**2.2. Functions of the endothelial glycocalyx**

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

GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; KS,

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

**of GAG-chains linked**

Glypicans 3/HS,CS GPI-anchored proteoglycans

Family of HSPGs

between 60 and 70 kDa

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

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

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

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

[27, 28, 57]. For example, CD34 functions as a L-selectin ligand mediating

) [40]. Thrombomodulin (TM) is an endothelial cell

**Comments Ref**

[50]

The glypican protein family has six members core protein of all glypicans is similar in size, approximately ranging

epitopes on O-glycans that are important for its

motif (SLe<sup>x</sup>

keratan sulfate; DSPG, dermatan sulfate proteoglycans; HSPGs, heparan sulfate proteoglycans.

structures contain one sialyl Le**<sup>x</sup>**

**Group Members Number/type** 

118 Endothelial Dysfunction - Old Concepts and New Challenges

function [33, 34].

proteoglycans).

is the sulfated SLe**<sup>x</sup>**

CD34 carries relatively abundant 6-sulfo SLe<sup>x</sup>

recognition by L-selectin [28].

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 impact of tangential stress of blood flow shift primarily to eGC; the latter accepts and scatters the load, created by fluid shear stress. Local spin moment, created by fluid shear stress, affects the proteoglycans chains, and further—the core proteins (syndecans and glypicans), causing actin cytoskeleton reorganization and transmission of the signal into the cell and the cell nucleus [70, 71]. The study of Fu and Tarbell (2013) aimed to determine the eGC role in mechanosensing and transduction, and measured the flow-induced production of NO *in vitro* [7]. It was found that compared to static conditions, the application of steady flow shear stress rapidly increased NO production from the baseline in bovine aortic endothelial cells. Enzymatic treatment of the key components of eGC (HS, HA) completely blocked flowinduced NO production without affecting receptor-mediated NO production, suggesting that the eGC has a direct effect on the NO production machinery [7]. Therefore, the eGC under physiological conditions (intact eGC) transforms hemodynamic effect into cell biochemical signals, which regulate the vascular tone.

modulation, caveolin-enriched microdomain regulation and interaction with endothelial HA binding proteins. Certain disease states, especially accompanied by SIR, increase hyaluronidase activity and reactive oxygen species (ROS) generation which break down high molecular weight HA to low molecular weight fragments causing damage to the eGC. Further, these HA fragments can activate specific HA binding proteins upregulated in vascular disease to promote actin cytoskeletal reorganization and inhibition of endothelial cell–cell contacts [77]. A glycocalyx-junction-break model, described by Curry and Adamson summarizes multiple studies and the role of the eGC in vascular permeability regulation [78]. According to this model, the layered structure of the endothelial barrier requires continuous activation of signaling pathways regulated by sphingosine-1-phosphate (S1P) and intracellular cAMP. These pathways modulate the adherens junction (*zonula adherens*), continuity of tight junction

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Third, the eGC forms anti-inflammatory and anti-adhesive barrier at the endothelial cells. Vascular protection via inhibition of coagulation and leukocyte adhesion is provided by maintenance of the composition permanence and balance of degradation under the impact of stress shift and synthesis of eGC components [73, 79]. Total negative charge, formed by carbohydrate residues of the glycoconjugates chains on cell surface, prevents adhesive interactions of blood cells with vascular wall, biologically active molecules with anti-thrombotic action, while eGC-associated molecules provide hemostasis [80, 81]. Also eGC plays a structural role, impeding adhesion by covering adhesion molecules on the surface of the cell and by creating steric hindrance, making leukocyte binding more challenging [82]. Under the effect of damaging factors, the structure and composition of eGC change, its thickness may reduce significantly, and carbohydrate residues, normally covert and masked, become apparent. Main damaging factors, affecting the eGC *in vivo,* are: inflammation, hyperglycemia, endotoxemia, septic shock, oxidized low-density lipoproteins, cytokines, natriuretic peptides, abnormal shift stress and damage due to ischemia-reperfusion [79]. Shedding of eGC components in response to cytokines and chemoattractants occurs in all compartments of microvasculature:

According to Lipowsky, the studies of leukocytic-endothelial adhesion in response to chemoattractants and cytokines, and shedding of constituents of the eGC, suggest that activation of extracellular proteases (matrix metalloproteases, MMPs) play a role in mediating the dynamics of leukocytes adhesion in response to inflammatory and ischemic stimuli [79]. Inhibition of MMP activation with sub-antimicrobial doses of doxycycline, or zinc chelators, have also inhibited leukocytes adhesion and shedding of glycans from the endothelial cells surface in response to the chemoattractant. Experiments by McDonald et al. have confirmed that under the enzymatic degradation of eGC with heparinase, endothelial cells developed a pro-inflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion [82]. The results show an up-regulation of ICAM-1 (expression increases in 3 times) with degradation compared to non-degraded controls, and attribute this effect to a down-regulation in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in response to flow; this suggests that eGC is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion [82]. Other mechanisms also contribute to the initiation

strands, and the balance of synthesis and degradation of eGC components [78].

arterioles [83], capillaries [83, 84] and venules [84–86].

