**3.2. Assessment of macrovascular function**

#### *3.2.1. Flow-mediated dilation*

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 endothelium injury [126].

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

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, response to nitroglycerine is used [127, 129, 130]. Nitroglycerine-induced vasodilation was significantly reduced in patients with cardiovascular disease [129]. Additionally, nitroglycerine-induced vasodilation was impaired in patients with atherosclerosis [131]. FMD should be interpreted as an index of vascular function reflecting both endothelium-dependent and -independent vasodilation in individuals with impaired nitroglycerine-induced vasodilation [129]. Furthermore, coronary artery dilation in response to nitroglycerine is impaired in patients with coronary heart disease which predicts long-term atherosclerotic disease progression and cardiovascular event rate [132]. These findings suggest that nitroglycerine-induced vasodilation *per se* may be a marker of the grade of atherosclerosis and predictor of cardiovascular events. However, the relationship between nitroglycerine-induced vasodilation and the risk for future cardiovascular events should still be established.
