Biochemical composition of endothelial glycocalyx—main components (part I: glycoproteins).
The endothelial glycocalyx is an intravascular compartment which consists of carbohydrate part of membrane glycoconjugates, free proteoglycans and associated proteins. It is thought to play an important role in the vascular tone regulation, vascular permeability and thromboresistance. It was suggested that the leading cause of endothelial dysfunction in various cardiovascular, inflammatory, and kidney diseases is the damage of the endothelial glycocalyx. This review presents the changes in the composition and structure of the endothelial glycocalyx in the settings of damage and under systemic inflammatory response, and the impact of these changes on the functions of endothelial cells and intercellular contacts, mediating the interaction of endothelium and the immune cells. The second issue, discussed in this article is a possible role of endothelial glycocalyx in the pathogenesis of preeclampsia—a complication of pregnancy associated with hypertension, proteinuria and edema. The reviewed data contribute a new insight in the endothelial dysfunction pathogenesis.
- endothelial glycome
- endothelial dysfunction
- glycobiology of inflammation
Preeclampsia (PE) is one of the main problems of modern obstetrics. PE develops in 2–9% of all pregnancies; it is the second most frequent cause of maternal morbidity and one of the leading causes of neonatal morbidity and mortality. PE is now regarded as a syndrome 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 complicated course of the disease .
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 . 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 —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.
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, 9, 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 not-anchored proteoglycans . 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, 12, 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 , with thickness ranging 2–4.5 μm  in different departments of the vascular system.
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) . 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 . 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 . 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 inTables 1 and 2 (parts I and II).
|Adhesion molecules||Е-selectin||Contains 11 potential N-glycosylation site|||
|Р-selectin||Contains 9 potential N-glycosylation sites|||
|Integrins: α1β1, α2β1, α3β1, α5β1, α6β1, α8β1, α9β1, αVβ1, αVβ3, α6β4, αVβ5||N-linked glycans||[14, 15]|
|VE-cadherin||Contains 7 potential N-glycosylation sites|||
|JAM-1||Contains 1 N-glycosylation site|||
|JAM-2||Contains 2 N-glycosylation sites|||
|JAM-3||Contains 2 N-glycosylation sites|||
|ICAM-1||Contains 8 N-glycosylation sites|||
|ICAM-2||Contains 6 N-glycosylation sites|||
|VCAM-1||Contains 6 N-glycosylation sites|||
|PECAM-1||Сontains 9 N-glycosylation sites|||
|ClyCAM-1||Mucin, containing predominantly O-linked carbohydrate chains (T-antigen and 6′ sulfated sialyl-Lewis-X)||[21, 22]|
|CD34||Mucin, O-glycosylation sites are more abundant than N-glycosylation sites|||
|MadCAM-1||Mucin; contain O-linked glycans (SLex)|||
|Coagulation and fibrinolysis regulators||Von Willebrand factor||Contains at least 10 potential N- and 10 O-glycosylation sites||[25, 26]|
|Thrombomodulin||Contains at least 4 N- and 1 O-glycosylation sites||[27, 28, 29, 30, 31, 32]|
|Antithrombin III||Contains 4 potential N-glycosylation sites||[33, 34]|
|Heparin cofactor II||Contains 3 potential N-glycosylation sites|||
|Group||Members||Number/type of GAG-chains linked||Comments||Ref|
|Glycosaminoglycans||HA||—||Anionic, nonsulfated glycosaminoglycan; structural unit of HA is a repeating disaccharide consisting of β-|||
|HS||—||The most common disaccharide unit within HS is composed of a mono-sulfated β-glucuronic acid linked to tri-sulfated α-N-acetylglucosamine|||
|CS||—||CS is a linear acidic polysaccharide, composed of repeating disaccharide units of β-glucuronic acid and β-N-acetyl-|||
|DS||—||Backbone of DS chains is a linear polymer composed of repeating disaccharide units of α-iduronic acid and β-N-acetyl-|||
|KS||—||Basic repeating disaccharide unit within keratan sulfate is units of β-|||
|Proteoglycans (extracellularly secreted)||Perlecan||3/HS,CS||A large basement membrane heparan sulfate proteoglycan; protein core of approximately 500 kDa|||
|Versican||10-30/CS,DS||Large aggregating chondroitin sulfate proteoglycan, core protein (at >350 kDa)||[41, 42]|
|Endocan||1/DS||Is a DSPG, small proteoglycan molecules (20 kDa) with a single DS chain; DS of endocan consists of about 32 disaccharide units|||
|Decorin||1/CS,DS||A prototype small leucine-rich proteoglycan (40 kDa); it has N-terminal attachment site for a single GAG chain of chondroitin or dermatan sulfate|||
|Biglycan||2/CS,DS||small leucine-rich proteoglycan (42 kDa protein core)||[45, 46]|
|Mimecan||2–3/KS||Small leucine-rich proteoglycan; (12–34 kDa protein core)||[47, 48]|
|Proteoglycans (associated with the cell surface)||Syndecans||5/HS,CS||Transmembrane proteoglycans|
Family of HSPGs, the syndecan protein family has four members.
