The authors would like to thank Dr George Balanos for his assistance in the flow-mediated dilatation technique.

NOTE: The protocol follows guidelines from Dudley Group NHS Foundation Trust&#8217;s Human Research Ethics Committee. Perform all described techniques in a temperature controlled laboratory (21 &#8211; 22&#160;oC), with stable lighting and absence of noise. Ask individuals undergoing assessments to refrain from food, drink, smoking and exercise 12 hr&#160;prior to the test. Withhold vasoactive medications for at least 12 hr&#160;when appropriate.
1. Laser Doppler Imaging with Iontophoresis
<ol>
	<li>Switch on Laser Doppler Imager (LDI) and allow the scanner to automatically stabilise for approximately 30 min. Start the LDI software and click &#8216;Measurement&#8217; (the software&#8217;s home screen will then be shown). On the home screen, select &#8216;Ionto Protocol&#8217; on the taskbar located at the top of the window.</li>
	<li>Manually enter the protocol (the protocol used in our laboratory involves a total of 13 scans, with electrical current for iontophoretic drug delivery set from scan 2 to scan 11 at a voltage of 30 &#181;a). Set scan 1 as a baseline scan with no electric current, and scan 12 &#38; 13 as recovery scans also with no electrical current. Click OK to confirm settings and return to home screen.</li>
	<li>Ask participant to relax in a semi-recumbent chair with their forearm resting 90 degrees on a comfortable, firm pillow, and place a black mat under the forearm. 
	NOTE: The mat helps to limit artefact measurements generated by background surfaces surrounding the tissue. It is important that the participants arm is strapped firmly to the pillow so that there is no movement and associated artefacts.</li>
	<li>Connect the wired plugs at the opposite end of each perspex chamber to the iontophoresis controller. Connect the chamber containing a 2.5 ml dose of 1% acetylcholine (ACh) to the anodal connection of the iontophoresis controller, and connect the second chamber containing a 2.5 ml dose of 1% sodium-nitroprusside (SNP) to the cathodal connection. Mix both agents in the chamber using 0.5% saline solution. Connect the two chambers to the volar aspect of the participant&#8217;s right forearm using double sided adhesive pads.</li>
	<li>Cover the chambers by 32 mm coverslips to prevent leakage of fluid.</li>
	<li>Before starting the scan, open the &#8216;Scanner Setup&#8217; window located on the top left of the home screen. Select the &#8216;Video and Distance&#8217; tab and select the &#8216;auto distance&#8217; function to measure the distance of the scanner head from the participants forearm.
	<ol>
		<li>Following completion of the auto distance measurement, select the &#8216;Image Scan&#8217; tab and determine the area that is to be scanned by clicking the &#8216;Mark&#8217; button in the bottom right corner of the window. If needed, change the size of the region of interest by manually entering in the size of the scanning area into the &#8216;Scan Area&#8217; section near the top of the window. Ensure that the region of interest includes the diameter of each of the iontophoresis chambers and is large enough to limit variability in skin blood flow.</li>
	</ol>
	</li>
	<li>Following completion of the assessment, save the data file. Open the data file using LDI image analysis software to perform measurements of perfusion.
	<ol>
		<li>Click &#8216;Image Review&#8217; on the main software window, and open the image file that is to be analysed.</li>
		<li>Use the software to mark out a region of interest around the outer diameters of each chamber. Adjust the region of interest so that it fits correctly on the area where the chambers were present. Then click the &#8216;statistics&#8217; icon and a column containing the median perfusion units for each chamber will be displayed. Note the baseline perfusion unit, as well as the highest perfusion unit from each of the preceding 12 scans for each chamber. 
		NOTE: This method of analysis is specific to our laboratory; however, other methods can be used to express data obtained from the LDI scan. For a comprehensive review please refer to guidelines from Roustit and Cracowski 12.</li>
	</ol>
	</li>
