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

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 struc-

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

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

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

**3. The development of systemic inflammatory response in pregnancy**

There is much experimental evidence of a so-called "physiological", controlled SIR during pregnancy. Similarly to the classic inflammatory response, physiological inflammatory response during pregnancy is a reaction to local damage (matrix remodeling, associated with implantation, placentation and angiogenesis in placenta) [92, 93] and foreign invaders (cells, microparticles and soluble factors of placental origin) [94, 95]. Humoral factors, cellular debris and subcellular particles of trophoblast are considered to be the triggers of SIR, but they can also play a role of adjuvants [95, 96]. Cells-effectors of the maternal innate immunity detect fetal products as pathogen/danger images, implementing cell and molecular protection mechanisms against allogeneic material [97]. The gene products inherited from the father can be

tures, which interact with cell adhesion molecules of leukocytes [18, 89].

122 Endothelial Dysfunction - Old Concepts and New Challenges

in blood vessels [80].

is shown at certain periods of normal pregnancy.

discoveries in the disease pathogenesis.

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 providing tethering and the leukocyte rolling along the vascular wall.

The counter-receptors for selectins are typically heavily glycosylated molecules, many of which bear terminal SLe<sup>x</sup> motifs (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc) [102]. P- and L-selectin, but 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 O-glycan expressing SLe<sup>x</sup> . Also, PSGL-1 is one of the physiological ligands for E-selectin, but E-selectin can also interact with several other glycoproteins that express the SLe<sup>x</sup> motif on either N- or O-glycans, including the E-selectin ligand-1, CD44, L-selectin (in humans), and possibly long-chain GSLs expressing the SLe<sup>x</sup> [68, 103]. Ligands for L-selectin that occur 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 6-sulfo-SLe<sup>x</sup> motif on mucin-type O-glycans and on N-glycans [104]. The ligands for E- and P-selectin are expressed on circulating leukocytes whereas L-selectin binds to ligands on both leukocytes and the endothelium [89].

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 pro-adhesive, whereas the neighboring sites may be anti-adhesive, underscoring the potential breadth of how ICAM-1 function may be regulated by N-glycosylation [106]. On the stage of firm adhesion an important aspect of inflammatory response is exposure of the active epitope of integrins, provided by chemokines, which are present on the endothelial cell surface, and are bound to HS. Glycosylation of chemokine receptors also contributes to the adequate dynamics of the inflammatory reaction, thus increasing the binding affinity of the chemokine to the receptor and protecting the latter from proteolytic cleavage (reviewed in [18, 89]).

Key molecules mediating leukocyte transmigration: PECAM-1, JAM-1, ICAM-2 and VE-cadherin, are highly-glycosylated. However, carbohydrates part in leukocyte transmigration is still not clear. The recent studies show that N-glycosylation of JAM-A is required for the protein's ability to reinforce barrier function [22]; sialic acid-containing glycan of PECAM-1 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface [52]; glycosylation status of ICAM-2 (hypo- or non-glycosylated variants) significantly affects the function of this protein in cell motility assays [107]; in pro-inflammatory conditions, modification of VE-cadherin glycans is observed [55]. This obviously requires further investigations. Molecules that mediate intercellular interactions during inflammation are presented in **Table 3**.

Many studies demonstrate modification of endothelial glycome (glycome is the entire complement of sugars, whether free or present in more complex molecules, of an organism) under inflammatory response. Modeling of inflammatory response *in vitro* on endothelial cell lines showed that an enhanced α2,6-sialylation was observed after TNF stimulation [108]. Proinflammatory stimuli increase hypoglycosylated (namely, high-mannose/hybrid) N-glycans on the cell surface as determined by lectin histochemistry, and cause an increase in genes encoding for fucosylation and sialylation (confirmed at specific staining with relevant lectins [18]; this correlates with increased monocyte adhesion [18]. Glycosylation of the endothelium has been proposed to act as a "zip code" for directing leukocyte subtype-specific recruitment in different vascular beds in response to specific stimuli [89].

