**3. Functional biomarkers of vascular (endothelial) function**

#### **3.1. Assessment of microvascular endothelial function**

#### *3.1.1. Coronary microvascular function assessment*

conditions such as tumor progression, hypertension, chronic kidney disease, and renal transplant rejection [48]. Tadzic et al. [49] have described an increased expression of cell adhesion molecules, intracellular adhesion molecule's (ICAM) and vascular cell adhesion molecule's (VACM) ligands, together with decrease of sCAMs and endocan in hypertensive patients on amlodipine therapy with reduction in blood pressure, suggesting de-activation of endothelium. Systolic and diastolic blood pressure was positively correlated with ICAM-1 and VCAM-1, and systolic blood pressure was negatively correlated with CD11a/LFA-1. Endocan

Diabetes is associated with increased circulating levels of endothelium-derived adhesion molecules and plasminogen activator inhibitor-1, which have pro-inflammatory and prothrombotic effects [50, 51]. In endothelial dysfunction, the endothelium can express adhesion molecules responsible for the withdrawal of leukocytes from vascular wall, such as VCAM-1 and ICAM-1 [36]. Also, E-selectin and platelet endothelial cell adhesion molecule have been expressed in atherosclerotic lesions and are involved in mononuclear cell adhesion to the vascular endothelium [52, 53]. The main difference in the activation of adhesion molecules is that the expression of ICAM-1 increases after cell activation, while E-selectin and VCAM-1 are only induced after cell activation. It is demonstrated that hyperglycemia results in the expression of adhesion molecules: endothelial-leukocyte adhesion molecule-1, VCAM-1, and ICAM-1 in human vascular endothelial cells [54]. In the rat mesenteric microcirculation, only intraperitoneal co-administration of IL-1β with D-glucose increased leukocyte rolling flux,

significantly positively correlated with ICAM-1 [49].

**Novel biomarkers System/cells Effect**

Myeloperoxidase (MPO) Activated neutrophils and

396 Endothelial Dysfunction - Old Concepts and New Challenges

Flow-mediated dilation (FMD) Blood vessels (endothelial

Intima-media thickness (IMT) Blood vessels (endothelial

**Table 1.** Potential novel biomarkers of atherosclerosis.

ADMA Inhibitor NOS Endothelial dysfunction

macrophages

IL-6, TNF-alfa lymphocytes Proinflammatory cytokines

NO metabolites (nitrates, nitrites) Endothelium (NO) Vasodilation (NO) and nitrosylation

Toll-like receptor 4 lymphocytes Innate immunity

function)

function + VSMC)

Intercellular matrix Intracellular matrix rearrangement

Lipids, activated proteins Reactive oxygen species and products

dependent

multifactorial

Production of oxidative stress

(with increased oxidative stress

NO dependent, or COX, EDHF, EDCF

**Metabolic/biochemical**

ox-LDL, 8-hydroxy-2′-deoxygunosine. MDA (lipid peroxidation), protein

MMP2, MMP9 TIMP2, TIMP9

carbonyl (PCO)

**Functional/structural**

In the past, coronary angiography (of larger conductance arteries, i.e., coronary vessels) was considered a gold standard for evaluation of the severity and extent of CAD. However, in the last two decades, the attention was shifted to the coronary microcirculation as the possible site of anatomical and functional abnormalities crucial for the development and progression of final myocardial ischemia. Thus, functional assessment of coronary microcirculation and its endothelial function became a challenge. For a long time, measurement of changes in coronary blood flow (CBF) during coronary angiography (Doppler wires) has been used as a surrogate parameter for coronary microvascular function assessment [65]. The final result of this measurement is assessment of coronary flow reserve (CFR) which presents the ratio between the maximal CBF during maximal coronary hyperemia (provoked by adenosine infusion, pacing, or exercise) and the resting CBF. It has been demonstrated that CFR is both endothelium-dependent and endothelium-independent, and CFR below 2.0 is considered abnormal [66]. For coronary microvascular endothelium-dependent vasodilation assessment, instead of maximal CBF, CBF in response to endothelium-dependent vasodilator (commonly ACh) infused at increasing concentrations is calculated. Another method for the assessment of coronary microvascular function includes the measurement of the number of cineangiographic frames that it takes to fill a distal vessel with proximal injection of contrast. This method is named Thrombolysis in Myocardial Infarction (TIMI) and provides semi-quantitative assessment of epicardial coronary blood flow [67]. The main advantage of the abovementioned methods is to measure microvascular endothelial function directly in this clinically important vascular bed. However, main limitations are the cost, invasive nature, and therefore a limited population in which these measurements can be actually performed (symptomatic individuals requiring invasive coronary angiography) [68].

