**3. Endothelium and vascular tone regulation: endothelial vasodilators**

One of the main functions of endothelium is its involvement in vascular tone regulation. In response to mechanical and pharmacological (ACh, histamine, bradykinin, VEGF, various hormones as estrogen, CGRP, substance P, insulin, and platelets products (serotonin, ADP)) stimuli, endothelium releases a number of vasoactive mediators which, by affecting VSMC, regulate vascular tone and thus help to adjust blood flow to tissue demands. There is a considerable and complex interplay between endothelial and other humoral vasoactive substances, as well as the sympathetic nervous system. Mostly investigated endothelial vasodilators are NO, PGI<sup>2</sup> , and endothelium-derived hyperpolarizing factor(s) (EDHF), whereas the main constrictors include endothelin, thromboxane, AngII, the cytochrome P450 (CYP)-hydroxylase-derived 20-hydroxyeicosatetraenoic acids (20-HETE) [113, 114], and constrictor prostanoids [115]. In a healthy state, vasodilators predominate whereas in endothelial dysfunction, reduced vasodilator bioavailability, in particular NO and an excessive release of vasoconstrictors result in an increased vascular tone. Moreover, in the settings of endothelial dysfunction, other vasodilators may compensate for the reduced NO bioavailability [116–118].

Of note is the dual role of eNOS: when the bioavailability of its substrates and/or cofactors is reduced and in conditions of increased oxidative stress, eNOS can get uncoupled and produces superoxide anion (O<sup>2</sup>−) instead of NO; O<sup>2</sup>− is a highly reactive radical which forms peroxynitrite (ONOO−) with NO, and in this way, it additionally decreases the bioavailability

Besides directly inducing vascular relaxations by activating the soluble guanylate cyclase

endothelin) affecting vascular tone [123, 124], and interferes with the sympathetic neurotrans-

NO exerts other effects: it inhibits platelet aggregation (interestingly, activated-plateletderived substances increase the activity of eNOS, thus producing more NO) and the adhesion of leucocytes to the vascular wall by decreasing the expression of adhesion molecules on endothelial surface [90]. Moreover, it interferes with cellular metabolism [125] by modulating mitochondrial function, and oxygen metabolism [106, 126]. As stated, NO forms ROS (ONOO−) with increased levels of O2− which, among others, impairs the mitochondrial respiratory chain [127]. On the other hand, the depolarization of mitochondrial membrane induced by mitochondrial KATP-channel activators has been reported to increase the activity of eNOS in rat cerebral arteries [128]. Other gaseous mediators involved in vascular tone regulation have

In the settings of hypoxia, NO could alternatively be derived from the reduction of ONOO<sup>−</sup>

A number of factors have been known to exert beneficial vasoprotective effects by interfering with eNOS activity and consequently increasing NO bioavailability, including female sex hormones (the protecting effects of estrogens have long been appreciated), insulin, glucagon-like peptide, thyroid hormones, erythropoietin, high density lipoproteins, etc., as well as endothe-

On the other hand, many factors negatively impact eNOS or scavenge NO, thereby reducing

levels of arginase, asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, cortisol, aldosterone, and numerous others. Moreover, environmental factors, such as smoking, radiation, increased salt intake or hyperglycemia, as well as other metabolic disturbances, such as insulin resistance and metabolic syndrome, hypercholesterolemia and obesity, homocysteinemia, uric acid, diminish the NO-dependent vasodilation and other NO-associated effects [130]. The latter settings predispose to endothelial dysfunction and the development of diseases, such as hypertension and atherosclerosis, and increase the risk for

Therefore, the biosynthetic pathways of NO and its downstream targets might all represent

Increased levels of ROS could be regarded as a common denominator of reduced NO bioavailability resulting in endothelial dysfunction. Fortunately, the endogenous ROS-scavenging

O2

thelial factors inducing VSMC hyperpolarization and vasodilation, respectively [75, 132, 133].

lial exposure to repetitive increases in shear stress as occurs in exercise [25, 130].

, EDHF,

Endothelium at a Glance

13

http://dx.doi.org/10.5772/intechopen.81286

or arginine, due to increased

) which is proposed as one of important endo-

(sGC) in VSMC, NO importantly modulates other endothelial autacoids (e.g., PGI<sup>2</sup>

of NO and contributes to endothelial dysfunction [43, 44, 90].

also been proposed to interfere with the mitochondrial function [127].

which may partly compensate for reduced eNOS activity [129].

the beneficial effects of NO: ROS, reduced availability of BH<sup>4</sup>

cardiovascular events.

potential therapeutic targets [90, 131].

enzymes convert O<sup>2</sup>− to hydrogen peroxide (H<sup>2</sup>

mission pre- and postsynaptically [89].

