**2.2. On endothelial barrier and mechanosensing**

*Paracellular transport pathways* across the capillary wall depend on endothelial permeability. Permeability is mainly determined by the integrity of endothelial glycocalyx and two types of inter-endothelial cell junctions, the distribution of which depends on the capillary network: tight junctions (TJs) and adherens junctions (AJs). The main component of AJs are cadherins (cadherin-VE being the most abundant), which belong to adhesion molecules and are also connected to actin cytoskeleton [31, 32]. Changes in VE-cadherin dynamics at AJs can lead to disassembly of AJs, increasing the junctional permeability; chronic vascular leakage that occurs in tumor vessels is associated with downregulation of VE-cadherin expression [33]. Interestingly, endothelial NO synthase (eNOS) hyperactivation in response to vascular endothelial growth factor (VEGF) or platelet aggregating factor (PAF) triggers S-nitrosylation of VE-cadherin, thereby causing destabilization of AJs, and leading to endothelial hyperpermeability [34]. In addition, ROS have been implicated in junctions disorganization [35]. As altered Ca2+ signaling has been implicated in inducing endothelial hyperpermeability, activation of transient receptor potential (TRP) channels which regulate Ca2+ entry has been associated with hyperpermeability states and transendothelial migration of leukocytes [31]. In addition, the integrity of AJs depends on small RhoGTPases that have been intensively investigated as potential drug targets [31]. Interestingly, statins (simvastatin) have been shown to prevent vascular injury associated with hyperpermeability and inflammation by affecting the RhoA-

*Transcellular transport mechanisms* are accomplished by membrane-bound vesicular carriers, such as caveolae and vesiculo-vacuolar organelles. Caveolae are plasma-membrane invaginations composed of lipids and caveolins, scaffolding proteins that are involved in transmembrane signaling. Caveolin has been used as molecular marker for the isolation of

With reference to endothelial heterogeneity, there are significant differences in the number of caveolae in endothelium along the vascular tree reaching up to 10,000 per cell in the capillaries [1]. Besides being involved in transcellular trafficking, caveolar network in endothelial cells affects many other endothelial functions. Caveolin can modulate signal transduction: G-protein-coupled receptors and receptor tyrosine kinases are localized into caveolae and interact with caveolin [37]. Caveolins have also been involved in cell migration, angiogenesis, and cancer [38]. VEGF is an important proangiogenic factor that promotes angiogenesis by increasing vascular permeability, endothelial cell migration, and the release of proteolytic matrix metalloproteinases (MMPs). VEGF receptors have been localized in caveolae and interact with caveolin-1 [39]. VEGF disrupts the interaction between the VEGF receptor (VEGFR)-2 and caveolin-1, thereby activating the downstream signaling [39]. Silencing caveolin-1 has been shown to affect MMPs activity and VEGF-induced angiogenesis [40]; moreover, it induced morphological alterations of endothelial cells, and reduced cell migration and tubulogenesis induced by VEGF, pointing to an important role of caveolae in angiogenesis. Additional evidence on the involvement of caveolae in vasculogenesis comes from studies showing that the recruitment of EPCs [41], which are now widely recognized as one of the key mechanisms in adult vasculogenesis, to the peripheral blood is dependent on the expression of VEGFR2 [42]. Finally, one should not forget one of the main effects of caveolin, namely inhibition of eNOS [43, 44]. As caveolin also interferes with adenosine receptor trafficking and the role of adenosine has been implicated in ischemia and inflammation, caveolar network

and Rac1-mediated cytoskeletal arrangements [36].

6 Endothelial Dysfunction - Old Concepts and New Challenges

may represent a potential therapeutic target [45].

caveolae-enriched membranes from *in vitro* cultured cells [37].

