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(Hypertension. 2007;49:748.)
© 2007 American Heart Association, Inc.

Tutorial

Methods for Evaluating Endothelial Function in Humans

From the Cardiovascular Research Institute, Washington Hospital Center, Washington, DC.

Correspondence to Julio A. Panza, Coronary Care Unit, Washington Hospital Center, 110 Irving St, NW, Suite 2A74, Washington, DC 20010. E-mail julio.a.panza{at}medstar.net

	Introduction

Top Introduction Methods for Assessing... Conclusions References

 
The endothelium is the main regulator of vascular wall homeostasis. Physiologically, endothelial cells maintain a relaxed vascular tone and low levels of oxidative stress, in part by releasing mediators, including NO, prostacyclin (PGI₂), and endothelin (ET-1), and controlling local angiotensin II activity.¹ In addition, the endothelium actively regulates vascular permeability to plasma constituents, platelet and leukocyte adhesion and aggregation, and thrombosis.¹ This state of balanced endothelial regulation of blood vessel function is, however, altered by a number of conditions. Thus, in response to a variety of noxious stimuli, the endothelium undergoes a phenotypic modulation to a nonadaptive state, commonly termed "endothelial dysfunction," characterized by loss or dysregulation of homeostatic mechanisms operative in healthy endothelial cells. This pathophysiological condition is associated with increased expression of adhesion molecules, increased synthesis of proinflammatory and prothrombotic factors, increased oxidative stress, and abnormal modulation of vascular tone, which may lead to different functional manifestations, including impaired endothelium-dependent vasodilation.²

Current evidence suggests that endothelial dysfunction occurs early in the process of atherogenesis and contributes to the formation, progression, and complications of the atherosclerotic plaque.³ Several studies from our and other laboratories have shown that patients with cardiovascular risk factors but no clinical evidence of atherosclerosis have endothelial dysfunction, indicated by an impaired response to endothelial vasodilator agents, such as acetylcholine and bradykinin.⁴ Combined with the pathophysiological role of the endothelium described above, these observations strongly suggest that endothelial dysfunction is a common mechanistic link between risk factors and the development of atherosclerosis. Furthermore, recent reports have demonstrated that endothelial dysfunction is an independent predictor of future cardiovascular events in patients with atherosclerotic risk factors,^5,6 in patients with stable ischemic heart disease,⁷ and in patients with acute coronary syndromes.⁸ Thus, endothelial dysfunction appears to be a systemic vascular process that not only mediates the development of the atherosclerotic plaque but may also modulate its clinical course. Finally, at least in patients with hypertension, an improvement in endothelial vasodilatory function with antihypertensive therapy correlates with a more favorable prognosis,⁹ suggesting that endothelial function may be used to assess the efficacy of treatments aiming at atherosclerotic risk reduction.

The initial approach in the discovery and evaluation of endothelial function was focused on the analysis of endothelium-dependent responses to pharmacological agents such as acetylcholine.¹⁰ However, with increased understanding of the numerous roles exerted by the endothelium, several novel techniques have been proposed to evaluate multiple aspects of endothelial function.^11–13 These new methods have broadened the number of research tools available to clinical investigators and have opened possibilities for implementation of endothelial function assessment in the evaluation of patients with increased cardiovascular risk. Therefore, a more in-depth knowledge of endothelial function and of the methodologies used for its assessment has become of critical importance for the understanding of vascular pathophysiology and its clinical implications.

The aim of this article is to review the more widely used techniques in the evaluation of endothelial function. Original studies are cited to provide examples of how investigators have used these methods and the conclusions that were reached. It is not within the scope of this article to provide an exhaustive review of different lines of investigations related to endothelial dysfunction or an in-depth analysis of the mechanisms underlying abnormal endothelial function.

	Methods for Assessing Endothelial Function

Top Introduction Methods for Assessing... Conclusions References

 
One of the major roles of the endothelium in the homeostasis of the cardiovascular system is the regulation of vascular tone. This action is exerted through the production and release of different vasodilator and vasoconstrictor substances that act on the underlying smooth muscle and control its contractile state. Different pharmacological and physical stimuli can trigger endothelial release of vasoactive factors. Evaluation of vascular responsiveness (by measuring changes either in vessel diameter or in blood flow) to these endothelium-dependent stimuli provides an important tool in studying endothelial vasoregulatory actions in vivo and, over the last 2 decades, has become a standard test of endothelial function.²

However, progress in our knowledge of the multiple roles that the endothelium plays in the maintenance of vascular wall homeostasis has broadened the scope of the term "endothelial dysfunction" to encompass dysregulation of other mechanistic processes not properly evaluated with the study of endothelium-dependent vasodilation. These processes include the control of vascular wall inflammation and smooth muscle proliferation, regulation of platelet adhesion, and aggregation, as well as modulation of thrombosis and fibrinolysis.¹⁴ In turn, awareness of the ever-expanding roles of the endothelium has resulted in an increased need to evaluate endothelial function in a comprehensive manner and beyond endothelium- dependent vascular tone regulation. In recent years, several novel techniques have been devised to explore different facets of endothelial function in vivo, including endothelial expression of inflammatory markers,¹⁵ adhesive properties of the endothelium with respect to the interactions with leukocytes and platelets,¹⁶ and factors involved in the regulation of thrombosis and fibrinolysis,¹⁷ as well as endothelial progenitor cells and their possible role in endothelial repair and maintenance of vascular homeostasis.^18,19 Together, these methodologies not only complement endothelium-dependent vasoreactivity testing but also provide means for a comprehensive assessment of multiple endothelial functions and their pathophysiological role in cardiovascular disease.

Endothelium-Dependent Vascular Tone
The pioneering investigations of Furchgott and Zawadzki first demonstrated the essential role of the endothelium in the modulation of vascular smooth muscle responses to acetylcholine.^10,20 Expanding on these seminal observations, various pharmacological (eg, acetylcholine and bradykinin) and physical (eg, shear stress) stimuli have been identified to act through the endothelial cells to release vasoactive substances and have subsequently become important research tools in investigating endothelial function. Because vascular tone is determined by both endothelial cell production of vasoactive substances and smooth muscle responsiveness, an abnormal vascular response to an endothelial stimulus may be secondary to either an endothelial cell defect or to changes in smooth muscle function. Therefore, to ascribe impaired responses to endothelial agonists to an abnormality at the level of the endothelium, it is necessary to directly test smooth muscle reactivity. This is commonly achieved by using endothelium-independent agents (eg, sodium nitroprusside and nitroglycerin) that bypass the endothelium to act directly on smooth muscle cells and, thus, cause vasorelaxation. It is, thus, the combination of information provided by the responses to endothelium-dependent and -independent stimuli that allows investigators to conclude that an impaired response is indeed related to abnormal endothelial function.

Vascular responses to endothelium-dependent and -independent stimuli can be tested in different vascular territories, thus allowing the study of both resistance and conduit vessels (Figure). This is relevant because, although endothelial cell actions are similar throughout the vascular tree, the specific roles exerted by the endothelium may vary according to the physiological function of the specific vascular bed. For example, assessment of the microcirculation permits the study of the role of the endothelium in the regulation of vascular resistance that, in turn, determines blood pressure and blood flow. Conversely, investigation of conduit vessels that are the target of atherosclerosis (eg, epicardial coronary arteries) allows the study of the processes that lead to plaque formation and progression.