Second, the negatively charged eGC forms a polyanionic hydrated mesh on the surface of endothelial cells, which acts as a selective permeability electrostatic barrier for plasma cells and proteins and serves as a selective permeability [72]. According to Salmon and Satchell, in both continuous and fenestrated microvessels, this eGC is acting as an integral component of the multilayered barrier provided by the walls of these microvessels (i.e., acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes) [73]. Dysfunction of any of these capillary wall components, including the eGC, can disrupt normal microvascular permeability. Disruption of eGC manifests with increased systemic microvascular permeability and albuminuria in the glomerulus [73]. Evidence from the experiments on Munich-Wistar-Fromter (MWF) rats, used as a model of spontaneous albuminuric chronic kidney disease (CKD), confirm that loss of eGC could contribute to both renal and systemic vascular dysfunction in proteinuric CKD [74]. Also, in the 5/6-nephrecomized rats model with CKD a significant decrease in eGC thickness and stiffness in the blood explants of aorta endothelial cell isolated from CKD rats was demonstrated [75]. An increase of the levels of the two major components of the eGC, namely syndecan-1 (Syn-1) and HA, in the blood of patients with CKD indicated the disease progression and correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), vWF and angiopoietin-2 [75]. The study of experimental eGC degradation in mice induced by long-term hyaluronidase infusion, including evaluation of the eGC thickness and composition by immunohistochemical methods and by transmission electron microscopy for complete and integral assessment of glomerular albumin passage, showed that glomerular fenestrae were filled with dense negatively charged polysaccharide structures that were largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact [76]. Thus, HA is a key component of the glomerular endothelial protein permeability barrier; reduction of the HA facilitates albumin passage across the endothelial layer and the glomerular basement membrane toward the epithelial compartment [76].

Regulation of selective permeability by eGC, and the role of its separate components in this, is still subject of discussion. According to Lennon and Singleton, the HA plays key role in supporting endothelial barrier function [77]. HA maintains vascular integrity through eGC modulation, caveolin-enriched microdomain regulation and interaction with endothelial HA binding proteins. Certain disease states, especially accompanied by SIR, increase hyaluronidase activity and reactive oxygen species (ROS) generation which break down high molecular weight HA to low molecular weight fragments causing damage to the eGC. Further, these HA fragments can activate specific HA binding proteins upregulated in vascular disease to promote actin cytoskeletal reorganization and inhibition of endothelial cell–cell contacts [77]. A glycocalyx-junction-break model, described by Curry and Adamson summarizes multiple studies and the role of the eGC in vascular permeability regulation [78]. According to this model, the layered structure of the endothelial barrier requires continuous activation of signaling pathways regulated by sphingosine-1-phosphate (S1P) and intracellular cAMP. These pathways modulate the adherens junction (*zonula adherens*), continuity of tight junction strands, and the balance of synthesis and degradation of eGC components [78].

impact of tangential stress of blood flow shift primarily to eGC; the latter accepts and scatters the load, created by fluid shear stress. Local spin moment, created by fluid shear stress, affects the proteoglycans chains, and further—the core proteins (syndecans and glypicans), causing actin cytoskeleton reorganization and transmission of the signal into the cell and the cell nucleus [70, 71]. The study of Fu and Tarbell (2013) aimed to determine the eGC role in mechanosensing and transduction, and measured the flow-induced production of NO *in vitro* [7]. It was found that compared to static conditions, the application of steady flow shear stress rapidly increased NO production from the baseline in bovine aortic endothelial cells. Enzymatic treatment of the key components of eGC (HS, HA) completely blocked flowinduced NO production without affecting receptor-mediated NO production, suggesting that the eGC has a direct effect on the NO production machinery [7]. Therefore, the eGC under physiological conditions (intact eGC) transforms hemodynamic effect into cell biochemical

