Eno E. Ebong

Imaging the Endothelial Glycocalyx In Vitro by Rapid Freezing/Freeze Substitution Transmission Electron Microscopy

Objective—Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function because of findings that GCX in vitro may be substantially thinner than GCX 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 (RF/FS) transmission electron microscopy (TEM), taking advantage of the 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 EC (BAEC) and rat fat pad EC were subjected to conventional or RF/FS-TEM. Conventionally preserved BAEC GCX was ≈0.040 &mgr;m in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ≈11 &mgr;m and rat fat pad EC GCX of ≈5 &mgr;m. 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 micrometers thick and is composed of extensive and well-integrated heparan sulfate, hyaluronic acid, and protein layers. Conclusion—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.

The Endothelial Glycocalyx: A Mechano-Sensor and -Transducer

A complex extracellular network of proteoglycans communicates mechanical stress to endothelial cells. 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.

The Structural Stability of the Endothelial Glycocalyx after Enzymatic Removal of Glycosaminoglycans

Rationale It 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. Objective To 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 Results Using 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. Conclusion All 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.

Fluid shear stress induces the clustering of heparan sulfate via mobility of glypican-1 in lipid rafts

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.

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1 , PI3K , FAK, and p38.

Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important antiatherogenic prostanoid and vasodilator produced in endothelial cells through the action of the cyclooxygenase (COX) isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5 h to examine shear stress-induced induction of COX-2/PGI2 Our results demonstrate the relationship between the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and the intracellular mechanoresponsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38 in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1-integrin, upregulation of COX-2, and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) substantially inhibited FSS activation of α5β1-integrin, upregulation of COX-2 gene and protein expression, and release of PGI2 in BAECs. Inhibition of integrin-associated FAK (PF573228) and MAPK p38 (SB203580) also inhibited the shear-induced upregulation of COX-2. Finally, a PECAM-1-/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared with wild-type mice, as well as complete inhibition of acute flow-induced PGI2 release compared with wild-type animals.NEW & NOTEWORTHY In this study we determined the major mechanotransduction pathway by which blood flow-driven shear stress activates cyclooxygenase-2 (COX-2) and prostaglandin I2 (PGI2) release in endothelial cells. Our work has demonstrated for the first time that COX-2/PGI2 mechanotransduction is mediated by the mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1).

Specificity in the participation of connexin proteins in flow-induced endothelial gap junction communication

Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx—Cx40 and Cx43—to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.

Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting

Cardiovascular diseases are facilitated by endothelial cell (EC) dysfunction and coincide with EC glycocalyx coat shedding. These diseases may be prevented by delivering medications to affected vascular regions using circulating nanoparticle (NP) drug carriers. The objective of the present study was to observe how the delivery of 10 nm polyethylene glycol-coated gold NPs (PEG-AuNP) to ECs is impacted by glycocalyx structure on the EC surface. Rat fat pad endothelial cells were chosen for their robust glycocalyx, verified by fluorescent immunolabeling of adsorbed albumin and integrated heparan sulfate (HS) chains. Confocal fluorescent imaging revealed a ~3 µm thick glycocalyx layer, covering 75% of the ECs and containing abundant HS. This healthy glycocalyx hindered the uptake of PEG-AuNP as expected because glycocalyx pores are typically 7 nm wide. Additional glycocalyx models tested included: a collapsed glycocalyx obtained by culturing cells in reduced protein media, a degraded glycocalyx obtained by applying heparinase III enzyme to specifically cleave HS, and a recovered glycocalyx obtained by supplementing exogenous HS into the media after enzyme degradation. The collapsed glycocalyx waŝ2 µm thick with unchanged EC coverage and sustained HS content. The degraded glycocalyx showed similar changes in EC thickness and coverage but its HS thickness was reduced to 0.7 µm and spanned only 10% of the original EC surface. Both dysfunctional models retained six- to sevenfold more PEG-AuNP compared to the healthy glycocalyx. The collapsed glycocalyx permitted NPs to cross the glycocalyx into intracellular spaces, whereas the degraded glycocalyx trapped the PEG-AuNP within the glycocalyx. The repaired glycocalyx model partially restored HS thickness to 1.2 µm and 44% coverage of the ECs, but it was able to reverse the NP uptake back to baseline levels. In summary, this study showed that the glycocalyx structure is critical for NP uptake by ECs and may serve as a passive pathway for delivering NPs to dysfunctional ECs.

Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target

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.

Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication

Vasculoprotective endothelium glycocalyx (GCX) shedding plays a critical role in vascular disease. Previous work demonstrated that GCX degradation disrupts endothelial cell (EC) gap junction connexin (Cx) proteins, likely blocking interendothelial molecular transport that maintains EC and vascular tissue homeostasis to resist disease. Here, we focused on GCX regeneration and tested the hypothesis that vasculoprotective EC function can be stimulated via replacement of GCX when it is shed. We used EC with [i] intact heparan sulfate (HS), the most abundant GCX component; [ii] degraded HS; or [iii] HS that was restored after enzyme degradation, by cellular self-recovery or artificially. Artificial HS restoration was achieved via treatment with exogenous HS, with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P). In these cells we immunocytochemically examined expression of Cx isotype 43 (Cx43) at EC borders and characterized Cx-containing gap junction activity by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Cellular self-recovery of HS restored baseline levels of Cx43 and dye transfer. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication.

Mechanisms of flow-dependent endothelial COX-2 and PGI 2 expression

The luminal surfaces of blood vessels are covered with several membrane-bound glycosaminoglycans (GAGs) that constitute the endothelial glycocalyx layer (EGL) of endothelial cells(ECs). ECs, like all anchorage-dependent cells, adhere to the extracellular matrix at focal adhesions. By virtue of their cellular location and signaling abilities, it has been suggested that focal adhesions and the EGL may function as mechanoreceptors, by sensing and facilitating the transfer of “outside in”, mechanical signals across the plasma membrane. However, the mechanism by which ECs detect fluid shear stress (FSS) and transduce signals across the membrane to activate signaling pathways such as the induction of COX-2 and release of PGI2 is still poorly understood. To directly test the role that focal adhesions (apical and basal) and the EGL play in mediating the “outside in”, COX-2 expression and PGI2 mechanotransduction, we first limited the formation of focal adhesions by culturing ECs on fibronectin in the presence of soluble fibronectin fragments H-Gly-Arg-Gly-Asp-Ser-Pro-OH (GRGDSP) peptide, which competitively disrupts the fibronectin-integrin interactions, and selectively degraded the glycocalyx component heparan sulfate (HS) using the EGL degrading enzyme Heparanase III (HepIII). ECs were then subjected to fluid shear stress and COX-2 expression and PGI2 release measured. Surprisingly, the expression and release of both COX-2 and PGI2 in response to FSS were not significantly decreased in ECs treated with either GRGDSP or EGL degrading enzyme HepIII. These results suggest that the “outside in” pathway is not the major mechanotransduction route by which shear stress activates COX-2 and release of PGI2 in ECs.