View larger version (84K): [in this window] [in a new window]   Methods for assessing endothelial regulation of vascular tone. The role of the endothelium in modulating vascular tone may be assessed in the coronary (top) and peripheral (bottom) circulations. Within the coronary circulation (A), the behavior of both the epicardial coronary arteries and the microarterioles may be assessed. Epicardial coronary arteries are imaged by means of coronary angiography (B), and the diameter can be accurately assessed by means of quantitative coronary angiography (QCA; C) to quantify the response to endothelium-dependent and -independent agents. The coronary microcirculation can be examined by cannulating a coronary artery with a Doppler wire (D) to measure blood flow velocity before and during administration of different agonists or antagonists. The blood flow velocity increases during the hyperemia induced by vasodilators (E). The peripheral circulation can be accessed both in the upper and the lower extremities (F). Imaging of the brachial artery with high frequency ultrasound (G) allows the assessment of flow-mediated dilation (ie, the increase in diameter of the conductance arteries during the hyperemia induced by a period of ischemia of the distal extremity (H). Other methods (eg, placement of the cuff on the forearm and use of a clamp to steady the ultrasound probe) have also been used. The forearm perfusion technique (I) requires insertion of a catheter into the brachial artery for drug infusion and assessment of the forearm blood blow response by means of strain gauge plethysmography. The forearm blood flow at rest and during the infusion of a pharmacological agent (eg, bradykinin) can be assessed by measuring the increase in arterial inflow measured during a brief period of venous outflow occlusion (J). Both flow-mediated dilation of the conductance arteries and assessment of the microcirculation with the perfusion technique may also be performed in the lower extremity (as represented by the dashed line arrows).

The techniques outlined below have been devised to investigate endothelium-dependent vascular responses in both the microvasculature and conduit vessels of the coronary and peripheral circulations. For the sake of clarity, these 2 vascular territories will be discussed separately.

Endothelium-Dependent Vasodilation
Coronary Studies
Coronary arteries represent one of the most critical sites of atherosclerotic plaque development; hence, the first studies of endothelial function in vivo evaluated endothelium-dependent reactivity of large epicardial vessels.²¹ In this model, different concentrations of acetylcholine and the endothelium-independent vasodilator nitroglycerin are infused into the coronary arteries, followed by angiographic measurements of coronary vessels diameter (Figure 1). Initial findings demonstrated that, in patients with coronary atherosclerosis, acetylcholine infusion results in paradoxical vasoconstriction of epicardial vessels in contrast to coronary vasodilation observed in patients without documented cardiovascular disease.²¹ A normal vasodilator response to nitroglycerin in both patient groups suggests that a defect in endothelial vasodilator function, and not in smooth muscle cell reactivity, was responsible for the abnormal acetylcholine-induced coronary vasorelaxation in patients with atherosclerosis. After these initial observations, different laboratories used responses to intracoronary acetylcholine infusion not only to demonstrate impaired epicardial vessel relaxation in patients with various cardiovascular risk factors,²² but also to identify the mediator(s) involved in abnormal endothelial vasodilatory function in these patients.²³

In addition to pharmacological agents (eg, acetylcholine and bradykinin), physical stimuli, such as increase in blood flow, have been used to investigate the responsiveness of coronary arteries. Adenosine and papaverine are endothelium-independent agents that act primarily on the coronary microvasculature to induce its vasodilation and thereby increase blood flow through the coronary arteries. This increase in flow, with the resulting increase in shear stress, leads to endothelial NO release and consequent proximal (epicardial) arterial vasodilation, a phenomenon called flow-mediated dilation (FMD). (FMD is commonly used in the assessment of peripheral conduit arteries, and it is described in more detail in the Peripheral Studies section.) By infusing either adenosine or papaverine into the midportion of the coronary artery of interest, followed by the angiographic measurement of its diameter, at the site proximal to the infusion, FMD can be assessed in the coronary vasculature.^24,25

Angiographic evaluation of changes in epicardial vessel diameter after infusion of pharmacological agents (eg, acetylcholine, bradykinin, and substance P) or FMD (induced by adenosine or papaverine) primarily evaluates large coronary conduit vessels. This assessment does not reflect the functional status of the coronary microcirculation that determines vascular resistance and, thus, blood flow to the myocardium. Because the coronary microvessels may be preferentially affected in certain cardiovascular conditions (eg, hypertension), it is critical to separately study their responses using dedicated methods. Endothelial function of the coronary microvasculature can be assessed by measuring changes in coronary blood flow.²⁶ This method involves placement of a Doppler wire into a coronary artery (usually the mid-left anterior descending artery) and measuring blood flow velocities after the infusion of endothelium-dependent (eg, acetylcholine) and endothelium-independent (eg, adenosine and papaverine) agents (Figure). Relative changes in coronary blood flow are calculated by multiplying changes in mean coronary blood flow velocity by changes in the estimated vessel cross-sectional area (determined by quantitative angiography).

To further assess endothelial function of coronary microvasculature and macrovasculature, and its role in coronary blood flow regulation, investigators have used more physiological stimuli, such as the cold pressor test and dynamic exercise. In the cold pressor test, sympathetic activation is achieved by immersing a patient’s hand and forearm in a slurry of iced water for 90 seconds, followed by intracoronary blood velocities measurements and quantitative angiography.²⁷ The supine dynamic exercise test has been performed using a bicycle ergometer with continuous hemodynamic monitoring and obtaining repeated coronary angiograms at baseline (before exercise), at peak exercise, after exercise, and after intracoronary nitroglycerin infusion.²⁸ Both dynamic exercise and the cold pressor test were reported to induce epicardial vasodilation and an increase in coronary blood flow in angiographically normal coronary arteries, whereas paradoxical vasoconstriction after these stimuli was observed in patients with evidence of coronary atherosclerosis.^27,28 These findings suggest that the dysfunctional endothelium in atherosclerosis might lead to increased coronary resistance and thereby contribute to clinical manifestations such as transient myocardial ischemia during exercise or sympathetic activation.

Measurements of the acetylcholine-induced and flow-mediated variations in coronary artery diameter allow direct assessment of endothelial function in the vascular district that is one of the most important targets for atherosclerosis. However, several limitations restrict their widespread use. Most importantly, the invasive nature of these studies not only confines their use to these patients undergoing coronary angiography for clinical reasons (thus introducing a selection bias) but also limits the possibility of repeated evaluations. Furthermore, measurements of coronary diameter, used in the assessment of endothelial function of large epicardial vessels, as well as in calculating changes in blood flow, are limited by the accuracy of coronary angiography and may pose technical difficulties in patients with atherosclerosis and potentially eccentric plaques. Therefore, less invasive techniques were developed to allow more widespread use of endothelial function testing. Importantly, impaired endothelial responses, characteristically found in coronary arteries of patients with cardiovascular risk factors, have also been confirmed in different peripheral circulatory territories of these patients.^29,30 This has led to the concept of a generalized nature of endothelial dysfunction and has facilitated endothelial function testing in more accessible vascular beds.