factors, and described it in hypercholesterolemia [78], diabetes mellitus [79], cigarette smoking [80], and aging [81], while the results in hypertensive patients were conflicting [3, 4, 82, 83]. Even though the method and pharmacologically induced vasodilation provide an insight into peripheral microvascular patho(physiology), venous occlusion plethysmography is characterized by several limitations and disadvantages, including its semi-invasive character, limited comparison between groups due to different initial blood pressure and forearm blood

The Markers of Endothelial Activation http://dx.doi.org/10.5772/intechopen.74671 399

RH-PAT is a noninvasive technique designed for assessment of peripheral microvascular function. This method reflects changes in finger pulse volume amplitude during reactive hyperemia (an equivalent to finger plethysmography). PAT device includes digital probes that are placed on the tip of each index finger and a blood pressure cuff (for provoking occlusion) that is placed around the upper arm of the study arm, while the other arm serves as a control [84, 85]. Vascular occlusion is provoked by inflation of the blood pressure cuff to a 50 mmHg above systolic blood pressure for 5 min. The PAT signal is recorded 10 min prior occlusion, and for 10 min after the cuff is deflated. The final result of this measurement is calculated as the ratio of average amplitude of the PAT signal over a period of 1 min, starting 1 min after cuff deflation to average amplitude of the PAT signal for 3 min at baseline (RH-PAT index) that is normalized to the control arm [84, 85]. Studies have shown that RH-PAT is at least partly NO dependent. Importantly, studies by Rubinshtein et al. and Akiyama et al. reported that RH-PAT may be a useful tool for prediction of future CV events in patients with CV risk [86, 87]. Advantages of this method are that it is noninvasive, it is very simple and reproducible, and that it is operator independent (RH-PAT index is measured automatically). Even though RH-PAT is very similar to FMD of the brachial artery, Framingham Heart Study has revealed that there was no significant correlation between RH-PAT and FMD [88]. Moreover, the same study reported that different CV risk factors contribute differently to changes in FMD and RH-PAT [89], suggesting that these two methods assess different vascular beds, and that macro- and microvascular endothelium is differently susceptible to various risk factors.

Because of its easy accessibility, the skin presents an appropriate site to study peripheral microcirculation, which was proposed as a suitable marker of systemic microvascular function in various diseases [89]. Therefore, in recent years, a number of simple and noninvasive methods have been developed in order to assess peripheral microcirculation. Still, it is an open question whether skin microcirculation is actually a representative indicator of the microvascular function of other organs. Despite that skin microvascular function was extensively used over the past 30 years to investigate vascular mechanisms in various diseases including hypertension [90, 91], obesity [92], diabetes [93, 94], aging, kidney disease [95], etc. The laser Doppler (LD) technique is based on the estimation of the flow rate in the skin microcirculation using the laser beam reflection from the erythrocyte in microcirculation and its wavelength change (Doppler's effect) [96]. Computer software determines the flow

flow, different sizes of the forearm, etc., [68].

*3.1.4. Laser Doppler (LD) flowmetry*

*3.1.3. Reactive hyperemia peripheral arterial tonometry (RH-PAT)*

In recent years, a number of other methods have been developed among them: (a) blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI), a functional test that detects a dissociation of tissue hemoglobin from blood flow, is shown to be a useful tool for coronary endothelial function assessment [69]; (b) positron-emission tomography (PET) myocardial perfusion imaging that is based on the assessment of regional myocardial blood flow both at rest and during various forms of vasomotor stress [70] and presents a powerful tool to evaluate the effects of CV risk factors on the health of the microvasculature and its endothelium [71]; and (c) myocardial perfusion echocardiography, a bedside method with relatively low cost that is capable to detect myocardial perfusion abnormalities and quantify regional and global coronary blood flow [72]. Despite the fact that these new methods manage to provide noninvasive evaluation of coronary microvasculature directly at the site, they are still unacceptable for routine screening due to their limited availability, expensive equipment and associated costs, and lack of experienced/trained staff. Considering that endothelial function is a systemic disorder, peripheral vascular beds and their microcirculation present a good alternative that provides an easier access and need less elaborate equipment.

