The authors thank the support of Institutional Program E015.

The institutional research ethics committee from the National Medical Center &#34;20 de Noviembre&#34; ISSSTE approved this protocol (ID No. 386.2013). All enrolled patients provided written informed consent. The details of the equipment and software used in this study are listed in the Table of Materials.
Patient inclusion/exclusion criteria: 
Eligible patients were older than 18 years and diagnosed with morbid obesity (Body Mass Index [BMI] &#62;40 kg/m&#178; or BMI &#62;35 kg/m&#178; with obesity-related health conditions, such as diabetes mellitus, hypertension, or obstructive sleep apnea/hypopnea) and candidates for bariatric surgery. Patients were excluded if they had used weight-reducing therapy during the 6 months prior to enrollment, had significant inflammatory diseases, severe renal and/or hepatic disease, active malignancy, pregnancy, or evidence of cardiovascular disease (either self-reported or diagnosed with ischemic heart disease, coronary artery disease, myocardial structural abnormalities, cardiac interventions, or being under treatment for any of these conditions).
1. Evaluation of cardiometabolic profile
NOTE: The study sample used for this experiment comprised 21 Metabolically Healthy Obese (MHO) and 25 Metabolically Unhealthy Obese (MUO) patients, determined by the absence or presence of metabolic syndrome, respectively. The participants were aged 43 &#177; 9 years old, with a BMI of 45 &#177; 7.8 kg/m&#178;, and 78% were female. The most prevalent co-morbidities were type 2 diabetes mellitus, systemic arterial hypertension, and/or dyslipidemia. The sample was intended to be age-matched.
<ol>
	<li>Evaluate cardiometabolic profile
	<ol>
		<li>Obtain demographic and anthropometric characteristics, such as age, gender, height, weight, chronic diseases (t2DM, hypertension, etc.), and medications consumed. Calculate BMI by dividing weight by the square of height. Obtain waist circumference by measuring between the lower point of the last rib and the iliac crest.</li>
		<li>Perform routine clinical biochemical tests, including glucose, insulin, liver function tests, and lipid profile22.</li>
		<li>Designate patients as MUO or MHO, as determined by the presence or absence of metabolic syndrome, respectively. 
		NOTE: Metabolic Syndrome is diagnosed according to the NCEP/ATP III criteria23. A patient is diagnosed with metabolic syndrome if they present at least three of the following five risk factors:</li>
		<li>Abdominal obesity (waist circumference &#62;102 cm in men or &#62;88 cm in women). (2) Serum triglyceride level &#8805;150 mg/dL (1.7 mmol/L). (3) HDL cholesterol level &#60;40 mg/dL (1.0 mmol/L) in men or &#60;50 mg/dL (1.3 mmol/L) in women. (4) Systolic blood pressure &#8805;130 mmHg or diastolic blood pressure &#8805;85 mmHg. (5) Fasting plasma glucose level &#8805;100 mg/dL (5.6 mmol/L).</li>
	</ol>
	</li>
</ol>
2. Vascular aging (arterial stiffness)
NOTE: Vascular aging may be evaluated in terms of aortic stiffness, which is determined by the central aortic pulse pressure and carotid-femoral pulse wave velocity (cfPWV). Nowadays, cfPWV is the gold standard for determining arterial stiffness13.
<ol>
	<li>Aortic pressure assessment 
	NOTE: Aortic pressure assessment is performed using a device for measuring arterial wave reflection and Pulse Wave Analysis (PWA), on which central aortic pressure waveform parameters are determined.
	<ol>
		<li>Create a profile of the patient in the device software and introduce data such as patient ID, name, birth date, gender, and height.</li>
		<li>Place the patient in a supine position for at least 5 min before beginning the assessment.</li>
		<li>Place a brachial cuff and secure it around the patient&#39;s arm, centered on the brachial artery, ensuring that the center of the cuff and the heart are at the same level.</li>
		<li>The device automatically performs PWA. Press the start button. The cuff will automatically inflate for the first time to determine brachial systolic and diastolic pressure. Then, the cuff will deflate and inflate again to capture the PWA waveform.</li>