Peripheral Studies
FMD
Shear stress acting on the endothelium likely represents the main physiological stimulus for the release of vasoactive factors and for the regulation of vascular tone. In experimental conditions in vivo, an increase in shear stress can be induced by the rise in blood flow that follows a short period of ischemia of the distal tissues. The endothelium responds dynamically to this stimulus by releasing NO, thus causing flow-mediated vasodilation (FMD). Based on this principle, the assessment of FMD was developed using ultrasound imaging.¹¹ This involves measurement of the change in the diameter of a conduit artery (typically brachial, although the radial and femoral arteries have also been used) in response to the increased blood flow induced by a period of ischemia applied to the distal part of the limb. The ultrasound system must be equipped with the vascular software for 2D imaging, color and spectral Doppler, an internal ECG, and a high-frequency vascular transducer.³¹ The subject is positioned supine, and the brachial artery is imaged above the antecubital fossa in the longitudinal plane (Figure). A segment with clear anterior and posterior intimal surfaces between the lumen and vessel wall is selected for continuous 2D grayscale imaging. A stereotactic probe-holding device can be helpful to maintain the same image of the artery throughout the study, and its use is highly recommended.³² The ischemic stimulus is created by placing a sphygmomanometric (blood pressure) cuff, either above the antecubital fossa or on the forearm, and inflating it to suprasystolic pressure (usually to 50 mm Hg above systolic pressure) for 5 minutes. After cuff release, reactive hyperemia produces a transient increase (6-fold) in blood flow through the conduit artery, with an increase in shear stress and conduit artery dilation (Figure 1). Two-dimensional images of the brachial artery and Doppler signals are acquired at baseline, before cuff inflation, and for 1 minute after cuff release (time of maximum vasodilation). FMD is the change in poststimulus diameter, usually expressed as a percentage of baseline diameter. In most studies, subjects are given a systemic vasodilator (usually a single dose of sublingual nitroglycerin) as a parallel experiment to assess endothelium-independent vasodilation.

Measurement of FMD is noninvasive and can evaluate the effects of cardiovascular risk factors on conductance vessels, the site of atherosclerotic plaque formation. In addition, given its simplicity, this technique can be used in relatively large populations of patients. This has led to the conduct of large-sized studies to investigate the effects of different pharmacological interventions on vascular function that have used FMD of the brachial artery as a surrogate end point of cardiovascular outcomes. However, there are important technical and interpretative limitations that must be taken into account when using FMD. This technique is highly dependent on the skills of the examiner. High-quality ultrasound images are essential for accurate analysis, typically requiring several months of hands-on training by experienced individuals, as well as continuous performance of the technique, to maintain optimal quality and consistency of the data. The interpretation of the images is subject to bias unless edge-detection techniques are used for the measurement of vessel diameter at critical time points. Computerized edge-detection and wall-tracking software systems have shown superiority to traditional manual measurements in reducing intraobserver variability,³³ suggesting that their use might improve reproducibility and allow for smaller sample sizes in intervention studies. However, there is still no consensus on the use of particular software system, and the lack of uniformly accepted standards in FMD testing makes comparison and reproducibility of the data from various laboratories very challenging. For example, positioning of the cuff (upper versus lower arm), duration of brachial artery occlusion, and timing for the detection of peak hyperemia tested, as well as the dose of nitroglycerin used to measure endothelium-independent dilation, still differ among investigators despite ongoing attempts for protocol standardization.^31,32,34 For example, when the cuff is positioned on the upper arm as opposed to the forearm, reactive hyperemia typically elicits a greater percentage of change in the diameter. This may be because of the greater recruitment of resistance vessels (NO-mediated event) or possibly because of direct effects of ischemia on the brachial artery (non-NO–mediated effects). In addition, upper arm occlusion has been reported to be technically more challenging, because the image is distorted by the collapse of the brachial artery during cuff inflation.³² Similarly, when assessing endothelium-independent vasodilation, the investigators can use either a 0.4-mg dose (old standard) of sublingual nitroglycerin or much smaller 0.025-mg dose that has been reported to be as effective in inducing vasodilation in healthy controls. Importantly, these and other interstudy variations add to the inherent biological variability in measurement, which can be affected by temperature, time of the day, diet, and drugs, as well as phase of the menstrual cycle. Baseline arterial diameter independently influences vasodilation, with small arteries dilating relatively more than the large arteries. Together, these variances in the technique may limit the interpretation of the study results, both in the interventional trials, which use time variations of repeated FMD measurements, as well as in the studies attempting to analyze correlations between FMD and cardiovascular risk in different populations.

Forearm Perfusion Technique
The forearm perfusion technique has been a well-established tool for the study of the human microcirculation for decades, because it permits the investigation of the peripheral vascular bed without the confounding effects resulting from the activation of systemic counterregulatory mechanisms. With this technique, a catheter is placed into the brachial artery, and drugs are infused, in small concentrations, directly into the forearm circulation. Blood flow is measured noninvasively, by means of strain gauge plethysmography, several times during infusion of the pharmacological agent (Figure). This technique is based on the principle that obstruction of the venous outflow (achieved by inflating a cuff around the upper part of the limb to a pressure below diastolic), in the setting of nondisturbed arterial inflow, leads to an increase in limb volume that is directly proportional to the rate of arterial inflow.³⁵ When the mercury strain gauge is placed around the forearm, the variations in limb volume are reflected in the changes of the circumference of the strain gauge (and, consequently, in its electrical resistance) that are recorded by the plethysmograph. With a properly calibrated strain gauge, the change in electrical resistance has a linear relationship with the change in forearm circumference and, hence, provides a quantitative estimate of volume and flow.³⁶ Venous occlusion plethysmography can be used in almost any limb segment; however, perfusion studies evaluating microvascular endothelial function have primarily focused on the forearm, given its steady blood flow under stable resting conditions. This is in contrast to the hand blood flow, which is strongly influenced by local skin temperature and fluctuates. Calf evaluations, on the other side, despite a relatively stable baseline blood flow, are rarely used, because significantly higher doses of drugs need to be infused (given the relatively larger size of the lower extremity), which commonly results in systemic effects and activation of autoregulatory mechanisms. With brachial artery infusion, systemic effects are not observed and, although several groups have used bilateral forearm plethysmography and forearm blood flow ratios (between the infused and noninfused or "control" arm)³⁷ to increase reproducibility and to adjust for any potential systemic changes or spontaneous changes in systemic vascular resistance, this approach has not been widely adopted.

The forearm perfusion technique has been extensively used in investigations of endothelial function of the peripheral microvasculature in patients with different atherosclerotic risk factors. The initial findings of blunted blood flow responses to acetylcholine in patients with hypertension^29,30 have been confirmed in the peripheral and coronary circulation of essential hypertensive patients,^38,39 as well as extended to patients with a secondary form of hypertension,⁴⁰ indicating that endothelial dysfunction is a generalized phenomenon associated with hypertensive conditions. Furthermore, given the critical role of the microvasculature in the regulation of vascular tone, these observations suggested that an endothelial defect may play a pathophysiological role in the increased vascular resistance characteristic of this condition. Similar findings have been described in patients with hypercholesterolemia and diabetes, and in smokers.^41–43 Importantly, forearm perfusion studies allow not only the detection of the impaired vasodilatory responses in patients with risk factors but also detailed investigation of the mechanisms underlying the observed abnormality. By infusing specific agonists and antagonists, the role of different pathways in endothelial regulation of vascular tone can be explored, and critical mediators of vasodilatory effects can be identified. This technique is highly reproducible and allows the construction of dose–response curves to assess efficacy, demonstrate antagonism, and compare responses between different groups of patients, thereby providing mechanistic insight into the pathophysiology of the endothelial dysfunction that cannot be easily explored in the coronary studies.

