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Objective Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function, due to findings that GCX in vitro may be substantially thinner than in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution transmission electron microscopy (RF/FS-TEM), taking advantage of high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS. Methods and Results Bovine aortic and rat fat pad endothelial cells (BAEC and RFPEC) were subjected to conventional- or RF/FS-TEM. Conventionally preserved BAEC GCX was ~0.040 μm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ~11 μm and RFPEC GCX of ~5 μm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several microns thick and is comprised of extensive and well integrated heparan sulfate, hyaluronic acid, and protein layers. Conclusions New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.

Mammalian epithelial cells are coated with a multifunctional surface glycocalyx (GCX). On vascular endothelial cells (EC), intact GCX is atheroprotective. It is degraded in many vascular diseases. GCX heparan sulfate (HS) is essential for healthy flow-induced EC nitric oxide (NO) release, elongation, and alignment. The HS core protein mechanisms involved in these processes are unknown. We hypothesized that the glypican-1 (GPC1) HS core protein mediates flow-induced EC NO synthase (eNOS) activation because GPC1 is anchored to caveolae where eNOS resides. We also hyphothesized that the HS core protein syndecan-1 (SDC1) mediates flow-induced EC elongation and alignment because SDC1 is linked to the cytoskeleton which impacts cell shape. We tested our hypotheses by exposing EC monolayers treated with HS degrading heparinase III (HepIII), and monolayers with RNA-silenced GPC1, or SDC1, to 3 to 24 hours of physiological shear stress. Shear-conditioned EC with intact GCX exhibited characteristic eNOS activation in short-term flow conditions. After long-term exposure, EC with intact GCX were elongated and aligned in the direction of flow. HS removal and GPC1 inhibition, not SDC1 reduction, blocked shear-induced eNOS activation. EC remodeling in response to flow was attenuated by HS degradation and in the absence of SDC1, but preserved with GPC1 knockdown. These findings clearly demonstrate that HS is involved in both centralized and decentralized GCX-mediated mechanotransduction mechanisms, with GPC1 acting as a centralized mechanotransmission agent and SDC1 functioning in decentralized mechanotransmission. This foundational work demonstrates how EC can transform fluid shear forces into diverse biomolecular and biomechanical responses.

RationaleIt is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.ObjectiveTo evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.Methods and ResultsUsing confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.ConclusionAll GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.

Background and aims Previous experiments suggest that both increased endothelial cell apoptosis and endothelial surface glycocalyx shedding could play a role in the endothelial dysfunction and inflammation of athero-prone regions of the vasculature. We sought to elucidate the possibly synergistic mechanisms by which endothelial cell apoptosis and glycocalyx shedding promote atherogenesis. Methods 4- to 6-week old male C57Bl/6 apolipoprotein E knockout (ApoE−/−) mice were fed a Western diet for 10 weeks and developed plaques in their brachiocephalic arteries. Results Glycocalyx coverage and thickness were significantly reduced over the plaque region compared to the non-plaque region (coverage plaque: 71±23%, non-plaque: 97±3%, p= 0.02; thickness plaque: 0.85±0.15 μm, non-plaque: 1.2±0.21 μm, p= 0.006). Values in the non-plaque region were not different from those found in wild type mice fed a normal diet (coverage WT: 92±3%, p= 0.7 vs. non-plaque ApoE−/−, thickness WT: 1.1±0.06 μm, p= 0.2 vs. non-plaque ApoE−/−). Endothelial cell apoptosis was significantly increased in ApoE−/− mice compared to wild type mice (ApoE−/− :64.3±33.0, WT: 1.1±0.5 TUNEL-pos/cm, p= 2×10−7). The number of apoptotic endothelial cells per unit length was 2 times higher in the plaque region than in the non-plaque region of the same vessel (p= 3×10−5). Increased expression of matrix metalloproteinase 9 co-localized with glycocalyx shedding and plaque buildup. Conclusions Our results suggest that, in concert with endothelial apoptosis that increases lipid permeability, glycocalyx shedding initiated by inflammation facilitates monocyte adhesion and macrophage infiltration that promote lipid retention and the development of atherosclerotic plaques.