#### **3.2. The glycobiology of immunoregulation**

The carbohydrate-protein interactions not only mediate the initial stages of inflammation, but also promote many cellular contacts, which regulate innate and adaptive immune response. The main carbohydrate binding proteins are endogenous lectins [109], widely present on the immune system cells and expressed both in membrane-linked and in soluble forms. Three main classes of endogenous lectins include:

А. C-type lectins, which, depending of specificity, are:


as a result of tissue damage: HA fragments or glycosaminoglycans of the extracellular cell matrix (ECM) and eGC [111], may act as pathogen/danger images for these lectins. The best known molecules related to С-type lectins are: selectins and myeloid range receptors (mannose-binding receptors DEC-205 and mannose receptor CD206); dectin-1 and dectin-2,

VCAM-1 • VLA-4 (α4β1-integrin) Mediates the stage of firm leucocytes adhesion of

LFA-1, lymphocyte function-associated antigen-1; Mac-1, macrophage-1 antigen; VLA-4, very late antigen-4.

**Table 3.** Molecules, mediating carbohydrate-protein interactions in inflammation site [80, 91, 92].

MadCAM-1, mucosal addressin cell adhesion molecule-1; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1; PCLP, podocalyxin-like protein; SLex, sialyl-Lewis X; PSGL-1, P-selectin glycoprotein ligand 1; ESL-1, E-selectin ligand-1;

B. **Galectins** are a family of 15 evolutionary conserved carbohydrate-binding proteins [89, 113], belonging to the glycoproteins and glycolipids of cell surface and ECM [114] and specifically

DC-SIGN (CD209), and langerin (CD207) [112].

**Cell adhesion molecules (proteins)**

L-selectin **1.** MadCAM-1

**2.** CD34 **3.** Sgp200 **4.** GlyCAM-1 **5.** Endoglycan **6.** Endomucin **7.** PCLP **8.** PSGL-1 **9.** 6-sulfo-SLe<sup>x</sup>

**Counter-receptors (carbohydrates) Comments**

Binding L-selectin with:

• SLe<sup>x</sup>

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

Mediates:

Mediate:

endothelium

inflammation;

inflammation;

• leukocyte capture and rolling

glycoproteins that express the SLe<sup>x</sup>

• leukocyte capture and rolling

leukocytes to endothelium

• peripheral node addressins (no. 1, 2, 3, 4, 5, 6, 7) mediates lymphocyte recirculation (homing);

9) mediates leukocyte capture and rolling

• leukocyte recruitment in both acute and chronic

either N- or O-glycans and possibly long-chain glycosphingolipids expressing the SLe<sup>x</sup>

• recruit leukocytes recruitment to sites of

Mediates the stage of firm adhesion of leukocytes to

antigen on

antigen;


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125

determinant is associated with

the MECA-79 epitope

**2.** heparin/heparin sulfate (binds weakly) **3.** some glycoproteins (mucins containing highly clustered glycans) that bear the SLe<sup>x</sup>

P-selectin **1.** PSGL-1 (major counter-receptor)

determinant

**4.** L-selectin (in humans)

**2.** Mac-1 (αMβ2-integrin)

**2.** ESL-1 **3.** CD44

ICAM-1 **1.** LFA-1 (αLβ2-integrin)

E-selectin **1.** PSGL-1

Lectins of С-type are present on macrophages, dendritic cells, natural killer cells, leukocytes. They act as pattern-recognition receptors and fulfill signaling and adhesion functions [110]. Glycoconjugates: bacterial lipooligosaccharides, peptidoglycans, and molecules emerged


pro-adhesive, whereas the neighboring sites may be anti-adhesive, underscoring the potential breadth of how ICAM-1 function may be regulated by N-glycosylation [106]. On the stage of firm adhesion an important aspect of inflammatory response is exposure of the active epitope of integrins, provided by chemokines, which are present on the endothelial cell surface, and are bound to HS. Glycosylation of chemokine receptors also contributes to the adequate dynamics of the inflammatory reaction, thus increasing the binding affinity of the chemokine to the receptor and protecting the latter from proteolytic cleavage (reviewed in [18, 89]).

Key molecules mediating leukocyte transmigration: PECAM-1, JAM-1, ICAM-2 and VE-cadherin, are highly-glycosylated. However, carbohydrates part in leukocyte transmigration is still not clear. The recent studies show that N-glycosylation of JAM-A is required for the protein's ability to reinforce barrier function [22]; sialic acid-containing glycan of PECAM-1 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface [52]; glycosylation status of ICAM-2 (hypo- or non-glycosylated variants) significantly affects the function of this protein in cell motility assays [107]; in pro-inflammatory conditions, modification of VE-cadherin glycans is observed [55]. This obviously requires further investigations. Molecules that mediate intercellular interactions during inflammation are presented in **Table 3**. Many studies demonstrate modification of endothelial glycome (glycome is the entire complement of sugars, whether free or present in more complex molecules, of an organism) under inflammatory response. Modeling of inflammatory response *in vitro* on endothelial cell lines showed that an enhanced α2,6-sialylation was observed after TNF stimulation [108]. Proinflammatory stimuli increase hypoglycosylated (namely, high-mannose/hybrid) N-glycans on the cell surface as determined by lectin histochemistry, and cause an increase in genes encoding for fucosylation and sialylation (confirmed at specific staining with relevant lectins [18]; this correlates with increased monocyte adhesion [18]. Glycosylation of the endothelium has been proposed to act as a "zip code" for directing leukocyte subtype-specific recruitment