Mechanosensing is accomplished by a complex mechanism involving parts of endothelial cells, which convey the mechanic signals sensed by surface cellular elements in the transduction intracellular signaling pathways, finally adjusting the endothelial response to alterations of shear-stress. Mechanosensing complex, located in AJs, is composed of endothelial glycocalyx and endothelial cilium, VE-cadherins, VEGFR2 and platelet endothelial cell adhesion molecules (PECAM-1 or CD31), and various ion channels: the role of Ca2+-dependent potassium channels (K<sup>+</sup> Ca) and TRP channels has extensively been investigated [10, 48]. However, certain predilection sites are prone to the generation of characteristic spatiotemporal shear-stress patterns favoring atherogenesis [9, 10, 48, 49].

*Glycocalyx*, a 50–100 μm thick structure anchored in the endothelial plasma membrane and composed of carbohydrate-rich proteoglycans, is located at the luminal surface of the endothelium [2, 30, 50]. It is importantly involved in mechanosensing, in the regulation of permeability, and inflammatory response [32, 48, 50] and has also been investigated as a potential therapeutic target [51, 52]. It prevents leucocyte and platelet adhesion [53] and mediates shear-stress-induced NO release, thus exerting vasculoprotective effects and contributing to vascular homeostasis [10, 54]. Besides being susceptible to pathological alterations in blood flow, other environmental alterations affect its composition and thus function, e.g., high glucose has been shown to be involved in glycocalyx degradation contributing to lower NO bioavailability in hyperglycemia [55, 56]. Recent concept has confirmed the existence of an additional thicker (0.5–1 μm) layer on the luminal endothelial surface (termed endothelial surface layer, ESL) which significantly impacts hemodynamic conditions, oxygen transport, vascular control, coagulation, inflammation, and atherosclerosis [2].

*Endothelial cilium* is a single hair-shaped projection from the cellular surface of endothelial cells. Besides being involved in sensing hemodynamic forces, it coordinates cell migration and division [30, 57]. Impaired function of the primary cilium, either genetically or due to environmental factors, results in developmental disorders or endothelial dysfunctions, respectively. The main components of the primary cilium are various membrane receptors (specific receptor tyrosine kinases, as platelet-derived growth factor (PDGF) receptor α, fibroblast growth factor receptor; insulin-like growth factor 1 (IGF-1) receptor; G-protein coupled receptors), channel proteins (polycystin-1 and -2), and special arrangement of the microtubules [30, 57]. Both polycystin proteins are importantly involved in intracellular signaling: polycystin-2, in turn, belongs to the TRP channel family which, by interacting with polycystin-1, increases intracellular Ca2+ leading to the activation of ryanodine receptors on the endoplasmic reticulum. The released Ca2+ subsequently activates a number of signaling pathways, among others the shearstress induced activation of eNOS and an increase in NO production [57, 58]. Alterations in the patterns and magnitude of biomechanical forces induce disorganization in cilia structure: prolonged exposure to increased shear stress induces disassembly of cilia, rearrangement of cytoskeleton, and increased acetylation of microtubules [59]. Dysfunctional cilia have been implicated in kidney disease, hypertension, and the development of atherosclerosis [60].

In addition to being part of endothelial cilium, microtubules polymerized with heterodimers of alpha- and beta-tubulins are important cytoskeletal proteins in endothelial cells. They regulate numerous cellular functions, including cell shape, adhesion, intracellular transport, mitosis, and migration and thus contribute to endothelial integrity [35, 61]. Increased level of tubulin acetylation has been shown to increase cell migration [62]. On the other hand, cyclic stretch and angiotensin II (AngII) have been shown to increase tubulin deacetylation and tubular reorganization, predisposing to the development of cardiovascular diseases [63]; potential role on the AngII type 1 receptor antagonist in positively affecting endothelial microtubular organization has been implicated [63]. The longevity regulator sirtuin 1 (SIRT1), a potential therapeutic target [64], has been implicated in tubulin deacetylation and the regulation of microtubule function [63].

