**2. Endothelium: its role in homeostasis and in pathology**

Vascular endothelium is a metabolically active neuroendocrine organ, which is spread in all tissues. The main functions of endothelium are: expression of receptor molecules, synthesis and secretion of biologically active molecules, vascular tone control, vascular permeability and new vessels formation, transportation of blood cells and soluble factors; homeostasis balance, participation in innate and adaptive immunity [8–10].

Supporting homeostasis, the endothelium is also subject to damage by factors, which cause endothelium pathology. Multiorganic dysfunction due to long-lasting activation under the effects of damaging factors lead to severe consequences.

Recent studies show that the homeostatic control over the cardiovascular and other systems is, among others, exerted by eGC, the outer above-membrane endothelium layer, which is formed by the sugar chains of transmembrane glycoconjugates and the associated notanchored proteoglycans [7]. However, there is limited data on eGC composition and its alterations under inflammatory and other pathological conditions.

#### **2.1. Endothelial glycocalyx structure and composition**

Endothelium surface layer is located on the luminal surface of the endothelium (endothelial surface layer—ESL). It is formed by the glycoproteins, proteoglycans and glycosphingolipids that are anchored in the membrane, as well as by secretory proteoglycans and glycosaminoglycans (GAGs), that are not anchored and are inter-connected by non-covalent interactions [11–13]. Their carbohydrate part contains a large amount of sialo and sulpho residues, forming overall negative charge of the endothelial cell surface. The outer segment of this layer (spreading out toward the vascular lumen), formed by the carbohydrate part of glycoconjugates, is a polysaccharide gel—eGC [14], with thickness ranging 2–4.5 μm [15] in different departments of the vascular system.

which is caused by disrupted adaptation to pregnancy and manifests with the development of complex, multiorganic and polysystemic insufficiency with clinical signs appearing after the 20th week of gestation [1, 2]. Despite of vigorous research in this area, pathogenesis of PE is still not clear. However, it is well known that the key factors of PE are immune system hyperactivation and the following excessive systemic inflammatory response (SIR), which initiate endothelium activation and cause endothelial dysfunction in cases of early onset and

Inflammatory response is accompanied by cell phenotype transformation (formation of activation cell status), leading to the generation of "danger" signals (generated from products of trauma, ischemia, necrosis or oxidative stress) [4, 5], which are recognizable by the immune system. It was found that the composition of endothelial glycocalyx (eGC) changed under excessive inflammatory response. Hypoglycosylated structures which may be perceived by immune system as neoantigens, appear оn the membrane of endothelial cells; also, antigens which are normally covert become apparent [6]. These events may promote autotolerance disruption and cause production of autoreactive antibodies damaging endothelial cells. In this regard, in this chapter a special attention is paid to eGC—functional layer of endothelial cells, which mediates all endothelial functions. Much evidence that under SIR, the alterations of eGC are associated with changes of cardiovascular system hemodynamics, vascular tone regulation, vascular permeability [7]—the main vectors of pathophysiological disorders in PE, and that alterations affect endothelial autoimmune phenotype formation, allow to assume that eGC may be one of the main targets of PE.

Vascular endothelium is a metabolically active neuroendocrine organ, which is spread in all tissues. The main functions of endothelium are: expression of receptor molecules, synthesis and secretion of biologically active molecules, vascular tone control, vascular permeability and new vessels formation, transportation of blood cells and soluble factors; homeostasis bal-

Supporting homeostasis, the endothelium is also subject to damage by factors, which cause endothelium pathology. Multiorganic dysfunction due to long-lasting activation under the

Recent studies show that the homeostatic control over the cardiovascular and other systems is, among others, exerted by eGC, the outer above-membrane endothelium layer, which is formed by the sugar chains of transmembrane glycoconjugates and the associated notanchored proteoglycans [7]. However, there is limited data on eGC composition and its alter-

Endothelium surface layer is located on the luminal surface of the endothelium (endothelial surface layer—ESL). It is formed by the glycoproteins, proteoglycans and glycosphingolipids

**2. Endothelium: its role in homeostasis and in pathology**

ance, participation in innate and adaptive immunity [8–10].

effects of damaging factors lead to severe consequences.

ations under inflammatory and other pathological conditions.

