**2.1. Biomarkers in pregnancy**

describe the ability of endothelium to release vasoactive substances and thereby regulate blood flow. The basic principle of vascular function is that healthy blood vessels dilate normally and the diseased blood vessels exhibit a dysfunctional vasodilation. Therefore, all methods for endothelial function assessment are based on the ability of endothelium to respond (vasodilation or vasoconstriction) to a specific stimulus (vascular occlusion, pharmacological vasodilators, heating, etc.).

Biomarkers are biological indicators of processes that are part of etiology of the diseases, and can, but do not have to be causal to diseases. One very important question is how specific and sensitive the marker is, since one molecule can appear in many conditions. Biomarkers of endothelial cell activation can be very diverse: biochemical/metabolic (such as plasma glucose, lipids, cytokines, asymmetric dimethylarginine (ADMA), high sensitive C-reactive protein (hsCRP), myeloperoxidase (MPO), cell adhesion molecules (CAMs), markers of coagulability, markers of oxidative stress, chemokines, microparticles, endothelial progenitor cells), functional biomarkers (such as flow-mediated dilation and other types of flowmetry, arteriographic measurements of vascular function) and structure (e.g., CIMT—carotid intima-

Over the last three decades, a number of methodological approaches were developed in order to evaluate and measure (patho)physiological function of the endothelium in humans [1, 2]. Evidently, these new methods intensified research and brought novelties in the field of vascular physiology and pathophysiology, but still are not implemented as clinical tools in daily practice. The approaches for endothelial function assessment were designed to provide insight into vascular/endothelial function in different sites (vascular beds) and different blood vessel types (conductive, resistant, and microcirculation). Earlier methods were more invasive (e.g., intracoronary infusion of acetylcholine (ACh), and later developed techniques that were less invasive have focused on peripheral circulation (forearm circulation) as a surrogate for coronary arteries [3–5]. As expected, all of these methods have their advantages and accepted limitations, and neither of the developed methods does present the absolute standard for the

There is an extensive body of evidence reporting that generalized endothelial dysfunction exhibited virtually in every arterial bed presents an early manifestation of a variety of cardiovascular diseases (CVDs) [6, 7]. Still, when investigating endothelial function in different CVDs, diverse (patho)physiological role of large conductance vessels and small microvascu-

There are many various molecules which have been denoted as vascular or endothelial markers, e.g., lipids, cytokines, ADMA, hsCRP, MPO, CAMs, markers of coagulability, markers of oxidative stress, chemokines, microparticles, and endothelial progenitor cells. It has been demonstrated that reduced bioavailability of nitric oxide (NO) plays a central role in impaired vascular/endothelial response (endothelial dysfunction) in conduit arteries, while NO in the microcirculation primarily modulates tissue metabolism [8]. On the other hand, a number of

**2. Biochemical biomarkers of vascular (endothelial) function**

evaluation of endothelial function, in both macro- and microcirculation.

media thickness, angiogenesis, or rarefaction).

392 Endothelial Dysfunction - Old Concepts and New Challenges

lature should be considered.

The importance of maternal vascular adaptation to pregnancy is to increase blood flow and to assure the proper development of the fetus. Several possible biochemical biomarkers have been proposed to evaluate vascular/endothelial function in pregnancy. First among them is NO, one of most important endothelial vasodilators, which is produced by NO synthase (NOS). It is well accepted that NOS-3 expression levels are increased in uterine artery endothelium in pregnancy [14]. Prostacyclin (PGI2) also plays an important role in vasodilator response, and its concentration is elevated in pregnancy [15]. In order to estimate the real impact of prostacyclin on vascular tone, determination of thromboxane a2 (TXA2)/PGI2 ratio is needed. Since both TXA2 and PGI2 have very short half-life, only indirect measures can be made of stable metabolites in the blood (thromboxane b2 (TXB2) and 6-keto-prostaglandin F2a (6-ketoPGF2A)), and there is no technique which allows their monitoring in real time. It has been demonstrated that cyclooxygenase 1 (COX1) is upregulated in endothelial cells during pregnancy, and therefore induces a PGI2 increment [14]. EDHF is the third major player in endothelial vasodilation in pregnancy, causing smooth muscle relaxation. As it is not a single factor and there is still ongoing research to identify its specific components, it is described as spectrum of responses that are neither NO nor PGI2 mediated. Another limiting problem is that there is no appropriate method for its tracking. Although EDHF may seem as unnecessary pathway beside NO and PGI2, a number of studies showed an important role of EDHF in endothelium vasodilation in pregnancy, suggesting that without EDHF, there would not be sufficient blood flow to the fetus [16].