On the downside, the forearm perfusion method is invasive and, although it is considered safe in the hands of an experienced investigator, it is inappropriate for large-scale trials or intervention studies that involve multiple repeated measurements. In addition, this technique evaluates primarily resistance vessels, thus limiting extrapolation of its findings to the conduit vessels, which are the primary site of atherosclerosis.

Beyond Impaired Vascular Reactivity and Into Specific Modulators of Vascular Tone
The finding of blunted vascular responsiveness to acetylcholine does not allow characterization of the mechanisms involved in the abnormal endothelial function. However, the availability of other investigational drugs for use in human studies greatly expanded the potential for exploring endothelial function both in the peripheral and coronary circulation. Some of the agents that have been used to delineate biochemical pathways and mediators involved in endothelial function are summarized here.

In Furchgott’s¹⁰ initial observation, acetylcholine was demonstrated to induce the release of a nonprostanoid vasodilator substance that was termed endothelium-derived relaxing factor. Later, NO was identified as the main mediator of endothelium-derived relaxing factor effects. NO is synthesized by the enzyme NO synthase (NOS) using the amino acid L-arginine as a substrate. Three distinct NOS isoforms have been identified: endothelial, neuronal, and inducible NOS. NO stimulates guanylyl cyclase in the smooth muscle cells, which leads to vasodilation, and is destroyed primarily by superoxide anion formed both intracellularly and extracellularly. The use of L-arginine analogues that competitively inhibit NO synthesis allowed the investigation of the NO pathway and the identification of NO as the principal mediator of endothelial vasodilator actions in vivo. For example, brachial infusion of N^G-monomethyl-L-arginine, the first L-arginine analogue used in humans, significantly decreased baseline blood flow and attenuated vasodilator responses to acetylcholine in normal individuals.⁴⁴ In contrast, in patients with hypertension,³⁷ as well as in patients with hypercholesterolemia,³⁸ the N^G-monomethyl-L-arginine–induced reduction in baseline blood flow was significantly smaller than in control subjects, indicating lower basal NO activity in the microvasculature of these patients. Similar results were observed in the coronary circulation.⁴⁵ Together, these observations supported the hypothesis that, in patients with cardiovascular risk factors, characterized by abnormal vascular reactivity, a defect in the NO pathway might be responsible for the observed impairment in vascular responsiveness. The use of other endothelial agonists, such as substance P, a tachykinin agonist, and bradykinin, has allowed the study of endothelium-dependent vasodilation beyond the muscarinic receptor.^46,47 Infusion of L-arginine, the substrate for NO formation, and of tetrahydrobiopterin cofactor essential for the catalytic activity of all 3 isoforms of the NOS enzyme^48,49 has also expanded our ability to investigate the NO system in vivo. Finally, the degradation of NO (effected by superoxide anions under physiological conditions)^50,51 has been studied with the use of copper–zinc superoxide dismutase, an extracellular superoxide anion scavenger, by oxypurinol, an inhibitor of the most important intracellular source of superoxide radicals, the xanthine oxidase system, which can be pharmacologically inhibited by oxypurinol, and by the direct infusion of vitamin C, a nonspecific water-soluble antioxidant. Elevated levels of asymmetrical dimethylarginine, an endogenous NOS inhibitor, have been described in patients with cardiovascular risk factors, such as arterial hypertension, hyperlipidemia, and diabetes, as well as chronic kidney disease,⁵² suggesting that impaired NO synthesis might account for the endothelial dysfunction that is present in many of these conditions.⁵³

Although the NO system plays a primary role in endothelial physiology, other biochemical pathways may exert significant vasoactive actions on the vasculature. In particular, the role of cyclooxygenase products in the maintenance of vascular tone and in the endothelium-dependent vasodilation has been investigated by infusing acetylsalicylic acid, a noncompetitive cyclooxygenase inhibitor, into the brachial artery of healthy subjects⁵⁴ and of patients with hypertension and hypercholesterolemia.⁵⁵ The existence of additional mechanism(s) mediating endothelium-dependent vasodilation, other than the NOS and the cyclooxygenase pathways, was suggested in the experiments on isolated blood vessels where the action of endothelial vasodilators was, at least in part, resistant to NO inhibitors.⁵⁶ Because of its association with the hyperpolarization of the vascular smooth muscle cells, this pathway was then attributed to a noncharacterized endothelial factor termed endothelium-derived hyperpolarizing factor.⁵⁷ To explore the role of endothelium-derived hyperpolarizing factor in the regulation of vascular responses in humans, investigators used ouabain, which inhibits Na⁺K⁺/ATPase and thereby depolarizes the membrane, preventing endothelium-derived hyperpolarizing factor effects on the smooth muscle.⁵⁸

In addition to important vasodilators, such as NO, PGI₂ and endothelium-derived hyperpolarizing factor, the endothelium also synthesizes and releases constricting factors, which help to maintain and modulate vascular tone. ET-1, first isolated in 1988 by Yanagisawa et al,⁵⁹ is the most potent known vasoconstrictor produced by the endothelium and is 1 of the 3 members of the ET-1 family. ET-1 exerts its biological activity by binding to 2 specific ET-1 receptor subtypes: endothelin-A and endothelin-B.^60,61 In vascular smooth muscle cells, both receptors appear to induce contraction,⁶² whereas, in endothelial cells, the ET_B subtype stimulates the release of NO and PGI₂, which cause smooth muscle relaxation.⁶³ ET-1 secretion by endothelial cells is polarized toward the underlying vascular smooth muscle, thus exerting primarily autocrine and paracrine effects and rendering plasma levels less physiologically relevant. The availability of selective and nonselective blockers of endothelin-A and endothelin-B receptors for human use has allowed assessment of the role of ET-1 in cardiovascular homeostasis in vivo.¹²