The apical surface of endothelial cells (ECs) is decorated with various membrane-bound macromolecules that constitute the glycocalyx (GCX). As the most apical structure on the EC, the GCX senses the force (shear stress) of flowing blood and transmits the force through the cytoskeleton to sites where transduction of force to biochemical response (mechanotransduction) may occur. In this presentation, we review the structure of the GCX and experiments that demonstrate its role in mechanotransduction and vascular remodeling. Experiments with enzymes that degrade specific glycosaminoglycan components showed that the GCX mediates the shear-induced production of nitric oxide, a central process in cardiovascular control, whereas the same enzyme treatments do not affect shear-induced production of prostacyclin, another hallmark of EC mechanotransduction. These experiments reinforce the concept of distributed sites of mechanotransduction in EC. The characteristic remodeling of the EC cytoskeleton and intercellular junctions in response to shear stress are dependent on the GCX as well, and we present the experiments and theories that support the role of the GCX in these processes. The GCX is a fascinating structure whose role in EC function is only beginning to be appreciated.

Purpose of ReviewThe cell surface-attached extracellular glycocalyx (GCX) layer is a major contributor to endothelial cell (EC) function and EC-dependent vascular health and is a first line of defense against vascular diseases including atherosclerosis. Here, we highlight our findings regarding three GCX-dependent EC functions, which are altered when GCX is shed and in atherosclerosis. We discuss why the GCX is a viable option for the prevention and treatment of atherosclerosis.Recent FindingsGCX regulated EC activities such as barrier and filtration function, active cell-to-cell communication, and vascular tone mediation contribute to function of the entire vascular wall. Atheroprone vessel regions, including bifurcation sites, exhibit breakdown in GCX. This GCX degradation allows increased lipid flux and thereby promotes lipid deposition in the vessel walls, a hallmark of atherosclerosis. GCX degradation also alters EC-to-EC communication while increasing EC-to-inflammatory cell interactions that enable inflammatory cells to migrate into the vessel wall. Inflammatory macrophages and foam cells, to be specific, appear in early stages of atherosclerosis. Furthermore, GCX degradation deregulates vascular tone, by causing ECs to reduce their expression of endothelial nitric oxide synthase (eNOS) which produces the vasodilator, nitric oxide. Loss of vasodilation supports vasoconstriction, which promotes the progression of atherosclerosis.SummaryCommon medicinal atherosclerosis therapies include lipid lowering and anti-platelet therapies. None of these treatments specifically target the endothelial GCX, although the GCX is at the front-line in atherosclerosis combat. This review demonstrates the viability of targeting the GCX therapeutically, to support proper EC functionality and prevent and/or treat atherosclerosis.

The endothelial glycocalyx plays important roles in mechanotransduction. We recently investigated the distribution and interaction of glycocalyx components on statically cultured endothelial cells. In the present study, we further explored the unknown organization of the glycocalyx during early exposure (first 30 min) to shear stress and tested the hypothesis that proteoglycans with glycosaminoglycans, which are localized in different lipid microdomains, respond distinctly to shear stress. During the initial 30 min of exposure to shear stress, the very early responses of the glycocalyx and membrane rafts were detected using confocal microscopy. We observed that heparan sulfate (HS) and glypican-1 clustered in the cell junctions. In contrast, chondroitin sulfate (CS), bound albumin, and syndecan-1 did not move. The caveolae marker caveolin-1 did not move, indicating that caveolae are anchored sufficiently to resist shear stress during the 30 min of exposure. Shear stress induced significant changes in the distribution of ganglioside GM1 (a marker for membrane rafts labeled with cholera toxin B subunit). These data suggest that fluid shear stress induced the cell junctional clustering of lipid rafts with their anchored glypican-1 and associated HS. In contrast, the mobility of CS, transmembrane bound syndecan-1, and caveolae were constrained during exposure to shear stress. This study illuminates the role of changes in glycocalyx organization that underlie mechanisms of mechanotransduction.