Endogenous eNOS inhibitor ADMA concentrations were found to be significantly lower in pregnant women. However, this did not explain the improved flow-mediated dilation (FMD) in the correlation analysis [17, 18]. Also, endothelial function in normal pregnancy was not attenuated despite the significant increase in hsCRP, and pregnancy-related changes in the concentrations of proinflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), were nonsignificant [19].

#### **2.2. Biomarkers in exercise**

It has been reported that the regulation of the NO-dependent pathway presents a key mechanism mediating endothelial adaptations to shear stress, including increased NO synthesis, increased expression and activity of antioxidative enzymes (e.g., superoxide dismutase (SOD) and catalase), and decreased oxidative stress level (reactive oxygen species (ROS) production) which all increases NO bioavailability. However, recent studies demonstrated that COX-dependent pathway and increased PGI2 synthesis take part in endothelial adaptations to shear stress, as well. Furthermore, a growing body of evidence suggest that increased shear stress generated by increased blood flow during exercise, presents a prime signal for decreased level of vasoconstrictor endothelin 1 (ET1), and inflammatory markers such as vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemotactic protein 1 (MCP-1) level [20]. Furthermore, increased endothelial NOS (eNOS) gene expression has been proposed to be a marker of anterograde shear stress-induced endothelial activation (result of repeated episodic increase in blood flow during exercise), and to have anti-atherogenic effect in endothelial cell cultures [21, 22].

3 months showed reduced serum ADMA levels for 30% [31]. Another study, from Stuhlinger et al. found that rosiglitazone reduced the level of ADMA by 30% in seven insulin-resistant

The Markers of Endothelial Activation http://dx.doi.org/10.5772/intechopen.74671 395

Toll-like receptors (TLRs), such as toll-like receptors TLR2 and TLR4 have been found to have elevated expression in T2DM patients, which could be a possible underlying mechanism of inflammation in T2DM [33]. TLR-2 and TLR-4 activation has also been found in murine models of atherosclerosis [33, 34]. There are many unanswered questions: the consequences of activation/blockade of TLRs in atherosclerosis, relationship between innate and adaptive responses in atherosclerosis, and mechanistic insight on the intricate balance of direct and risk

The over expression of TNF-alpha and its inflammatory and immunomodulatory effects have been implicated in the pathogenesis of CAD and myocardial dysfunction. Cardiovascular complications may be influenced by TNF-alpha gene polymorphisms. Certain studies failed to find a significant association between the TNF-alpha gene polymorphisms and CVD [35].

IL-6 is associated with the process of inflammation and coronary artery disease. Patients with high levels of IL-6 show worse in-hospital outcome following treatment in case of unstable angina. An association has been shown between the IL-6 promoter polymorphism −174G/C

Endothelial cells also express chemotactic factors: MCP-1, proinflammatory cytokines (macrophage colony-stimulating factor) and tumor necrosis factor-beta (TNF-β) [36]. Hyperglycemia promotes MCP-1 expression in vascular endothelial cells and has a pivotal role in the pathogenesis of diabetic vasculopathy [37]. Patients with diabetes mellitus or obesity have increased circulating levels of inflammatory markers, including C reactive protein (CRP), TNF-α, and IL-6 [38–40]. Blood level of CRP, as independent predictor of diabetes, is increased in both Type I and Type II diabetes [41, 42]. TNF-α can induce cytokines such as IL-6 which regulates the expression of CRP. They can impair endothelial function and contribute to atherothrombosis especially in patients with Type II diabetes, alone or in combination [43]. It was also found in male diabetic patients that increased levels of inflammatory markers predict cardio-

Microparticles, the membrane vesicles released by various cell types and circulating endothelial cells represent novel biomarkers of endothelial injury, associated with atherosclerosis and related complications (thrombosis, inflammation, and apoptosis). Microparticles are suggested to be biomarkers of vascular injury and inflammation [45]. Changes in circulating levels of microparticles might give an important clinical information in healthy subjects or patients with CVDs as a surrogate marker of vascular function, but it is still not clear whether

