Glycocalyx Thickness Research Articles

Impaired glycocalyx barrier properties contribute to enhanced intimal low-density lipoprotein accumulation at the carotid artery bifurcation in mice

The glycocalyx contributes to the barrier properties of vascular endothelium, and recently, we reported using electron microscopy that glycocalyx is diminished at lesion prone sites in arterial bifurcations in mice. In the present study, we examined using confocal microscopy the dimension and composition of the endothelial glycocalyx at low- and high-risk atherogenic regions within the common carotid (common) and internal carotid branch (sinus) of C57BL/6J mice and compared dimensional variations with its ability to limit transendothelial leakage of low-density lipoprotein (LDL). Confocal laser scanning microscopy of arterial surfaces stained for heparan sulfate and hyaluronan revealed thinner glycocalyces at the sinus region (2.2 +/- 0.7 and 2.3 +/- 0.7 microm, respectively; P < 0.05) than the glycocalyx thickness at the common region (4.3 +/- 1.6 and 4.3 +/- 1.6 microm, respectively). This thinner glycocalyx was associated with impaired LDL retention by the glycocalyx resulting in a two to three times increase in intimal accumulation of LDL 15 min after i.v. bolus administration: 10.8 +/- 5.6 vs. 4.0 +/- 1.9 x 10,000 a.u. (sinus vs. common, P < 0.05). These results indicate that impaired glycocalyx barrier properties may contribute to transendothelial leakage of atherogenic LDL at lesion prone arterial sites.

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-α (TNFα) plays a role in this process. Design, subjects and intervention Healthy male volunteers received low-dose endotoxin (1 ng/kg) intravenously, with ( n = 8) or without ( n = 13) pre-treatment with the soluble TNFα 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 ± 0.1 μm, p < 0.01). Concomitantly, plasma levels of the principal glycocalyx constituent hyaluronan (62 ± 18 to 85 ± 24 ng/mL, p < 0.05), monocyte activation and coagulation activation increased (F1 + 2; 0.3 ± 0.1 to 2.8 ± 1.5 nmol/L, p < 0.05 and d-dimer; from 0.2 ± 0.1 to 0.4 ± 0.1 mg/L, p < 0.05 compared to baseline). Inhibition of TNFα by etanercept attenuated loss of microvascular glycocalyx thickness (0.54 ± 0.1 to 0.35 ± 0.1 μm, p < 0.05). Changes in hyaluronan (58 ± 13 to 46 ± 10 ng/mL, p < 0.05) and coagulation activation were also attenuated (F1 + 2; 0.3 ± 0.1 to 2.1 ± 0.9 nmol/L and d-dimer; from 0.2 ± 0.1 to 0.3 ± 0.1 mg/L, p < 0.05 compared to baseline). Conclusions These data suggest that inflammatory activity, in part mediated by TNFα, leads to perturbation of the endothelial glycocalyx in humans. This may contribute to the vascular vulnerability induced by inflammation.

The recovery time course of the endothelial glycocalyx after enzymatic and cytokine degradation.

The glycocalyx surface layer on vascular endothelial cells is thought to be a dynamic extracellular matrix, which exhibits changes in thickness and composition in response to local conditions. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. While reconstitution experiments have been performed, the endogenous recovery time of the glycocalyx after degradation has not been addressed. Endothelium with a partially or completely degraded glycocalyx is at risk for increased exposure to blood‐borne free radicals, which, under chronic conditions, could in turn contribute to cardiovascular disease. It is therefore important to ascertain the time course of endogenous recovery of the glycocalyx after degradation. To track glycocalyx recovery, we use micro‐particle image velocimetry in cremaster‐muscle venules to estimate the hydrodynamically relevant thickness of the glycocalyx in mice that have previously been treated with an enzyme (through systemic tail‐vein injection) or an inflammatory cytokine (through scrotal injection). Mice are injected with either hyaluronidase, heparinase III, or TNF‐ α and the glycocalyx thickness is estimated after 1, 3, 5, and 7 days. Preliminary data indicate that it takes over five days for the endothelial glycocalyx to restore itself to its native dimensions of ~ 0.5 μ m. Supported by NIH grant R01 HL76499.