	<li>To calculate percentage change in perfusion in response to ACh and SNP, subtract baseline perfusion from the peak perfusion, divide by baseline perfusion and then multiply by 100. 
	NOTE: In our lab, changes in perfusion relative to baseline have shown good intra-observer coefficient of variation for ACh (7%) and SNP (6%).</li>
</ol>
2. Flow-mediated Dilatation and Glyceryl trinitrate-mediated Dilatation
<ol>
	<li>Switch on the Doppler ultrasound machine and networked PC containing vascular image analysis (VIA) software. 
	NOTE: The VIA software captures a live image (at 25 frames per sec) and provides information on the vessel diameter as well as the quality of the vascular borders being detected by the ultrasound machine. Other software packages are available which may contain additional features and settings. It is advisable to consult operating manuals for specific software.</li>
	<li>Ask participant to relax in a semi-recumbent armchair and place their arm on a comfortable pillow out to their side but level with the heart. Place a blood pressure cuff around the participant&#8217;s wrist. 
	NOTE: The patient should be asked to keep their arm as still as possible to prevent movement artefacts during the measurement.</li>
	<li>Secure the linear array transducer from the ultrasound machine into a stereotactic clamp, and tighten the clamp using the wingnuts so that the ultrasound transducer remains in a fixed position. 
	NOTE: The clamp will ensure that the ultrasound transducer will remain stable once the blood vessel is located.</li>
	<li>On the ultrasound machine, scroll into the &#8216;Menu&#8217; and set the scanning frequency at 5 MHz and optimize the depth (the recommended depth setting is 3.5 cm) and gain settings on the ultrasound machine. Adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel.</li>
	<li>Using the linear array transducer, locate the brachial artery which is usually found 2-10 cm above the antecubital fossa in the longitudinal scanning plane. Make any adjustments to clarify the image quality at this stage. To help identify the artery, turn on the colour Doppler to help show pulsatile arterial blood flow and distinguish it from continuous venous blood flow. View the brachial artery horizontally across the screen; it should appear as two solid parallel lines, separated by a clear area in between the lines which represents the lumen of the vessel.</li>
	<li>To allow the VIA software to automatically record vessel diameter, use the cursor to mark a predetermined region of interest to detect and track the anterior and posterior walls of the artery. 
	NOTE: The size of the region of interest can be increased or decreased using the &#8216;x&#8217; and &#8216;y&#8217; buttons located on the main software screen.</li>
	<li>Click &#8216;Start&#8217; on the VIA software and image the artery for 2 min. Following this, press &#8216;Inflate&#8217; on the VIA software and simultaneously inflate the blood pressure cuff placed around the wrist to suprasystolic pressures (usually above 220 mmHg) for 5 min. 
	NOTE: The purpose of the wrist cuff is to occlude blood flow to the hand.</li>
	<li>After 5 min&#160;deflate the blood pressure cuff to induce reactive hyperaemia which, in a healthy vessel, will stimulate NO-mediated vasodilatation. 
	NOTE: Peak dilatation can occur up to 180 sec&#160;following cuff deflation, so it is advisable to continue recording vascular diameters for 3 min&#160;after cuff release.</li>
	<li>Following a 10 min rest period, re-locate the brachial artery using the linear array transducer and record a 2 min&#160;baseline diameter reading in the same manner as step 2.7.</li>
	<li>Then ask the participant to place a 500 &#181;g sublingual glyceryl-trinitrate (GTN) tablet under their tongue and continue to measure the brachial artery diameter for a further 5 min. After this period, ask the participant to remove the GTN tablet and monitor the participant to make sure they do not experience any adverse effects to the drug.</li>
	<li>Carry out all analysis of data offline. Twenty-five data points are available for each second of the assessment; collapse this data into one-second epochs in Microsoft Excel. Export the data to a digital signal analysis package and filter with a 3 sec moving average filter.</li>
