Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

Summary

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

Abstract

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.

Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.

The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.

There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.

This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Introduction

The human vascular endothelium provides structural and functional roles within the body. In histological sections, the endothelium appears small, comprising a thin layer of cells 1-2 microns thick sitting atop a layer of smooth muscle cells (the media) and a thick layer of connective tissue (the adventitia). Taken as a whole, the endothelium provides a wide area for the exchange of information between the blood and vascular smooth muscle tissue. By one estimate, a cross sectional area of 700 m² and a mass of 1,000-1,500 grams in a 70 kg man, is comparable in mass to the liver¹. A healthy endothelium allows for mechanical to chemical signal transduction to maintain homeostasis of the blood vessel. Endothelial dysfunction is an imbalance of these mediators and the first step in vascular disease, present prior to histological evidence of atherosclerosis. A non-invasive, in vivo method for quantifying the vasodilatory function of human artery exists. This method, endothelium-dependent, flow-mediated vasodilation (FMD) is widely used in clinical trials.

The endothelium acts as a structural component of the vasculature and manufactures components of the extracellular matrix such as glycosaminoglycans and fibronectin². Long term changes in blood flow and acute injury to the artery may lead to structural changes. Functionally, the vascular endothelial cells participate in regulation of vessel tone, inflammatory processes, antithrombosis, and anticoagulation. Endothelial cells affect vasoconstriction through endothelin while vasodilation is mediated by nitric oxide (NO), prostacyclin, and endothelial derived hyperpolarizing factor (EDHF)^3-6.

Endothelial dysfunction is an impairment of any of these mediators and the first step in atherosclerosis. Not surprisingly, as a mechanism of disease, it is associated with a number of clinically important conditions such as coronary artery disease, hypertension and diabetes mellitus^7-11. Importantly, endothelial dysfunction can be observed in individuals without diagnosed cardiovascular disease and is predictive of future cardiovascular events^7,12,13. One measure of endothelial dysfunction, in combination with the Framingham score, can provide additional prognostic information above either measure alone¹⁴.

Measures of endothelial dysfunction may involve the direct infusion of a pharmacological agent. Intercoronary infusion of acetylcholine, for example, combined with quantitative angiography demonstrates vasodilation in subjects with an intact endothelium. However, individuals with endothelial damage experience paradoxical vasoconstriction.¹⁵ In peripheral arteries, infusion of a pharmacological agent with measurement of flow by gauge-strain plethysmography is possible¹⁶.

Agents that directly affect the endothelium and elicit a chemical signal are termed endothelium-dependent vasodilators. Acetylcholine, for example, acts on muscarinic receptors on endothelial cells, leading to increased intra-cellular calcium concentration, activation of nitric oxide synthase and vasodilation. Agents that affect vasodilation without involvement of the endothelium are called endothelium-independent agents. Nitroglycerin, for example, activates soluble guanyl cyclase and cyclic guanosine-3’,-5’-monophasphate (cGMP) which mediates vasodilation in the vessel wall through protein kinases regulating intracellular calcium concentrations¹⁷.

There is a non-invasive, in vivo method for quantifying endothelial dysfunction introduced by Celermajer and associates called “flow-mediated, endothelium-dependent vasodilation” (FMD)¹⁸. Briefly, changes to arterial blood flow open shear stress sensitive ion channels in the endothelium. The signal is tranduced via a second-messenger cascade and activates endothelial nitric oxide synthase (eNOS), generating NO. This species diffuses across the cell membrane to neighboring smooth muscle cells (SMC). Within the SMC, the signal is transduced, lowering intracellular calcium concentration and affecting vasorelaxation¹⁹. The diameter of the artery lumen increases, leading to an increase in blood flow consistent with the Hagen-Poiseullie equation. The effect of FMD may be abolished with administration of an NO synthase inhibitor such as mono-methylarginine (L-NMMA)²⁰.

