Endothelial glycocalyx of blood circulation system. I. Detection,components, and structural organization

The luminal surface of a blood vessel accommodates a complex multicomponent system of mainly carbohydrates and proteins called glycocalyx. According to the concept of the double protective layer, glycocalyx is the first protection barrier of the vascular wall. The structure of glycocalyx is determined by a group of proteoglycans, glycoproteins, and glycosaminoglycans. Two groups of molecules are distinguished within the glycocalyx constituents, that is, membrane proteoglycans (syndecans and glypicans bound to endothelial cell membranes) and soluble proteoglycans (perlecan, biglycan, versican, decorin, and mimecan). There are five types of glycosaminoglycan chains; these are heperan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. There is a dynamic equilibrium between the soluble components of glycocalyx and flowing blood, which allows for separation of the endothelial surface layer. Due to its complexity and location at the interface of blood circulation system, glycocalyx is involved in the maintenance of vascular homeostasis. Here, the molecular composition of glycocalyx, properties of its components, biosynthesis, and common structural features are discussed.     

Endothelial glycocalyx as a critical signalling platform integrating the extracellular haemodynamic forces and chemical signalling

The glycocalyx covers the human mammalian cells and plays important roles in stroke, inflammation and atherosclerosis. It has also been shown to be involved in endothelial mechanotransduction of shear stress. Shear stress induces the remodelling of the major component of the glycocalyx including glypican‐1, a cell membrane heparan sulphate proteoglycan. Other factors, such as sphingosine‐1‐phosphate (S1P), protect the glycocalyx against syndecan‐1 ectodomain shedding and induce the synthesis of heparan sulphate. In this study, we reviewed the role of shear stress and S1P in glycocalyx remodelling and revealed that the glycocalyx is a critical signalling platform, integrating the extracellular haemodynamic forces and chemical signalling, such as S1P, for determining the fate of endothelial cells and vascular diseases. This review integrated our current understanding of the structure and function of the glycocalyx and provided new insight into the role of the glycocalyx that might be helpful for investigating the underlying biological mechanisms in certain human diseases, such as atherosclerosis.     

High glucose causes dysfunction of the human glomerular endothelial glycocalyx

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.     

The role of syndecans in disease and wound healing.

Syndecans are a family of transmembrane heparan sulfate proteoglycans widely expressed in both developing and adult tissues. Until recently, their role in pathogenesis was largely unexplored. In this review, we discuss the reported involvement of syndecans in human cancers, infectious diseases, obesity, wound healing and angiogenesis. In some cancers, syndecan expression has been shown to regulate tumor cell function (e.g. proliferation, adhesion, and motility) and serve as a prognostic marker for tumor progression and patient survival. The ectodomains and heparan sulfate glycosaminoglycan chains of syndecans can also act as receptors/co-receptors for some bacterial and viral pathogens, mediating infection. In addition, syndecans bind to obesity-related factors and regulate their signaling, in turn modulating food consumption and weight balance. In vivo animal models of tissue injury and in vitro data also implicate syndecans in processes necessary for wound healing, including fibroblast and endothelial proliferation, cell motility, angiogenesis, and extracellular matrix organization. These new insights into the involvement of syndecans in disease and tissue repair coupled with the emergence of syndecan-specific molecular tools may lead to novel therapies for a variety of human diseases.     

Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys¹³⁹ (6-kDa linker), Lys¹⁰⁷, and Lys¹⁶¹, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.     

The Adaptive Remodeling of Endothelial Glycocalyx in Response to Fluid Shear Stress

The endothelial glycocalyx is vital for mechanotransduction and endothelial barrier integrity. We previously demonstrated the early changes in glycocalyx organization during the initial 30 min of shear exposure. In the present study, we tested the hypothesis that long-term shear stress induces further remodeling of the glycocalyx resulting in a robust layer, and explored the responses of membrane rafts and the actin cytoskeleton. After exposure to shear stress for 24 h, the glycocalyx components heparan sulfate, chondroitin sulfate, glypican-1 and syndecan-1, were enhanced on the apical surface, with nearly uniform spatial distributions close to baseline levels that differed greatly from the 30 min distributions. Heparan sulfate and glypican-1 still clustered near the cell boundaries after 24 h of shear, but caveolin-1/caveolae and actin were enhanced and concentrated across the apical aspects of the cell. Our findings also suggest the GM₁-labelled membrane rafts were associated with caveolae and glypican-1/heparan sulfate and varied in concert with these components. We conclude that remodeling of the glycocalyx to long-term shear stress is associated with the changes in membrane rafts and the actin cytoskeleton. This study reveals a space- and time- dependent reorganization of the glycocalyx that may underlie alterations in mechanotransduction mechanisms over the time course of shear exposure.     

Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns.

The syndecans are a gene family of four transmembrane heparan sulfate proteoglycans that bind, via their HS chains, diverse components of the cellular microenvironment. To evaluate the expression of the individual syndecans, we prepared cDNA probes to compare mRNA levels in various adult mouse tissues and cultured mouse cells representing various epithelial, fibroblastic, endothelial, and neural cell types and B cells at various stages of differentiation. We also prepared antibody probes to assess whether the extracellular domains of the individual syndecans are shed into the conditioned media of cultured cells. Our results show that all cells and tissues studied, except B-stem cells, express at least one syndecan family member; most cells and tissues express multiple syndecans. However, each syndecan family member is expressed selectively in cell-, tissue-, and development-specific patterns. The extracellular domain of all syndecan family members is shed as an intact proteoglycan. Thus, most, if not all, cells acquire a distinctive repertoire of the four syndecan family members as they differentiate, resulting in selective patterns of expression that likely reflect distinct functions.     

Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 microm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.     

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer. Stiffness was measured before and after removal of specific glycocalyx components. The mean stiffness of BLMVEC glycocalyx was found to be ~7.5 kT/nm(2) (or ~31 pN/nm). Enzymatic digestion of the glycocalyx with pronase or hyaluronan with hyaluronidase increased the mean effective stiffness of the glycocalyx; however, the increase of the mean stiffness on digestion of heparan sulfate with heparinase III was not significant. The results imply that hyaluronan chains act as a cushioning layer to distribute applied forces to the glycocalyx structure. Effective stiffness was also measured for the glycocalyx exposed to 0.1%, 1.0%, and 4.0% BSA; glycocalyx compliance increased at two extreme BSA concentrations. The RICM images indicated that glycocalyx thickness increases with BSA concentrations. Results demonstrate that RICM is sensitive to detect the subtle changes of glycocalyx compliance at the fluid-fiber interface.     

The glycocalyx is present as soon as blood flow is initiated and is required for normal vascular development

The glycocalyx, and the thicker endothelial surface layer (ESL), are necessary both for endothelial barrier function and for sensing mechanical forces in the adult. The goal of this study is to use a combination of imaging techniques to establish when the glycocalyx and endothelial surface layer form during embryonic development and to determine the biological significance of the glycocalyx layer during vascular development in quail embryos. Using transmission electron microscopy, we show that the glycocalyx layer is present as soon as blood flow starts (14 somites). The early endothelial glycocalyx (14 somites) lacks the distinct hair-like morphology that is present later in development (17 and 25 somites). The average thickness does not change significantly (14 somites, 182nm±33nm; 17 somites, 218±30nm; 25 somites, 212±32nm). The trapping of circulating fluorescent albumin was used to evaluate the development of the ESL. Trapped fluorescent albumin was first observed at 25 somites. In order to assess a functional role for the glycocalyx during development, we selectively degraded luminal glycosaminoglycans. Degradation of hyaluronan compromised endothelial barrier function and prevented vascular remodeling. Degradation of heparan sulfate down regulated the expression of shear-sensitive genes but does not inhibit vascular remodeling. Our findings show that the glycocalyx layer is present as soon as blood flow starts (14 somites). Selective degradations of major glycocalyx components were shown to inhibit normal vascular development, examined through morphology, vascular barrier function, and gene expression.     

Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy

The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ～18 μm) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components. The modulus-indentation depth profiles showed the cells becoming progressively stiffer with increased indentation. Three different enzymes were used: heparinases III and I and hyaluronidase. The main effects of heparinase III and hyaluronidase enzymes were that the elastic modulus in the cell junction regions increased more rapidly with the indentation than in BLMVEC controls, and that the effective thickness of glycocalyx was reduced. Cytochalasin D abolished the modulus increase with the indentation. The confocal profiling of heparan sulfate and hyaluronan with atomic force microscopy indentation data demonstrated marked heterogeneity of the glycocalyx composition between cell junctions and nuclear regions.     