#### *3.1.2. Venous occlusion plethysmography*

Venous occlusion plethysmography presents a semi-invasive technique (arterial puncture) for assessment of forearm blood flow (and the corresponding microcirculation) changes before and after infusion of vasoactive substances into a cannulated brachial artery [3]. The method was introduced 90 years ago by Hewlett and van Zwaluwenburg [73], and the basic methodology has changed little since its first description. Basic principle of this method is to stop the return of venous blood from the forearm (inflating the cuff over the diastolic pressure value) with the preserved arterial blood inflow to the forearm, leading to a linear increase in blood flow at a given time, which is proportional to the arterial blood inflow. Another cuff excludes the blood flow through the hand to reduce the temperature fluctuations of the blood flow depending on the temperature. Changes in the flow are recorded by changing the electrical resistance of the plethysmograph located around the longest part of the forearm [74]. The main advantage of this method is that it provides assessment of endothelium-dependent and -independent vasodilation and mechanisms mediating it by intra-arterial infusion of vasoactive substances (e.g., ACh or sodium nitroprusside, and SNP), hormones, and drugs. However, its important limitation is that it could not strictly discern between macro- and microcirculation. Final results are expressed as ratio between blood flow changes in both arms and are well reproducible [75]. Regarding the mechanisms, some studies reported that ACh-induced dilation was inhibited by a NOS inhibitor, L-NG-monomethyl Arginine citrate (L-NMMA) [76], suggesting that NO is the main vasodilator mediating endothelium-dependent vasodilation in this vascular bed. On the other hand, others reported that EDHF has a crucial role in mediating microvascular endothelial-dependent vasodilation, especially in population with multiple CV risk factors [77]. A large number of studies used venous occlusion plethysmography to assess the association between endothelial dysfunction and CV risk factors, and described it in hypercholesterolemia [78], diabetes mellitus [79], cigarette smoking [80], and aging [81], while the results in hypertensive patients were conflicting [3, 4, 82, 83]. Even though the method and pharmacologically induced vasodilation provide an insight into peripheral microvascular patho(physiology), venous occlusion plethysmography is characterized by several limitations and disadvantages, including its semi-invasive character, limited comparison between groups due to different initial blood pressure and forearm blood flow, different sizes of the forearm, etc., [68].

#### *3.1.3. Reactive hyperemia peripheral arterial tonometry (RH-PAT)*

vascular bed. However, main limitations are the cost, invasive nature, and therefore a limited population in which these measurements can be actually performed (symptomatic individu-

In recent years, a number of other methods have been developed among them: (a) blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI), a functional test that detects a dissociation of tissue hemoglobin from blood flow, is shown to be a useful tool for coronary endothelial function assessment [69]; (b) positron-emission tomography (PET) myocardial perfusion imaging that is based on the assessment of regional myocardial blood flow both at rest and during various forms of vasomotor stress [70] and presents a powerful tool to evaluate the effects of CV risk factors on the health of the microvasculature and its endothelium [71]; and (c) myocardial perfusion echocardiography, a bedside method with relatively low cost that is capable to detect myocardial perfusion abnormalities and quantify regional and global coronary blood flow [72]. Despite the fact that these new methods manage to provide noninvasive evaluation of coronary microvasculature directly at the site, they are still unacceptable for routine screening due to their limited availability, expensive equipment and associated costs, and lack of experienced/trained staff. Considering that endothelial function is a systemic disorder, peripheral vascular beds and their microcirculation present a good

alternative that provides an easier access and need less elaborate equipment.