**2.1. Endothelial glycocalyx structure and composition**

complicated course of the disease [3].

114 Endothelial Dysfunction - Old Concepts and New Challenges

The base of the eGC is formed by carbohydrate-protein conjugates—transmembrane and secretory proteins; their carbohydrate part is represented by both short (2–15 monosaccharide residues) branched oligosaccharides, often decorated with sialic acid and sulfate (in glycoproteins), and by high-molecular glycans, often ending with highly sulfated residues (in proteoglycans) [16]. The glycoproteins can contain N-linked (Asn-linked) and/or O-linked (Ser/Thr-linked) glycans of variable length and composition. Complex hybrid and high-mannose glycans are usually present in the glycoproteins [17]. The main glycoproteins of endothelial cells are cell adhesion molecules (selectins, integrins, immunoglobulin superfamily molecules, endothelial mucins and addressins) which provide homing, migration and interaction between cells in different processes, and secretory molecules associated with eGC, participating in vascular homeostasis support, fibrinolysis and coagulation (thrombomodulin, von Willebrand factor (vWF)), antithrombin III, etc.). These molecules expression depends on factors, altering endothelium activation [16]. Under inflammatory response, the glycans modification occurs, leading to alteration of intercellular contacts, hemostasis and blood rheology. Biochemical eGC composition (the main structural and associated molecules) is presented in **Tables 1** and **2** (parts I and II).

It was found that the carbohydrate part is crucially important for glycoprotein function. N-linked glycans, particularly high-mannose chains, determine specific interactions of different molecules from the intercellular adhesion molecule (ICAM) family with the receptors [17]. N-glycans of the junctional adhesion molecule-A (JAM-A) regulate leukocyte adhesion and lymphocyte function-associated antigen-1 (LFA-1) binding [22]. Platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD31), a membrane highly glycosylated protein (~30% of molecular mass), has N-linked glycans represented by neutral and sialylated glycans [51, 52]. E-selectin is heavily glycosylated protein with hybrid/complex type N-linked oligosaccharides [53]. Cadherin of the vascular endothelium (VE-cadherin, CD144)—is the main transmembrane protein of adhesion contacts; its carbohydrate part is presented mainly by sialylated biantennary N-glycans of a complex type, and sialylated hybride N-chains (~40 and 28% of all identified glycans, respectively). Branched tri- and tetraantennary N-glycans, as well as N-glycans of high-mannose type are represented in smaller quantity in N-glycans of VE-cadherin [21, 54]. In the presence of antiinflammatory factors (such as tumor necrosis factor-α, TNF-α) the quantity of glycans ending with α2,6-sialic acid residues and fucose-α1,2-galactose-β1,4-N-acetylglucosamine increases, as well as the expression of N-glycans of high-mannose and hybrid type, which mediate intercellular contacts of monocytes with endothelium in the rolling and adhesion, particularly at the intercellular connections sites [55].

Hemostasis controlling proteins associated with outer eGC are also highly-glycosylated. VWF is a key component for maintenance of normal hemostasis, acting as the carrier protein of


**Group Members Number/type** 

Proteoglycans (extracellularly

Proteoglycans (associated with

the cell surface)

secreted)

**of GAG-chains linked**

Glycosaminoglycans HA — Anionic, nonsulfated glycosaminoglycan;

**Comments Ref**

http://dx.doi.org/10.5772/intechopen.75043

[9]

117

[36]

[37]

[38]

[39]

[40]

[41, 42]

[43]

[44]

[45, 46]

[47, 48]

[49]

structural unit of HA is a repeating disaccharide consisting of β-d-glucuronic acid and β-N-acetyl-d-glucosamine;

within HS is composed of a monosulfated β-glucuronic acid linked to tri-sulfated α-N-acetylglucosamine

residues at different positions

polymer composed of repeating disaccharide units of α-iduronic acid and β-N-acetyl-d-galactosamine. These sugar residues can be modified by ester sulfate

keratan sulfate is units of β-d-galactose and β-N-acetyl-d-galactosamine

sulfate proteoglycan; protein core of

proteoglycan, core protein (at >350 kDa)

(20 kDa) with a single DS chain; DS of endocan consists of about 32 disaccharide

proteoglycan (40 kDa); it has N-terminal attachment site for a single GAG chain of chondroitin or dermatan sulfate