Endothelial vasodilators mediate the endothelium-dependent vasodilation. Yet, it must be noted that the contribution of each to the regulation of vascular tone differs between the vascular beds: while NO has mainly been implicated in the regulation of larger vessels, other vasodilators seem to play the prominent role in microcirculation [116, 117, 119].

FMD denotes endothelium-dependent decrease of vascular tone in response to increased blood flow, which is noted down—as well as upstream of the vessel tree, and has been used as a surrogate marker of endothelial function in the clinics. It is mostly tested by performing a transient occlusion of a distal (or proximal) artery to induce local ischemia and assessing blood flow increase after reperfusion. The phenomenon of postocclusive hyperemia (PORH) is a good example to explain the mechanism of FMD: endothelium-dependent vasodilation elicited by increased shear stress due to increased flow as a consequence of the vasorelaxant effect of locally accumulated metabolites additionally increases the flow to meet tissue metabolic demands and simultaneously oppose the pressure-induced myogenic vasoconstrictory response [116].

#### **3.1. Nitric oxide exerts many vasoprotective functions**

NO is constantly being formed from the amino acid L-arginine by constitutively expressed eNOS. It can also be produced by other isoforms of NOS, namely inducible (iNOS) and neuronal (nNOS), present in various cell types (endothelial cells, platelets, neurons and VSMC, macrophages, polymorphonucleated leukocytes) which are important in the settings of endothelial activation and/or dysfunction [43]. eNOS is a calmodulin-dependent enzyme requiring cofactors as Ca2+, tetrahydrobiopterin (BH<sup>4</sup> ), nicotinamide adenine dinucleotide phosphate (NADP), etc. Its activity is regulated by complex interactions of the endothelial microdomain proteins whereby its association with the heat-shock protein increases and with the caveolin-1 decreases its activity [44]. Moreover, it is modulated posttranslationally in a Ca2+-dependent way through the activation of various Ca2+-channels on the cell membrane and by Ca2+-independent way which is the main mechanism of shear-stress-induced eNOS activation. The Ca2+-independent pathway mainly affects the phosphorylation of eNOS by Akt kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), or AMPK, depending on the stimulus; a variety of stimuli, including hormones, growth factors, purines, histamine, bradykinin, serotonin, noradrenaline, etc., affect G-protein-coupled receptors and finally activate the corresponding target to subsequently phosphorylate eNOS and increase its activity [43]. On the other hand, hyperglycemia has been suggested to adversely affect eNOS phosphorylation thus attenuating NO production [120]. Another common posttranslational modification of eNOS activity is acetylation/deacetylation: for example, SIRT-1, a class III histone deacetylase enhances the ACh- and shear-stress-induced-NO production by deacetylating eNOS, thereby enhancing its binding to calmodulin [121]. Moreover, aspirin has been shown to enhance eNOS activation by acetylating eNOS, independently of its effect on cyclooxygenase inhibition [122].

Of note is the dual role of eNOS: when the bioavailability of its substrates and/or cofactors is reduced and in conditions of increased oxidative stress, eNOS can get uncoupled and produces superoxide anion (O<sup>2</sup>−) instead of NO; O<sup>2</sup>− is a highly reactive radical which forms peroxynitrite (ONOO−) with NO, and in this way, it additionally decreases the bioavailability of NO and contributes to endothelial dysfunction [43, 44, 90].

(CYP)-hydroxylase-derived 20-hydroxyeicosatetraenoic acids (20-HETE) [113, 114], and constrictor prostanoids [115]. In a healthy state, vasodilators predominate whereas in endothelial dysfunction, reduced vasodilator bioavailability, in particular NO and an excessive release of vasoconstrictors result in an increased vascular tone. Moreover, in the settings of endothelial dysfunction, other vasodilators may compensate for the reduced NO bioavailability [116–118]. Endothelial vasodilators mediate the endothelium-dependent vasodilation. Yet, it must be noted that the contribution of each to the regulation of vascular tone differs between the vascular beds: while NO has mainly been implicated in the regulation of larger vessels, other

FMD denotes endothelium-dependent decrease of vascular tone in response to increased blood flow, which is noted down—as well as upstream of the vessel tree, and has been used as a surrogate marker of endothelial function in the clinics. It is mostly tested by performing a transient occlusion of a distal (or proximal) artery to induce local ischemia and assessing blood flow increase after reperfusion. The phenomenon of postocclusive hyperemia (PORH) is a good example to explain the mechanism of FMD: endothelium-dependent vasodilation elicited by increased shear stress due to increased flow as a consequence of the vasorelaxant effect of locally accumulated metabolites additionally increases the flow to meet tissue metabolic demands and simultaneously oppose the pressure-induced myogenic vasoconstrictory