Endocan or endothelial cell specific molecule-1 (ESM-1) is a novel endothelium-derived soluble proteoglycan [46]. It binds to a wide range of bioactive molecules associated with cellular signaling and adhesion. It is involved in regulation of proliferation, differentiation, migration, and adhesion of different types of cells in health and disease. The endocan concentration is related to endothelial activation and neovascularization [47]. Endocan levels are elevated in

and hypertension, left ventricular hypertrophy and ischemic heart disease CAD [35].

non-diabetic hypertensive individuals [32].

factor-mediated effects of TLRs in CVD [33, 34].

vascular risk in diabetic patients [44].

it is a cause or effect of atherosclerosis [45].

Further studies are required to resolve this controversy.

On the other hand, rhythmic stretching (cyclic strain) provoked by systolic blood pressure changes during exercise affects endothelial cell growth and NO- and EDHF-dependent vasodilation pathway, and its effect depends on the blood pressure increment during exercise (e.g., >135 mmHg elicits inhibition of endothelial cell growth) [23]. Surprisingly, further studies on endothelial cell cultures have reported that rhythmic stretching can induce ROS production and increase the expression of cell adhesion molecules. On the other hand, ROS produced by cyclic strain may indirectly increase expression of eNOS [24]. It became evident that the time of exposure to high blood pressure/cyclic strain (continuous or pulsatile) is crucial for its final effect on endothelial function. Brief increases in blood pressure and ROS production associated with bouts of exercise may signal an increase in eNOS production and other beneficial effects resulting in improved endothelial function. Chronic increases in cyclic strain (e.g., hypertension) may elevate ROS chronically and finally provoke development of endothelial dysfunction. Thus, beside abovementioned endothelial biomarkers of inflammation and endothelial dysfunction, measurement of oxidative stress level and antioxidant capacity present suitable and commonly used markers of endothelial response to different exercise modes and patterns (shear stress) in both health and disease.

#### **2.3. Biomarkers in cardiometabolic diseases**

Oxidation of low density lipoproteins (oxLDL) and NO synthesis contribute to endothelial dysfunction, vascular aging, and disease. OxLDL and NO exert contradictory actions within the vascular endothelium such as: leukocyte adhesion, platelet aggregation, and vascular smooth muscle cell proliferation and migration [25, 26]. While oxLDL—an oxidative stress biomarker—has been identified as a pro-atherogenic risk factor for coronary artery disease (CAD), NO is a free radical signal-transducing molecule that maintains vasodilation, modulates *in vitro* lipid peroxidation reactions and alters pro-inflammatrory gene expression. Both are part of complex atherosclerotic process, from initiation to plaque destabilization and coronary artery disease [25, 26].

As already mentioned, ADMA is an endogenous inhibitor of NO synthase [27] and thus may cause endothelial dysfunction [28]. Increased plasma levels of ADMA are related with hyperlipidemia, hypertension, coronary artery disease, unstable angina, stroke and end-stage renal disease and diabetes [28]. Reduced plasma levels of ADMA after percutaneous coronary intervention could be indicative of a reduced risk of recurrent cardiovascular events. Although ADMA was significantly associated with all-cause mortality in patients with acute coronary syndrome and ischemic heart disease, there is no clear association between ADMA and cardiovascular disease incidence [29]. Type II diabetes has been associated with increased ADMA levels. ADMA and NO have been found to be significant determinants of insulin resistance [30]. A study performed in type 2 diabetes patients that used antidiabetic metformin for 3 months showed reduced serum ADMA levels for 30% [31]. Another study, from Stuhlinger et al. found that rosiglitazone reduced the level of ADMA by 30% in seven insulin-resistant non-diabetic hypertensive individuals [32].

decreased level of vasoconstrictor endothelin 1 (ET1), and inflammatory markers such as vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemotactic protein 1 (MCP-1) level [20]. Furthermore, increased endothelial NOS (eNOS) gene expression has been proposed to be a marker of anterograde shear stress-induced endothelial activation (result of repeated episodic increase in blood flow during exercise), and to have anti-atherogenic effect in endo-