		<li>Obtain PWA by applanation tonometry, which allows recording peripheral pulse and generation of central aortic pressure waveforms. Peripheral locations for applanation tonometry include brachial or radial arteries.</li>
		<li>After scaling the arterial pressure waveform, analyze the systolic part of the waveform, also considering biometric and demographic data. 
		NOTE: The software calculates based on the formula K&#183;Psa&#183;(1 + Ts/Td), where Psa is the area under the systolic part of the curve above end-diastolic pressure, Ts and Td are the durations of systole and diastole, respectively, and K is a constant related to stroke volume24,25.</li>
		<li>Calculate the Augmentation Index following the NOTE below. 
		NOTE: An early incident arterial wave is produced after left ventricular ejection, with a subsequent reflected wave originating from the periphery. The velocity of these waves increases according to arterial stiffness, and if the reflected wave arrives at the central aorta early, aortic systolic pressure will increase. This is known as Augmented Pressure, whereas its percentage relative to the Pulse Pressure is known as the Augmentation Index (Aix).</li>
		<li>Obtain an automatic report of the test, containing aortic parameters, including average central pressure waveform: SP (aortic Systolic Pressure), DP (aortic Diastolic Pressure), PP (Aortic Pulse Pressure), MAP (Mean Arterial Pressure), and HR (Heart Rate); clinical parameters which are represented on a bar graph; as well as Aix.</li>
	</ol>
	</li>
	<li>Perform cfPWV determination through applanation tonometry method (SphygmoCor)
	<ol>
		<li>Place a femoral cuff around the patient&#39;s thigh, as high as possible, ensuring the tube is centered at the top of the leg.</li>
		<li>Find the carotid pulse on the patient&#39;s neck below the jaw. Ask the patient to slightly rotate their head aside; if required, place a pillow below the neck to provide support. Once the carotid pulse is found at the strongest perceived site, place an indicator mark on the patient&#39;s skin.</li>
		<li>Obtain three measurements. First, the distance between the carotid pulse and the suprasternal notch; second, the distance between the suprasternal notch and the femoral cuff; and finally, the distance between the femoral artery by palpating the pulse on the patient&#39;s groin and the femoral cuff. 
		NOTE: All distances shall be taken in straight lines (avoiding the patient&#39;s body curves) and must be submitted in millimeters.
		<ol>
			<li>Then, introduce information on the three distances into the cfPWV software.</li>
		</ol>
		</li>
		<li>Place the tip of the tonometer on the site where the carotid pulse was previously located. Then press the START button. The sensor will automatically detect the carotid pulse and will record the shape of the pulse once a regular pattern is registered. The device will sync both pulses (carotid and femoral) to determine the wave pulses and estimate the cfPWV and Pulse Transit Time (Figure 1).</li>
		<li>Ensure that the software automatically performs velocity calculation by computing D/&#916;t, where D is the transit distance between carotid and femoral pulse sites, and &#916;t is the time delay from the peak ECG R-wave to the foot of the corresponding pressure waveform between the carotid and femoral waveforms.</li>
		<li>Evaluate a quality control test, as reported by the device, to determine whether the measurements are acceptable. 
		NOTE: Hold the tonometer tip like a pencil to ensure maximum stability. Pressure and position adjustments on the tonometer should be delicate and soft to obtain accurate measurements, which will be indicated by a green or yellow color of an upper line on the screen and waveforms; otherwise, the color will turn red, indicating a need for a reduction in tonometer pressure (if the indicator of pressure level goes up) or an increase in tonometer pressure (if the indicator goes down). The operator must ensure that there is a well-defined upstroke on the carotid waveforms, as it is an important feature that will be used to determine the cfPWV. It is important to locate the screen of the computer in a convenient place for the operator to view prior to the determination. If the screen is in a place where the operator finds it difficult to view, it can make the data capture challenging. The device will need a minimum of 10 s of consistent simultaneous waveforms of the carotid and femoral pulses and will automatically capture the waveforms; nevertheless, some circumstances will require the operator to capture the pulse waves manually (Figure 2).</li>
