**2.1. On endothelial permeability, inter-endothelial cell junctions, and caveolae**

Given the pivotal role of the microvascular endothelium in supplying tissues with nutrients and oxygen, endothelial integrity is crucial for tissue-fluid homeostasis. While basal permeability mainly governs water and solutes transport across the capillary wall of a healthy endothelium, the term inducible (or induced) permeability refers to alterations in endothelial permeability associated with inflammation and occurs predominantly at the site of postcapillary venules [1]. The latter occurs as a consequence of endothelial cell retraction and intercellular gap formation by a variety of agonists (histamine, serotonin, bradykinin, substance P, vascular endothelial growth factor (VEGF)) [28], or according to more recent speculations, due to transcellular vascular leakage of macromolecules [29].

In general, microvascular endothelial structure directs the capillary dynamics: fluids and small solutes (less than 3 nm) move passively across the endothelium mainly via paracellular transport mechanisms or transcellularly by simple diffusion (nonpolar substances and gases) whereas larger macromolecules are transported by transcellular mechanisms, including receptor-dependent and receptor-independent transcytosis [30].

*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 RhoAand Rac1-mediated cytoskeletal arrangements [36].

In addition, being part of the mechanosensory complex, caveolae and AJs are involved in mechanosensing [31, 46]: exposure of endothelial cells to shear stress has been shown to affect

Endothelium at a Glance

7

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

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

predilection sites are prone to the generation of characteristic spatiotemporal shear-stress pat-

*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,

*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].

vascular control, coagulation, inflammation, and atherosclerosis [2].

Ca) and TRP channels has extensively been investigated [10, 48]. However, certain

the number and distribution of caveolae [47, 48].

**2.2. On endothelial barrier and mechanosensing**

terns favoring atherogenesis [9, 10, 48, 49].

channels (K<sup>+</sup>

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

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 may represent a potential therapeutic target [45].

In addition, being part of the mechanosensory complex, caveolae and AJs are involved in mechanosensing [31, 46]: exposure of endothelial cells to shear stress has been shown to affect the number and distribution of caveolae [47, 48].