Second, the negatively charged eGC forms a polyanionic hydrated mesh on the surface of endothelial cells, which acts as a selective permeability electrostatic barrier for plasma cells and proteins and serves as a selective permeability [72]. According to Salmon and Satchell, in both continuous and fenestrated microvessels, this eGC is acting as an integral component of the multilayered barrier provided by the walls of these microvessels (i.e., acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes) [73]. Dysfunction of any of these capillary wall components, including the eGC, can disrupt normal microvascular permeability. Disruption of eGC manifests with increased systemic microvascular permeability and albuminuria in the glomerulus [73]. Evidence from the experiments on Munich-Wistar-Fromter (MWF) rats, used as a model of spontaneous albuminuric chronic kidney disease (CKD), confirm that loss of eGC could contribute to both renal and systemic vascular dysfunction in proteinuric CKD [74]. Also, in the 5/6-nephrecomized rats model with CKD a significant decrease in eGC thickness and stiffness in the blood explants of aorta endothelial cell isolated from CKD rats was demonstrated [75]. An increase of the levels of the two major components of the eGC, namely syndecan-1 (Syn-1) and HA, in the blood of patients with CKD indicated the disease progression and correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), vWF and angiopoietin-2 [75]. The study of experimental eGC degradation in mice induced by long-term hyaluronidase infusion, including evaluation of the eGC thickness and composition by immunohistochemical methods and by transmission electron microscopy for complete and integral assessment of glomerular albumin passage, showed that glomerular fenestrae were filled with dense negatively charged polysaccharide structures that were largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact [76]. Thus, HA is a key component of the glomerular endothelial protein permeability barrier; reduction of the HA facilitates albumin passage across the endothelial layer and the glomerular basement

Regulation of selective permeability by eGC, and the role of its separate components in this, is still subject of discussion. According to Lennon and Singleton, the HA plays key role in supporting endothelial barrier function [77]. HA maintains vascular integrity through eGC

signals, which regulate the vascular tone.

120 Endothelial Dysfunction - Old Concepts and New Challenges

membrane toward the epithelial compartment [76].

Third, the eGC forms anti-inflammatory and anti-adhesive barrier at the endothelial cells. Vascular protection via inhibition of coagulation and leukocyte adhesion is provided by maintenance of the composition permanence and balance of degradation under the impact of stress shift and synthesis of eGC components [73, 79]. Total negative charge, formed by carbohydrate residues of the glycoconjugates chains on cell surface, prevents adhesive interactions of blood cells with vascular wall, biologically active molecules with anti-thrombotic action, while eGC-associated molecules provide hemostasis [80, 81]. Also eGC plays a structural role, impeding adhesion by covering adhesion molecules on the surface of the cell and by creating steric hindrance, making leukocyte binding more challenging [82]. Under the effect of damaging factors, the structure and composition of eGC change, its thickness may reduce significantly, and carbohydrate residues, normally covert and masked, become apparent. Main damaging factors, affecting the eGC *in vivo,* are: inflammation, hyperglycemia, endotoxemia, septic shock, oxidized low-density lipoproteins, cytokines, natriuretic peptides, abnormal shift stress and damage due to ischemia-reperfusion [79]. Shedding of eGC components in response to cytokines and chemoattractants occurs in all compartments of microvasculature: arterioles [83], capillaries [83, 84] and venules [84–86].

According to Lipowsky, the studies of leukocytic-endothelial adhesion in response to chemoattractants and cytokines, and shedding of constituents of the eGC, suggest that activation of extracellular proteases (matrix metalloproteases, MMPs) play a role in mediating the dynamics of leukocytes adhesion in response to inflammatory and ischemic stimuli [79]. Inhibition of MMP activation with sub-antimicrobial doses of doxycycline, or zinc chelators, have also inhibited leukocytes adhesion and shedding of glycans from the endothelial cells surface in response to the chemoattractant. Experiments by McDonald et al. have confirmed that under the enzymatic degradation of eGC with heparinase, endothelial cells developed a pro-inflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion [82]. The results show an up-regulation of ICAM-1 (expression increases in 3 times) with degradation compared to non-degraded controls, and attribute this effect to a down-regulation in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in response to flow; this suggests that eGC is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion [82]. Other mechanisms also contribute to the initiation of lymphocytes adhesion to the endothelial cells after reduction of eGC layer: decrease of NO production, which is capable to inhibit leukocyte-endothelial cell adhesion [87]; appearance of eGC fragments, (such as low-molecular-weight HA), which show their pro-inflammatory properties, affecting the maturity of dendritic cells and stimulating them to produce cytokines [14, 88]; and exposure and synthesis under inflammatory response of hypoglycosylated structures, which interact with cell adhesion molecules of leukocytes [18, 89].