Celermajer et al.’s innovative work has allowed the use of high resolution B-mode ultrasound to assess the change in artery diameter during the reactive hyperemia that follows ischemia. In this technique, a human subject rests supine and the diameter of the brachial artery is measured in a longitudinal plane. A blood-pressure cuff is used to produce ischemia in the limb. Following release of the blood pressure cuff the diameter of the artery is measured again. The rapid change in shear stress is the stimulus for NO mediated vasodilation. A simple equation describes the change in the diameter relative to the baseline diameter (Equation 1). A full discussion of the parameters of this equation, hyperemia and baseline diameter, can be found in the Protocol and Results sections.

In multiple studies, percent FMD has been found to predict cardiovascular events in patient with established cardiovascular disease^21-24. A correlation between brachial artery percent FMD and coronary artery FMD was established by Anderson et al., demonstrating a link between peripheral measurements and the more clinically-relevant ischemic changes to the heart²⁵. FMD does not demonstrate the maximum vasodilation of the vessel. To evaluate this, FMD can be followed by endothelium-dependent, nitroglycerin-mediated vasodilation of the same vessel.

There are technical issues affecting the measurement of percent FMD. Since the introduction of the technique, several studies showed a high degree of within-subject and inter-operator variability²⁶. It has been shown that physiological factors such as cigarette smoking, antihypertensive medications, time of day, and fasting state affect percent FMD. Likewise, technical choices such as the position of the cuff relative to the site of measurement and duration of occlusion have been shown to affect the measurement^27,28. Guidelines have been published that describe the current consensus and allow for standardization of technique between laboratories^19,29.

Despite the evolving consensus on technique, flow-mediated vasodilation remains heavily operator dependent with a long learning curve. Corretti, for example, recommends the sonographer complete 100 scans under the supervision of an experienced investigator before operating independently. To maintain a level of adequate expertise, it is recommended the technician complete 100 scans annually. For investigators with a small sample population and limited resources, the learning curve presents a barrier to entry. This article will demonstrate a method for flow-mediated vasodilation of the brachial artery in the upper arm and offer technical suggestions to reduce intra-operator variability.

Protocol

The following procedure, developed as part of an investigator-initiated study, was reviewed and approved by the University of California, San Francisco (UCSF) Committee on Human Research (CHR) and all participants gave informed consent.

1. Equipment

1. Use an EKG gated image capture system to record and analyze the FMD. Connect a Philips HD11 ultrasound to a desktop PC.
2. Connect a video signal from the ultrasound with a special frame-grabber card on the PC.
3. Relay an audio signal from the ultrasound to an EKG gating module which amplifies the signal. Carry the amplified signal to the PC, to allow the image capture software to identify and record images at a consistent point in the cardiac cycle. Generate the signal from the sharp deflection of the R-wave in the EKG.
4. Use a 5-12 MHz linear array transducer to optimize resolution at the depth of the brachial artery.

2. Subject Preparation

1. Ensure that participants fast and avoid exercise for 8 hr before the exam as well as avoid caffeine or nicotine for at least four fours. Ensure that participants avoid medications affecting vascular tone or cardiac output for four half-lives.
   NOTE: Diet, medications, and time of day may affect results.
2. Conduct the exam in a quiet, darkened room at 21 °C. When conducting longitudinal studies, hold repeat exams at the same time of day.