	<li>Establish the baseline diameter from the 120 sec&#160;of data prior to the cuff-inflation. Visually inspect the baseline region and exclude artefacts. Average the remaining baseline regions to produce the baseline diameter.</li>
	<li>For the flow-mediated dilatation (FMD) analysis, use the software to automatically scan the post cuff-deflation region for peak dilation and use the cursor to mark this peak for visual inspection. If the peak has been misidentified, use the cursor to select a more confined region within which the peak could then be identified. Record the peak value as peak diameter.</li>
	<li>For the GTN data, adopt an identical procedure to that used with FMD, except search for peak dilation in the region following the 5&#160;min&#160;of drug administration.</li>
	<li>To calculate FMD % and GTN %, subtract baseline diameter from the peak diameter, divide by baseline diameter and then multiply by 100. 
	NOTE: In our laboratory, the intra-observer coefficient of variation is 11% for FMD, and 12% for GTN.</li>
</ol>
3. Carotid Intima-media Thickness
<ol>
	<li>Ask participant to lie comfortably on a bed, and place a pillow under the head to offer support to the neck.</li>
	<li>Connect the electrocardiogram (ECG) leads to the Doppler ultrasound and then attach them onto the patient limbs. Only a basic ECG trace is required, so place the appropriate leads on the left and right arms, and on the left ankle.</li>
	<li>Prepare the ultrasound machine by scrolling through the &#8216;menu&#8217; and setting the scanning frequency at 10 MHz and optimizing depth (the recommended depth setting is 3 &#8211; 4 cm) and gain settings. Adjust the gain settings to ensure that there is symmetrical brightness for the near and far wall of the vessel.</li>
	<li>Ask participant to tilt their head slightly to the left, and using the linear array transducer, scan the right carotid artery along all its sections (common, internal and external carotid artery) using the longitudinal scanning plane to identify the presence of any plaques. Save images that display any evidence of plaque. To help identify the artery, look for a bifurcation point in the vessel, as this shows the common carotid artery bifurcating into the internal and external carotid arteries.</li>
	<li>For measurement of carotid intima-media thickness (cIMT), attain at least 3 images of a section of the common carotid artery that is free of plaque, and is 1 cm proximal to the carotid bulb. Attain all images at the peak of the R wave on the ECG as this corresponds to ventricular diastole and the point at which the vessel is under the least amount of shear stress.</li>
	<li>Repeat steps 3.4 and 3.5 in the left carotid artery. Ask the participant to tilt their head slightly to the right for this measurement.</li>
	<li>To assist in attaining clear images of the near and far walls, carefully manipulate the ultrasound probe during the assessment to ensure the vessel is perpendicular to the ultrasound beam. Achieve this by subtly changing the tilt and rotation of the transducer along with minor adjustments to the pressure applied to the proximal-to-distal angle (heel-toe movement) of the probe.</li>
	<li>Carry out analysis of images offline using Artery Measurement Software (AMS) to detect the vascular boundary according to the lines of Pignoli. Load up the image to be analyzed, and then using the cursor, create a region of interest in a section of the vessel that is free from plaque. Click &#8216;detect&#8217; on the software and record the values displayed on the screen for cIMT and lumen diameter. 
	NOTE: accurate readings can only be obtained from the far wall, so ignore readings from the near wall.</li>
	<li>Take three measurements for each side, and then average these to give the mean cIMT for the right and left carotid arteries separately. Further average the cIMT from both sides to give the overall cIMT. 
	NOTE: The intra-observer coefficient of variation for this technique in our laboratory is 9%.</li>
	<li>Perform measurement of any plaque using the same software by manually marking out the plaque using the cursor. Click &#8216;classify&#8217; on AMS to automatically calculate the echogenicity of the plaque and grade according to its susceptibility for rupture. Click on the &#8220;Plaque Characteristics&#8221; window to see this information.</li>
</ol>