metabolism [74] and inflammatory response and modifies vascular tone regulation by secreting protective adipokines which exert paracrine actions directly and indirectly by stimulating the release of endothelial vasodilators on VSMC [75, 76]. Moreover, healthy PVAT has been shown to enhance insulin-induced vasodilation by releasing adipokines [77], as well as pros-

tissue inflammation [78]. However, these anti-contractile and anti-inflammatory properties of PVAT are blunted in disease, such as obesity, and hypertension where an imbalance of secreted adipokines from PVAT and alterations in metabolism predispose to inflammation and the development of endothelial dysfunction [79]. Hypoxia is one of the main mechanisms increasing the release of inflammatory cytokines (IL-6 and TNFα) from PVAT [80], and inducing macrophage activation which all favors a pro-inflammatory and pro-contractile state [81]. Moreover, in response to vascular injury [82] or high fat diet [83], PVAT has been shown to express pro-inflammatory phenotype and a significant reduction of adiponectin levels leading to atherosclerosis. Interestingly, the inflammatory profile of PVAT has been shown to differ between distinct fat depots [84]. Independent human studies have proven beneficial effects of exercise and diet on PVAT, and consequently also on endothelial dysfunction [85, 86].

Endothelial and VSMC are abundantly innervated with the fibers of *the sympathetic nervous system*, and there is a considerable and complex cross talk between various neurotransmitters released from these fibers and endothelial autacoids [87–89]. Besides noradrenaline, that binds on various adrenoceptors on endothelial cells and VSMC, other transmitters (such as adenosine triphosphate (ATP), calcitonin gene-related peptide (CGRP) acetylcholine (ACh), and neuropeptide Y) are released from nerve varicosities, thereby profoundly modulating

thelium, thereby increasing NO production [89, 90], while the co-transmitted ATP mediates endothelial hyperpolarization by acting on endothelial purinoceptors (P2Y) [88]; both effects finally induce vasorelaxation and, respectively, modulate the "classical" sympathetic vasoconstrictor effects on VSMC. Moreover, NO affects neurotransmission at the level of blood

Endothelial dysfunction and autonomic nervous system imbalance often coexist in the development of cardiovascular diseases [88]. Indeed, an inverse relationship between markers of endothelial function and the sympathetic activity in healthy conditions is well known: in young adults, acute increase of the sympathetic activity, as assessed by measuring the plasma noradrenaline concentration, has been shown to impair endothelium-dependent dilation [91, 92]. Sympathetic nervous system activity is also proposed to be an important factor contributing

Endothelium maintains blood fluidity, preventing intravascular coagulation and thrombus formation, respectively; endothelial cells express a variety of intraluminal surface proteins (such as thrombomodulin) and secrete molecules with anticoagulant and antithrombotic properties: ectonucleotidases and protein C and S as well as substances which inhibit platelet

**2.4. Other endothelial functions: involvement in hemostasis, inflammation, and** 

and NO).


Endothelium at a Glance

9

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


endothelial function. Noradrenaline was shown to activate β<sup>3</sup>

thetic neurotransmission in the central nervous system [87].

vessels, acting on presynaptic α<sup>2</sup>

to endothelial dysfunction with age [92].

adhesion and aggregation (PGI<sup>2</sup>

**angiogenesis**

) [74]. Adiponectin, the most abundant adipokine, has been shown to decrease

tacyclin (PGI<sup>2</sup>

Disturbed flow patterns and increase of shear-stress have been acknowledged to affect the above targets, thus contributing to endothelial dysfunction in many ways, including increased generation of ROS, promoting cytoskeletal disassembly, increasing cellular permeability, expression of adhesion molecules and inflammation, as well as inducing mitogenic signaling pathways through extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) involved in vascular remodeling which all favor atheroma formation [65]. Moreover, there is ample evidence that nonlaminar flow can result in gene expression of pro-inflammatory molecules in the vascular wall [49, 65].

#### **2.3. On endothelium and inter-cellular communication**

As mentioned, endothelial homeostasis involves mutual communication with other cells.