Perfusate composition is a determinant of the hydrodynamically relevant endothelial glycocalyx thickness in vivo.

The endothelial‐cell glycocalyx surface layer is a highly hydrated structure that is constantly bathed in the perfusing fluid, typically blood in vivo. It is proposed that there is a constant exchange of molecules between the blood and the endothelial glycocalyx that is essential to the integrity of the layer. To test the effect of different prefusate compositions on the endothelial glycocalyx in vivo, a micromanipulator is used to introduce a micropipette into the lumen of one of the primary feed arterioles in a mouse cremaster‐muscle preparation. The micropipette then steadily perfuses the microvascular network fed by that arteriole with either native plasma, cell‐culture media containing 20% serum, or saline. Micro‐particle image velocimetry data is then collected from downstream microvessels. Velocity profiles extracted from the μ‐PIV data are then used to estimate the effective hydrodynamically relevant thickness of the glycocalyx. Preliminary data indicate that vessels perfused with saline have no detectable hydrodynamically relevant layer. Supported by NIH grant R01 HL76499.

Characterization of the hepatic endothelial glycocalyx of sialidase deficient mice in response to TNFα

Evidence exists that components of the venular glycocalyx are shed and sialic acid is cleaved during inflammation. The objective of this study was to examine the role of sialidase in altering the hepatic glycocalyx. Female C57Bl/6 (WT) or sialidase deficient (B6Sm−/−) mice were injected with 500 ng of TNFα or saline and the hepatic microcirculation examined by intravital confocal microscopy at 4h. Sugars present on the vasculature were detected by IV injection of FITC‐labeled Bandeiraea simplicifolia (BS‐1) (detects α‐N‐acetylgalactosamine and α‐galactose residues) or Texas‐Red‐labeled Lycopersicon esculentum (TL) (detects N‐acetylglucosamine residues). Both lectins labeled the sinusoidal endothelium with no significant difference between the groups in the mean fluorescence intensity of the BS‐1 or TLlabeled glycocalyx. However, the saline treated WT mice had a thicker glycocalyx as compared to the saline treated B6Sm−/− mice (876 ±37 nm and 777 ± 18 nm, respectively) and TNFα stimulation resulted in a decrease of the glycocalyx thickness (772 ± 45 nm and 658 ± 23 nm, respectively). Our data supports the idea that components of the glycocalyx are shed during inflammation resulting in loss of glycocalyx height. Surprisingly, these sugars were expressed primarily on the sinusoids rather than venules and in contrast to other vascular beds, were not removed during TNFα‐induced hepatic inflammation.

Measuring endothelial glycocalyx dimensions in humans: a potential novel tool to monitor vascular vulnerability

The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as a measure of glycocalyx thickness, and the transient widening of the erythrocyte column on glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy men using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r = 0.63). Measurements in humans were reproducible (0.58 +/- 0.16 and 0.53 +/- 0.15 microm, coefficient of variance 15 +/- 5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r = 0.45), fasting plasma glucose (r = 0.43), and high-density lipoprotein-cholesterol (r = 0.40) and correlated negatively with low-density lipoprotein-cholesterol (r = -0.41) as well as body mass index (r = -0.45) (all P < 0.05). In conclusion, the dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.

Shedding of the Endothelial Glycocalyx in Patients Undergoing Major Vascular Surgery With Global and Regional Ischemia