The authors declare that they have no competing financial interests.

The present manuscript details the methodology of several distinct assessments of vascular function and morphology which can be performed in the peripheral vasculature. Each assessment provides information on the distinct stages of atherosclerosis, and help to characterize the vascular profile of different vascular territories.
We have previously reported that microvascular endothelial function is independent from large vessel endothelial function in a population of rheumatoid arthritis patients at increased risk of CVD 39. Moreover, assessments of vascular function and morphology were also independent from each other in the same group of patients and in patients with CVD 40,41. These findings can be explained by the heterogeneity of function and structure of endothelial cells in different vascular territories 38, as well as a possible time lag for progression of functional alterations to morphological abnormalities in the vessel. A study by Hashimoto and colleagues 42 revealed that several participants with atherosclerosis had decreased FMD values but normal cIMT values. These findings suggest that examination of subclinical atherosclerosis using a variety of methods is important to decipher the global effects of CVD.
The importance of the microvasculature in health and disease is gaining increasing attention in the medical literature. The microvessels form a much larger surface area than large vessels making them significant targets for damage from injurious stimuli 43. It has been hypothesized that microvessels might be the primary source of inflammatory mediators which infiltrate the endothelium of the larger vessels leading to lesion formation 43. In type II diabetics, microvascular disease often precedes large vessel disease 44, and in other populations with increased risk for CVD such as rheumatoid arthritis, interventions which reduce the CVD risk improve microvascular, but not large vessel, endothelial function 45,46. Collectively, these findings suggest that examination of microvascular function may help in understanding the complex mechanisms which initiate atherosclerosis.
In the present work, assessment of microvascular endothelial function was performed using LDI with iontophoresis of vasoactive agents. Several other assessments can be used to assess microvascular function including nailfold capillaroscopy and venous occlusion plethysmography. However, the former assessment provides information on microvascular morphology only, while the latter is time consuming and in some protocols invasive due to administration of intra-brachial vasoactive agents 1. In contrast, LDI offers a simple, time-efficient approach to measure microvascular perfusion of skin blood vessels in response to vasoactive agents which are administered non-invasively. The measurement of skin blood flow has gained widespread acceptance in the literature due to its ease of accessibility and strong correlation with established CVD 12. Moreover, the advantage of LDI over other Doppler techniques such as laser Doppler flowmetry, is that it can simultaneously scan multiple points in a given area and can therefore account for cellular movement artefacts and spatial differences of skin blood flow, both of which can affect the perfusion of the vessel 47,48.
Despite the obvious advantages of iontophoresis, it is important to note that current induced vasodilatation (CIV) from iontophoresis may confound the effects of the vasoactive agents especially at the cathode. The choice of vehicle for drug delivery could help to reduce this effect, with 0.5% sodium chloride (as used in the current protocol) effective in limiting CIV 18. Furthermore, use of larger diameter chambers and low electrical currents (as used in the current protocol) all help to reduce CIV 18. Use of a control site has also been recommended 12. Biological and behavioural factors can also affect the reliability and repeatability of the technique. For example, circadian variation and smoking have been shown to influence microvascular endothelial function 49,50. Strict recording conditions must be adhered to in order to obtain accurate results and established guidelines should be followed when designing protocols 12.
Measurement of FMD and GTN-mediated dilatation provides information on endothelial dysfunction in the large blood vessels, and is used widely in non-invasive vascular research. The FMD technique provides surrogate information on NO bioavailability and is a useful prognostic marker of cardiac events in different clinical populations 7-9. In the present work, the protocol presented accounts for many of the factors that are necessary for adequate stimulation of NO-mediated vasodilatation 25. For example, the occluding cuff was placed distal to the ultrasound probe and around the wrist 27, the duration of ischemia was 5 min&#160;26 and adequate time was allowed to record the &#8216;true&#8217; peak diameter following reactive hyperaemia 28. Unfortunately, the protocol did not include characterization of the shear stress profile as the automatic edge detection software did not allow simultaneous recording of vessel diameter and pulse wave velocity signal. The calculation of shear stress is integral to accurate measurement of FMD 26 and we recommended that, where possible, vascular research groups use software which allows such measurements to be performed.
Assessments of FMD and GTN-mediated dilatation are also susceptible to environmental and biological variations 24, as small changes in vascular diameter can elicit large FMD/GTN responses. For example, typical FMD values for healthy participants range from 5-10% 51, which corresponds to a 0.25 &#8211; 0.5mm change in arterial diameter for an artery with a diameter of 5mm. Given such small changes to the arterial diameter, careful attention must be paid to technical and biological factors that may influence the measurement. Indeed, FMD can be affected by a variety of biologic and behavioural factors such as sympathetic activation 52, sleep deprivation 53, caffeine consumption 54, smoking 55, antioxidant therapy 56 and time of day 57. Accordingly, it is important to control for these factors by utilising information from established guidelines 24,25.