Venous occlusion plethysmography presents a semi-invasive technique (arterial puncture) for assessment of forearm blood flow (and the corresponding microcirculation) changes before and after infusion of vasoactive substances into a cannulated brachial artery [3]. The method was introduced 90 years ago by Hewlett and van Zwaluwenburg [73], and the basic methodology has changed little since its first description. Basic principle of this method is to stop the return of venous blood from the forearm (inflating the cuff over the diastolic pressure value) with the preserved arterial blood inflow to the forearm, leading to a linear increase in blood flow at a given time, which is proportional to the arterial blood inflow. Another cuff excludes the blood flow through the hand to reduce the temperature fluctuations of the blood flow depending on the temperature. Changes in the flow are recorded by changing the electrical resistance of the plethysmograph located around the longest part of the forearm [74]. The main advantage of this method is that it provides assessment of endothelium-dependent and -independent vasodilation and mechanisms mediating it by intra-arterial infusion of vasoactive substances (e.g., ACh or sodium nitroprusside, and SNP), hormones, and drugs. However, its important limitation is that it could not strictly discern between macro- and microcirculation. Final results are expressed as ratio between blood flow changes in both arms and are well reproducible [75]. Regarding the mechanisms, some studies reported that ACh-induced dilation was inhibited by a NOS inhibitor, L-NG-monomethyl Arginine citrate (L-NMMA) [76], suggesting that NO is the main vasodilator mediating endothelium-dependent vasodilation in this vascular bed. On the other hand, others reported that EDHF has a crucial role in mediating microvascular endothelial-dependent vasodilation, especially in population with multiple CV risk factors [77]. A large number of studies used venous occlusion plethysmography to assess the association between endothelial dysfunction and CV risk

als requiring invasive coronary angiography) [68].

398 Endothelial Dysfunction - Old Concepts and New Challenges

*3.1.2. Venous occlusion plethysmography*

RH-PAT is a noninvasive technique designed for assessment of peripheral microvascular function. This method reflects changes in finger pulse volume amplitude during reactive hyperemia (an equivalent to finger plethysmography). PAT device includes digital probes that are placed on the tip of each index finger and a blood pressure cuff (for provoking occlusion) that is placed around the upper arm of the study arm, while the other arm serves as a control [84, 85]. Vascular occlusion is provoked by inflation of the blood pressure cuff to a 50 mmHg above systolic blood pressure for 5 min. The PAT signal is recorded 10 min prior occlusion, and for 10 min after the cuff is deflated. The final result of this measurement is calculated as the ratio of average amplitude of the PAT signal over a period of 1 min, starting 1 min after cuff deflation to average amplitude of the PAT signal for 3 min at baseline (RH-PAT index) that is normalized to the control arm [84, 85]. Studies have shown that RH-PAT is at least partly NO dependent. Importantly, studies by Rubinshtein et al. and Akiyama et al. reported that RH-PAT may be a useful tool for prediction of future CV events in patients with CV risk [86, 87]. Advantages of this method are that it is noninvasive, it is very simple and reproducible, and that it is operator independent (RH-PAT index is measured automatically). Even though RH-PAT is very similar to FMD of the brachial artery, Framingham Heart Study has revealed that there was no significant correlation between RH-PAT and FMD [88]. Moreover, the same study reported that different CV risk factors contribute differently to changes in FMD and RH-PAT [89], suggesting that these two methods assess different vascular beds, and that macro- and microvascular endothelium is differently susceptible to various risk factors.

#### *3.1.4. Laser Doppler (LD) flowmetry*

Because of its easy accessibility, the skin presents an appropriate site to study peripheral microcirculation, which was proposed as a suitable marker of systemic microvascular function in various diseases [89]. Therefore, in recent years, a number of simple and noninvasive methods have been developed in order to assess peripheral microcirculation. Still, it is an open question whether skin microcirculation is actually a representative indicator of the microvascular function of other organs. Despite that skin microvascular function was extensively used over the past 30 years to investigate vascular mechanisms in various diseases including hypertension [90, 91], obesity [92], diabetes [93, 94], aging, kidney disease [95], etc.