Family of HSPGs, the syndecan protein

Core protein of all glypicans is ranging

at various positions

approximately 500 kDa

(12–34 kDa protein core)

family has four members.

between 198 to 346 kDa

KS — Basic repeating disaccharide unit within

Perlecan 3/HS,CS A large basement membrane heparan

Versican 10-30/CS,DS Large aggregating chondroitin sulfate

Endocan 1/DS Is a DSPG, small proteoglycan molecules

units

Biglycan 2/CS,DS small leucine-rich proteoglycan (42 kDa protein core)

Decorin 1/CS,DS A prototype small leucine-rich

Mimecan 2–3/KS Small leucine-rich proteoglycan;

Syndecans 5/HS,CS Transmembrane proteoglycans

composed of repeating disaccharide units of β-glucuronic acid and β-N-acetyl-dgalactosamine and modified with sulfate

contains no core protein

HS — The most common disaccharide unit

Endothelial Dysfunction as a Consequence of Endothelial Glycocalyx Damage: A Role in the…

CS — CS is a linear acidic polysaccharide,

DS — Backbone of DS chains is a linear

MadCAM-1, mucosal addressin cell adhesion molecule-1; JAM-1, junctional adhesion molecule-1; JAM-2, junctional adhesion molecule-2; JAM-3, junctional adhesion molecule-3; ICAM-1, inter-cellular adhesion molecule-1; ICAM-2, inter-cellular adhesion molecule-2; VCAM-1, vascular cell adhesion molecule-1; PECAM-1, platelet/endothelial cell adhesion molecule-1; SLe<sup>x</sup> , sialyl-Lewis-X.

**Table 1.** Biochemical composition of endothelial glycocalyx—main components (part I: glycoproteins).

the coagulant Factor VIII and mediating platelet adhesion at the sites of vascular injury [31]. VWF is heavily glycosylated by N- and O-linked oligosaccharides, and glycosylation affects many of its functions [30]. Antithrombin is a major inhibitor of the blood coagulation cascade. Endothelial Dysfunction as a Consequence of Endothelial Glycocalyx Damage: A Role in the… http://dx.doi.org/10.5772/intechopen.75043 117


the coagulant Factor VIII and mediating platelet adhesion at the sites of vascular injury [31]. VWF is heavily glycosylated by N- and O-linked oligosaccharides, and glycosylation affects many of its functions [30]. Antithrombin is a major inhibitor of the blood coagulation cascade.

**Table 1.** Biochemical composition of endothelial glycocalyx—main components (part I: glycoproteins).

**Group Members Comments References**

site

Р-selectin Contains 9 potential N-glycosylation sites

VE-cadherin Contains 7 potential N-glycosylation sites

ClyCAM-1 Mucin, containing predominantly

CD34 Mucin, O-glycosylation sites are more

(SLe<sup>x</sup> )

MadCAM-1 Mucin; contain O-linked glycans

Thrombomodulin Contains at least 4 N- and 1

Von Willebrand factor Contains at least 10 potential N- and

Antithrombin III Contains 4 potential N-glycosylation sites

Heparin cofactor II Contains 3 potential N-glycosylation sites

MadCAM-1, mucosal addressin cell adhesion molecule-1; JAM-1, junctional adhesion molecule-1; JAM-2, junctional adhesion molecule-2; JAM-3, junctional adhesion molecule-3; ICAM-1, inter-cellular adhesion molecule-1; ICAM-2, inter-cellular adhesion molecule-2; VCAM-1, vascular cell adhesion molecule-1; PECAM-1, platelet/endothelial cell

O-linked carbohydrate chains (T-antigen and 6′ sulfated

abundant than N-glycosylation sites

sialyl-Lewis-X)

10 O-glycosylation sites

O-glycosylation sites

JAM-1 Contains 1 N-glycosylation site [17] JAM-2 Contains 2 N-glycosylation sites [18] JAM-3 Contains 2 N-glycosylation sites [19] ICAM-1 Contains 8 N-glycosylation sites [13] ICAM-2 Contains 6 N-glycosylation sites [20] VCAM-1 Contains 6 N-glycosylation sites [13] PECAM-1 Сontains 9 N-glycosylation sites [13]

[13]

[13]

[16]

[21, 22]

[23]

[24]