Assessment of advanced but subclinical atherosclerosis was done using cIMT. The technique has been utilised in several clinical populations and provides great detail on arterial structure when compared to more sophisticated techniques such as magnetic resonance imaging 34. As with the other vascular techniques, measurement of cIMT requires careful consideration of technical factors which can affect the measurement. Generally, cIMT should be performed in areas free from plaque, in the far wall of the common carotid artery. Similar to FMD, measurement of cIMT is performed using high-resolution ultrasonography and so is highly user-dependent. Reported coefficient of variation (CofV) range from 2.4 &#8211; 18.3% 58, while for FMD it is 1 &#8211; 84% 59. However, even when both techniques are performed by competent ultrasonographers with external factors well-controlled, there remains a high CofV 58,60,61. One reason for this could be that analysis of vascular borders are carried out using manual methods 60,61. Such analysis can reduce reliability as imaging artefacts such as false borders, noise from the ultrasound signal, and distortion of vessels can affect interpretation of the image 22.
Recent developments in continuous automated edge-detection software have greatly improved the detection of vascular wall boundaries 21,22. In the present study, VIA software was used to measure the brachial artery diameter, while AMS was used to detect cIMT. The use of these software greatly reduces operator dependence, yet in the case of AMS, some degree of operator control is still available in situations where image quality may be poor 62. Laboratories which use automated edge detection software generally tend to have low CofV 58,63,64, thus it should be the aim of all vascular research laboratories to incorporate automated measurement of vascular boundaries in order to ensure accuracy of the results. It is also good practice to report results of reproducibility studies for specific protocols when publishing study outcomes.
Although assessments of vascular function are routinely used in clinical research, a limitation of the techniques is that normative values for LDI with iontophoresis and FMD do not exist. It is therefore important that healthy age- and sex-matched control groups are examined to compare findings with the experimental group. Even though these techniques associate with poor prognosis in a variety of populations with evidence of CVD 6-9, there is still a dearth of studies which have examined the relationship between poor endothelial function and adverse cardiovascular outcomes such as myocardial infarction and stroke. Further prospective studies are required to address these concerns. Another limitation is the use of human operators to perform the assessments and carry out the analysis. This introduces a potential source of bias; however, this can be limited by blinding the operator to the results or ensuring that the reader is different from the operator. It is also important to ensure that the reader follows a standardised protocol for data analysis, so that all data is analysed consistently.
In summary, the present manuscript provides detailed information on the methodological steps necessary to successfully perform assessments of microvessel and large vessel endothelial function as well as vascular morphology of the peripheral circulation. When used together, the assessments provide global information on the different stages of atherosclerosis. Further prospective studies examining the potential diagnostic role of these techniques are warranted.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis via the regulation of a multitude of vasoactive processes. Disruption to these processes may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease (CVD) 1. Peripheral endothelial function is a good indicator of early abnormalities in the vascular wall 2. Furthermore, measures of peripheral endothelial function have been shown to reflect coronary endothelial function 3-5, and as such are regarded as good predictors of cardiovascular disease 6-9. This is perhaps hardly surprising given that atherosclerosis is now broadly appreciated to be a systemic disorder 10. Assessments of peripheral endothelial function typically quantify the vasodilatory response of the vessel to a specific stimulus, with an attenuation of the dilatory response indicative of endothelial dysfunction 11, and can be measured in different vascular beds. Assessments of advanced structural changes in the vessel can be characterised by ultrasound examination of the intima-media thickness.
In the microcirculation, laser Doppler flowmetry (LDF) and Laser Doppler imaging (LDI) with iontophoresis of vasodilator agonists can provide useful information on microvascular perfusion 12. Both techniques measure the Doppler shift created by scattered light from moving red blood cells. Perfusion is represented as blood flux rather than blood flow (ml/min), with blood flux reflecting average red blood cell velocity and concentration. Measurement of blood flux is linearly associated with actual blood flow 13. The assessment of LDI offers considerable advantages over LDF, because unlike LDF, LDI can scan over a vast area thus accounting for heterogeneity in skin blood flow and increasing the reproducibility of the technique 12.