On the other hand, rhythmic stretching (cyclic strain) provoked by systolic blood pressure changes during exercise affects endothelial cell growth and NO- and EDHF-dependent vasodilation pathway, and its effect depends on the blood pressure increment during exercise (e.g., >135 mmHg elicits inhibition of endothelial cell growth) [23]. Surprisingly, further studies on endothelial cell cultures have reported that rhythmic stretching can induce ROS production and increase the expression of cell adhesion molecules. On the other hand, ROS produced by cyclic strain may indirectly increase expression of eNOS [24]. It became evident that the time of exposure to high blood pressure/cyclic strain (continuous or pulsatile) is crucial for its final effect on endothelial function. Brief increases in blood pressure and ROS production associated with bouts of exercise may signal an increase in eNOS production and other beneficial effects resulting in improved endothelial function. Chronic increases in cyclic strain (e.g., hypertension) may elevate ROS chronically and finally provoke development of endothelial dysfunction. Thus, beside abovementioned endothelial biomarkers of inflammation and endothelial dysfunction, measurement of oxidative stress level and antioxidant capacity present suitable and commonly used markers of endothelial response to different exercise

Oxidation of low density lipoproteins (oxLDL) and NO synthesis contribute to endothelial dysfunction, vascular aging, and disease. OxLDL and NO exert contradictory actions within the vascular endothelium such as: leukocyte adhesion, platelet aggregation, and vascular smooth muscle cell proliferation and migration [25, 26]. While oxLDL—an oxidative stress biomarker—has been identified as a pro-atherogenic risk factor for coronary artery disease (CAD), NO is a free radical signal-transducing molecule that maintains vasodilation, modulates *in vitro* lipid peroxidation reactions and alters pro-inflammatrory gene expression. Both are part of complex atherosclerotic process, from initiation to plaque destabilization and coro-

As already mentioned, ADMA is an endogenous inhibitor of NO synthase [27] and thus may cause endothelial dysfunction [28]. Increased plasma levels of ADMA are related with hyperlipidemia, hypertension, coronary artery disease, unstable angina, stroke and end-stage renal disease and diabetes [28]. Reduced plasma levels of ADMA after percutaneous coronary intervention could be indicative of a reduced risk of recurrent cardiovascular events. Although ADMA was significantly associated with all-cause mortality in patients with acute coronary syndrome and ischemic heart disease, there is no clear association between ADMA and cardiovascular disease incidence [29]. Type II diabetes has been associated with increased ADMA levels. ADMA and NO have been found to be significant determinants of insulin resistance [30]. A study performed in type 2 diabetes patients that used antidiabetic metformin for

modes and patterns (shear stress) in both health and disease.

**2.3. Biomarkers in cardiometabolic diseases**

nary artery disease [25, 26].

thelial cell cultures [21, 22].

394 Endothelial Dysfunction - Old Concepts and New Challenges

Toll-like receptors (TLRs), such as toll-like receptors TLR2 and TLR4 have been found to have elevated expression in T2DM patients, which could be a possible underlying mechanism of inflammation in T2DM [33]. TLR-2 and TLR-4 activation has also been found in murine models of atherosclerosis [33, 34]. There are many unanswered questions: the consequences of activation/blockade of TLRs in atherosclerosis, relationship between innate and adaptive responses in atherosclerosis, and mechanistic insight on the intricate balance of direct and risk factor-mediated effects of TLRs in CVD [33, 34].

The over expression of TNF-alpha and its inflammatory and immunomodulatory effects have been implicated in the pathogenesis of CAD and myocardial dysfunction. Cardiovascular complications may be influenced by TNF-alpha gene polymorphisms. Certain studies failed to find a significant association between the TNF-alpha gene polymorphisms and CVD [35]. Further studies are required to resolve this controversy.

IL-6 is associated with the process of inflammation and coronary artery disease. Patients with high levels of IL-6 show worse in-hospital outcome following treatment in case of unstable angina. An association has been shown between the IL-6 promoter polymorphism −174G/C and hypertension, left ventricular hypertrophy and ischemic heart disease CAD [35].

