Hans Vink

The endothelial glycocalyx: composition, functions, and visualization.

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Mechanotransduction and flow across the endothelial glycocalyx

In this inaugural paper, we shall provide an overview of the endothelial surface layer or glycocalyx in several roles: as a transport barrier, as a porous hydrodynamic interface in the motion of red and white cells in microvessels, and as a mechanotransducer of fluid shearing stresses to the actin cortical cytoskeleton of the endothelial cell. These functions will be examined from a new perspective, the quasiperiodic ultrastructural model proposed in Squire et al. [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239–255] for the 3D organization of the endothelial surface layer and its linkage to the submembranous scaffold. We shall show that the core proteins in the bush-like structures comprising the matrix have a flexural rigidity, EI, that is sufficiently stiff to serve as a molecular filter for plasma proteins and as an exquisitely designed transducer of fluid shearing stresses. However, EI is inadequate to prevent the buckling of these protein structures during the intermittent motion of red cells or the penetration of white cell microvilli. In these cellular interactions, the viscous draining resistance of the matrix is essential for preventing adhesive molecular interactions between proteins in the endothelial membrane and circulating cellular components.

The Endothelial Glycocalyx Protects Against Myocardial Edema

Myocardial tissue edema attributable to increased microvascular fluid loss contributes to cardiac dysfunction after myocardial ischemia, cardiopulmonary bypass, hypertension, and sepsis. Recent studies suggest that carbohydrate structures on the luminal surface of microvascular endothelium are essential to prevent tissue edema. We carefully preserved these structures for visualization with electron microscopy, revealing that the rat myocardial capillary endothelial surface is coated with a 0.2- to 0.5-&mgr;m-thick carbohydrate layer and that its degradation instantly results in notable myocardial tissue edema.

Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface

Objective—A thick endothelial glycocalyx provides the endothelial surface with a nonadherent shield. Oxidized LDL (Ox-LDL) degrades the endothelial glycocalyx. We hypothesized that glycocalyx degradation stimulates leukocyte-endothelial cell adhesion, whereas intravascular supplementation with sulfated polysaccharides reconstitutes the endothelial glycocalyx and attenuates Ox-LDL-induced leukocyte-endothelial cell adhesion. Methods and Results—Degradation of the endothelial glycocalyx by local microinjection of heparitinase (10 to 50 U/mL) into mouse cremaster venules dose-dependently increased the number of adherent leukocytes. Systemic administration of Ox-LDL (0.4 mg/100 g body weight) induced 10.1±0.9 adherent leukocytes/100 &mgr;m at 60 minutes. In the venules perfused with 500-kDa dextran sulfate (1 mg/mL), the number of adherent leukocytes at 60 minutes after Ox-LDL bolus application was not influenced (9.2±1.0 leukocytes/100 &mgr;m). However, the venules locally perfused with heparan sulfate (10 mg/mL) or heparin (1 mg/mL) displayed a significantly lower number of adherent leukocytes induced by Ox-LDL: 5.1±0.7 and 5.4±0.9 leukocytes/100 &mgr;m, respectively (P <0.05). Fluorescently labeled heparan sulfate and heparin, but not dextran sulfate, attached to the venule luminal surface after Ox-LDL administration. Conclusions—Endothelial glycocalyx degradation stimulates leukocyte immobilization at the endothelial surface. Circulating heparan sulfate and heparin attach to the venule wall and attenuate Ox-LDL-induced leukocyte immobilization.

The endothelial glycocalyx: a potential barrier between health and vascular disease.

Purpose of review Although cardiovascular prevention has improved substantially, we still face the challenge of finding new targets to reduce the sequelae of atherosclerosis further. In this regard, optimizing the vasculoprotective effects of the vessel wall itself warrants intensive research. In particular, the endothelial glycocalyx, consisting of proteoglycans, glycoproteins and adsorbed plasma proteins, may play an essential role in protecting the vessel wall from atherosclerosis. Recent developments In this review, we will discuss the different vasculoprotective effects exerted by the endothelial glycocalyx, the factors that damage it, and the first preliminary data on the glycocalyx dimension in humans. Whereas most glycocalyx research has traditionally focused on the microvasculature, more recent data have underscored the importance of the glycocalyx in protecting the macrovasculature against pro-atherogenic insults. It has been shown that glycocalyx loss is accompanied by a wide array of unfavourable changes in both small and larger vessels. Pro-atherogenic stimuli increase the shedding of glycocalyx constituents into the circulation, contributing to the progressive loss of the vasculoprotective properties of the vessel wall. Novel techniques have facilitated reproducible measurements of systemic glycocalyx volume in humans. Consistent with experimental data, the volume of the human glycocalyx is also severely perturbed by exposure to atherogenic risk factors. Summary Cumulating evidence suggests that an intact glycocalyx protects the vessel wall, whereas disruption of the glycocalyx upon atherogenic stimuli increases vascular vulnerability for atherogenesis.

Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation

The endothelium holds a pivotal role in cardiovascular health and disease. Assessment of its function was until recently limited to experimental designs due to its location. The advent of novel techniques has facilitated testing on a more detailed basis, with focus on distinct pathways. This review presents available in-vivo and ex-vivo methods for evaluating endothelial function with special focus on more recent ones. The diagnostic modalities covered include assessment of epicardial and microvascular coronary endothelial function, local vasodilation by venous occlusion plethysmography and flow-mediated dilatation, arterial pulse wave analysis and pulse amplitude tonometry, microvascular blood flow by laser Doppler flowmetry, biochemical markers and bioassays, measurement of endothelial-derived microparticles and progenitor cells, and glycocalyx measurements. Insights and practical information on the theoretical basis, methodological aspects, and clinical application in various disease states are discussed. The ability of these methods to detect endothelial dysfunction before overt cardiovascular disease manifests make them attractive clinical tools for prevention and rehabilitation.

Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress

The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of coagulation and leucocyte adhesion, by contributing to the vascular permeability barrier and by mediating shear stress‐induced NO release. In this review, we will focus on the relationship between fluid shear stress and the endothelial glycocalyx. We will address the hypothesis that modulation of glycocalyx synthesis by fluid shear stress may contribute to thinner glycocalyces, and therefore more vulnerable endothelium, at lesion‐prone sites of arterial bifurcations. Finally, we will discuss the effects of known atherogenic stimuli such as hyperglycaemia on whole body glycocalyx volume in humans and its effect on endothelial function.

Damage of the Endothelial Glycocalyx in Dialysis Patients

Damage to the endothelial glycocalyx, which helps maintain vascular homeostasis, heightens the sensitivity of the vasculature to atherogenic stimuli. Patients with renal failure have endothelial dysfunction and increased risk for cardiovascular morbidity and mortality, but the state of the endothelial glycocalyx in these patients is unknown. Here, we used Sidestream Darkfield imaging to detect changes in glycocalyx dimension in dialysis patients and healthy controls from in vivo recordings of the sublingual microcirculation. Dialysis patients had increased perfused boundary region and perfused diameters, consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties. These patients also had higher serum levels of the glycocalyx constituents hyaluronan and syndecan-1 and increased hyaluronidase activity, suggesting the shedding of these components. Loss of residual renal function had no influence on the imaging parameters but did associate with greater shedding of hyaluronan in blood. Furthermore, patients with higher levels of inflammation had more significant damage to the glycocalyx barrier. In conclusion, these data suggest that dialysis patients have an impaired glycocalyx barrier and shed its constituents into blood, likely contributing to the sustained endothelial cell activation observed in ESRD.

Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas

A technique is presented for the 3D visualisation of the coronary arterial tree using an imaging cryomicrotome. After the coronary circulation of the excised heart was filled with a fluorescent plastic, the heart was frozen and mounted in the cryomicrotome. The heart was then sliced serially, with a slice thickness of 40μm, and digital images were taken from each cutting plane of the remaining bulk material using appropriate excitation and emission filters. Using maximum intensity projections over a series of images in the cutting plane and perpendicular plane, the structural organisation of intramural vessels was visualised in the present study. The branching end in the smallest visible vessels, which define tissue areas that are well delineated from each other by 1–2 mm wide bands populated only by vessels less than 40 μm in diameter. The technique presented here allows further quantification in the future of the 3D structure of the coronary arterial tree by image analysis techniques.

Tumor necrosis factor-α inhibition protects against endotoxin-induced endothelial glycocalyx perturbation

OBJECTIVE Inflammatory stimuli profoundly increase the vulnerability of the vessel wall to atherogenesis. The endothelial glycocalyx, a layer of glycosaminoglycans and proteoglycans covering the luminal side of the vasculature, has recently emerged as an orchestrator of vascular homeostasis. In the present study, we investigated whether endotoxin-induced inflammatory reactions lead to a decrease of endothelial glycocalyx thickness in humans and whether tumor necrosis factor-alpha (TNFalpha) plays a role in this process. DESIGN, SUBJECTS AND INTERVENTION Healthy male volunteers received low-dose endotoxin (1ng/kg) intravenously, with (n=8) or without (n=13) pre-treatment with the soluble TNFalpha receptor etanercept. Endothelial glycocalyx thickness and related parameters were determined after endotoxin challenge. RESULTS Endotoxin resulted in a profound reduction in microvascular glycocalyx thickness (from 0.60+/-0.1 to 0.30+/-0.1microm, p<0.01). Concomitantly, plasma levels of the principal glycocalyx constituent hyaluronan (62+/-18 to 85+/-24ng/mL, p<0.05), monocyte activation and coagulation activation increased (F1+2; 0.3+/-0.1 to 2.8+/-1.5nmol/L, p<0.05 and d-dimer; from 0.2+/-0.1 to 0.4+/-0.1mg/L, p<0.05 compared to baseline). Inhibition of TNFalpha by etanercept attenuated loss of microvascular glycocalyx thickness (0.54+/-0.1 to 0.35+/-0.1mum, p<0.05). Changes in hyaluronan (58+/-13 to 46+/-10ng/mL, p<0.05) and coagulation activation were also attenuated (F1+2; 0.3+/-0.1 to 2.1+/-0.9nmol/L and d-dimer; from 0.2+/-0.1 to 0.3+/-0.1mg/L, p<0.05 compared to baseline). CONCLUSIONS These data suggest that inflammatory activity, in part mediated by TNFalpha, leads to perturbation of the endothelial glycocalyx in humans. This may contribute to the vascular vulnerability induced by inflammation.