The laser Doppler (LD) technique is based on the estimation of the flow rate in the skin microcirculation using the laser beam reflection from the erythrocyte in microcirculation and its wavelength change (Doppler's effect) [96]. Computer software determines the flow size, which is rather an index of skin perfusion (flux) than direct measure of skin blood flow. Results are commonly expressed in arbitrary units (perfusion units, PU) or as cutaneous vascular conductance (CVC; flux divided by arterial pressure in mV/mmHg) [96]. The first developed technique was the laser Doppler flowmetry (LDF) that measures blood flow in a single point and thus over a small volume but with a high sampling frequency. A major limitation of this technique is its spatial variability, due to regional heterogeneity of skin perfusion and blood flow measurement in a single point [97]. Later, laser Doppler imaging (LDI) was developed, which provides a 2D image of skin microvascular perfusion using the same principle as LDF. Since this method assess flow over larger surface than LDF, it managed to reduce spatial variability, but it appears to be much slower than LDF, making rapid changes in blood flow difficult to record [98]. Both techniques are commonly used for microvascular reactivity assessment in response to various stimuli, including iontophoresis of vasoactive drugs, postocclusive reactive hyperemia (PORH), and thermal challenges [98].

used as vehicle than distilled water; pre-treatment with anesthetic should be considered; and,

The Markers of Endothelial Activation http://dx.doi.org/10.5772/intechopen.74671 401

PORH refers to an increase in (micro)vascular blood flow due to transient short vascular occlusion, and represents a test that is commonly used for assessment of microvascular reactivity [98]. According to the literature, several mechanisms are involved in microvascular PORH response, including sensory nerves involvement via neural axon reflex [110], metabolic and myogenic component, and endothelial-dependent vasodilators production. Regarding endothelium, EDHF was suggested as an important mediator of PORH [9], while the role of prostaglandins is still not clarified [11, 12]. Studies have reported that eNOS inhibition does not alter PORH, suggesting that NO is not normally involved in forearm microvascular PORH [111]. It has been suggested that inhibition of COX inhibition may unmask the NO dependence of PORH in human cutaneous circulation [12]. Despite an evident role of endothelium-derived vasoactive mediators in skin microvascular PORH, it should be used as a tool for assessment of general microvascular reactivity, rather than a measure for microvascular endothelial function [89]. PORH can be used in conjunction with both LDF and LDI, but an advantage is given to the LDF, because LDI is considered too slow to track microvascular kinetics during PORH. Moreover, inter-day reproducibility of single-point LDF was excellent when the probe was placed on exactly the same site from one day to another [112]. While recording skin microvascular PORH homogenizing both skin and room temperature is important, since temperature plays a key role in regulation of baseline flux [97]. Another issue is related to the PORH measurement, and that is heterogeneity in study design, especially vascular occlusion duration (from 1 to 15 min) [113] and different cuff pressures used, ranging between 160 and 220 mmHg [114]. Although it is accepted as a good tool for microvascular reactivity assess-

Local thermal hyperemia (LTH) presents peripheral skin microvascular response to local heating mediated by joint effect of neural-dependent and NO-dependent vasodilator pathway [98]. LTH is characterized by initial peak (within the first 5 min) which depends on sensory nerves, and by sustained plateau which is mostly NO-dependent [115]. LTH has better reproducibility in conjunction with LDI, rather than a single-point LDF, and this reproducibility depends on the site of measurement too [97]. Similar to PORH, there is heterogeneity in the study design using LTH, including local warming temperature (42–43°C) [116], the time of heating, and the nature of the device used to heat the skin [89]. Another used thermal stimulus is local cooling that induces an initial vasoconstriction followed by transient vasodilation, and finally, prolonged vasoconstriction [116]. It has been demonstrated that initial vasoconstriction depends on norepinephrine, and prolonged vasoconstriction involved both norepinephrine and inhibition of NO system [116]. Results have shown that this method has

the best reproducibility when the cooling protocol lasts for 30 min at 15°C [97].

vascular function assessment, especially when coupled with PORH and/or LTH [89].

Laser speckle contrast imaging is a novel technique that combines advantages of LDF and LDI, with very good inter-day reproducibility for both PORH and LTH measurements [117, 118]. This method is based on speckle contrast analysis that provides an index of blood blow. A potential limitation of this technique is its sensitivity to movements and potential challenging data analysis, but despite limitations, this method is expected to be a remarkable tool for micro-

finally, skin resistance should be reduced as much as possible.

ment, this method still requires standardization.