*Gap junctions* in turn enable a direct transmission of electrical and chemical signals and thus exchange of ions and other small molecules between endothelial cells, VSMC, and pericytes [8, 30, 66]. The main components of endothelial gap junctions are connexins (Cx) which have been shown to be defective in diseased states and thus represent a potential therapeutical target. A total of 21 connexins have been identified in humans displaying cellular specificity [67]. Healthy endothelium mainly expresses Cx-37 and Cx-40 [67, 68]. Alterations in connexin expression might be associated with endothelial dysfunction and increased susceptibility to atherosclerosis: altered expression of Cx-37 was shown to decrease NO bioavailability by decreasing eNOS activity [69], whereas endothelial-specific deletion of Cx-40 was reported to increase CD73-dependent leukocyte adhesion to endothelium [70].

*Pericytes* are contractile cells that wrap around the endothelial cells of capillaries and venules by sharing their basement membrane [71, 72]. As there is a considerable paracrine signaling between both cell lineages (via the release of transforming growth factor, angiopoietins, PDGF, sphingosine-1-phosphate), pericytes have been implicated in the regulation of capillary blood flow, and in the maturation and survival of endothelial cells by modulating apoptosis, and promoting angiogenesis [71–73]. Disturbance in pericyte-endothelial communication induces various pathological processes, associated with increased proneness to hemorrhage, apoptosis, impaired (tumor) angiogenesis, and endothelial hyperplasia [73].

An important communication exists between the *perivascular adipose tissue* (PVAT), endothelial cells, and VSMCs: PVAT secretes a number of adipokines (tumor necrosis factor, TNFα, interleukin-6, IL-6, resistin, irisin) with various pro- and antiatherogenic properties [8, 11]. The level of adipocytokines has been suggested as an independent predictor of endothelial dysfunction in healthy subjects [17]. In a healthy individual, PVAT importantly influences metabolism [74] and inflammatory response and modifies vascular tone regulation by secreting protective adipokines which exert paracrine actions directly and indirectly by stimulating the release of endothelial vasodilators on VSMC [75, 76]. Moreover, healthy PVAT has been shown to enhance insulin-induced vasodilation by releasing adipokines [77], as well as prostacyclin (PGI<sup>2</sup> ) [74]. Adiponectin, the most abundant adipokine, has been shown to decrease tissue inflammation [78]. However, these anti-contractile and anti-inflammatory properties of PVAT are blunted in disease, such as obesity, and hypertension where an imbalance of secreted adipokines from PVAT and alterations in metabolism predispose to inflammation and the development of endothelial dysfunction [79]. Hypoxia is one of the main mechanisms increasing the release of inflammatory cytokines (IL-6 and TNFα) from PVAT [80], and inducing macrophage activation which all favors a pro-inflammatory and pro-contractile state [81]. Moreover, in response to vascular injury [82] or high fat diet [83], PVAT has been shown to express pro-inflammatory phenotype and a significant reduction of adiponectin levels leading to atherosclerosis. Interestingly, the inflammatory profile of PVAT has been shown to differ between distinct fat depots [84]. Independent human studies have proven beneficial effects of exercise and diet on PVAT, and consequently also on endothelial dysfunction [85, 86].

In addition to being part of endothelial cilium, microtubules polymerized with heterodimers of alpha- and beta-tubulins are important cytoskeletal proteins in endothelial cells. They regulate numerous cellular functions, including cell shape, adhesion, intracellular transport, mitosis, and migration and thus contribute to endothelial integrity [35, 61]. Increased level of tubulin acetylation has been shown to increase cell migration [62]. On the other hand, cyclic stretch and angiotensin II (AngII) have been shown to increase tubulin deacetylation and tubular reorganization, predisposing to the development of cardiovascular diseases [63]; potential role on the AngII type 1 receptor antagonist in positively affecting endothelial microtubular organization has been implicated [63]. The longevity regulator sirtuin 1 (SIRT1), a potential therapeutic target [64], has been implicated in tubulin deacetylation and the regulation of microtubule function [63].