[25, 26]

[27–32]

[33, 34]

[35]

N-linked glycans [14, 15]

Adhesion molecules Е-selectin Contains 11 potential N-glycosylation

Integrins: α1β1, α2β1, α3β1, α5β1, α6β1, α8β1, α9β1, αVβ1, αVβ3, α6β4,

αVβ5

116 Endothelial Dysfunction - Old Concepts and New Challenges

Coagulation and fibrinolysis regulators

adhesion molecule-1; SLe<sup>x</sup>

, sialyl-Lewis-X.


five types of GAG chains: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (hyaluronic acid, HA). They are linear polymers of disaccharides with variable lengths that are modified by sulfation and/or (de)acetylation to a variable extent [15]. In the human body the GAGs are present in a protein bound form (i.e., in proteoglycans composition) and do not exist in a free form, except for HA. Besides playing structuring and supporting roles, proteoglycans are involved in cell signaling, regulation of cell proliferation, adhesion, migration, differentiation [55]. Key eGC glycans are heparan sulfate proteoglycans (HSPGs), which compose about 50–90% of the total amount of proteoglycans present in the eGC, and HA—the main supporting glycan [14, 15]. Main

Endothelial Dysfunction as a Consequence of Endothelial Glycocalyx Damage: A Role in the…

http://dx.doi.org/10.5772/intechopen.75043

Glycosphingolipids (GSLs), a class of ceramide-based glycolipids, are also a significant part of eGC. Glycosphingolipids are subclassified as neutral (no charged sugars or ionic groups), sialylated (gangliosides), or sulfated [58]. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid raft [59]. Cultured human umbilical vein endothelial cells (HUVEC)

Cer), but the most abundant neutral GSL is lactosylceramide (LacCer, CDw17) [60]. LacCer can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis [61, 62]. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18 integrin as well as being associated with several key cellular processes [63]. Endothelium activa-

expression; interferon gamma (IFNγ) has a striking effect on the surface expression of GSLs; IL-1 increases the cell content of neutral and acidic GSLs but does not alter their surface expression [55]. Cytokines TNF-α and IL-1 can potentiate the toxic effect of verocytotoxin (Shiga-like toxin-produced by *Escherichia coli* and the main cause of hemolytic uremic syndrome) to human

Acidic GSLs of human endothelial cells are: monosialoganglioside or GM3—the major ganglioside of endothelial cells, and it constitutes about 90% of the whole ganglioside fraction [67], and sulfoglucuronyl paragloboside (SGPG), a minor GSL in endothelial cells, is a ligand for L-selectin [55]. Although GIyCAM-1 and CD34 constitute the major L-selectin ligand on

Cer (CD77) binds to the verocytotoxin and injures human endothelial cells [66].

The eGC is considered as an intravascular compartment which has various functions.

First, eGC mediates the endothelial mechanotransduction of shear stress and performs regulation of shear stress-induced nitric oxide (NO) production [69]. This is provided by the

Cer and

119

Cer [64]

Cer and Gb4

Cer synthesis in these cells [65], because

gangliosides may also play a role, since L-selectin can

proteoglycans of the eGC and their characteristics are given in **Table 2** (part II).

appeared to contain complex lacto and globo series compounds (lactosylceramide, Gb3

tion by pro-inflammatory cytokines, particularly by TNF-α, affect the Gb<sup>3</sup>

GSLs under physiologic flow conditions [68].

endothelial cells by inducting an increase in the Gb3

venous endothelium, endothelial SLe**<sup>x</sup>**

**2.2. Functions of the endothelial glycocalyx**

Gb4

Gb3

also bind SLe<sup>x</sup>

GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; KS, keratan sulfate; DSPG, dermatan sulfate proteoglycans; HSPGs, heparan sulfate proteoglycans.

**Table 2.** Biochemical composition of endothelial glycocalyx—main components (part II: glycosaminoglycans and proteoglycans).