Core protein of all glypicans is ranging between 198 to 346 kDa
Family of HSPGs
The glypican protein family has six members core protein of all glypicans is similar in size, approximately ranging between 60 and 70 kDa
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 . N-glycans of the junctional adhesion molecule-A (JAM-A) regulate leukocyte adhesion and lymphocyte function-associated antigen-1 (LFA-1) binding . 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 . 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 anti-inflammatory 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 .
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 the coagulant Factor VIII and mediating platelet adhesion at the sites of vascular injury . VWF is heavily glycosylated by N- and O-linked oligosaccharides, and glycosylation affects many of its functions . Antithrombin is a major inhibitor of the blood coagulation cascade. 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 . 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 Lex motif (SLex) . 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 . Major capping group in GlyCAM-1, CD34 and MadCAM-1 is the sulfated SLex [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 SLex epitopes on O-glycans that are important for its recognition by L-selectin .
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 . 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 . 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 . GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid raft . Cultured human umbilical vein endothelial cells (HUVEC) appeared to contain complex lacto and globo series compounds (lactosylceramide, Gb3Cer and Gb4Cer), but the most abundant neutral GSL is lactosylceramide (LacCer, CDw17) . 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 . Endothelium activation by pro-inflammatory cytokines, particularly by TNF-α, affect the Gb3Cer and Gb4Cer  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 . 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 Gb3Cer synthesis in these cells , because Gb3Cer (CD77) binds to the verocytotoxin and injures human endothelial cells .
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 , and sulfoglucuronyl paragloboside (SGPG), a minor GSL in endothelial cells, is a ligand for L-selectin . Although GIyCAM-1 and CD34 constitute the major L-selectin ligand on venous endothelium, endothelial SLex gangliosides may also play a role, since L-selectin can also bind SLex GSLs under physiologic flow conditions .
2.2. Functions of the endothelial glycocalyx
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 . 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 . 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 flow-induced NO production without affecting receptor-mediated NO production, suggesting that the eGC has a direct effect on the NO production machinery . Therefore, the eGC under physiological conditions (intact eGC) transforms hemodynami
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 . 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) . 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 . 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 . 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 . 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 . 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 . 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 .
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 . 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 . A glycocalyx-junction-break model, described by Curry and Adamson summarizes multiple studies and the role of the eGC in vascular permeability regulation . 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 .
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 . 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 . Shedding of eGC components in response to cytokines and chemoattractants occurs in all compartments of microvasculature: arterioles , capillaries [83, 84] and venules [84, 85, 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 . 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 . 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 . 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 ; 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].
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 .
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.
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 . The gene products inherited from the father can be regarded as exogenous factors, while endogenous factors are gene products, resulting from trauma, ischemia, necrosis or oxidative stress . 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 ; 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 . 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 cells [100, 101].
3.1. The glycan-mediated processes in inflammation
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 SLex motifs (Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc) . 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 SLex. 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 SLex 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 SLex [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-SLex motif on mucin-type O-glycans and on N-glycans . The ligands for E- and P-selectin are expressed on circulating leukocytes whereas L-selectin binds to ligands on both leukocytes and the endothelium .