	</ol>
	</li>
</ol>
3. Endothelial dysfunction (flow-mediated dilation [FMD])
NOTE: The Flow Mediated Dilation (FMD) test is a non-invasive technique to evaluate vascular health; it is specifically useful for assessing endothelial function and has been described as a useful tool to predict future cardiovascular events21. It is performed using ultrasonography with a linear transducer.
<ol>
	<li>Perform the FMD protocol according to international recommendations26. Use a sphygmomanometer and place the cuff around the right forearm.</li>
	<li>Place a high-resolution linear transducer in B-mode coupled to the compatible analysis software on the brachial artery.</li>
	<li>Scan longitudinally 5-10 cm above the elbow. Acquire the clearest B-mode image of the anterior and posterior intimal interfaces and determine the baseline artery diameter.</li>
	<li>Hold the transducer at the same point to ensure consistency of the site of measurement.</li>
	<li>Occlude for 5 min using the blood pressure cuff, reaching a pressure of 30-50 mmHg above the determined systolic pressure.</li>
	<li>After cuff deflation, perform a continuous record of the longitudinal image of the brachial artery for 3 min, and determine the diameter again.</li>
	<li>Calculate FMD as the percentage change relative to the vessel diameter before cuff inflation, as follows: (peakdiameter &#8722; baselinediameter)/baselinediameter (peak diameter &#8722; baseline diameter) / baseline diameter (peakdiameter &#8722; baselinediameter)/ baselinediameter x 100 (Figure 3). FMD% measures the ability of the arteries to respond with endothelial nitric oxide release during reactive hyperemia (flow-mediated)27.</li>
	<li>Further evaluate endothelial dysfunction by determining plasma NO (Nitric Oxide assay kit, commercially available) as measured by enzyme-linked immunosorbent assay.
	<ol>
		<li>Prepare the standard curve and samples, then add them to wells in the assay plate.</li>
		<li>Add nitrate reductase and enzyme cofactor and incubate for 60 min at room temperature to convert nitrate to nitrite.</li>
		<li>Add enhancers and Griess reagents; then develop at room temperature for 10 min, allowing nitrite to turn into a deep purple azochromophore compound, which accurately reflects NO amounts.</li>
		<li>Analyze in a microplate reader by measuring at an optical density of 540 nm.</li>
	</ol>
	</li>
</ol>
4. Subclinical atherogenesis (carotid intima-media thickness [CIMT])
NOTE: Patients must be placed in a supine position comfortably, with the head rotated to expose the jugular vein and the carotid artery; a rolled-up towel or pillow under the neck can be used to better expose the carotid.
<ol>
	<li>Perform the carotid intima-media thickness (CIMT) measurement protocol according to expert Consensus28,29 as follows: apply a 4.0 MHz ultrasonographic probe to identify neck vascular structures, such as the carotid artery and jugular vein, as well as the thyroid gland, in a transverse orientation at the base of the neck.</li>
	<li>Locate the right carotid artery using a transverse orientation of the transducer, moving in a cephalic direction until the carotid bulb and the bifurcation of the internal and external carotid artery are identified. Then, rotate the transducer 90&#176; to achieve a longitudinal view of the carotid bulb.</li>
	<li>Determine carotid intima-media thickness by measuring the distance between the intima-lumen and media-adventitia interfaces along a 1 cm range distal from the carotid bulb (Figure 4). 
	NOTE: The protocol does not include follow-up, medications, or interventions.</li>
</ol>

