*3.2.2. New method for assessment of endothelial function—measurement of ezFMD*

Since FMD requires an expensive ultrasound system and high levels of technical skills, a novel method for measurement of endothelial function, namely, measurement of enclosedzone flow-mediated dilatation (ezFMD) was developed [133]. ezFMD is a noninvasive method which assesses the level of vasodilatation from the oscillation signals transmitted to a sphygmomanometer cuff attached to the upper arm. In patients with cardiovascular diseases, ezFMD was significantly lower than in age- and gender-matched healthy individuals. In addition, cardiovascular risk factors were independent predictors of ezFMD. ezFMD was significantly correlated with conventional FMD [134]. Conventional measurement of FMD by ultrasound is measured by the change in vascular diameter, whereas ezFMD is based on the change in vascular volume. Both methods are equally valuable for assessing endothelial function, however, measurement of ezFMD is easier and less biased than measurement of FMD.

#### *3.2.3. Coronary epicardial vasoreactivity*

*3.1.5. Fingertip digital thermal monitoring (DTM)*

402 Endothelial Dysfunction - Old Concepts and New Challenges

**3.2. Assessment of macrovascular function**

*3.2.1. Flow-mediated dilation*

thelium injury [126].

flow-mediated constriction), and low costs [68].

Fingertip digital thermal monitoring (DMT) of vascular reactivity represents a noninvasive, reproducible, operator-independent technique based on changes in fingertip temperature during cuff-occlusive reactive hyperemia [119]. This method relies on a premise that changes in fingertip temperature during and after vascular occlusion that reflects changes in blood flow and thus microvascular and endothelial function [120]. So far, studies have reported that vascular function measured by DTM correlate with Framingham Risk Score and coronary artery calcium score (a measurement of the amount of calcium in the walls of the coronary arteries using a special computed tomography (CT) scan of heart) independently of age, sex, and traditional cardiac risk factors [121]. Although clinical implications of DTM are promising, more studies on the mechanisms mediating this vascular response and large prospective

FMD of the brachial artery is the most widely used noninvasive *in vivo* method for an indirect assessment of endothelial function of conduit vessels introduced by Celermajer and colleagues [5]. It provides decisive information about the ability of the endothelium to respond to particular stimulus (reactive hyperemia). In this method, an arterial occlusion cuff is placed to the forearm and inflated to stop the anterograde blood flow, thus generating ischemia. Consequently, distal from that the occlusion, in the resistance arteries, vasodilation occurs, and when the sphygmomanometer is deflated, reactive hyperemia occurs in the brachial artery. The method involves ultrasound arterial imaging in two conditions, at rest (baseline) and during reactive hyperemia after 5 min arterial occlusion, and FMD is expressed as the % difference between that two measured diameters [122]. The exact mechanism mediating FMD during reactive hyperemia has not been fully elucidated; it is considered that shear stress-induced NO is the main mediator [76, 85], but also other endothelium-derived vasodilator factors may also contribute [123]. Because reactive hyperemia flow, induces increased shear stress on endothelium challenges FMD, it might be a significant measure of peripheral microvascular function because reactive hyperemia is greatly dependent on maximal forearm resistance [124]. Furthermore, peripheral endothelial function as assessed by FMD correlates with vascular function of coronary artery [125]. In addition, impaired FMD is one of the early manifestations of vascular disease, and may be an important indicator of endo-

However, although the principle of this technique seems simple, its application is technically challenging and requires comprehensive practicing and standardization [127, 128]. Easy access of this noninvasive method is one of the main advantages of this method, while other advantages being a good correlation with invasive epicardial vascular function assessment, possibility to assess other important parameters (i.e., flow, baseline arterial diameters and

To ensure that impaired FMD is not due to underlying vascular smooth muscle dysfunction or alterations in vascular structure but truly a consequence of endothelial dysfunction,

trials are needed to establish the real research and clinical value of this method.

Quantitative coronary angiography (QCA) or intravascular ultrasound are methods for imaging vasomotor responses of epicardial coronary arteries, which enable tracing of changes in vessel diameters in response to endothelium-dependent interventions, e.g., intracoronary infusion of drugs or substances, such as acetylcholine [2]. Vessels with an intact endothelium vasodilate in response to ACh infusion, whereas segments with dysfunctional endothelial cells display abnormal vascular response [2]. Estimation of coronary endothelial function with intracoronary ACh provides diagnostic and prognostic data in patients with suspected coronary microvascular dysfunction.

Some of advantages of this method is direct assessment of the coronary vascular bed and represents gold standard for assessment of epicardial macrovasculature, while its disadvantage is invasiveness and limitation to those patients undergoing coronary angiography [68].