The stimulus for increasing blood flux during LDI are provided by iontophoresis of vasodilator agonists acetylcholine (ACh) and sodium nitroprusside (SNP), which assess endothelium-dependent and endothelium-independent function respectively, into the skin using a weak electrical current 14. Once through the skin, ACh binds to endothelial cell muscarinic receptors releasing the vasodilator nitric oxide (NO). The use of SNP directly activates smooth muscle cell receptors to allow for maximum vasodilatation of the vessel and examination of smooth muscle integrity 15. There is some uncertainty on whether ACh-mediated dilatation involves NO at all, as ACh may stimulate non-NO pathways such as cycloxygenase-mediated pathways12. Nevertheless, we have previously reported that ACh and SNP responses are impaired in patient populations at increased risk of CVD 16 and that exercise interventions known to improve NO bioactivity also improve ACh-mediated blood flux using LDI 17. The vehicle for transporting the agents into the skin microvessels often include sodium chloride or deionised water 18,19. Microvascular endothelial function can be quantified using different approaches, with cutaneous vascular conductance &#8211; a product of flux divided by arterial pressure, used in studies where blood pressure may change over the study duration (i.e., during exercise or anti-hypertensive treatment) 12. Another commonly used quantification is to calculate the area under the curve for blood flux or express the percentage increase in flux from baseline. It is important to note that there are no established guidelines for presenting data, but investigators should utilise an approach which shows good reproducibility.
In the large vessels, flow-mediated dilatation (FMD) and glyceryl-trinitrate mediated dilatation (GTN) are performed to assess endothelium-dependent and endothelium-independent function respectively 20. FMD is typically carried out in the brachial artery where a cuff is used to occlude arterial blood flow for 5 min; release of the cuff causes a sudden increase in blood flow (reactive hyperaemia) through the brachial artery resulting in shear-stress mediated dilatation of the vessel. The baseline and post-cuff release diameter are quantified by ultrasound imaging of the vessel with subsequent assessments of the vessel diameter performed manually 20 or using automated edge detection software 21,22. The use of GTN helps to determine if abnormalities in vasodilatation are due to a loss in smooth muscle cell integrity, or impaired release of NO from the endothelial cells23. FMD and GTN are expressed as the percentage increase in post-stimulus vessel diameter relative to the baseline diameter.
The correct assessment of FMD requires a number of important considerations in the study protocol 24,25. The duration of cuff occlusion must be carefully timed; 5 min&#160;of cuff occlusion is sufficient for NO-mediated dilatation while longer cuff occlusion results in non-NO mediated dilatation 26. Similarly, placement of the occluding cuff around the wrist and distal from the ultrasound probe predominantly invokes NO-mediated dilatation, whereas cuff placement on the upper arm and proximal to the probe only partially stimulates NO 27. It is also important to measure peak dilatation following cuff deflation over a protracted period of time, as measurement of peak diameter within the first 60 sec&#160;following cuff deflation can underestimate FMD by 25 &#8211; 40% 28. Indeed, a period of 180 sec&#160;is likely to be sufficient in capturing true peak diameter, with most peak values occurring within the first 120 sec&#160;28.
The stimulus for FMD involves the production of shear stress, which activates specific endothelial receptors to release NO 29. However, shear stress may also activate several other vasoactive factors (some of which may cause vasoconstriction) 30, making it essential that the evoked shear stress stimulus reflects vasodilation from NO pathways 26. It is also important to account for the shear stress stimulus during FMD, with calculation of shear rate (velocity/diameter) serving as an adequate measure of shear stress, but not necessarily reflecting peak flow 31. Recent physiological recommendations suggest that the shear stress profile should always be characterised when ultrasound systems allow simultaneous measurement of pulse wave velocity and active B-mode imaging in duplex mode 25.
Assessment of the carotid arteries using B-mode ultrasound can provide information on the carotid intima-media thickness (cIMT), and was first described in 1986 by Pignoli and colleagues 32. Assessment of cIMT reflects proliferation of smooth muscle cells into the intima of the vessel and is a useful predictor of clinical events in early atherosclerosis 33. Carotid ultrasound can often predict arterial structure better than similar techniques (such as magnetic resonance imaging or radiographic assessments) 34. In addition, cIMT associates with a number of classical CVD risk factors including ageing, hypertension, and dyslipidemia 35. Changes to the walls of the carotid artery are usually initiated by a reduction in NO bioavailability which promotes inflammation within the vessel 36. The common carotid artery, internal carotid artery and carotid bifurcation points can be used to determine cIMT, as each site can similarly predict cardiovascular events 37.
In the present manuscript, we provide detailed methodology on assessment of microvascular endothelial function (LDI with iontophoresis), large vessel endothelial function (FMD and GTN) and vascular morphology (cIMT). Atherosclerosis is a multi-stage process which begins with endothelial dysfunction and ends with focal atherosclerotic lesions in the large arteries. The rationale for choosing the above assessments is that they reflect the different stages of atherosclerosis and help to account for the heterogeneous nature of the vasculature 38. Furthermore, we have previously shown that in a population of patients at increased risk for CVD, microvascular endothelial function was independent from large vessel endothelial function 39, and functional assessments were independent from structural assessments of the vasculature 40. Therefore, global assessments of the vasculature can help to decipher the different stages of atherosclerosis.