3. Baseline Measurements

1. Ask the subject to lie supine on an exam table. Attach a 3-lead EKG in a standard position. Address any orthopedic issues to ensure the subject will be comfortable and refrain from movement during the exam.
2. Allow the subject to rest for 10 min before the start of the exam. After 5 min rest, measure the subject's blood pressure by an oscillometric, non-invasive blood-pressure monitor.
   1. Apply a 5 cm tourniquet cuff in either a proximal or distal position to demonstrate the upper arm technique.
   2. Extend the subject’s arm laterally and maintain at the level of the heart.
   3. Depending upon the operator's preference, use a table and pillow to constrain the subject’s arm.
   4. Place the arm of the operator in a position that resists fatigue and provides support for the wrist. Try to minimize extension of the wrist and keep the forearm in the anatomic neutral position.
3. Conduct a cross-sectional scan of the brachial artery, beginning at the insertion of the bicep and proceeding proximally. Use color flow imaging to verify the brachial artery and to locate collateral vessels that may serve as landmarks.
4. When a suitable position is found, rotate the probe 90° so that the proximal edge appears on the left of the ultrasound screen. Maintain position on the artery using substantial practice and a delicate touch. Verify the orientation by pushing the tissue near the distal edge. Mark the subject’s skin along the distal edge of the probe.
5. Align the focus setting of the probe with the deep or “far” wall of the brachial artery to improve lateral resolution of the image. Vary probe settings on the axial resolution with a higher frequency improving axial resolution.
6. Adjust the angle of the probe to optimize contrast resolution of both the near and the far walls. Small changes to the angle can result in improved contrast. Estimate the angle with a simple protractor if serial exams are conducted on the subject.
7. To ensure quality measurements, ensure that the vessel is horizontal and aligned with the longitudinal axis. Make small changes in pressure (heeling one edge of the probe) to help align the artery. Overall, keep pressure light to help prevent operator fatigue.
8. When optimized, ensure that the “Double Lines of Pignoli” can be seen in both walls, corresponding to the intima-media thickness. Use gain adjustments to reduce echo in the vessel lumen. Allow at least 2 cm of intima-mediate thickness (IMT) on both sides for accurate diameter measurements.

4. Baseline Measurements

1. Record the baseline velocity using 2D Doppler mode. Place the sample gates in the middle of the lumen and maintain an insonation angle of 60°. Collect 60 sec of data.

5. Occlusion Phase

1. Inflate the cuff to 50 mm Hg above the subject’s systolic blood pressure. Using a 5 cm tourniquet cuff will overestimate the systolic pressure. Use the 2D Doppler mode to verify occlusion.
2. Use a timer to track the duration of occlusion as many blood pressure cuffs will slowly lose pressure over 5 min. Use 2D Doppler mode to verify complete occlusion.
3. After 4:30 of occlusion, place the 2D-Doppler gate slightly superficial to the longitudinal axis of the artery. Adjust the vertical scale to account for velocities 2-3x higher than baseline.
4. Adjust the settings on the image capture software for 3:10 of recording.
5. Begin recording 10 sec before cuff release to capture the time of cuff release, an important parameter when measuring time to peak diameter during data analysis.

6. Hyperemia

1. Release the cuff. As the artery may shift superficially after cuff release, make small changes to the probe's position while listening for amplification of the sound to help compensate for the shift. Reposition the Doppler sample gate and insonation angle if the artery shifts.
2. After 30 sec of velocity recording, switch the ultrasound to B-Mode.
3. Since it is common for the probe to slide proximally during an exam, use vessel landmarks or the marking on the subjects' skin to verify probe position. This phase of the exam is critical for obtaining accurate results.
4. Adjust the probe position or angle to optimize the IMT on both walls as small changes can substantially improve the image. Record the diameter for 3 min.
5. If repeat measurements are planned, use the marking on the subject's skin to record distance from the antecubital fossa. Ask the subject to bend their arm 90° and mark the crease. Measure from this line to the line made earlier.

Results

The key variables of flow-mediated vasodilation are shown in Table 1.

Variable	Description
Mean Velocity (cm/sec)	The mean arterial velocity of blood in the middle 50% of the lumen during one cardiac cycle estimated from Doppler spectral waveforms, proportional to blood flow and inversely proportional to cross-sectional area (see Fi...

Discussion

Endothelial dysfunction is an imbalance in the chemical mediators affecting vessel tone and an early step in the development of atherosclerosis. Measuring the reactivity of an artery is a way to assess the state of these chemical pathways. Both direct and indirect methods of assessing reactivity exist for the different vascular beds, ranging from direct infusion of an endothelium agonist in the coronary circulation to non-invasive, pulse waveform analysis in the index finger³⁸.

Brac...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Philips HD 11XE ultrasound	Philips Healthcare
5-12 MHz linear array transducer	Philips Healthcare	L12-5
Ultrasound gel	Parker Laboratories
Vascular Research Tools v.5.0	Medical Imaging Applications, LLC
MIA Gating module	Medical Imaging Applications, LLC
Windows XP	Microsoft, Inc
Hand-held aneroid manometer	Welch Allyn	DS66