Physiologically, endothelium-dependent vasodilation occurs in response to exercise or tachycardia as a replacement for exercise, but also pacing induced tachycardia, and leads to increased flow-mediated endothelium-dependent vasomotion of the epicardial vessels that is impaired in atherosclerosis [68]. In healthy isolated intramyocardial porcine coronary resistance arteries, bradykinin, serotonin, and the alpha 2-adrenergic agonist clonidine evoked endothelium-dependent relaxations, which were fully (clonidine) or partially (serotonin) mediated by NO, while vasodilator response to bradykinin seems to be mediated by some other endothelium-derived mediator, different from NO [135]. Further, cold pressor test (CPT), in which the subject puts his hand into ice water, is another mode to assess epicardial vasoreactivity. In the study by Nabel et al., the response to CPT was assessed in patients with angiographically normal coronary arteries, in patients with mild coronary atherosclerosis and in patients with advanced coronary stenosis, using quantitative angiography and Doppler flow velocity measurements. Normal vessels exhibited vasodilation (partly related to betaadrenergic receptor stimulation and partly due to flow-mediated dilation or alpha-2 adrenergic receptor activation) while atherosclerotic vessels exhibited vasoconstriction in response to CPT, possibly due to altered sensitivity to adrenergic stimulation and/or some other impairment of endothelium-dependent vasodilation [136].

coronary artery disease [143] and cerebrovascular disease [144]. CIMT represents the combined width of the intima and media; in healthy individuals, it is composed almost entirely of media, with a progressive intimal thickening or hypertrophy of media, determined by age, gender, and hypertension [145]. The major advantage of CIMT is that it is noninvasive and reproducible, relatively inexpensive to perform, also widely available

The Markers of Endothelial Activation http://dx.doi.org/10.5772/intechopen.74671 405

The baseline pathogenic process in cardiovascular diseases, such as atherosclerosis and coronary artery disease, is an endothelial dysfunction with complex underlying mechanisms: oxidative stress, diminished vasoreactivity, hemostatic disturbances, and inflammation leading to the disease progression by modulating the arterial wall, promoting lipoprotein retention, plaque formation and possibly its destabilization. Endothelial dysfunction is characterized by endothelial dysfunction, impaired vascular homeostasis and reduced "anti"-mechanisms (-oxidant, -inflammatory, -thrombotic) and activated "pro"-mechanisms. Diagnostic tools for detecting endothelial dysfunction in humans are limited. They should be safe, cost-effective, noninvasive, repeatable, reproducible, and standardized. Current diagnostic methods are FMD, forearm plethysmography, finger-pulse plethysmography, PWV analysis, and coronary angiography. However, there is a need for additional diagnostic tools, biomarkers. For everyday clinical use, more and larger human-based studies are necessary to validate clinical

To find a specific and sensitive biomarker for any disease sometimes looks like a search for the Holy Grail—something precious but impossible to find. The reason for that could be those cardiometabolic diseases, all having a common point—endothelial dysfunction and it is likely that they have common underlying mechanisms leading to endothelial dysfunction. These mechanisms may be redundant and not activated at the same time and the same order, but certainly end up with impaired endothelium, and inappropriate vascular response to physiological stimuli with inability to compensate for pathophysiological events, finally leading to manifested disease and organ damage. One can only take with "a grain of salt" as many different biomarkers as possible and build up a picture of their relationship to the disease's

This work is supported by the European Structural and Investment Funds grant for the Croatian National Scientific Center of Excellence for Personalized Health Care, University of

and well standardized [146].

usefulness of biomarkers [147, 148].

etiopathogenesis, development, and prognosis.

Josip Juraj Strossmayer Osijek (grant #KK.01.1.1.01.0010).

**4. Conclusion**

**Acknowledgements**

*3.2.6. Functional endothelial biomarkers in cardiovascular diseases*

#### *3.2.4. Pulse wave velocity*

Pulse wave velocity (PWV) is the velocity at which the pulse pressure wave spreads from the left ventricle (at the end of ventricular ejection) to the periphery. It results in an earlier return of the reflected wave which increases the pressure and subsequently the afterload of the left ventricle and reduces coronary artery perfusion pressure during diastole. One of the most frequently used noninvasive methods for the assessment of aortic stiffness is carotidfemoral (aortic) PWV [137]. It is a simple, noninvasive, and reproducible method which has been used as a gold standard and provides a predictive value of aortic stiffness for future cardiovascular events [138]. PWV has been used as significant marker of cardiovascular risk. Data indicate that increased arterial stiffness is being independently predictive of coronary artery disease, stroke, and cardiovascular events in general [139]. While PWV values are lower in healthy young individuals, the values of PWV increase with reduction of arterial elasticity [140].

Applanation tonometry is another method that is used for pulse wave analysis. Rather than directly assessing aortic pulse wave, it estimates aortic pulse wave from the common carotid artery or the radial artery pulse waves. As the measurement is easier, radial artery tonometry has been the most commonly recommended approach [137]. Since the method can detect changes that might be related to vascular health, even before the onset of signs and symptoms, yet, the PWV analysis occupies an important place in clinical practice [141]. This method has some limitations that are related to associated comorbidities, such as metabolic syndrome, obesity, and diabetes, because the femoral pressure waveform may be difficult to record accurately in these patients [137].

#### *3.2.5. Intima-media thickness*

Carotid intima-media thickness (CIMT) is a method that evaluates extra-cranial carotid arteries by high-resolution ultrasound, and represents an important marker of subclinical atherosclerosis [142]. CIMT is increased in atherosclerosis and also correlates with coronary artery disease [143] and cerebrovascular disease [144]. CIMT represents the combined width of the intima and media; in healthy individuals, it is composed almost entirely of media, with a progressive intimal thickening or hypertrophy of media, determined by age, gender, and hypertension [145]. The major advantage of CIMT is that it is noninvasive and reproducible, relatively inexpensive to perform, also widely available and well standardized [146].