Pulse Wave Analysis
Recently, alternative noninvasive techniques, based on the analysis of the arterial pulse waveform, have been implemented in the assessment of endothelial function. Applanation tonometry is a method that involves positioning the tonometer (a pencil-shaped probe) over the maximal arterial pulsation of the artery under study (typically a superficial artery, such as a radial, brachial, and femoral) to minimally flatten or applanate the arterial wall. This normalizes the circumferential stresses in the arterial wall thereby allowing accurate recording of the pressure waveform (through the changes of the electrical resistance of a piezoelectrical crystal within the tonometer).⁶⁴ The obtained pulse-waveform shape provides information about arterial compliance and serves as the basis for the calculation of the augmentation index (a ratio between the pulse pressure at the second systolic peak and the pulse pressure at the first systolic peak), which is commonly used as a measure of arterial stiffness. The changes in the peripheral pressure waveform, as measured by tonometry and quantified using augmentation index, in response to ß-2 adrenergic stimulation, have been tested in the assessment of global endothelial function.^65,66 In these studies, systemically given ß-2 receptor agonists (inhaled albuterol and salbutamol or subcutaneously injected terbutaline) diminished the reflected wave (or second systolic peak) of the pulse-wave contour and reduced the augmentation index in a similar fashion to sublingual nitroglycerin. Importantly, ß-2 agonist-induced, but not nitroglycerin-induced, changes in the arterial pressure waveform could be blunted by the N^G-monomethyl-L-arginine infusion, suggesting that ß-2 agonists, in part, mediate their effects on wave reflection through the endothelial NO release and that pulse wave methodology might be applied to the assessment of endothelial function.^65,66 In addition to applanation tonometry, pulse wave amplitude of the peripheral microvasculature can be assessed by measuring changes in digital pulse volume using a finger photopletysmograph (pulse contour analysis).⁶⁷ Endothelial NO production, stimulated by ß-2 receptor stimulation, results in characteristic changes in digital pulse volume, the response of which can be blocked by N^G-monomethyl-L-arginine and is impaired in patients with type 2 diabetes.⁶⁷ Reactive hyperemia peripheral artery tonometry also uses plethysmography to record digital volume changes accompanying pulse waves.⁶⁸ Digital hyperemic response assessed by reactive hyperemia peripheral artery tonometry has been shown to correlate with brachial artery FMD measurements,⁶⁸ as well as with coronary blood flow response in patients without significant angiographic evidence of coronary disease,⁶⁹ thus suggesting that it may be used as a noninvasive tool to identify patients with coronary microvascular endothelial dysfunction. This method was patented as Endo PAT 2000 and approved by the Food and Drug Administration for use as a diagnostic aid in patients with signs and symptoms of ischemic heart disease. Recently, FMD, pulse wave analysis and pulse contour analysis have been compared in the assessment of endothelial function and detection of inflammation-induced changes in vascular function in children and adults.⁷⁰ In this study, FMD was found to be the most reproducible method, followed by pulse wave analysis, whereas pulse contour analysis showed greater variability, particularly in children. The authors concluded that FMD remains the noninvasive technique of choice for the study of endothelial function in adults and in children, whereas pulse wave analysis and pulse contour analysis remain promising techniques which, until their reproducibility is improved, require much larger patient populations to achieve reproducible results.⁷⁰

Laser Doppler Flowmetry of the Skin
Laser Doppler flowmetry has been implemented to evaluate endothelial function of the skin microvasculature using postocclusive hyperemia, local thermal hyperemia, and acetylcholine iontophoresis. (For detailed description of these specialized techniques, refer to a recent review by Cracowski et al.⁷¹). Despite being noninvasive and, therefore, attractive for routine clinical and research use, these techniques have certain limitations. First, because the skin is a critical thermoregulatory organ, there are extreme variations in basal blood flux, which, in turn, dictate the need to use maximal vasodilatation (by either local warming of the skin or local sodium nitroprusside infusion) to normalize submaximal flux values. Second, poor interassay and intra-assay reproducibility and lack of standardization (eg, site of the skin measurement) limit within-patient and across-studies comparisons. Third, and most important, recent insights into the mechanisms of the postocclusive hyperemia⁷² and acetylcholine-mediated dilatation⁷³ indicate that these phenomena are not primarily NO mediated, suggesting that they might represent a summation of complex, microvascular responses involving sensory nerves and metabolic and endothelial (independent from NO) vasodilators. Therefore, rather than representing specific markers of endothelial function, these tests provide a more global form of assessing microvascular function.

Markers of Thrombosis
Under physiological conditions, the endothelium prevents thrombus formation through a number of mechanisms. Thrombomodulin, protein S, heparin sulfate, and tissue factor pathway inhibitor are all endothelium-derived inhibitors of coagulation, whereas PGI₂, NO, and surface-bound CD39 inhibit platelet aggregation.⁷⁴ However, when endothelial function is perturbed, for example, with altered shear stress or inflammation, the vascular lining rapidly converts from a nonthrombogenic surface to a procoagulant one. This shift is because of the downregulation of the anticoagulant factors, as well as the activation of prothrombotic mediators. For instance, induced expression of tissue factor (normally not expressed by endothelial cells), plasminogen activator inhibitor (PAI-1), as well as increased secretion of von Willebrand factor, have been described in atherosclerotic vessels, where they may impair fibrinolysis and potentiate thrombus formation.^17,75

The plasma levels of several procoagulant mediators have been shown to increase with endothelial damage, suggesting that they could represent reliable markers of endothelial dysfunction. Furthermore, an alteration in the production of these molecules by the endothelium could directly contribute to atherotrombotic disease. However, prospective epidemiological studies aiming to evaluate the association between plasma levels of different hemostatic markers and the risk for cardiovascular disease are relatively sparse and often with inconclusive results.

von Willebrand factor, a large glycoprotein produced mainly by vascular endothelial cells, enhances thrombosis by acting as an important cofactor in platelet adhesion and aggregation, as well as the carrier for factor VIII.⁷⁶ The commercially available ELISA for detection of von Willebrand factor antigen in plasma or serum has been used in several large population studies that investigated the association between von Willebrand factor levels and the risk of future coronary disease or stroke.^13,77

Tissue plasminogen activator (t-PA) and PAI-1 are 2 other major, endothelium-derived, coagulation markers of which the plasma levels have been associated with increased cardiovascular risk. PAI-1 is a member of the family of serine protease inhibitors that is synthesized by the liver and adipose tissue in addition to the endothelium.⁷⁸ Through binding to serine proteases, such as t-PA, PAI-1 inhibits cleavage of plasminogen, promotes clot stabilization, and potently attenuates the fibrinolytic cascade. Overexpression of human PAI-1 in transgenic mice was shown to induce coronary artery thrombosis and subendothelial infarction⁷⁹ supporting the hypothesis that abnormal fibrinolysis could play a role in cardiovascular disease in humans. However, 2 prospective population studies in men and women with a high prevalence of coronary artery disease reported that, although levels of PAI-1 appeared to positively correlate with the risk for cardiovascular events, this association was not statistically significant after the adjustment for traditional risk factors.^80,81 Commercially available ELISA tests (used in epidemiological studies) measure either the plasma concentration or the activity of circulating PAI-1 antigen, both of which have been shown to be influenced by numerous factors, such as circadian rhythm, insulin, glucose, and neurohormonal mediators, as well as acute-phase reactants. In turn, these inflammatory, metabolic, and neurohormonal processes may independently or synergistically contribute to endothelial damage, raising the question of whether the increased PAI-1 antigen levels are the marker of prevalent endothelial dysfunction or whether they represent a net activation of endogenous procoagulant response to the underlying atherosclerosic process. Investigators have tried to answer this question in part by evaluating the levels of its counterpart in the fibrinolytic system, t-PA. t-PA is primarily synthesized and secreted by the vascular endothelium, and its plasma levels do not show the circadian variation characteristic of PAI-1. Multiple epidemiological studies have demonstrated that elevated plasma levels of t-PA correlate with increased cardiovascular risk, which may appear counterintuitive, given the profibrinolytic role of t-PA in the process of hemostasis; however, increased t-PA antigen levels (typically measured by ELISA) are a correlate of increased plasma PAI-1 activity and are, thus, associated with increased cardiovascular risk.

