveExerciseGold standardTime intensivePacingLimited to those undergoing coronary angiographyCPTChallenging for serial measurementsCoronary microvascular function–Doppler wiresCoronary microvascularAssessment directly in the coronary microvasculatureInvasiveAchResistance arteriesExpensiveAdenosineTime intensivePapaverineLimited to those undergoing coronary angiographyChallenging for serial measurementsFMDBrachial arteryEasy accessChallenging to perform wellReactive HyperemiaConduit arteryCorrelation with invasive epicardial vascular functionDisparate protocols for performance and standardizationsMany outcome studiesNeed for standardizationInexpensivePossibility to assess other important parameters (flow, baseline arterial diameters, FMC)Venous occlusion plethysmographyForearm vasculatureEasy accessInvasive (cannulation of the brachial artery)Ach and other vasoactive substancesMicrovasculatureVasoactive substances infused to generate a dose-response relationshipTime consumingContralateral arm as a controlEndoPATFingerEasy to access and performExpense of disposable finger probesReactive hyperemiaMicrovasculatureAutomatedPAT signal influenced by variable non endothelial factorsLow interobserver and intraobserver variabilityCorrelation with invasive microvascular vascular functionQCA indicates quantitative coronary angiography; Ach, acetylcholine; CPT, cold pressor test; FMD, flow-mediated dilation; FMC, flow-mediated constriction; and PAT, peripheral arterial tonometry.Methods to Assess Vascular FunctionThe first demonstration of endothelial dysfunction in atherosclerotic coronary arteries using intracoronary infusion of acetylcholine and quantitative coronary angiography dates back to 1986 by Ludmer and colleagues.9 Their seminal studies heralded an important shift in paradigm in the understanding of human atherosclerosis, which had previously been regarded as a purely structural disease. Their research drew attention to the functional manifestations of atherosclerosis such as exaggerated vasoconstriction as a consequence of poorly functioning endothelium. Later, less invasive techniques were developed using mainly the forearm circulation as a surrogate for coronary arteries.6,10,11 All approaches have their advantages and disadvantages; most important, different vascular beds are examined (Figure). The basic principle, however, is similar: Healthy arteries such as the coronary or brachial arteries dilate in response to reactive hyperemia (flow-mediated vasodilatation) or after pharmacological stimuli, including intra-arterial infusion of endothelium-dependent vasodilators such as acetylcholine, bradykinin, or serotonin, via release of NO and/or other endothelium-derived vasoactive substances.2 In disease states, such endothelium-dependent dilatation is reduced or absent. However, regardless of which technique is applied, vascular responses are determined not only by the functional status of the vasculature at the place of measurement but also by the structural condition of the resistance arteries in the microvasculature. Furthermore, to differentiate endothelium-dependent from endothelium-independent responses, exogenous NO donators (eg, glycerol-trinitrate) or direct non–NO donators such as adenosine can be applied. Impaired endothelial-independent function is associated with structural vascular alterations and alterations in smooth muscles cells rather than changes in the endothelium.Download figureDownload PowerPointFigure. The principles of the most commonly used methods to assess endothelial function. FMD indicates flow-mediated vasodilatation.Coronary Epicardial and Microvascular FunctionTo assess coronary endothelial function, a functional test is performed to measure epicardial and resistance vessel endothelial function. Although these methods are limited by the invasive nature, their advantage is to measure endothelial function directly in this clinically important vascular bed.Epicardial Endothelial FunctionTo image vasomotor responses of epicardial coronary arteries, quantitative coronary angiography or intravascular ultrasound is used, and changes in vessel diameters and cross-sectional areas in response to endothelium-dependent interventions are documented. After acetylcholine infusion, vessels and segments with an intact endothelium vasodilate, whereas vessels and segments with dysfunctional or disrupted endothelium will respond with vasoconstriction as a result of direct activation of muscarinic receptors on vascular smooth muscle cells.9 Similar induced functional changes in vascular reactivity have been demonstrated with salbutamol12 and other substances (Table 2) and with more physiological interventions, eg, increased coronary blood flow. However, dose titration is more difficult. Physical measures of endothelium-dependent responses include exercise13,14 or pacing-induced tachycardia as a surrogate for exercise15,16 and induce an increase in coronary blood flow and thus shear stress on the coronary circulation, which leads to flow-mediated endothelium-dependent vasomotion of the epicardial vessels. Similar responses can be seen in response to mental stress.17 The observation of endothelium-dependent flow-mediated dilatation in the coronary epicardial vessels and its impairment in atherosclerosis18,19 provided the rationale