<table><tbody><tr><td>Laser Doppler Imager</td><td>Moor Instruments, Devon, UK</td><td>moorLDI2</td><td></td></tr><tr><td>Iontophoresis Controller</td><td>Moor Instruments, Devon, UK</td><td>MIC2</td><td></td></tr><tr><td>Miochol-E 20 mg</td><td>Novartis UK</td><td>Prescribed by physician</td><td>Acetylcholine for endothelium-dependent function</td></tr><tr><td>Nitroprussiat Fides 50 mg</td><td>Rottapharm Spain</td><td>Prescribed by physician</td><td>Sodium nitroprusside for endothelium-independent function</td></tr><tr><td>Doppler Ultrasound</td><td>Siemens PLC, Camberley UK</td><td>Accuson Antares</td><td></td></tr><tr><td>Glyceryl Trinitrate 500 mcg</td><td>Alpharma, Barnstaple, UK</td><td>Prescribed by physician</td><td></td></tr></tbody></table>

a methodological approach to non-invasive assessments of vascular function and morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

Laser Doppler Imaging with Iontophoresis
The median blood flux units following the laser Doppler imaging scans from a healthy middle aged female free from CVD are shown in Figure 1. There was a marked increase in median blood flux for both ACh and SNP. Baseline blood flux was 48 perfusion units for ACh, and 67 perfusion units for SNP. Peak blood flux in response to ACh was 455 perfusion units, and for SNP 446 perfusion units. This yielded an 831% and 566% increase in perfusion (relative to baseline) for ACh and SNP respectively. The values that are provided are highly-dependent on the equipment used to examine skin blood flux along with environmental factors.
Flow-mediated dilatation and Glyceryl trinitrate-mediated Dilation
Figure 2 displays the baseline and peak diameters for FMD and GTN assessments from a healthy young male free from CVD. The baseline diameter of the brachial artery was 3.0 mm for the FMD and GTN assessments. The peak diameter in the FMD test was 3.3 mm, while for the GTN assessment it was 3.9 mm, which corresponds to a 10 and 30% increase in blood flow respectively, relative to baseline.
Carotid Intima-media Thickness
Figure 3 shows the left carotid artery of a healthy individual. Calculation of cIMT values is performed using automated edge-detection software. The cIMT in the far wall was 0.83mm and the lumen diameter of the vessel was 7.71mm. The results for the right carotid artery in the same individual were 0.87mm for cIMT, and 7.80mm for the lumen diameter. When averaging the reading from both sides, cIMT was 0.85mm, and lumen diameter was 7.76mm.
<img alt="Figure 1" src="/files/ftp_upload/52339/52339fig1highres.jpg" /> 
Figure 1. Changes in blood flux in response to laser Doppler imaging with iontophoresis. After completion of a baseline scan to measure baseline blood flux, 10 scans (scan 1 to 10) with iontophoresis of ACh and SNP using a 30 &#181;a electrical current were performed. Following iontophoresis, 2 recovery scans were performed. ACh = acetylcholine; SNP = sodium nitroprusside.
<img alt="Figure 2" src="/files/ftp_upload/52339/52339fig2highres.jpg" /> 
Figure 2. Flow-mediated and glyceryl trinitrate-mediated dilatation. The graph displays the baseline diameter and a clear increase in peak diameters following application of the flow-mediated and glyceryl trinitrate-mediated dilatation stimuli. FMD = flow-mediated dilatation; GTN = glyceryl trinitrate-mediated dilatation.
<img alt="Figure 3" src="/files/ftp_upload/52339/52339fig3highres.jpg" /> 
Figure 3. Ultrasound scan of the carotid artery. An ultrasound scan of the left carotid artery is shown with a region of interest placed 1cm from the carotid bulb (point of bifurcation). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52339/52339fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