Using Arterial Stiffness and Endothelial Function for Cardiovascular Assessment

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K., Christian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+intima-media+thickness+is+associated+with+obesity+and+hypertension+in+young+people.">Carotid intima-media thickness is associated with obesity and hypertension in young people.</a> Hypertension. 79 (6), 1177-1179 (2022).</li><li>Mansiroglu, A. K., Seymen, H., Sincer, I., Gunes, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+endothelial+dysfunction+in+COVID-19+with+flow-mediated+dilatation.">Evaluation of endothelial dysfunction in COVID-19 with flow-mediated dilatation.</a> Arq Bras Cardiol. 119 (2), 319-325 (2022).</li><li>Sincer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+serum+total+antioxidant+status+and+flow-mediated+dilation+in+patients+with+systemic+lupus+erythematosus:+an+observational+study.">Association between serum total antioxidant status and flow-mediated dilation in patients with systemic lupus erythematosus: an observational study.</a> Anatol J Cardiol. 15 (11), 913-918 (2015).</li><li>Sincer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+correlation+between+uric+acid+levels+and+flow-mediated+dilatation+in+patients+with+masked+hypertension.">Significant correlation between uric acid levels and flow-mediated dilatation in patients with masked hypertension.</a> Clin Exp Hypertens. 36 (5), 315-320 (2014).</li><li>Nakanishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+intima-media+thickness+and+subclinical+left+heart+dysfunction+in+the+general+population.">Carotid intima-media thickness and subclinical left heart dysfunction in the general population.</a> Atherosclerosis. 305, 42-49 (2020).</li><li>Klobu&#269;ar, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+between+endothelial+lipase,+high-density+lipoprotein,+and+endothelial+function+differ+in+healthy+volunteers+and+metabolic+syndrome+patients.">Associations between endothelial lipase, high-density lipoprotein, and endothelial function differ in healthy volunteers and metabolic syndrome patients.</a> Int J Mol Sci. 24 (3), 2073 (2023).</li><li>Sandoo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+functional+and+morphological+assessments+of+endothelial+function+in+patients+with+rheumatoid+arthritis:+a+cross-sectional+study.">The association between functional and morphological assessments of endothelial function in patients with rheumatoid arthritis: a cross-sectional study.</a> Arthritis Res Ther. 15 (5), R107 (2013).</li><li>Toyoda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+brachial+flow-mediated+dilation+and+carotid+intima-media+thickness+in+patients+with+coronary+artery+disease.">Relationship between brachial flow-mediated dilation and carotid intima-media thickness in patients with coronary artery disease.</a> Int Angiol. 39 (5), 433-442 (2020).</li></ol>

The authors have nothing to disclose.

Addressing vascular health and understanding and managing cardiovascular risk are essential for the prevention, early intervention, and reduction of the global burden of cardiovascular diseases. In this regard, the combined use of methods to assess the elasticity and compliance of the arterial wall (including aortic hemodynamic parameters, cfPWV for arterial stiffness, and Augmentation Index), endothelial nitric oxide production, and atherosclerosis provide a more comprehensive evaluation. These methods are highly useful...