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 . Also, there is experimental evidence that high-mannose ICAM-1 can function in leukocyte firm-adhesion . 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 . 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 ; sialic acid-containing glycan of PECAM-1 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface ; glycosylation status of ICAM-2 (hypo- or non-glycosylated variants) significantly affects the function of this protein in cell motility assays ; in pro-inflammatory conditions, modification of VE-cadherin glycans is observed . This obviously requires further investigations. Molecules that mediate intercellular interactions during inflammation are presented in Table 3.
|Cell adhesion molecules (proteins)||Counter-receptors (carbohydrates)||Comments|
|L-selectin||Binding L-selectin with:|
|E-selectin||glycoproteins that express the SLex antigen on either N- or O-glycans and possibly long-chain glycosphingolipids expressing the SLex antigen;|
|ICAM-1||Mediates the stage of firm adhesion of leukocytes to endothelium|
|VCAM-1||Mediates the stage of firm leucocytes adhesion of leukocytes to endothelium|
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 . Pro-inflammatory 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 ; this correlates with increased monocyte adhesion . 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 .
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 , 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:
Specific to mannose (Man-) and/or fucose (Fuc-) terminated glycans;
Specific to galactose (Gal-) or N-acetylgalactosamine (GalNAc-)/N-acetylglucosamine (GlcNAc-)
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 . Glycoconjugates: bacterial lipooligosaccharides, peptidoglycans, and molecules emerged as a result of tissue damage: HA fragments or glycosaminoglycans of the extracellular cell matrix (ECM) and eGC , 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) .
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  and specifically binding mainly to N-acetyllactosamine. The main ligands are Galβ1-3GlcNAc- or Galβ1-4GlcNAc- . Galectins are involved in many cell activities: cell cycle regulation, migration, cell signals transmission, effectory functions, apoptosis, immunoregulation . 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 .
C. Siglecs are a family of 17 known lectins, which specifically bind the glycans structures with terminal sialic acid . Sialyl Tn (Neu5Acα2,6GalNAcα-) is a common ligand for all members of this family. Glycan 6′ sulfated SLex is a ligand for Siglec-8, and is important for selectin-dependent cell adhesion . 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 . 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 .
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) . Molecular patterns, containing sialic acid and heparin/HS are supposed to act as self-images . 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 ; 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, 126, 127]. Excessive SIR developing in pathologic pregnancy is characterized by compensatory reactions and development of various dysfunctions, resulting in organic or multi-organic failure .
4. Endothelial activation and endothelial dysfunction
As a rule, in the studies dedicated to determination of endothelium role in different pathologies, the authors use terms “endothelial activation” and “endothelial dysfunction” . 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, but also may cause multiorgan failure.
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 . 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 , 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 . 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 .
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 damage of the endothelial cells, their apoptosis and necrosis.
5. Preeclampsia as a manifestation of excessive systemic inflammatory response, accompanied by endothelial activation/dysfunction
PE is a multisystemic pathologic condition, manifesting after the 20th week of pregnancy. PE clinical signs are: an increase of systolic blood pressure (SBP) above 140 mm Hg, diastolic blood pressure (DBP) above 90 mm Hg for the first time noted during pregnancy; proteinuria (≥0.3 g/L) in daily urine, edema, manifestation of multiorganic/polysystemic dysfunction/insufficiency . Severe PE is accompanied by acute renal failure, eclampsia, pulmonary edema, HELLP (hemolysis, elеvated liver enzymes, and lоw plаtelet соunt)-syndrome .
Etiology of PE is not clear; genetic, immunological and microenvironment may play a role [134, 135, 136, 137, 138]. Currently two phenotypic variations of PE are distinguished: early manifestation of the symptoms (before the 34th week of gestation) and later manifestation (after the 34th week of gestation) . Pathophysiological mechanisms of PE development are distinguished accordingly . The first—“fetal” pathway—is characterized by inadequate or microcellular invasion of trophoblast cells into the uterine spiral arteries and lack or incompleteness of the phase of substitution of placental smooth muscle elastic fibers with fibrinoid [140, 141]. In this mechanism, physiological remodeling and transformation of spiral arteries is lacking, and this affects the uterine-placentary blood flow quality [142, 143, 144]. Fetal mechanism of PE development presents with severe disease course and frequent complications in the neonate. The second pathway is “maternal”, where the deficiency of uterine-placental blood flow appears as a result of spiral arteries damage due to certain maternal diseases, especially thrombophilias (genetic or acquired). In this case, the study of placental morphology testifies adequate gestational reorganization of spiral arteries. Maternal pathway usually implies later manifestation and a milder course. Some also distinguish the third (or “mixed”) pathway, where the arteries are both affected and poorly reorganized [145, 146].