Disturbed flow patterns and increase of shear-stress have been acknowledged to affect the above targets, thus contributing to endothelial dysfunction in many ways, including increased generation of ROS, promoting cytoskeletal disassembly, increasing cellular permeability, expression of adhesion molecules and inflammation, as well as inducing mitogenic signaling pathways through extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) involved in vascular remodeling which all favor atheroma formation [65]. Moreover, there is ample evidence that nonlaminar flow can result in gene expression of pro-inflammatory molecules in the vascular wall [49, 65].

As mentioned, endothelial homeostasis involves mutual communication with other cells.

*Gap junctions* in turn enable a direct transmission of electrical and chemical signals and thus exchange of ions and other small molecules between endothelial cells, VSMC, and pericytes [8, 30, 66]. The main components of endothelial gap junctions are connexins (Cx) which have been shown to be defective in diseased states and thus represent a potential therapeutical target. A total of 21 connexins have been identified in humans displaying cellular specificity [67]. Healthy endothelium mainly expresses Cx-37 and Cx-40 [67, 68]. Alterations in connexin expression might be associated with endothelial dysfunction and increased susceptibility to atherosclerosis: altered expression of Cx-37 was shown to decrease NO bioavailability by decreasing eNOS activity [69], whereas endothelial-specific deletion of Cx-40 was reported to

*Pericytes* are contractile cells that wrap around the endothelial cells of capillaries and venules by sharing their basement membrane [71, 72]. As there is a considerable paracrine signaling between both cell lineages (via the release of transforming growth factor, angiopoietins, PDGF, sphingosine-1-phosphate), pericytes have been implicated in the regulation of capillary blood flow, and in the maturation and survival of endothelial cells by modulating apoptosis, and promoting angiogenesis [71–73]. Disturbance in pericyte-endothelial communication induces various pathological processes, associated with increased proneness to hemorrhage, apopto-

An important communication exists between the *perivascular adipose tissue* (PVAT), endothelial cells, and VSMCs: PVAT secretes a number of adipokines (tumor necrosis factor, TNFα, interleukin-6, IL-6, resistin, irisin) with various pro- and antiatherogenic properties [8, 11]. The level of adipocytokines has been suggested as an independent predictor of endothelial dysfunction in healthy subjects [17]. In a healthy individual, PVAT importantly influences

**2.3. On endothelium and inter-cellular communication**

8 Endothelial Dysfunction - Old Concepts and New Challenges

increase CD73-dependent leukocyte adhesion to endothelium [70].

sis, impaired (tumor) angiogenesis, and endothelial hyperplasia [73].

Endothelial and VSMC are abundantly innervated with the fibers of *the sympathetic nervous system*, and there is a considerable and complex cross talk between various neurotransmitters released from these fibers and endothelial autacoids [87–89]. Besides noradrenaline, that binds on various adrenoceptors on endothelial cells and VSMC, other transmitters (such as adenosine triphosphate (ATP), calcitonin gene-related peptide (CGRP) acetylcholine (ACh), and neuropeptide Y) are released from nerve varicosities, thereby profoundly modulating endothelial function. Noradrenaline was shown to activate β<sup>3</sup> -adrenergic receptors on endothelium, thereby increasing NO production [89, 90], while the co-transmitted ATP mediates endothelial hyperpolarization by acting on endothelial purinoceptors (P2Y) [88]; both effects finally induce vasorelaxation and, respectively, modulate the "classical" sympathetic vasoconstrictor effects on VSMC. Moreover, NO affects neurotransmission at the level of blood vessels, acting on presynaptic α<sup>2</sup> -adrenergic receptors, and it also interferes with the sympathetic neurotransmission in the central nervous system [87].

Endothelial dysfunction and autonomic nervous system imbalance often coexist in the development of cardiovascular diseases [88]. Indeed, an inverse relationship between markers of endothelial function and the sympathetic activity in healthy conditions is well known: in young adults, acute increase of the sympathetic activity, as assessed by measuring the plasma noradrenaline concentration, has been shown to impair endothelium-dependent dilation [91, 92]. Sympathetic nervous system activity is also proposed to be an important factor contributing to endothelial dysfunction with age [92].