Two isoforms exist in the circulation, α-antithrombin and β-antithrombin, which differ in the glycosylation of the polypeptide chain; β-antithrombin lacks the carbohydrate present at Asn135 in α-antithrombin. Of the two forms, β-antithrombin has the higher affinity for heparin due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky glycan in α-antithrombin [56]. The carbohydrate structures of heparin cofactor II (member of serpin superfamily) circulating in blood are complex-type biantennary and triantennary chains in a ratio of 6:1 with the galactose being >90% sialylated with α2-6-linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sialyl Le**<sup>x</sup>** motif (SLe<sup>x</sup> ) [40]. Thrombomodulin (TM) is an endothelial cell surface glycoprotein (contains N- and O-linked glycans) that directly inhibits the procoagulant activities of thrombin and the TM-thrombin complex accelerates the thrombin catalyzed activation of protein C. Moreover, the GAG O-linked chains of TM contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. Soluble TM in urine has no GAG chain which could promote its anticoagulant activities. Studies of the rabbit recombinant ТМ have shown that addition of a GAG chain may increase its anticoagulant function [33, 34].

Endothelial mucins (CD34; glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1); mucosal addressin cell adhesion molecule-1 (MadCAM-1)) contact leukocytes by their binding to L-selectin. This interaction facilitates leukocytes transportations from blood to lymphoid organs and inflamed tissues [28]. Major capping group in GlyCAM-1, CD34 and MadCAM-1 is the sulfated SLe**<sup>x</sup>** [27, 28, 57]. For example, CD34 functions as a L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope. CD34 can exist in 2 glycoforms: the L-selectin-binding (L-B-CD34) and nonbinding (L-NB-CD34) glycoforms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represents less than 10% of total CD34. It has been shown, that a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe<sup>x</sup> epitopes on O-glycans that are important for its recognition by L-selectin [28].

The eGC mostly consists of proteoglycans—highly glycosylated proteins (glycans account for 90–95% of the molecular mass); GAGs branches form their carbohydrate part. There are five types of GAG chains: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (hyaluronic acid, HA). They are linear polymers of disaccharides with variable lengths that are modified by sulfation and/or (de)acetylation to a variable extent [15]. In the human body the GAGs are present in a protein bound form (i.e., in proteoglycans composition) and do not exist in a free form, except for HA. Besides playing structuring and supporting roles, proteoglycans are involved in cell signaling, regulation of cell proliferation, adhesion, migration, differentiation [55]. Key eGC glycans are heparan sulfate proteoglycans (HSPGs), which compose about 50–90% of the total amount of proteoglycans present in the eGC, and HA—the main supporting glycan [14, 15]. Main proteoglycans of the eGC and their characteristics are given in **Table 2** (part II).

Glycosphingolipids (GSLs), a class of ceramide-based glycolipids, are also a significant part of eGC. Glycosphingolipids are subclassified as neutral (no charged sugars or ionic groups), sialylated (gangliosides), or sulfated [58]. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid raft [59]. Cultured human umbilical vein endothelial cells (HUVEC) appeared to contain complex lacto and globo series compounds (lactosylceramide, Gb3 Cer and Gb4 Cer), but the most abundant neutral GSL is lactosylceramide (LacCer, CDw17) [60]. LacCer can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis [61, 62]. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18 integrin as well as being associated with several key cellular processes [63]. Endothelium activation by pro-inflammatory cytokines, particularly by TNF-α, affect the Gb<sup>3</sup> Cer and Gb4 Cer [64] expression; interferon gamma (IFNγ) has a striking effect on the surface expression of GSLs; IL-1 increases the cell content of neutral and acidic GSLs but does not alter their surface expression [55]. Cytokines TNF-α and IL-1 can potentiate the toxic effect of verocytotoxin (Shiga-like toxin-produced by *Escherichia coli* and the main cause of hemolytic uremic syndrome) to human endothelial cells by inducting an increase in the Gb3 Cer synthesis in these cells [65], because Gb3 Cer (CD77) binds to the verocytotoxin and injures human endothelial cells [66].

Acidic GSLs of human endothelial cells are: monosialoganglioside or GM3—the major ganglioside of endothelial cells, and it constitutes about 90% of the whole ganglioside fraction [67], and sulfoglucuronyl paragloboside (SGPG), a minor GSL in endothelial cells, is a ligand for L-selectin [55]. Although GIyCAM-1 and CD34 constitute the major L-selectin ligand on venous endothelium, endothelial SLe**<sup>x</sup>** gangliosides may also play a role, since L-selectin can also bind SLe<sup>x</sup> GSLs under physiologic flow conditions [68].