Endothelial cells also express chemotactic factors: MCP-1, proinflammatory cytokines (macrophage colony-stimulating factor) and tumor necrosis factor-beta (TNF-β) [36]. Hyperglycemia promotes MCP-1 expression in vascular endothelial cells and has a pivotal role in the pathogenesis of diabetic vasculopathy [37]. Patients with diabetes mellitus or obesity have increased circulating levels of inflammatory markers, including C reactive protein (CRP), TNF-α, and IL-6 [38–40]. Blood level of CRP, as independent predictor of diabetes, is increased in both Type I and Type II diabetes [41, 42]. TNF-α can induce cytokines such as IL-6 which regulates the expression of CRP. They can impair endothelial function and contribute to atherothrombosis especially in patients with Type II diabetes, alone or in combination [43]. It was also found in male diabetic patients that increased levels of inflammatory markers predict cardiovascular risk in diabetic patients [44].

Microparticles, the membrane vesicles released by various cell types and circulating endothelial cells represent novel biomarkers of endothelial injury, associated with atherosclerosis and related complications (thrombosis, inflammation, and apoptosis). Microparticles are suggested to be biomarkers of vascular injury and inflammation [45]. Changes in circulating levels of microparticles might give an important clinical information in healthy subjects or patients with CVDs as a surrogate marker of vascular function, but it is still not clear whether it is a cause or effect of atherosclerosis [45].

Endocan or endothelial cell specific molecule-1 (ESM-1) is a novel endothelium-derived soluble proteoglycan [46]. It binds to a wide range of bioactive molecules associated with cellular signaling and adhesion. It is involved in regulation of proliferation, differentiation, migration, and adhesion of different types of cells in health and disease. The endocan concentration is related to endothelial activation and neovascularization [47]. Endocan levels are elevated in


adhesion, and migration, indicating that pro-inflammatory environment in diabetes is a criti-

The Markers of Endothelial Activation http://dx.doi.org/10.5772/intechopen.74671 397

Increased concentration of plasma glucose activates the endothelium [56–58]. Exposure of arterial tissue to increased glucose level induces superoxide production and impairs NO bioavailability in the vascular wall which leads to increased oxidative stress in these conditions [59]. In diabetes mellitus, the production of superoxide and NADPH oxidase activity are increased [60, 61] which promote activation of the pro-inflammatory transcription factor NFκB [56]. The transcription factor NFκB is one of key regulator of endothelial activation and is included in insulin resistance [62, 63]. This is supported by study in obese persons [64]. Salsalate (an anti-inflammatory drug) increased expression of the inhibitor of NF-κB and reduced NFκB activation in freshly isolated endothelial cells taken from obese persons. Salsalate increased brachial artery flow-mediated dilation and reduced nitrotyrosine and expression of NADPH oxidase p47(phox) in these endothelial cells [64]. **Table 1** presents some of the proposed novel biomarkers for atherosclerosis, which could also be related to

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

cal factor in pro-atherosclerotic effects of hyperglycemia [54, 55].

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

**3.1. Assessment of microvascular endothelial function**

*3.1.1. Coronary microvascular function assessment*

other cardiometabolic diseases.

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

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 significantly positively correlated with ICAM-1 [49].

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, adhesion, and migration, indicating that pro-inflammatory environment in diabetes is a critical factor in pro-atherosclerotic effects of hyperglycemia [54, 55].

Increased concentration of plasma glucose activates the endothelium [56–58]. Exposure of arterial tissue to increased glucose level induces superoxide production and impairs NO bioavailability in the vascular wall which leads to increased oxidative stress in these conditions [59]. In diabetes mellitus, the production of superoxide and NADPH oxidase activity are increased [60, 61] which promote activation of the pro-inflammatory transcription factor NFκB [56]. The transcription factor NFκB is one of key regulator of endothelial activation and is included in insulin resistance [62, 63]. This is supported by study in obese persons [64]. Salsalate (an anti-inflammatory drug) increased expression of the inhibitor of NF-κB and reduced NFκB activation in freshly isolated endothelial cells taken from obese persons. Salsalate increased brachial artery flow-mediated dilation and reduced nitrotyrosine and expression of NADPH oxidase p47(phox) in these endothelial cells [64]. **Table 1** presents some of the proposed novel biomarkers for atherosclerosis, which could also be related to other cardiometabolic diseases.