Microdialysis is a technique based on intradermal insertion of small fibers for continuous delivery of drugs into a small area of tissue. This type of drug delivery provides avoiding its systemic effect [99] and it provides controlled drug application and absence of currentinduced vasodilation, compared to iontophoresis. However, microdyalisis is invasive and painful, and justifies the use of local anesthesia which might also affect the blood flow and thus impact the results. It was commonly used to assess the role of NO in PORH and the thermal hyperemia response of skin microcirculation measured with LDF [98].

Iontophoresis is a method for noninvasive transdermal drug delivery (charged molecules) using low-density electric current. ACh and SNP iontophoresis are widely used for assessment of endothelium-dependent and endothelium-independent vasodilation of skin microcirculation [98, 100]. Regarding endothelium-dependent dilation, studies reported that ACh-induced dilation seems to be predominantly mediated by COX metabolites (although results are still conflicting) [101, 102], and NO does not extensively contribute to such dilation [103] in skin microcirculation. Beside endothelial-dependent vasodilation, ACh administration induces neural axon reflex-mediated dilation as well [104]. Iontophoresis is associated with several issues: (a) current itself may induce nonspecific vasodilation, which could interfere with the vasodilation potency of administrated drug, and it was suggested that it depends on the delivered electrical charge and the current delivery pattern [105]; (b) current-induced dilation also may depend on vehicles that have been used to dilute drugs (e.g., tap water, distilled water, deionized water, and saline), but this was not observed for ACh and SNP [106]; (c) skin resistance may influence drug delivery, and thus reduce skin resistance which was suggested as a part of good practice [100]; (d) spatial variability of ACh and SNP, suggesting that monitoring larger areas using LDI, rather than LDF provides better reproducibility [107, 108]; and (e) site of iontophoresis, since for example SNP-induced dilation could not be provoked on finger pulp, but it was provoked on the dorsum of the finger [109]. To summarize, ACh and SNP iontophoresis is widely used for endothelium-dependent and -independent microvascular vasodilation assessment in both healthy and various diseases. However, when interpreting results, complexity of mechanisms involved in these responses should be taken into account. Moreover, studies using iontophoresis should be carefully designed to reduce non-specific current-induced dilation by using low intensity current; saline should be rather used as vehicle than distilled water; pre-treatment with anesthetic should be considered; and, finally, skin resistance should be reduced as much as possible.

size, which is rather an index of skin perfusion (flux) than direct measure of skin blood flow. Results are commonly expressed in arbitrary units (perfusion units, PU) or as cutaneous vascular conductance (CVC; flux divided by arterial pressure in mV/mmHg) [96]. The first developed technique was the laser Doppler flowmetry (LDF) that measures blood flow in a single point and thus over a small volume but with a high sampling frequency. A major limitation of this technique is its spatial variability, due to regional heterogeneity of skin perfusion and blood flow measurement in a single point [97]. Later, laser Doppler imaging (LDI) was developed, which provides a 2D image of skin microvascular perfusion using the same principle as LDF. Since this method assess flow over larger surface than LDF, it managed to reduce spatial variability, but it appears to be much slower than LDF, making rapid changes in blood flow difficult to record [98]. Both techniques are commonly used for microvascular reactivity assessment in response to various stimuli, including iontophoresis of vasoactive drugs, post-

Microdialysis is a technique based on intradermal insertion of small fibers for continuous delivery of drugs into a small area of tissue. This type of drug delivery provides avoiding its systemic effect [99] and it provides controlled drug application and absence of currentinduced vasodilation, compared to iontophoresis. However, microdyalisis is invasive and painful, and justifies the use of local anesthesia which might also affect the blood flow and thus impact the results. It was commonly used to assess the role of NO in PORH and the ther-