Another approach to examine more dynamic and potentially informative aspects of t-PA release and its association with endothelial function and atherosclerosis is in vivo assessment of acute t-PA release.⁸² In this model, different stimuli, such as desmopressin⁸³ or bradykinin,⁸⁴ are used to stimulate endothelial cells to release t-PA. To avoid confounding hemodynamic effects related to the systemic infusion, as well as the activation of the sympathetic nervous system and concomitant release of other mediators, investigators have primarily focused on the measurement of the t-PA release in individual vascular beds. Regional forearm t-PA release in response to intrabrachial infusions has been assessed by using 2 similar methodologies: the arteriovenous technique,⁸⁵ based on the differences in plasma concentrations of t-PA between the inflowing arterial and outflowing venous plasma of a single arm, and the venovenous technique,⁸⁶ based on the differences in venous plasma concentrations between the 2 arms. Both methods use strain gauge plethysmography to measure forearm blood flow which, multiplied by the difference in the t-PA measured and corrected for hematocrit, gives the net t-PA release in the forearm. Similarly, dynamic t-PA release can be calculated in the coronary circulation by infusing the agents into the coronary artery and measuring coronary blood flow by intracoronary Doppler combined with quantitative coronary angiography or intravascular ultrasound.⁸⁷ An impairment in acute endothelial t-PA release has been reported in smokers⁸⁸ and in patients with hypertension⁸⁹ despite preserved endothelium-dependent vasodilation suggesting that reduced t-PA release may be a more sensitive marker of endothelial dysfunction. The limitations of this technique are primarily related to its invasive nature. In particular, coronary t-PA release, although likely to be of greatest relevance to coronary pathophysiology, can be performed only in subjects undergoing coronary angiography. Forearm assessments, similar to the ones evaluating endothelium-dependent vasodilation, can be performed more widely but are not suitable for large epidemiological investigations or longitudinal studies requiring repetitive measurements.

Markers of Inflammation
Strong evidence suggests that atherosclerotic risk factors are often associated with systemic inflammation, which is a key player in the development and progression of atherosclerosis.³ The endothelium not only actively produces proinflammatory and anti-inflammatory molecules, but it also represents the actual target for the circulating inflammatory mediators that are synthesized by other cell types, including platelets, leukocytes, hepatocytes, and adipocytes.¹⁵ In this way, diverse systemic inflammatory processes may lead to the impairment in endothelial function, which then further contributes to the proinflammatory milieu by increasing the expression of cellular adhesion molecules (CAMs), synthesizing cytokines (eg, interleukin-8 and monocyte chemoattractant protein-1), and downregulating NO and PGI₂ release. Importantly, these markers of inflammation may be predictive of clinical disease. The CAMs, including P- and E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1, mediate leukocyte rolling (selectins) and attachment (vascular cell adhesion molecule-1 and intercellular adhesion molecule-1) to the endothelium. Although increased cell surface expression of these molecules is difficult to quantify in vivo, soluble forms can now be quantitated in the circulation by the enzyme immunoassays.¹⁶ This is particularly useful in large-scale, prospective studies in which stored, frozen peripheral blood samples can be used for the measurement of different markers. Using this approach, baseline plasma levels of intercellular adhesion molecule-1 and E-selectin have been shown to be associated with increased cardiovascular risk in generally healthy population,⁹⁰ whereas, in the population with previously documented coronary artery disease, elevated circulating vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 values were predictors of adverse outcome.⁹¹ However, the results from other large studies indicated that, after the adjustments for other cardiovascular risk factors, the association of CAMs with coronary heart disease was not statistically significant.^92,93 Together, these observations suggest that soluble forms of adhesion markers may reflect a generalized inflammatory state and that further investigations are needed to determine the role of CAMs in the development and progression of atherosclerosis. C-reactive protein is the best characterized of the currently available biomarkers and has emerged as a potential marker for cardiovascular risk.⁹⁴ It is mainly synthesized in the liver, as an acute phase reactant, but is also produced in smooth muscle cells within human coronary arteries and is expressed preferentially in diseased vessels.⁹⁵ C-reactive protein can be measured with several standardized, validated, and inexpensive high-sensitivity assays and is the only biomarker ready for clinical use.⁹⁶ In fact, high-sensitivity C-reactive protein measurements seem to add predictive value above the currently established risk factors.⁹⁷ Other various inflammatory markers have been investigated for their association with cardiovascular disease and potential clinical use in atherosclerotic risk assessment; for detailed reviews, refer to the American Heart Association/Centers for Disease Control and Prevention Scientific Statement⁹⁷ and similar specialized publications.⁹⁸

Endothelial Progenitor Cells
In healthy subjects, the endothelium undergoes a low basal turnover, which is reflected in very low amounts of circulating, vessel wall–derived endothelial cells.⁹⁹ In situations of acute and chronic injury, which trigger endothelial cell apoptosis and consequent denudation of the endothelial layer, the number of circulating endothelial cells rapidly increases, suggesting that these cells may play a role in the regeneration of the endothelium in areas of damage.¹⁰⁰ However, mature endothelial cells are terminally differentiated cells with a low proliferative potential, and their capacity to substitute damaged endothelium is limited. Therefore, the endothelial repair may depend on the presence of other cell types. Accumulating evidence indicates that peripheral blood of adult humans contains a unique subtype of circulating, bone marrow–derived cells with properties similar to those of embryonal angioblasts.¹⁰⁰ These cells, commonly referred to as endothelial progenitor cells (EPCs), have the potential to proliferate and to differentiate into mature endothelial cells.¹⁰⁰ Intriguingly, EPCs may provide a circulating pool of cells that could form a cellular patch at the site of denuding injury and serve as a cellular reservoir to replace dysfunctional endothelium. In support of this hypothesis, recent data suggest that, in patients with cardiovascular risk factors, the number of EPCs may be a surrogate biologic marker for vascular function and cumulative cardiovascular risk.¹⁹ Furthermore, the results of a recent study in patients with coronary artery disease indicate that low levels of circulating EPCs may be associated with the occurrence of cardiovascular events and death from cardiovascular causes.⁹⁹ In turn, this would suggest that increasing the number of circulating EPCs might result in improved cardiovascular outcomes.

Genetic Markers
With the recent completion of the Human Genome Project, there has been an increasing focus on elucidating genetic susceptibility to the common multifactorial diseases, such as atherosclerosis and cardiovascular disease.¹⁰¹ The identification of genetic markers in these polygenic diseases is, however, difficult, because they may involve large number of genes acting in complex networks, thus resulting in a small individual gene effect size on the disease state. In addition, the precise characterization of the reproducible study phenotype (if possible, by using detailed physiological testing) represents an important prerequisite for the investigation of genetic variants, but may prove challenging in complex diseases, such as hypertension, dyslipidemia, and atherosclerosis, which encompass a wide spectrum of phenotypic presentations. The investigation of genetic markers linked to endothelial dysfunction overcomes some of these difficulties. The endothelial function, which, in genetic associations analysis, is often referred to as an intermediate phenotype of atherosclerosis (ie, an early, subclinical phenotype that occurs over the natural course of disease progression), may be assessed by fine physiological measurements, thus allowing for the precise definition of the phenotype studied.¹⁰¹ Moreover, endothelial dysfunction occurs early in the disease process, when the secondary changes in the disease phenotype, which often obscure genetic association, are much less likely to be present. In addition, from a clinical perspective, the investigation of subclinical stages in the pathogenesis of atherosclerosis offers an important advantage of identifying younger, healthy subjects without symptoms who are at risk for the development of the disease.