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16.3K Views.  Bangor University. The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

Video: A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

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

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.

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.

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

Quantitative MRI of Endothelial Permeability and Dysfunction in Atherosclerosis

We have developed an accurate, non-invasive, and easy-to-use method to quantify endothelial permeability and dysfunction in the arteries using Magnetic Resonance Imaging (MRI), named qMETRIC. This technique enables assessing vascular damage and cardiovascular risk associated with atherosclerosis in preclinical models and humans.

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of ...

Bioengineering

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Neuroscience

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Biology

Using Arterial Stiffness and Endothelial Function for Cardiovascular Assessment

Pulse-Wave Velocity, Flow-Mediated Dilation, and Carotid Intima-Media Thickness to Assess Cardiovascular Risk in Population with Metabolic Syndrome

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical settings to assess arterial stiffness, endothelial function, and subclinical atherogenesis. These measurements may reflect vascular disease and atherosclerotic progression, which are major causes of adverse cardiovascular events. These methods are particularly valuable in determining cardiovascular dysfunction among populations with different risk factors, such as diabetes mellitus, hypertension, and other metabolic dysfunction-related conditions. They provide a non-invasive and reliable source of information that complements clinical practice. Early detection, risk assessment, and therapeutic decisions regarding cardiovascular disease can be achieved, ultimately contributing to improved patient outcomes. Traditional tools for evaluating cardiovascular disease do not reveal whether metabolic syndrome affects early subclinical cardiovascular disease in patients with obesity. Recent research has highlighted the importance of including arterial stiffness and endothelial function in a comprehensive cardiovascular assessment. Therefore, the aim of the present study is to describe methods that provide information on early subclinical vascular aging, endothelial dysfunction, and atherogenic disease, enabling vascular-targeted risk stratification among populations with obesity and different metabolic profiles.

Author Spotlight: Assessing the Cardiovascular Profile of Patients with Metabolic Syndrome

Endothelial/vascular aging and atherogenesis are key mechanisms that promote the development of cardiovascular diseases. The present protocol describes methods to evaluate arterial stiffness, endothelial dysfunction, and atherogenesis in patients with related risk factors, which are highly valuable in the cardiovascular research field.

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

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