The astonishing thickness of the endothelial glycocalyx, which rivals that of endothelial cells in the microvasculature, was disclosed in the last 15 years. As already demonstrated, this structure plays a key role in the regulation of inflammation and vascular permeability. Two components of the glycocalyx, syndecan-1 and heparan sulfate, were measured in arterial blood of 18 patients undergoing surgery of the ascending aorta with cardiopulmonary bypass (n=12 with and n=6 without deep hypothermic circulatory arrest) and of 14 patients undergoing surgery for infrarenal aortic aneurysm. Basal values of syndecan-1 (1.2 microg/dL) and heparan sulfate (590 microg/dL) of patients were similar to those of control subjects. Anesthesia and initiation of surgery caused no changes. Global ischemia with circulatory arrest (n=12) was followed by transient 42- and 10-fold increases in syndecan-1 and heparan sulfate, respectively, during early reperfusion (0 to 15 minutes). After regional ischemia of heart and lungs (cardiopulmonary bypass; n=6), syndecan-1 increased 65-fold, and heparan sulfate increased 19-fold. Infrarenal ischemia was followed by 15- and 3-fold increases, respectively (n=14). The early postischemic rises were positively correlated (r=0.76, P<0.001). Plasma concentrations of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 did not change. Circulating polymorphonuclear granulocytes and the level of postischemic heparan sulfate corresponded negatively. Immunohistochemical imaging and immunoassay of isolated hearts (guinea pig) substantiated syndecan-1 and heparan sulfate as components of the endothelial glycocalyx released into the coronary venous effluent. Electron microscopy revealed shedding of the glycocalyx after ischemia/reperfusion. This study provides the first evidence in humans for shedding of the endothelial glycocalyx during ischemia/reperfusion procedures.

Using micro‐particle image velocimetry to interrogate the endothelial‐cell glycocalyx in vitro after hyaluronin and hyaluronidase treatments

Since the glycocalyx surface layer on vascular endothelial cells is likely to have important implications for endothelial-cell function, it is necessary to determine if a physiologically typical layer can be grown and maintained on cultured endothelial cells and what the precise conditions are under which this does and does not occur. To make this determination, we use micro-particle image velocimetry (μ-PIV) in cylindrical collagen tubes (~120 μm diameter), which are steadily perfused with media and lined on their luminal surface with a confluent monolayer of HUVECs. Velocity profiles extracted from the μ-PIV data were used to estimate the effective hydrodynamic thickness of glycocalyx. Using this method we have determined that no hydrodynamically relevant layer exists under standard culture conditions in vitro, while this same approach estimates the hydrodynamically relevant layer thickness to be ~0.5 μm in post-capillary venules in vivo. However, our preliminary μ-PIV analysis has shown that a pharmacological dose of hyaluronan and chrondroitin sulfate added to the culture media can result in a hydrodynamically relevant layer on the surface of the HUVECs that is ~0.5 μm thick. Supported by NIH grant R01 HL76499.

Using fluorescent micro‐particle image velocimetry to interrogate the surface glycocalyx on cultured endothelial cells in collagen microchannels

Numerous studies of endothelial cells in culture have been undertaken that are intended to elucidate mechanisms of endothelial-cell function spanning a broad range of fields in cardiovascular physiology. Since the surface glycocalyx on vascular endothelial cells is likely to have important implications for endothelial-cell function, it is necessary to determine if a physiologically typical surface glycocalyx can be grown and maintained on cultured endothelial cells and required conditions are. To make this determination, we use fluorescent micro- particle image velocimetry (μ-PIV) in cylindrical collagen lined on their luminal surface with a confluent monolayer of human umbilical-cord vascular endothelial cells (HUVECs). From μ-PIV data of the velocity and positions of microspheres in an optical section through the median plane of the tube, a velocity profile of the local microfluidics in the vicinity of the HUVEC luminal surface is generated. This velocity profile was used to estimate the effective hydrodynamic thickness of glycocalyx. By modulating the composition of the perfusate solution, the μ-PIV data can be used to assess whether molecular adsorption from the perfusate is an important determinant of the hydrodynamic properties of the glycocalyx. Results will be shown for a variety of perfusate compositions before and after treatments to degrade the glycocalyx. Supported by NIH grants R01 HL76499.

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.

Effects of aging and HIF‐1α deficiency on permeability of hippocampal vessels

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.