Iontophoresis is a method for noninvasive transdermal drug delivery (charged molecules) using low-density electric current. ACh and SNP iontophoresis are widely used for assessment of endothelium-dependent and endothelium-independent vasodilation of skin microcirculation [98, 100]. Regarding endothelium-dependent dilation, studies reported that ACh-induced dilation seems to be predominantly mediated by COX metabolites (although results are still conflicting) [101, 102], and NO does not extensively contribute to such dilation [103] in skin microcirculation. Beside endothelial-dependent vasodilation, ACh administration induces neural axon reflex-mediated dilation as well [104]. Iontophoresis is associated with several issues: (a) current itself may induce nonspecific vasodilation, which could interfere with the vasodilation potency of administrated drug, and it was suggested that it depends on the delivered electrical charge and the current delivery pattern [105]; (b) current-induced dilation also may depend on vehicles that have been used to dilute drugs (e.g., tap water, distilled water, deionized water, and saline), but this was not observed for ACh and SNP [106]; (c) skin resistance may influence drug delivery, and thus reduce skin resistance which was suggested as a part of good practice [100]; (d) spatial variability of ACh and SNP, suggesting that monitoring larger areas using LDI, rather than LDF provides better reproducibility [107, 108]; and (e) site of iontophoresis, since for example SNP-induced dilation could not be provoked on finger pulp, but it was provoked on the dorsum of the finger [109]. To summarize, ACh and SNP iontophoresis is widely used for endothelium-dependent and -independent microvascular vasodilation assessment in both healthy and various diseases. However, when interpreting results, complexity of mechanisms involved in these responses should be taken into account. Moreover, studies using iontophoresis should be carefully designed to reduce non-specific current-induced dilation by using low intensity current; saline should be rather

occlusive reactive hyperemia (PORH), and thermal challenges [98].

400 Endothelial Dysfunction - Old Concepts and New Challenges

mal hyperemia response of skin microcirculation measured with LDF [98].

PORH refers to an increase in (micro)vascular blood flow due to transient short vascular occlusion, and represents a test that is commonly used for assessment of microvascular reactivity [98]. According to the literature, several mechanisms are involved in microvascular PORH response, including sensory nerves involvement via neural axon reflex [110], metabolic and myogenic component, and endothelial-dependent vasodilators production. Regarding endothelium, EDHF was suggested as an important mediator of PORH [9], while the role of prostaglandins is still not clarified [11, 12]. Studies have reported that eNOS inhibition does not alter PORH, suggesting that NO is not normally involved in forearm microvascular PORH [111]. It has been suggested that inhibition of COX inhibition may unmask the NO dependence of PORH in human cutaneous circulation [12]. Despite an evident role of endothelium-derived vasoactive mediators in skin microvascular PORH, it should be used as a tool for assessment of general microvascular reactivity, rather than a measure for microvascular endothelial function [89]. PORH can be used in conjunction with both LDF and LDI, but an advantage is given to the LDF, because LDI is considered too slow to track microvascular kinetics during PORH. Moreover, inter-day reproducibility of single-point LDF was excellent when the probe was placed on exactly the same site from one day to another [112]. While recording skin microvascular PORH homogenizing both skin and room temperature is important, since temperature plays a key role in regulation of baseline flux [97]. Another issue is related to the PORH measurement, and that is heterogeneity in study design, especially vascular occlusion duration (from 1 to 15 min) [113] and different cuff pressures used, ranging between 160 and 220 mmHg [114]. Although it is accepted as a good tool for microvascular reactivity assessment, this method still requires standardization.

Local thermal hyperemia (LTH) presents peripheral skin microvascular response to local heating mediated by joint effect of neural-dependent and NO-dependent vasodilator pathway [98]. LTH is characterized by initial peak (within the first 5 min) which depends on sensory nerves, and by sustained plateau which is mostly NO-dependent [115]. LTH has better reproducibility in conjunction with LDI, rather than a single-point LDF, and this reproducibility depends on the site of measurement too [97]. Similar to PORH, there is heterogeneity in the study design using LTH, including local warming temperature (42–43°C) [116], the time of heating, and the nature of the device used to heat the skin [89]. Another used thermal stimulus is local cooling that induces an initial vasoconstriction followed by transient vasodilation, and finally, prolonged vasoconstriction [116]. It has been demonstrated that initial vasoconstriction depends on norepinephrine, and prolonged vasoconstriction involved both norepinephrine and inhibition of NO system [116]. Results have shown that this method has the best reproducibility when the cooling protocol lasts for 30 min at 15°C [97].

Laser speckle contrast imaging is a novel technique that combines advantages of LDF and LDI, with very good inter-day reproducibility for both PORH and LTH measurements [117, 118]. This method is based on speckle contrast analysis that provides an index of blood blow. A potential limitation of this technique is its sensitivity to movements and potential challenging data analysis, but despite limitations, this method is expected to be a remarkable tool for microvascular function assessment, especially when coupled with PORH and/or LTH [89].