Syndecans as receptors and organizers of the extracellular matrix

Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as “co-receptors”. However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.     

Molecular and cellular mechanisms of syndecans in tissue injury and inflammation

The syndecan family of heparan sulfate proteoglycans is expressed on the surface of all adherent cells. Syndecans interact with a wide variety of molecules, including growth factors, cytokines, proteinases, adhesion receptors and extracellular matrix components, through their heparan sulfate chains. Recent studies indicate that these interactions not only regulate key events in development and homeostasis, but also key mechanisms of the host inflammatory response. This review will focus on the molecular and cellular aspects of how syndecans modulate tissue injury and inflammation, and how syndecans affect the outcome of inflammatory diseases in vivo.     

The Circulating Glycosaminoglycan Signature of Respiratory Failure in Critically Ill Adults

Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.     

The role of endothelial glycocalyx components in mechanotransduction of fluid shear stress

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.     

Syndecans-2 and -4; close cousins, but not identical twins

The vertebrate syndecans, which make up a four-member family of small type I transmembrane heparan sulfate proteoglycans, constitute evolutionarily conserved family proteins. In particular, sequences in the transmembrane and cytoplasmic domains are a unifying feature within the family. However, the extracellular domain sequences are molecule-specific, implying that different syndecans have evolved to carry out similar, but non-identical, functions. While all four syndecans have been implicated in regulation of the cytoskeleton, their roles are clearly complex. Recent developments indicate that the closely related syndecan-2 and -4 have separable functions, though both bind a number of ligands through their heparan sulfate chains. The specification of these activities is probably core protein related, but is it due to a distinct expression pattern or molecule-specific regulatory mechanisms? Although there is not yet enough data to provide unambiguous answers, here we shall review the known functions and regulatory mechanisms of syndecan-2 and -4.     

Heparan sulfates mediate pressure-induced increase in lung endothelial hydraulic conductivity via nitric oxide/reactive oxygen species

We investigated the nonlinear dynamics of the pressure vs. hydraulic conductivity (L(p)) relationship in lung microvascular endothelial cells and demonstrate that heparan sulfates, an important component of the endothelial glycocalyx, participate in pressure-sensitive mechanotransduction that results in barrier dysfunction. The pressure vs. L(p) relationship was complex, possessing both time- and pressure-dependent components. Pretreatment of lung capillary endothelial cells with heparanase III completely abolished the pressure-induced increase in L(p). This extends our (7) previous observation regarding heparan sulfates as mechanotransducers for shear stress. Inhibition of nitric oxide (NO) synthase with L-NAME (N(G)-nitro-L-arginine methyl ester HCl) and intracellular scavenging of reactive oxygen species (ROS) by TBAP [tetrakis-(4-benzoic acid) porphorin] significantly attenuated the pressure-induced L(p) response. Intracellular NO/ROS were visualized using the fluorescent dye, 2'7'-dichlorofluorescein diacetate (DCFA), and cells demonstrated a pressure-induced increase in intracellular fluorescence. Heparanase pretreatment significantly reduced the pressure-induced increase in intracellular fluorescence, suggesting that cell-surface heparan sulfates directly participate in mechanotransduction that results in NO/ROS production and increased permeability. This is the first report to demonstrate a role for heparan sulfates in pressure-mediated mechanotransduction and barrier regulation. These observations may have important clinical implications during conditions where pulmonary microvascular pressure is elevated.     

Syndecans play dual roles as cell adhesion receptors and docking receptors

Syndecan are a family of cell surface heparan sulfate proteoglycans that act as cell surface receptors. Most cell surface receptors have a limited number and type of ligand interactions, responding only to the binding of (a) specific ligand(s). In contrast, syndecans can interact with various numbers and types of ligands, and thus play more diverse roles than others. Various syndecan functions have not yet been fully classified and categorized, but we herein review previous studies suggesting that syndecans play dual function as cell surface receptors by acting as both adhesion receptors and docking receptors. Through this dual regulatory function, syndecans are capable of regulating both intra- and extracellular activities, potentially altering a variety of cell behaviors.