NO is constantly being formed from the amino acid L-arginine by constitutively expressed eNOS. It can also be produced by other isoforms of NOS, namely inducible (iNOS) and neuronal (nNOS), present in various cell types (endothelial cells, platelets, neurons and VSMC, macrophages, polymorphonucleated leukocytes) which are important in the settings of endothelial activation and/or dysfunction [43]. eNOS is a calmodulin-dependent enzyme

phosphate (NADP), etc. Its activity is regulated by complex interactions of the endothelial microdomain proteins whereby its association with the heat-shock protein increases and with the caveolin-1 decreases its activity [44]. Moreover, it is modulated posttranslationally in a Ca2+-dependent way through the activation of various Ca2+-channels on the cell membrane and by Ca2+-independent way which is the main mechanism of shear-stress-induced eNOS activation. The Ca2+-independent pathway mainly affects the phosphorylation of eNOS by Akt kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), or AMPK, depending on the stimulus; a variety of stimuli, including hormones, growth factors, purines, histamine, bradykinin, serotonin, noradrenaline, etc., affect G-protein-coupled receptors and finally activate the corresponding target to subsequently phosphorylate eNOS and increase its activity [43]. On the other hand, hyperglycemia has been suggested to adversely affect eNOS phosphorylation thus attenuating NO production [120]. Another common posttranslational modification of eNOS activity is acetylation/deacetylation: for example, SIRT-1, a class III histone deacetylase enhances the ACh- and shear-stress-induced-NO production by deacetylating eNOS, thereby enhancing its binding to calmodulin [121]. Moreover, aspirin has been shown to enhance eNOS activation by acetylating eNOS, independently of its effect on

), nicotinamide adenine dinucleotide

vasodilators seem to play the prominent role in microcirculation [116, 117, 119].

**3.1. Nitric oxide exerts many vasoprotective functions**

12 Endothelial Dysfunction - Old Concepts and New Challenges

requiring cofactors as Ca2+, tetrahydrobiopterin (BH<sup>4</sup>

cyclooxygenase inhibition [122].

response [116].

Besides directly inducing vascular relaxations by activating the soluble guanylate cyclase (sGC) in VSMC, NO importantly modulates other endothelial autacoids (e.g., PGI<sup>2</sup> , EDHF, endothelin) affecting vascular tone [123, 124], and interferes with the sympathetic neurotransmission pre- and postsynaptically [89].

NO exerts other effects: it inhibits platelet aggregation (interestingly, activated-plateletderived substances increase the activity of eNOS, thus producing more NO) and the adhesion of leucocytes to the vascular wall by decreasing the expression of adhesion molecules on endothelial surface [90]. Moreover, it interferes with cellular metabolism [125] by modulating mitochondrial function, and oxygen metabolism [106, 126]. As stated, NO forms ROS (ONOO−) with increased levels of O2− which, among others, impairs the mitochondrial respiratory chain [127]. On the other hand, the depolarization of mitochondrial membrane induced by mitochondrial KATP-channel activators has been reported to increase the activity of eNOS in rat cerebral arteries [128]. Other gaseous mediators involved in vascular tone regulation have also been proposed to interfere with the mitochondrial function [127].

In the settings of hypoxia, NO could alternatively be derived from the reduction of ONOO<sup>−</sup> which may partly compensate for reduced eNOS activity [129].

A number of factors have been known to exert beneficial vasoprotective effects by interfering with eNOS activity and consequently increasing NO bioavailability, including female sex hormones (the protecting effects of estrogens have long been appreciated), insulin, glucagon-like peptide, thyroid hormones, erythropoietin, high density lipoproteins, etc., as well as endothelial exposure to repetitive increases in shear stress as occurs in exercise [25, 130].

On the other hand, many factors negatively impact eNOS or scavenge NO, thereby reducing the beneficial effects of NO: ROS, reduced availability of BH<sup>4</sup> or arginine, due to increased levels of arginase, asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, cortisol, aldosterone, and numerous others. Moreover, environmental factors, such as smoking, radiation, increased salt intake or hyperglycemia, as well as other metabolic disturbances, such as insulin resistance and metabolic syndrome, hypercholesterolemia and obesity, homocysteinemia, uric acid, diminish the NO-dependent vasodilation and other NO-associated effects [130]. The latter settings predispose to endothelial dysfunction and the development of diseases, such as hypertension and atherosclerosis, and increase the risk for cardiovascular events.