to study similar responses in the peripheral vasculature later (see below). Another “physiological” test to assess epicardial vasoreactivity is the use of the cold pressor test in which the subject puts his or her hand into ice water. The activation of the sympathetic nervous system leads to release of NO and endothelium-derived hyperpolarizing factors via stimulation of endothelial α2-adrenergic receptors and consequently vasodilation in healthy arteries.20 However, in dysfunctional endothelium, α1-adrenergic–mediated constriction of smooth muscle cells will dominate,21 closely mirroring the responses to acetylcholine.21,22Table 2. Pharmacological Triggers for the Assessment of Coronary Vascular FunctionEpicardial VesselsMicrocirculationEndothelium-dependent vascular functionAcetylcholineAcetylcholineSalbutamolSalbutamolSerotoninBradykininSubstance PCalcitonin gene-related peptideEndothelium-independent vascular functionNitroglycerinAdenosineNitroprussideDipyridamolePapaverineNitroprussidePapaverineCoronary Microvascular FunctionChanges in coronary (or myocardial) blood flow can be used as a surrogate parameter for microvascular function.23 Coronary flow reserve is the ratio of maximal coronary blood flow during maximal coronary hyperemia with provocative stimuli (such as adenosine infusion, pacing, or exercise) divided by the resting coronary blood flow. This maximal blood flow response (coronary flow reserve) is both endothelium- and non–endothelium-dependent, and a coronary flow reserve <2.0 is considered abnormal.24 To measure endothelium-dependent microvascular function, the percent increase in coronary blood flow in response to endothelium-dependent vasodilators (commonly acetylcholine) infused at increasing concentrations is analyzed.Other methods to estimate microvascular function have been introduced, eg, the measurement of the number of cineangiographic frames that it takes to fill a distal vessel with proximal injection of contrast. The corrected Thrombolysis in Myocardial Infarction frame count provides a semiquantitative assessment of epicardial coronary blood flow.25 Taking the main disadvantage—the invasive nature of the above-mentioned tests—into account, noninvasive functional tests to assess the coronary microvasculature have been developed, among them positron emission tomography,26 myocardial perfusion imaging,27 blood oxygen level–dependent magnetic resonance imaging,28 and echocardiography29; however, a detailed discussion of these tests is beyond the scope of this review.Peripheral Techniques to Assess Endothelial FunctionThe aforementioned techniques to measure coronary epicardial vascular function and to assess the coronary microcirculation are very well suited for patients requiring a coronary angiogram for clinical indications. However, to assess vascular function and health in the asymptomatic patient, performing an invasive functional coronary angiogram is usually not appropriate. Therefore, noninvasive or less invasive surrogate techniques to assess macrovascular and microvascular endothelial function have been developed. Although they do not measure vascular function in the coronary circulation directly, they have been shown to correlate reasonably with its more invasive counterparts.30–32 Whereas all these techniques assess the generalized function of the vasculature, one has to keep in mind that certain phenomena cannot be explained by systemic endothelial dysfunction; it is likely that local factors (eg, flow patterns) and local vascular dysregulation observed at branch points related to disturbed shear stresses also contribute to disease.33–37Plethysmography of the Forearm CirculationAlthough still limited by its semi-invasive nature (arterial puncture), this technique measures changes in forearm blood flow by venous plethysmography in both arms before and after infusion of vasoactive substances into a cannulated brachial artery.10 The main advantage is that vasoactive molecules, hormones, or drugs (eg, acetylcholine or nitroglycerin) can be infused, thus quantifying endothelium-dependent and endothelium-independent vasodilation in a dose-dependent manner. The dosages required have limited systemic effects, allowing the contralateral limb to serve as an internal control. The results are expressed as the ratio of the changes in flow measured in both arms and are reproducible.38 The response to acetylcholine is significantly reduced by intra-arterial infusion of NG-monomethyl-l-arginine (but not by acetylsalicylic acid),39 demonstrating a key role for NO. However, it has to be taken into account that, especially in patients with multiple risk factors, endothelium-derived hyperpolarization factors also play an essential role for resting microvascular tone40 and for agonist-stimulated vasodilation.41,42 The technique is well suited to measure differences in blood flow to various stimuli or inhibitors in a single patient. However, because of different initial arterial pressures, forearm blood flow, different sizes of the forearm, and other factors, comparisons between groups or serial studies in the same patient are of limited value.43 Although pharmacologically induced vasodilation with this technique gives interesting insights into microvascular pathophysiology, the response not necessarily mimics micro