The authors would like to thank the many subjects and patients who have participated in our studies in which we have evaluated endothelial function using the FMD test.

NOTE: The following FMD procedure is routinely conducted during vascular assessment studies in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP). All procedures followed the principles of the Declaration of Helsinki and were approved by the Institutional Review Board at Georgia Regents University. All participants were informed of the objectives and possible risks of the technique before written consent for participation was obtained. Figure 1 illustrates a schematic summary of the essential elements tha...

Introduced in 198920, the FMD test has been widely used in humans as a non-invasive measure of endothelial function. FMD has not only been shown to predict future vascular-related disease risk19,52,53, lower FMD values have been show to strongly correlate with cardiovascular impairments24,25,54. Although there are other techniques to assess endothelial function, both invasively (coronary angiography) and non-invasively (venous plethysmography and finger plethysmography), FMD has been the ...

Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Dysfunction of the vascular endothelium represents an initial phase toward the development of multiple vascular-related diseases1. Hence, an accurate assessment of endothelial function in humans represents an important technique that could help in understanding the etiology of multiple cardiovascular pathologies, with the ultimate goal of improving the efficacy of the treatment and prevention of disease.
Endothelium
The endothelium is a monolayer of cells that synthesizes numerous vasoactive substances, such as nitric oxide (NO), prostacyclins, endothelins, endothelial cell growth factor, interleukins, and plasminogen inhibitors2. Such factors contribute to the endothelium&#39;s function to regulate blood fluidity, vascular tone, platelet aggregation, permeability of plasma components and vessel wall inflammation2-4. Additionally, NO plays a key anti-atherogenic role in promoting vasodilation and maintaining endothelial integrity. NO regulates vessel tone and diameter through controlling the equilibrium between the delivery of oxygen to the tissues and their metabolic demand3,5. There are multiple endogenous, exogenous, and mechanical stimulator factors that induce endothelial NO synthase (eNOS) which synthesizes NO from L-arginine6,7. The most notable mechanical stimulus is shear stress. Wall shear stress contributes to greater activation of eNOS, resulting in NO production and subsequent smooth muscle relaxation4. For that reason the decrease in NO bioavailability is often used as a measure of endothelial dysfunction8.
Endothelium dysfunction
The imbalance between vasodilator and vasoconstrictor factors leads to a dysfunctional endothelium2. In addition, the release of inflammatory mediators and altered local shear forces may enhance the synthesis of endothelial derived reactive oxygen species (ROS). This upregulation in redox signaling not only modifies the integrity of the endothelium and reduces the synthesis of NO9, it can uncouple eNOS resulting in direct production of additional free radicals. Ultimately, this amelioration in NO bioavailability promotes vasoconstriction, vascular stiffness, and reduced arterial distensibility4.
The degree of dysfunction of the endothelium has been related with the severity of several pathologies such as hypertension10, atherosclerosis11, ischemic stroke12, diabetes13, preeclampsia14 or kidney diseases15 among others. Hence, there is vast interest to not only evaluate changes in endothelial function over time, but also following therapeutic interventions. Different methods have been used for the clinical assessment of endothelial function both invasively (cardiac catheterization and venous occlusion plethysmography3,16) and non-invasively (flow mediated dilation, radial artery tonometry and pulse contour analysis4,17,18) in coronary and peripheral circulations19.
Flow-mediated dilation
Flow mediated dilation (FMD) is a non-invasive, ultrasonic evaluation of endothelial function and has been correlated with the development of vascular health problems. Since its inception in 198920, FMD has been widely utilized as a reliable, in vivo method to evaluate predominately NO-mediated endothelial function in humans19,21,22. Indeed, the brachial artery FMD test has been associated with other invasive techniques23 and numerous investigations have described a strong inverse relationship between FMD and cardiovascular injury24,25 such that individuals with more vascular pathology exhibit a lower FMD25. Accordingly, these data emphasize the prognostic information that this technique can provide as it relates to future cardiovascular disease in asymptomatic subjects26-30.
During the FMD test, the diameters of the brachial artery are continuously measured at baseline and after the release of a circulatory arrest of the forearm. Upon cuff release, the induced-reactive hyperemia promotes an increase in shear stress mediated NO release and subsequent vasodilation19,31. FMD is expressed as the percent increase in arterial diameter following the release of the cuff compared with the diameter at baseline (FMD%).
Despite the increasing clinical interest in this technique, the FMD test is a physiological assessment and therefore, several variables need to be considered in order to conduct a precise assessment of endothelial function in humans. This article describes a standardized protocol and the recommended methodology to minimize the technical and biological issues to help improve the accuracy, reproducibility and interpretation of the FMD test.

<table>
	<tbody>
		<tr>
			<td>Doppler ultrasound</td>
			<td>GE Medical Systems&#160;</td>
			<td>Logiq 7</td>
			<td>Essential to include Duplex mode for simultaneous acquisition of B-mode and Doppler</td>
		</tr>
		<tr>
			<td>Electrocardiographic (ECG) gating&#160;</td>
			<td>Accusync Medical Research</td>
			<td>Accusync 72</td>
			<td></td>
		</tr>
		<tr>
			<td>12-MHz Linear array transducer&#160;</td>
			<td>GE Medical Systems</td>
			<td>11L-D</td>
			<td>A high-resolution linear array probe is essential</td>
		</tr>
		<tr>
			<td>Forearm occlusion cuff&#160;</td>
			<td>D.E. Hokanson</td>
			<td>SC5</td>
			<td>5 x 84 cm</td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel&#160;</td>
			<td>Parker</td>
			<td>01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid cuff inflator</td>
			<td>D.E. Hokanson</td>
			<td>E-20 AG101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterotactic-probe holder</td>
			<td>Flexabar&#160;</td>
			<td>18047</td>
			<td>Magnetic base fine adjustor</td>
		</tr>
		<tr>
			<td>Edge detection analysis software</td>
			<td>Medical Imaging Applications</td>
			<td>Brachial Analyzer 5</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrasound assessment of endothelial function: a technical guideline of the flow-mediated dilation test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

Baseline characteristics from an apparently healthy cohort group are presented in Table 1. The most common variables of FMD testing conducted in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP) are presented in Table 2. The following variables are considered the main FMD parameters to analyze by the published FMD tutorial4 and guidelines36.
Baseline an...

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Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...