Therefore, the biosynthetic pathways of NO and its downstream targets might all represent potential therapeutic targets [90, 131].

Increased levels of ROS could be regarded as a common denominator of reduced NO bioavailability resulting in endothelial dysfunction. Fortunately, the endogenous ROS-scavenging enzymes convert O<sup>2</sup>− to hydrogen peroxide (H<sup>2</sup> O2 ) which is proposed as one of important endothelial factors inducing VSMC hyperpolarization and vasodilation, respectively [75, 132, 133]. The bad ROS are turned into the good ones thus ameliorating their damaging effects [133]. Indeed, increased levels of H2 O2 have also been suggested to overtake the role of NO in the settings of reduced NO bioavailability also in humans [134].

large-conductance Kca channels; inward rectifier K<sup>+</sup>

capillaries [153].

channels [158].

**4. Conclusion**

[148] have been implicated in finally inducing EDH. To this end, the corresponding channels have been investigated as potential targets to affect EDH [147, 149]. Endothelial hyperpolarization can spread via myoendothelial gap junctions to directly induce VSMC hyperpolarization: the role of gap junctions has been implicated also in humans *in vitro* and *in vivo* as carbenoxolone, a nonspecific gap junction blocker, diminished conducted vasodilation in isolated human coronary arterioles [150], and, moreover, reduced FMD in the brachial artery of healthy volunteers [151]. The term conducted dilation has been used to denote electrotonic transmission of local hyperpolarization, and may spread several mm upstream independent of alterations in shear stress [116, 119, 152]. It reflects the involvement of gap junctions and enables a coordinated adjustment of vascular resistance in larger and smaller arterioles and

Age and gender might affect the EDHF-mediated response as male animals were reported to exhibit smaller EDHF-mediated endothelium-dependent relaxation compared to females; similarly, aging affected EDH. A reduced SKCa activity and a reduced expression of gap junction proteins, Cx-40 and Cx-43, have been suggested to account for these differences [154]. Moreover, 17b-estradiol has been proposed to activate AMPK and/or SIRT1, both implicated to be involved in increasing the EDHF-mediated signaling [154]. Alteration of the EDH con-

Finally, other endothelium-derived vasodilators should be listed: adenosine and purines, various peptides, including CGRP, C-type natriuretic peptide (CNP), adrenomedullin, endo-

carbon monoxide (CO) which have been suggested to compensate for decreased NO bioavailability [127, 155, 156]. Adenosine, one of the most potent vasodilators (exerting also other vasculoprotective effects) is mainly formed from extracellular nucleotides (ATP, AMP) by the action of ectonucleotidases, expressed in endothelial cells and also investigated as potential therapeutic targets [157]. Interestingly, circulating ATP itself has been proposed to mediate vasodilation in humans *in vivo* by inducing vascular hyperpolarization via activation of KIR

By spreading throughout the vascular system and exerting pleiotropic functions, the endothelium could be regarded as one of the main players in cardiovascular physiology. The integrity of endothelium is crucial for vascular homeostasis and health. On the other hand, endothelial cells are susceptible to changes in blood composition and hemodynamic forces and as such vulnerable to developing endothelial dysfunction. Endothelial dysfunction nowadays is acknowledged a key initiating event in atherosclerosis, and connected to several pathological conditions and cardiovascular events. Accordingly, understanding endothelial function and dysfunction is crucial for recognition and treatment, or, optimally, for prevention of the development of cardiovascular diseases, the leading cause of death worldwide. To this end,

cannabinoids, and gaseous transmitters other than NO, namely hydrogen sulfide (H<sup>2</sup>

tributes to endothelial dysfunction observed in various pathologies [145, 154].

channels (KIR) [147], and TRPV4 channels

http://dx.doi.org/10.5772/intechopen.81286

Endothelium at a Glance

15

S), and

#### **3.2. Endothelium-dependent relaxations beyond NO: hyperpolarization**

Shear stress and various agonists also stimulate the production of other vasodilators, among which the derivatives of arachidonic acid (AA) play an important role.

AA is liberated from membrane phospholipids by the action of Ca2+-stimulated phospholipase A2 , and subsequently metabolized into biologically active eicosanoids with a variety of functions. Three main complex enzymatic pathways of AA metabolism are the cyclooxygenase pathway, the cytochrome (CYP)-P450 pathway, and the lipoxygenase pathway; however, AA can be transformed into isoprostanes nonenzymatically by ROS [135]. Regarding vascular tone regulation, AA metabolites include a number of vasodilators and vasoconstrictors [113–115, 135–138].