Disrupted trophoblast invasion initiates ischemic and hypoxic damage of placental cells and tissues, leading to increase of cell debris and microparticles of fetal origin contents in the mother’s blood. These processes result in the mother’s immune cells activation and inflammatory cytokines synthesis induction , leading to the development of generalized endothelial activation/dysfunction with development of multiorganic insufficiency  (Figure 1). Trophoblast debris was also found in the mother’s is blood in a normal pregnancy and it was primarily apoptotic. Particles of trophoblast debris range from polynuclear aggregates of the syncytium cells to subcellular micro and nanoparticles. In vitro co-culturing of trophoblast debris, obtained from women with normal pregnancy, with macrophages and endothelial cells leads to tolerogenic М2-phenotype of macrophages [149, 150]. Trophoblast debris becomes more necrotic when in vitro system is supplemented with antiphospholipid antibodies or IL-6. Phagocytosis of the necrotic debris by the endothelial cells is accompanied by their activation . Activation of endothelial cells is also caused by the addition of the trophoblast debris isolated from patients with preeclampsia to the culture of the endothelial cells .
5.1. Endothelium activation markers in preeclampsia
Numerous studies have shown that in PE, manifestations of excessive SIR are observed due to the loss of control over the balance of production of pro/anti-inflammatory cytokines. This leads to an increase in the synthesis and expression of key molecules that mediate intercellular contacts between leukocytes and endothelium [147, 153, 154]. In this context, it has been shown that in PE, the plasma levels of sE-selectin, sVCAM-1 and sICAM-1 were significantly elevated [100, 155, 156, 157], and that cultivation of endothelial cells with the blood serum of PE women significantly increased the expression of ICAM-1 by the endothelial cells .
It was found that the expression of E-selectin and P-selectin in the endothelial cell culture was significantly higher after administration of trophoblast cells from the PE patients, than after cultivation of endothelial cells with trophoblast cells isolated from placental tissue of healthy women . We have shown in a prospective longitudinal study that in patients with severe PE, the levels of sE-selectin, sVCAM-1 and sICAM-1 were increased from the 8th week of pregnancy until the appearance of clinical symptoms of the disease . In a similar design study, it was shown that joint determination of sICAM-1 and sVCAM-1 levels measured in peripheral blood within 22–29 weeks of gestation, was of high predictive value and capable to detect up to 55% of women with a pathologic pregnancy . The increased levels of sICAM-1 and sVCAM-1 in blood during PE significantly correlated with the signs of the acute phase of inflammation and PE: hypertension, proteinuria, increase of hepatic enzymes levels . Also it was noted that high levels of sVCAM-1 and sE-selectin in women with PE could result in adverse perinatal outcome and endothelial dysfunction in fetus, as confirmed by negative correlation between sVCAM-1 and endogenous NO synthesis by HUVECs, isolated from the umbilical cord after birth .
5.2. Alteration of endothelial glycocalyx in preeclampsia
The signs of endothelial activation are the expression of activation markers by endothelial cells and increased plasma concentrations of the soluble forms of CAMs and of the factors, regulating angiogenesis and blood clotting. However, the main feature of the evolving endothelial activation is alteration, damage and shedding of the eGC and an increase of its components concentration in blood. Currently, there are limited studies of this phenomena in PE, but available reports show significant alteration of eGC composition in the placental structures in PE . The most prominent alteration of the eGC composition was found in the placentas of women with severe PE. Alterations take place also in the eGC capillaries of terminal placental villi: the content of glycans with terminal β-galactosyl and α-mannosyl residues increase, while the content of α2,3-linked sialic acids decrease in the glycome in severe PE . These alterations are supposed to point to the exposure of glycans bearing the “danger signals” and being the counter-receptors for endogenous lectins; interaction with these activate maternal immune system [166, 167] (REF). Such studies, performed by immunohistochemistry of placenta after childbirth and using the lectins panel or monoclonal antibodies to carbohydrates antigens, give an idea of alterations of the placental glycome and its separate structures, including capillary endothelium, and provide evidence obtained by direct eGC visualization [165, 168]. Since direct visualization of the eGC is impossible in clinical trials where no surgical tissue sampling is implied, in these cases, an indirect assessment of the content of the degradation products of eGC is used.