regarded as exogenous factors, while endogenous factors are gene products, resulting from trauma, ischemia, necrosis or oxidative stress [97]. Also there are some reports on generation of various new antigens due to inflammatory response; they are variations of the "modified own"; of the neoantigens formed as a result of the post-translational proteins modification [98]; and of antigens, mobilized to membrane from cytoplasm and the inner cell compartments interacting with membrane proteins or phospholipids, and acting as images of danger [99]. The enhanced pro-inflammatory background in normal pregnancy is evidenced by an increase of the level of the soluble cell adhesion molecules (sCAM) in blood, indicating the activation of leukocytes (increase of sE-selectin, sVCAM-1, sICAM-1 levels) and endothelial

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Central event of the inflammatory response is the contact between leukocytes and endothelium, with subsequent migration of immune cells to the inflammatory lesion. At early stages of inflammatory response endothelial selectins (Е-selectin and Р-selectin) and lymphocytic L-selectin form reversible bonds with carbohydrate counter-receptors on the partner cell, thus

The counter-receptors for selectins are typically heavily glycosylated molecules, many of which

not E-selectin, bind to some forms of heparin/HS. However, each of the selectins binds with higher affinity to its specific macromolecular ligands. Many of the known ligands are mucins containing sialylated fucosylated O-glycans. The major ligand for P-selectin, named P-selectin glycoprotein ligand-1 (PSGL-1), has sulfated tyrosine residues adjacent to a core-2 based

on either N- or O-glycans, including the E-selectin ligand-1, CD44, L-selectin (in humans),

within specialized endothelia termed high endothelial venules (HEV; HEV are specialized post-capillary venous swellings characterized by plump endothelial cells as opposed to the usual thinner endothelial cells found in regular venules. HEVs enable lymphocytes circulating in the blood to directly enter a lymph node by crossing through the HEV) contain

P-selectin are expressed on circulating leukocytes whereas L-selectin binds to ligands on both

At the firm adhesion stage, following the leukocyte capture stage and rolling, N-linked glycans on ICAM-1 regulate binding to its integrin ligands—macrophage-1 antigen (Mac-1) and LFA-1. Moreover, it was found that Mac-1 binds with higher avidity to molecules of ICAM-1 with smaller N-linked oligosaccharide chains, since the binding with the ligand increased after the use of α-mannosidase inhibitor deoxymannojirimicin (DMJ). In contrast, LFA-1 binds with higher affinity to glycoforms of ICAM-1, which has a more complex carbohydrate chain [89]. Also, there is experimental evidence that high-mannose ICAM-1 can function in leukocyte firm-adhesion [105]. It is speculated that some N-glycan-binding sites on ICAM-1 may be

motif on mucin-type O-glycans and on N-glycans [104]. The ligands for E- and

but E-selectin can also interact with several other glycoproteins that express the SLe<sup>x</sup>

motifs (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc) [102]. P- and L-selectin, but

. Also, PSGL-1 is one of the physiological ligands for E-selectin,

[68, 103]. Ligands for L-selectin that occur

motif

cells [100, 101].

bear terminal SLe<sup>x</sup>

6-sulfo-SLe<sup>x</sup>

O-glycan expressing SLe<sup>x</sup>

**3.1. The glycan-mediated processes in inflammation**

and possibly long-chain GSLs expressing the SLe<sup>x</sup>

leukocytes and the endothelium [89].

providing tethering and the leukocyte rolling along the vascular wall.

Modulation of eGC structure under effects of damaging factors, including inflammation, shows a thromboresistance loss [90, 91]. This occurs due to destabilization of heparin sulfate chains, the binding sites for coagulation inhibitor factors (antithrombin-III, the protein C system, and tissue factor pathway inhibition); this leads to a reduction of their local concentration at the vascular wall. In turn, a concentration gradient of protective and regulative molecules, associated with eGC (albumin, fibrinogen, orosomucoid, extracellular superoxide dismutase, fibronectin, vitronectin, collagens, thrombospondin-1 and other), and of growth factors (fibroblast growth factors, vascular endothelial growth factors, transforming growth factor-β, platelet-derived growth factors) is also decreased, facilitating pathological processes in blood vessels [80].

Therefore, the eGC is a labile structure; its composition changes under effects of damaging factors. This determines development of pathophysiological processes of endothelium activation/dysfunction with loss of vascular tone regulation, hemostasis and barrier function. Endothelium activation/dysfunction is induced by inflammation and accompanies it, thus forming a vicious cycle, which can be overcome only under normal immune system functioning. Inflammatory response of various degree accompanies not only pathologic processes, it is also observed under physiological conditions, for example, a pro-inflammatory background is shown at certain periods of normal pregnancy.

Understanding the mechanisms of disruption of maternal immunology tolerance to fetus, causes of transition of physiologic inflammatory reaction to systemic and excessive inflammatory response (as in PE), accompanied by endothelial activation/dysfunction, and revelation of the contribution of eGC damage to preeclampsia development may be subject of new discoveries in the disease pathogenesis.