One of the most investigated AA metabolites is PGI<sup>2</sup> , formed by prostacyclin synthase, which belongs to the CYP-450 family and is highly expressed in endothelial cells and associated with cyclooxygenase (COX)-2 [115, 135–137]. Its vasodilator effects mainly involve binding to prostacyclin (IP) receptors and activation of adenylate cyclase increasing cAMP level in VSMC and subsequent relaxation. Yet, the role of PGI<sup>2</sup> as endogenous mediator of endothelium-dependent vasodilation *in vivo* has often been questioned and its other effects, such as inhibition of platelet adhesion and aggregation, and reduction of oxidative stress [135, 139] might rather account for its vasculoprotective effects. Moreover, COX-2-derived PGI<sup>2</sup> might play a compensatory role for the decreased NO bioavailability [117, 118]. To this end, it must be noted that other prostanoids from the COX metabolic pathways also affect vascular tone: due to various metabolic pathways as well as various prostaglandin receptors coupled to different signaling pathways, they might either induce vasoconstriction or vasodilation [115, 135, 137–139]. It is the delicate balance between vasoconstrictors and vasodilators which enables normal functioning of healthy endothelium; in endothelial dysfunction, the effect of vasoconstrictive prostanoids predominates, predisposing to the development of hypertension, atherosclerosis, and various other diseases.

Besides NO and PGI<sup>2</sup> , endothelial hyperpolarization (EDH) accounts for endotheliumdependent, flow-mediated vasodilation; by blocking eNOS (by L-NMMA) and COX (by more or less specific COX inhibitors), the role of non-NO-non-PGE-dependent vasodilation has unequivocally been confirmed not only in *in vitro* assays and in animal models but also *in vivo* in various human vessels during resting [140–143] and exercise [144].

Many of endothelial mediators and signals are known to induce the hyperpolarization of VSMC [8, 130, 135, 136, 145, 146]: epoxyeicosatrienoic acids (EETs) produced in the CYP-450 dependent metabolism of AA [135, 136]; the above mentioned H<sup>2</sup> O2 [133, 140], potassium ions released from the endothelial cells via Kca channels, and direct transmission of endothelial cell hyperpolarization by myoendothelial gap junctions. Thus, EDHF comprises a variety of factors which activate various potassium channels: small (SKCa), intermediate, and large-conductance Kca channels; inward rectifier K<sup>+</sup> channels (KIR) [147], and TRPV4 channels [148] have been implicated in finally inducing EDH. To this end, the corresponding channels have been investigated as potential targets to affect EDH [147, 149]. Endothelial hyperpolarization can spread via myoendothelial gap junctions to directly induce VSMC hyperpolarization: the role of gap junctions has been implicated also in humans *in vitro* and *in vivo* as carbenoxolone, a nonspecific gap junction blocker, diminished conducted vasodilation in isolated human coronary arterioles [150], and, moreover, reduced FMD in the brachial artery of healthy volunteers [151]. The term conducted dilation has been used to denote electrotonic transmission of local hyperpolarization, and may spread several mm upstream independent of alterations in shear stress [116, 119, 152]. It reflects the involvement of gap junctions and enables a coordinated adjustment of vascular resistance in larger and smaller arterioles and capillaries [153].

Age and gender might affect the EDHF-mediated response as male animals were reported to exhibit smaller EDHF-mediated endothelium-dependent relaxation compared to females; similarly, aging affected EDH. A reduced SKCa activity and a reduced expression of gap junction proteins, Cx-40 and Cx-43, have been suggested to account for these differences [154]. Moreover, 17b-estradiol has been proposed to activate AMPK and/or SIRT1, both implicated to be involved in increasing the EDHF-mediated signaling [154]. Alteration of the EDH contributes to endothelial dysfunction observed in various pathologies [145, 154].

Finally, other endothelium-derived vasodilators should be listed: adenosine and purines, various peptides, including CGRP, C-type natriuretic peptide (CNP), adrenomedullin, endocannabinoids, and gaseous transmitters other than NO, namely hydrogen sulfide (H<sup>2</sup> S), and carbon monoxide (CO) which have been suggested to compensate for decreased NO bioavailability [127, 155, 156]. Adenosine, one of the most potent vasodilators (exerting also other vasculoprotective effects) is mainly formed from extracellular nucleotides (ATP, AMP) by the action of ectonucleotidases, expressed in endothelial cells and also investigated as potential therapeutic targets [157]. Interestingly, circulating ATP itself has been proposed to mediate vasodilation in humans *in vivo* by inducing vascular hyperpolarization via activation of KIR channels [158].