The carbohydrate-protein interactions not only mediate the initial stages of inflammation, but also promote many cellular contacts, which regulate innate and adaptive immune response. The main carbohydrate binding proteins are endogenous lectins [109], widely present on the immune system cells and expressed both in membrane-linked and in soluble forms. Three

• Specific to galactose (Gal-) or N-acetylgalactosamine (GalNAc-)/N-acetylglucosamine

Lectins of С-type are present on macrophages, dendritic cells, natural killer cells, leukocytes. They act as pattern-recognition receptors and fulfill signaling and adhesion functions [110]. Glycoconjugates: bacterial lipooligosaccharides, peptidoglycans, and molecules emerged

in different vascular beds in response to specific stimuli [89].

**3.2. The glycobiology of immunoregulation**

124 Endothelial Dysfunction - Old Concepts and New Challenges

main classes of endogenous lectins include:

(GlcNAc-)

А. C-type lectins, which, depending of specificity, are:

• Specific to mannose (Man-) and/or fucose (Fuc-) terminated glycans;

MadCAM-1, mucosal addressin cell adhesion molecule-1; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1; PCLP, podocalyxin-like protein; SLex, sialyl-Lewis X; PSGL-1, P-selectin glycoprotein ligand 1; ESL-1, E-selectin ligand-1; LFA-1, lymphocyte function-associated antigen-1; Mac-1, macrophage-1 antigen; VLA-4, very late antigen-4.

**Table 3.** Molecules, mediating carbohydrate-protein interactions in inflammation site [80, 91, 92].

as a result of tissue damage: HA fragments or glycosaminoglycans of the extracellular cell matrix (ECM) and eGC [111], may act as pathogen/danger images for these lectins. The best known molecules related to С-type lectins are: selectins and myeloid range receptors (mannose-binding receptors DEC-205 and mannose receptor CD206); dectin-1 and dectin-2, DC-SIGN (CD209), and langerin (CD207) [112].

B. **Galectins** are a family of 15 evolutionary conserved carbohydrate-binding proteins [89, 113], belonging to the glycoproteins and glycolipids of cell surface and ECM [114] and specifically binding mainly to N-acetyllactosamine. The main ligands are Galβ1-3GlcNAc- or Galβ1- 4GlcNAc- [115]. Galectins are involved in many cell activities: cell cycle regulation, migration, cell signals transmission, effectory functions, apoptosis, immunoregulation [116]. Galectins may regulate inflammatory reaction both positively (Gal-3, Gal-8, Gal-9) and negatively (Gal-1). The endothelium may be a source of Gal-1, which then targets the neutrophils to inhibit cell recruitment, аnd Gal-3, Gal-8, Gal-9 promote neutrophil and eosinophil adhesion [89].

**4. Endothelial activation and endothelial dysfunction**

damage of the endothelial cells, their apoptosis and necrosis.

but also may cause multiorgan failure.

As a rule, in the studies dedicated to determination of endothelium role in different pathologies, the authors use terms "endothelial activation" and "endothelial dysfunction" [129]. Activation should be distinguished from activity because in its resting state, endothelium is a metabolically active organ, which produces vasodilatory substances and bears anticoagulative and antiadhesive phenotype. Activation of endothelium under various pathophysiologic processes leads to alterations of its phenotype and function. These events may be reversible,