Obesity is a major health problem worldwide due to its associated complications, such as hypertension, dyslipidemia, liver disease, atherosclerosis, insulin resistance, and type 2 diabetes mellitus (T2DM), as well as an increased risk of cardiovascular diseases (CVD)1.
The constellation of these conditions, known as Metabolic Syndrome (MS), has been reported to be a major cause of CVD pathogenesis, which is one of the leading causes of death, accounting for up to 30% of all deaths worldwide2. Obese individuals have a higher requirement for oxygen and nutrients throughout the body due to an increased blood supply demand, leading to significant hemodynamic changes. These changes can result in decreased nitric oxide (NO) availability, increased oxidative stress, and vascular endothelial dysfunction3,4,5.
Atherosclerotic diseases are major cardiovascular conditions and represent the leading cause of death worldwide. This is a clinical manifestation of multiple possible factors, including genetic and environmental factors6. People with metabolic abnormalities, such as insulin resistance or prediabetes, have been shown to have a significantly higher prevalence and incidence of coronary atherosclerosis than healthy people. Furthermore, congested blood vessels with highly lipidic plaque have been found even before the appearance of clinical manifestations of metabolic dysfunction7,8,9,10.
Arterial stiffness, endothelial dysfunction, and atherogenesis have been described as important factors in the development of cardiovascular diseases. These processes are related to vascular aging and atherogenic plaque formation in critical vessels like coronary, carotid, or limb arteries. Translational research has evidenced that arterial stiffness, endothelial dysfunction, and atherogenesis are related to common vascular damage induced by chronic inflammation, lower NO production, and oxidative stress11,12.
Measurement of Carotid-femoral pulse-wave velocity (cfPWV) represents the gold standard method to measure arterial stiffness. cfPWV can be measured using a carotid tonometer simultaneously with a leg cuff to capture blood pressure waveforms at the carotid and femoral sites. Then, a software can perform velocity calculation by computing D/&#916;t, where D is the transit distance between carotid and femoral pulse sites, and &#916;t is the time delay from the peak ECG R-wave to the foot of the corresponding pressure waveform between the carotid and femoral waveforms. Increased stiffness of central arteries, like the aorta, causes a higher speed of the ejected pulse from the left ventricle through the arteries, as well as a faster return of the reflected pressure, with a consequent elevation of pressure during left ventricular ejection, which potentially decreases coronary artery perfusion. Therefore, cfPWV may be useful as a marker of coronary artery disease, stroke, and cardiovascular diseases13,14.
Likewise, Pulse Wave Analysis (PWA) is a non-invasive vascular parameter that assesses central pressure wave characteristics, where aortic systolic and diastolic blood pressures are the main variables. By measuring arterial stiffness and elastic compliance, PWA reflects arterial distensibility, which is closely related to cardiovascular risk. This method allows for measuring parameters like the Augmentation Index, which has the ability to predict the severity of cardiovascular and coronary artery diseases. The Augmentation Index may be described as follows: an early incident arterial wave is produced after left ventricular ejection, with a subsequent reflected wave originating from the periphery. The velocity of these waves increases according to arterial stiffness, and if the reflected wave arrives at the central aorta early, aortic systolic pressure will increase. This is known as Augmented Pressure (AP), whereas its percentage relative to Pulse Pressure is known as the Augmentation Index. PWA can be measured through the applanation tonometry method, involving a slight compression of the brachial artery so that its transmural pressure is zero. At this point, Mean Arterial Pressure can be measured. After scaling the arterial pressure waveform, the systolic part of the AP waveform is analyzed, also considering biometric and demographic data15,16,17. Particularly, the applanation tonometry method (SphygmoCor) has shown acceptable repeatability and significant correlation with invasive aortic catheterization in determining aortic PWV, as well as good agreement with the Artery Society Guidelines18,19,20.
Other vascular tests like flow-mediated dilation (FMD) and carotid intima-media thickness (CIMT) represent non-invasive techniques performed by ultrasonography with linear transducers. These assessment procedures are useful for evaluating vascular health, specifically endothelial dysfunction and subclinical atherogenesis, respectively. Both have shown prognostic ability for cardiovascular events. FMD is commonly considered a reflection of endothelium-dependent arterial function, primarily mediated by nitric oxide. It serves as a surrogate marker for vascular health and has been utilized non-invasively to compare subject groups and assess the effects of interventions on individuals21.
The aim of the present study is to describe the use of methods that yield the determination of markers reflecting early subclinical vascular aging, endothelial dysfunction, and atherogenic disease. Such information allows risk stratification among populations with obesity and different metabolic profiles. These methods might be useful to determine cardiovascular damage and prognosis, as well as to evaluate vascular and atherogenic responses to pharmacologic and non-pharmacologic interventions, particularly among populations with metabolic risk factors.

<table><tbody><tr><td>Device for measuring arterial wave reflection and Pulse Wave Analysis</td><td>ATCOR</td><td>SphygmoCor</td><td>Analyzer of pulse wave for central pressure. It contains a brachial cuff and a femoral cuff</td></tr><tr><td>Microplate reader for absorbance, Sunrise</td><td>Tecan&amp;nbsp;</td><td>30190079</td><td>Detection Mode: Absorbance; Wavelength Range: 340 nm - 750 nm; Filter Wavelength: 405 nm,&amp;nbsp;450 nm,&amp;nbsp;492 nm,&amp;nbsp;620 nm; Plate Format 96 well plates</td></tr><tr><td>Nitric oxide assay kit&amp;nbsp;</td><td>Abcam</td><td>ab65328</td><td>Nitric Oxide Assay Kit, Colorimetric, Abcam Cat. ab65328 for 96-well plates</td></tr><tr><td>Portatil ultrasound to measure FMD</td><td>Sonolife</td><td>MED 36-13</td><td>Ultrasonography linear transducer</td></tr><tr><td>Software for FMD WirelessUSG</td><td>SonoStarMed Technologies</td><td>WirelessUSG v. 3.6.52</td><td>Software used to measure artery diameter for FMD</td></tr><tr><td>Software used to calculate vascular parameters from Waveform Analysis</td><td>ATCOR</td><td>SphygmoCor XCEL</td><td>Software used to integrate patient profile, waveform analysis, calculation of PWA, PWV and other vascular parameters</td></tr><tr><td>Sphygmomanometer</td><td>Homecare</td><td>ANEROIDE 1000</td><td>100% cotton self-adjustable bracelet with hook, Adult artery indicator cuff.</td></tr><tr><td>Ultrasound to measure CIMT</td><td>Philips&amp;nbsp;</td><td>EPIQ7L12-3 Broadband Linear Array Transducer</td><td>Linear transducer (Broadband Linear Array Transducer)</td></tr></tbody></table>