Indirect methods have significant limitations, but they are the only possibility to evaluate the eGC in vivo. Indirect assessment of the eGC by ELISA show that in PE, the plasma content of the structural proteoglycans (endocan-1, syndecan-1, decorin and HA) and the GAGs of eGC increase [169, 170, 171]. Serum endocan concentrations were significantly elevated in women with PE versus normotensive controls, and concentrations seem to be associated with the severity of the disease . Median maternal plasma endocan concentrations were higher in PE patients and lower in acute pyelonephritis with bacteremia than in uncomplicated pregnancy. No significant difference was observed in the median plasma endocan concentration between other obstetrical syndromes and uncomplicated pregnancies . It is suggested that in PE, the maternal endothelium is a source of GAGs in blood, and intensive eGC shedding thus indicates a manifestation of endothelial dysfunction [169, 170, 171, 172, 173, 174]. Also, patients with PE show GAGs excretion in urine; this is thought to be linked with the eGC proteoglycans alterations and with the glomerular basement membrane changes, and associated with proteinuria . In vitro and in vivo experimental studies, using cell and animal models is another opportunity of indirect eGC evaluation. This approach was used to study CKD [74, 75], cardio-vascular and inflammatory diseases [13, 176], cancer [13, 176, 177]—the conditions manifestating with hypertension, proteinuria, edema, SIR, thrombosis. The results of such studies provide some keys to PE, which is less studied, but exhibits similar clinical signs. Experimental models allow to evaluate not only the degree of the eGC damage by various factors (SIR being the most significant), but also the molecular changes of the eGC composition. This moment is a crucial point because SIR is not a specific process; it accompanies almost any pathology and promotes the generation of neoantigens, acting as an adaptive response trigger and provoking autoimmune reactions.
Endothelial dysfunction represents the central link in the pathogenesis of various diseases and complications, and is a subject of intensive research. On the background of the progress in understanding the mechanisms of development, diagnosis and treatment of endothelial dysfunction, many studies in the recent years have been focused on the eGC as an early indicator of endothelial injury and a potential marker of vascular injury.
Alterations of the phenotype of endothelial cells, secretion and release of various activation markers into the bloodstream and dysfunction of the endothelium are directly related to the damage of eGC. This damage is the initiating factor and the initial stage in the development of endothelial activation/dysfunction, but this stage has for a long time been obscure due to the difficulties of eGC visualization and diagnosis.
By now, the main criteria for eGC damage assessment have been defined. In addition to the appearance of eGC components in the blood, the degree of manifestation of the SIR is also an important criterium of the damage, since endothelial inflammation and dysfunction are inseparably related processes. In this regard, the molecular mechanism of the inflammatory reaction is based on the ligand-receptor, carbohydrate-protein interaction of the immune cells and endothelium, and alteration of glycome/glycocalyx is a crucial factor in the development of inflammation and endothelial dysfunction. Therefore, the pathogenesis of endothelial activation/dysfunction should be envisioned from the point of damage of the intravascular compartment—the eGC, which regulates the functions of the endothelium.
Expanding research of the eGC role in the development of endothelial dysfunction may be a subject of new discoveries in the pathogenesis of a large group of diseases, including pregnancy pathology and PE, especially since PE is a classic example of the immune system hyperactivation, manifestation of SIR and development of endothelial dysfunction. Undoubtedly, future studies of the eGC will evoke an absolutely new insight in the development and progression of endothelial dysfunction.
We thank Alexey Komarovsky and Natalia Baranova for their help in the manuscript preparation.
Conflict of interest
The authors report no conflicts of interest.
The study has been carried out within the State Contract (№ 116082210002) “Investigation of diagnostic and prognostic value of molecular-genetic, immunologic, epigenetic factors in pre-eclampsia.”