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There are two stages in endothelial activation: endothelial stimulation (early events) and endothelial activation (later events). The latter can be subdivided in endothelial activation of types I and II, respectively [130, 131]. Endothelial activation of type I follows the stimulation stage and manifests with shedding of the adhesion molecules and molecules with antithrombotic properties, such as Р-selectin, thrombin, heparin, antithrombin ІII and thrombomodulin, from the surface of the endothelial cells. In the same time, the endothelial cells of the venules and small veins decrease in volume, and the contacts between the cells become distorted, resulting in hemorrhages, edema, and increase of vessels permeability [131]. Endothelial activation of type II is a slightly delayed process, which depends on gene transcription activation and protein synthesis *de novo*. As a result, the genes coding for the adhesion molecules, chemokines and procoagulative factors: Е-selectin, vWF, IL-8, thrombocytes activating factor [132], are activated. Also, the secretion of NO and prostacyclin increases. Morphologic changes show protrusion of the endothelial cells into the vessel lumen, cell hypertrophy and an increase of cell permeability. The result of this stage is leukocyte contact with activated endothelium through lectin-carbohydrate interactions, extravasation, transendothelial migration, and, possibly, leucocyte binding with Fc-receptors (FcR) of endothelial cells with immune complexes disposition [131]. Alterations of phenotype, accompanying endothelial cells activation, manifest also with the change of the carbohydrate composition of the molecules forming the eGC. Therefore, endothelial activation implies an alteration of the endothelial cells phenotype under the activation factors (cytokines, endotoxins, etc.) impact, inducing shedding and modification of the vasculoprotective surface layer associated with the membrane, and expression of the activation antigens. This correlates with pro-adhesive, antigen-presenting and procoagulative properties of the endothelial cells. Activation reflects an ability of endothelial cells to perform new functions, but this status does not presume a cell damage or their uncontrolled division. Endothelium activation is a reverse process with a possibility to return to a state of active reposing cells [131]. Endothelium dysfunction, on the other hand, is a stage following the endothelium activation and manifesting with cell functional activity change; it leads to loss of the ability of endothelium to perform its function, and to a disbalance of factors, which provide homeostasis and a normal course of all processes, mediated by endothelium [8, 129, 131]. Endothelial dysfunction is a consequence of chronic, permanent endothelial activation and may lead to non-reversible

C. Siglecs are a family of 17 known lectins, which specifically bind the glycans structures with terminal sialic acid [117]. Sialyl Tn (Neu5Acα2,6GalNAcα-) is a common ligand for all members of this family. Glycan 6′ sulfated SLe<sup>x</sup> is a ligand for Siglec-8, and is important for selectin-dependent cell adhesion [118]. The majority of this family members are inhibitory receptors as they bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their structure, and they are mainly expressed on immune cells [119]. Siglecs participate in regulation/restriction of an excessive activation response to inflammatory reaction, initiated via recognition of pathogen associated molecular patterns, and damage-associated molecular patterns, with following phagocytosis of cells, bearing these patterns [120, 121]. Siglecs regulate cell proliferation, differentiation, apoptosis, adhesion, cytokines synthesis and negative regulation of В-lymphocyte signaling [122].

Some endogenous lectins are capable, like autoantibodies, to interact with the body's unchanged antigens (glycans), so-called own self-images (SAMPs-self-associated molecular patterns) [111]. Molecular patterns, containing sialic acid and heparin/HS are supposed to act as self-images [111]. Also it is thought that interaction of lectins, recognizing SAMPs, (mainly siglecs), with ligands, inhibits the immune response to foreign/damaging effects [111, 120].

It is known that presence of terminal sialic acid is very important: this substance provides the overall negative charge of cell surface, glycoconjugates conformation stabilization, production of glycoconjugates, and cells protection from recognition and degradation. Sialylation protective properties manifest not only with sialylated structures interaction with inhibited receptors, but also with masking of sugar residues which are the antigen determinants [123, 124]. For example, at desialylation, the unmasked residues of Galβ-, GalNAc-, and mannose, interacting with lectins from galectins family and С-type lectins [120]; these interactions are important for metastasis and SIR development.

Therefore, inflammatory response regulation is implemented under direct involvement of the glycan binding proteins (endogenous lectins) and glycans; composition and structure of these vary significantly under physiological and pathophysiological conditions, providing evidence of the eGC modification at inflammation, and of formation of the carbohydrate "zip code", which acts as navigator for immune cells. Inflammatory reactions in pregnancy are initiated by pathogenic and danger images, which are formed at the fetal-mother cell contact; this activates innate and adaptive immunity. SIR may be enhanced or restricted through mechanisms based on carbohydrate-protein interaction [125–127]. Excessive SIR developing in pathologic pregnancy is characterized by compensatory reactions and development of various dysfunctions, resulting in organic or multi-organic failure [128].