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.

Subjects were classified as MHO and MUO based on their cardiometabolic profiles. The MUO group exhibited a higher prevalence of chronic diseases, such as systemic arterial hypertension, type 2 diabetes mellitus (t2DM), and dyslipidemia. Similarly, the MUO phenotype showed elevated levels of glucose and HbA1c, as well as differences in triglycerides and total cholesterol (Table 1).
Vascular aging was then assessed, reflecting arterial stiffness and endothelial dysfunction, dete...

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, 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 ...

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787 Views.  ISSSTE. Vasculaire veroudering en atherogenese zijn belangrijke mechanismen die hart- en vaatziekten bevorderen, met name in aanwezigheid van risicofactoren zoals diabetes, hypertensie en andere metabole disfuncties, polsgolfsnelheid, stroomgemedieerde dilatatie en dikte van de intimamedia van de halsslagader, die evolueren naar arteriële stijfheid, endotheeldisfunctie en subklinische atherogenese, en zeer waardevolle informatie verschaffen over het cardiovasculaire...

Video: Auteur in de schijnwerpers: beoordeling van het cardiovasculaire profiel van patiënten met metabool syndroom

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 ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

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 ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

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 ...

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.

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.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

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.

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 ...

Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively by the measurement of carotid to femoral pulse wave velocity (cf-PWV) (Class I; Level of Evidence A). In literature, numerous community-based or disease-specific studies have reported that higher cf-PWV is associated with increased cardiovascular risk. Here, we discuss strategies to evaluate arterial stiffness with cf-PWV. Following the well-defined steps detailed here, e.g., proper position operator, distance measurement, and tonometer position, we will obtain a standard cf-PWV value to evaluate arterial stiffness. In this paper, a detailed stepwise method to record a good quality PWV and pulse wave analysis (PWA) using a non-invasive tonometry-based device will be discussed.

Measuring the Carotid to Femoral Pulse Wave Velocity Cf-PWV to Evaluate Arterial Stiffness

This protocol describes a method to standardize the measurements of carotid-femoral pulse wave velocity to evaluate arterial stiffness.

For the elderly, arterial stiffening is a good marker for aging evaluation and it is recommended that the arterial stiffness be determined noninvasively ...

Ultrasound Assessment of Aortic and Carotid Artery Intima-media Thickness in Children and Adolescents

Carotid intima-media thickness (IMT), measured using high-resolution B-mode ultrasonography, is a widely utilized surrogate marker of subclinical atherosclerosis, the pathophysiological process underlying most clinical cardiovascular disease events. Atherosclerosis is a gradual disease that originates early in life, thus, there has been increased interest in measuring carotid IMT in childhood and adolescence to assess structural change in the arterial vasculature in response to adverse exposures. However, the timing of atherosclerosis varies across the vascular tree. Primordial atherosclerotic lesions are present in the abdominal aorta as early as infancy, compared to mid-adolescence for the common carotid. Measurement of IMT at either site is susceptible to several technical challenges that need to be considered, especially in younger children. In this paper, we provide a detailed stepwise method for high-quality assessment of IMT of the abdominal aorta and common carotid artery in the young. We also provide insight into the appropriateness of either site when exploring the associations between early-life exposures and later-life cardiovascular disease.

The present article describes the methodological considerations for the noninvasive assessment of abdominal aortic and carotid intima-media thickness using B-mode ultrasonography. This technique is commonly used in the developmental origins of health and disease research as a surrogate for early arterial changes.

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

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