A number of candidate genes involved in endothelial function have been tested in the genetic association studies using primarily single nucleotide polymorphisms (or variants at a single DNA base pair). Given the critical importance of NO in endothelial function, genes involved in NO synthesis and/or degradation were of particular interest. To date, more than 100 polymorphisms have been identified in, or the in vicinity, of the NOS 3 gene.¹⁰² However, it is unlikely that individual polymorphism, such as endothelial NOS Asp298, may make a useful contribution in identifying asymptomatic individuals at increased risk for atherosclerosis, because genetic contribution to cardiovascular disease is likely mediated through small-to-moderate effects of many genes.¹⁰³ Similar conclusions were drawn from the studies on the polymorphisms of other genes of which the products have been implicated in endothelial dysfunction (eg, methylene hydrofolate reductase, bradykinin receptor, interleukin-6, leukocyte adhesion molecule-1, and angiotensin-converting enzyme) that were either limited to a single investigation or yielded inconsistent results.¹⁰⁴ Future genetic association studies, whether assessing clinical end points, or intermediate phenotypes such as endothelial function, need to extend their focus on the analysis of multiple genes and their combined effects on the classical cardiovascular risk profile. The development of new high-throughput genotyping technologies, as well as the use of alternative approaches that allow genomewide analysis (as opposed to single molecule approach), such as DNA microarrays and serial analysis of gene expression, represent great potential for further clarification of the molecular pathways involved in endothelial dysfunction and atherosclerosis.

Prognostic Implications of Assessing Endothelial Function
Endothelial dysfunction is the earliest detectable stage in the development of atherosclerosis³ and may contribute to the clinical expression and outcome of atherosclerotic disease.^9,105–108 An impaired endothelium-dependent vasodilation is a marker of adverse prognosis in patients with risk factors without atherosclerosis,^6,7 in patients with stable ischemic heart disease,^6,109 and in patients with acute coronary syndromes.^5,8 Importantly, both coronary^7,109 and forearm^7,8,109 blood flow responses to intra-arterial infusion of acetylcholine independently predict cardiovascular events, including death from cardiovascular causes, myocardial infarction, ischemic stroke, coronary angioplasty, and coronary or peripheral bypass operation in patients with documented coronary artery disease. Thus, endothelial dysfunction appears to be a systemic vascular process that identifies patients who have increased risk for cardiovascular events in the short and long term. A recent review identified several studies that support the concept that impaired endothelium-dependent vasodilation is associated with poor prognosis.¹⁰⁵ Although these studies investigated different vascular beds and used different methods, including coronary responses to the acetylcholine infusion⁷ and cold-pressor test,¹⁰⁷ as well as venous occlusion plethysmography^6,109 and brachial ultrasound,^9,108 in assessment of the peripheral microvasculature and macrovasculature, respectively, they all demonstrated that endothelial vasodilatory function independently predicts future cardiovascular events.

Other markers of endothelial function, such as circulating procoagulant, prothrombotic, and proinflammatory mediators, have also been shown to be associated with increased risk for atherosclerosis but, except for C-reactive protein, the evidence for their independent value in predicting increased cardiovascular risk is still lacking. This is also the case for EPCs and genetic markers of endothelial function. Future studies are needed to determine which individual marker or combination of markers provides independent information about increased risk for atherosclerotic disease and how it relates to traditional cardiovascular risk factors.

	Conclusions

Top Introduction Methods for Assessing... Conclusions References

 
Endothelial dysfunction is an early process in several cardiovascular conditions, which may contribute to the progression and clinical manifestations of atherosclerotic disease. Therefore, studies of endothelial function provide insight into the mechanisms of atherosclerosis and may help identify individuals who are at increased risk. Several techniques have been developed and applied to the investigation of endothelial function and dysfunction in humans. Together, they allow a comprehensive assessment of the different roles of the endothelium, such as the regulation of the vascular tone, control of leukocyte and platelet adhesion and aggregation, and anti-inflammatory and antithrombotic endothelial function. These methods have been used in mechanistic studies and in investigations using endothelial function as a surrogate marker for the evaluation of interventions that reduce cardiovascular risk. However, most studies evaluating the utility of endothelial function as a screening test were relatively small; therefore, their findings still await confirmation from large epidemiological trials. Similarly, the assessment of endothelial function for the management of individual patients, such as using FMD to identify patients who might benefit from more intensive treatment, holds great promise, but there is insufficient evidence to support these applications at the present time. The use of the standardized approaches and their implementation in large-scale outcome studies, accompanied by further development of both established and new methods of assessing endothelial function, will be critical in determining its potential role in the clinical arena.

	Acknowledgments

 
Disclosures

None.

Received September 20, 2006; first decision October 9, 2006; accepted January 24, 2007.

	References

Top Introduction Methods for Assessing... Conclusions References

 

1. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990; 323: 27–36.[Medline] [Order article via Infotrieve]
2. Drexler H. Endothelial dysfunction: clinical implications. Prog Cardiovasc Dis. 1997; 39: 287–324.[CrossRef][Medline] [Order article via Infotrieve]
3. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.[Free Full Text]
4. Panza JA. Endothelial dysfunction in essential hypertension. Clin Cardiol. 1997; 20 (suppl 2): II26–II33.
5. Halcox JP, Quyyumi AA. Coronary vascular endothelial function and myocardial ischemia: why should we worry about endothelial dysfunction? Coron Artery Dis. 2001; 12: 475–484.[CrossRef][Medline] [Order article via Infotrieve]
6. Perticone F, Ceravolo R, Pujia A, Ventura G, Iacopino S, Scozzafava A, Ferraro A, Chello M, Mastroroberto P, Verdecchia P, Schillaci G. Prognostic significance of endothelial dysfunction in hypertensive patients. Circulation. 2001; 104: 191–196.[Abstract/Free Full Text]
7. Halcox JP, Schenke WH, Zalos G, Mincemoyer R, Prasad A, Waclawiw MA, Nour KR, Quyyumi AA. Prognostic value of coronary vascular endothelial dysfunction. Circulation. 2002; 106: 653–658.[Abstract/Free Full Text]
8. Fichtlscherer S, Breuer S, Zeiher AM. Prognostic value of systemic endothelial dysfunction in patients with acute coronary syndromes: further evidence for the existence of the "vulnerable" patient. Circulation. 2004; 110: 1926–1932.[Abstract/Free Full Text]
9. Modena MG, Bonetti L, Coppi F, Bursi F, Rossi R. Prognostic role of reversible endothelial dysfunction in hypertensive postmenopausal women. J Am Coll Cardiol. 2002; 40: 505–510.[Abstract/Free Full Text]
10. Furchgott RF. The discovery of endothelium-dependent relaxation. Circulation. 1993; 87 (suppl 5): V3–V8.
11. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992; 340: 1111–1115.[CrossRef][Medline] [Order article via Infotrieve]
12. Cardillo C, Kilcoyne CM, Waclawiw M, Cannon RO, 3rd, Panza JA. Role of endothelin in the increased vascular tone of patients with essential hypertension. Hypertension. 1999; 33: 753–758.[Abstract/Free Full Text]
13. Folsom AR, Wu KK, Rosamond WD, Sharrett AR, Chambless LE. Prospective study of hemostatic factors and incidence of coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study. Circulation. 1997; 96: 1102–1108.[Abstract/Free Full Text]
14. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation. 2004; 109 (suppl 1): III27–III32.[Medline] [Order article via Infotrieve]
15. Bhatt DL. Coronary artery inflammation. In: Topol EJ, ed. Acute Coronary Syndromes. New York, NY: Marcell Dekker, Inc; 2005: 1–31.
16. Gearing AJ, Newman W. Circulating adhesion molecules in disease. Immunol Today. 1993; 14: 506–512.[CrossRef][Medline] [Order article via Infotrieve]
17. Padro T, Steins M, Li CX, Mesters RM, Hammel D, Scheld HH, Kienast J. Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants. Cardiovasc Res. 1997; 36: 28–36.[CrossRef][Medline] [Order article via Infotrieve]
18. Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Bohm M, Nickenig G. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005; 353: 999–1007.[Abstract/Free Full Text]
19. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 348: 593–600.[Abstract/Free Full Text]
20. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.[CrossRef][Medline] [Order article via Infotrieve]
21. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986; 315: 1046–1051.[Abstract]
22. Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD, Yeung AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. Circulation. 1990; 81: 491–497.[Abstract/Free Full Text]
23. Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation. 1993; 88: 2541–2547.[Abstract/Free Full Text]
24. Cox DA, Vita JA, Treasure CB, Fish RD, Alexander RW, Ganz P, Selwyn AP. Atherosclerosis impairs flow-mediated dilation of coronary arteries in humans. Circulation. 1989; 80: 458–465.[Abstract/Free Full Text]
25. Drexler H, Zeiher AM, Wollschlager H, Meinertz T, Just H, Bonzel T. Flow-dependent coronary artery dilatation in humans. Circulation. 1989; 80: 466–474.[Abstract/Free Full Text]
26. Treasure CB, Klein JL, Vita JA, Manoukian SV, Renwick GH, Selwyn AP, Ganz P, Alexander RW. Hypertension and left ventricular hypertrophy are associated with impaired endothelium-mediated relaxation in human coronary resistance vessels. Circulation. 1993; 87: 86–93.[Abstract/Free Full Text]
27. Nabel EG, Ganz P, Gordon JB, Alexander RW, Selwyn AP. Dilation of normal and constriction of atherosclerotic coronary arteries caused by the cold pressor test. Circulation. 1988; 77: 43–52.[Abstract/Free Full Text]
28. Gordon JB, Ganz P, Nabel EG, Fish RD, Zebede J, Mudge GH, Alexander RW, Selwyn AP. Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise. J Clin Invest. 1989; 83: 1946–1952.[Medline] [Order article via Infotrieve]
29. Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE. Abnormal endothelium- dependent vascular relaxation in patients with essential hypertension. N Engl J Med. 1990; 323: 22–27.[Abstract]
30. Linder L, Kiowski W, Buhler FR, Luscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo. Blunted response in essential hypertension. Circulation. 1990; 81: 1762–1767.[Abstract/Free Full Text]
31. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002; 39: 257–265.[Abstract/Free Full Text]
32. Deanfield J, Donald A, Ferri C, Giannattasio C, Halcox J, Halligan S, Lerman A, Mancia G, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti A, Schiffrin EL, Taddei S, Webb DJ. Endothelial function and dysfunction. Part I: Methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. J Hypertens. 2005; 23: 7–17.[CrossRef][Medline] [Order article via Infotrieve]
33. Woodman RJ, Playford DA, Watts GF, Cheetham C, Reed C, Taylor RR, Puddey IB, Beilin LJ, Burke V, Mori TA, Green D. Improved analysis of brachial artery ultrasound using a novel edge-detection software system. J Appl Physiol. 2001; 91: 929–937.[Abstract/Free Full Text]
34. Kuvin JT, Patel AR, Karas RH. Need for standardization of noninvasive assessment of vascular endothelial function. Am Heart J. 2001; 141: 327–328.[CrossRef][Medline] [Order article via Infotrieve]
35. Whitney RJ. The measurement of volume changes in human limbs. J Physiol. 1953; 121: 1–27.[Free Full Text]
36. Greenfield AD, Whitney RJ, Mowbray JF. Methods for the investigation of peripheral blood flow. Br Med Bull. 1963; 19: 101–109.[Medline] [Order article via Infotrieve]
37. Calver A, Collier J, Moncada S, Vallance P. Effect of local intra-arterial NG-monomethyl-L-arginine in patients with hypertension: the nitric oxide dilator mechanism appears abnormal. J Hypertens. 1992; 10: 1025–1031.[Medline] [Order article via Infotrieve]
38. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Role of endothelium- derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation. 1993; 87: 1468–1474.[Abstract/Free Full Text]
39. Brush JE Jr, Faxon DP, Salmon S, Jacobs AK, Ryan TJ. Abnormal endothelium-dependent coronary vasomotion in hypertensive patients. J Am Coll Cardiol. 1992; 19: 809–815.[Abstract]
40. Taddei S, Virdis A, Mattei P, Salvetti A. Vasodilation to acetylcholine in primary and secondary forms of human hypertension. Hypertension. 1993; 21: 929–933.[Abstract/Free Full Text]
41. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest. 1990; 86: 228–234.[Medline] [Order article via Infotrieve]
42. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AD. Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest. 1996; 97: 2601–2610.[Medline] [Order article via Infotrieve]
43. Heitzer T, Yla-Herttuala S, Luoma J, Kurz S, Munzel T, Just H, Olschewski M, Drexler H. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia. Role of oxidized LDL. Circulation. 1996; 93: 1346–1353.[Abstract/Free Full Text]
44. Vallance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet. 1989; 2: 997–1000.[Medline] [Order article via Infotrieve]
45. Quyyumi AA, Mulcahy D, Andrews NP, Husain S, Panza JA, Cannon RO 3rd. Coronary vascular nitric oxide activity in hypertension and hypercholesterolemia. Comparison of acetylcholine and substance P. Circulation. 1997; 95: 104–110.[Abstract/Free Full Text]
46. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA. Impaired endothelium- dependent vasodilation in patients with essential hypertension: evidence that the abnormality is not at the muscarinic receptor level. J Am Coll Cardiol. 1994; 23: 1610–1616.[Abstract]
47. Panza JA, Garcia CE, Kilcoyne CM, Quyyumi AA, Cannon RO 3rd. Impaired endothelium-dependent vasodilation in patients with essential hypertension. Evidence that nitric oxide abnormality is not localized to a single signal transduction pathway. Circulation. 1995; 91: 1732–1738.