Estimation of viscosity profiles using velocimetry data from parallel flows of linearly viscous fluids: application to microvascular haemodynamics

An approach is presented that uses velocimetry data to estimate accurately the spatial distribution of viscosity in steady laminar parallel flows of incompressible linearly viscous fluids. The approach is generally applicable to Newtonian fluids with spatially varying viscosity or to particle-suspension flows where a non-uniform distribution of the particles contributes to spatial variations in the local effective viscosity of the suspension. Emphasis is placed on the application of these methods to steady axisymmetric blood flow in cylindrical glass capillary tubes and microvessels. In this context, the spatial variations in viscosity over the vessel cross-section are predicted where it is assumed that the rheological properties associated with a heterogeneous red blood cell suspension can be well approximated by a continuous generalized linearly viscous fluid having a spatially non-uniform viscosity. For such a fluid, an expression for the viscosity profile over the vessel cross-section is derived that satisfies the conservation principles of mass and momentum and depends upon the a priori determined velocity distribution, which is extracted from fluorescent micro-particle image velocimetry data obtained from microvessels in vivo. These profiles provide useful information about dynamic, kinematic and rheological properties of the flow that include expressions for the axial pressure-gradient component, the local shear stress distribution, and the relative apparent viscosity. In microvessels, the effect of the glycocalyx surface layer on the vessel wall is also accounted for in the analysis by modelling the layer as a uniformly thick porous medium. Velocimetry data are presented from in vivo measurements made in venules after the application of a light-dye treatment to degrade the glycocalyx. Results reveal that these methods are sufficiently sensitive to detect a reduction in glycocalyx thickness of –70% when compared with results from a separately published data set obtained from venules having an intact glycocalyx.

Observation and characterisation of the glycocalyx of viable human endothelial cells using confocal laser scanning microscopy

This paper describes the use of confocal laser scanning microscopy (CLSM) to observe and characterise the fully hydrated glycocalyx of human umbilical vein endothelial cells (HUVECs). Viable HUVECs in primary culture were studied at room temperature in HEPES-buffered, phenol red- and serum-free CS-C cell culture medium. A fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC) (2 μg ml−1, 30 min) was used to detect N-acetylneuraminic (sialic) acid, which is a significant component of the endothelial glycocalyx. Single confocal sections, less than 1.3 μm thick, were collected at intervals of 0.5 μm, scanning through the entire z-axis of a series of cells. Cell-surface associated staining was observed, which enabled the glycocalyx thickness to be deduced as 2.5 ± 0.5 μm. This dimension is significantly greater than that measured by electron microscopy, for glutaraldehyde-fixed cells (0.10 ± 0.04 μm). The specificity of WGA-FITC staining was demonstrated by treatments with several enzymes, known to degrade glycocalyx (heparatinase, chondroitinase, hyaluronidase and neuraminidase), of which neuraminidase (1 U ml−1, 30–60 min) was the most effective, removing up to 78 ± 2% of WGA-FITC binding to HUVECs. Cell viability was assessed simultaneously with ethidium homodimer-1 staining and confirmed by standard colorimetric 3-[4,5]dimethylthiazol-2,5diphenyltetrazolium bromide (MTT) test. CLSM thus provides a useful approach for in situ visualisation and characterisation of the endothelial glycocalyx in viable preparations, revealing a thickness that is an order of magnitude greater than found in ex situ measurements on fixed cells.

Cell-Cell Affinity of Senescent Human Erythrocytes

During their 120-day life span, human red blood cells (RBC) undergo several physicochemical changes, including an increased tendency to aggregate in plasma or polymer solutions. This study was designed to examine potential associations between age-related differences in RBC mobility, aggregation, and membrane glycocalyx properties for cells suspended in buffer and in 3 g/dl solutions of 70.3 kDa dextran. A recent model for depletion-mediated RBC aggregation was employed to calculate the changes of glycocalyx properties that were consistent with experimental electrophoretic mobility (EPM) and aggregation data. Young and old cells were obtained by density separation, after which aggregation and EPM were determined versus ionic strength; old cells exhibited a two- to threefold greater aggregation in dextran. EPM of old cells was identical to young cells in polymer-free media yet was 4% greater in dextran. The greater EPM for old RBC indicates a larger polymer depletion layer, which could be explained either by a 10–15% decrease of their glycocalyx thickness or a similar percentage decrease of polymer penetration into their glycocalyx. The larger depletion layer leads to markedly elevated cell-cell affinities for old cells, with the computed affinity increases consistent with enhanced old RBC aggregation. These results provide a rational explanation for the aggregation and EPM behavior of old RBC, and raise the possibility of depletion-mediated interactions contributing to senescent cell removal from the circulation.

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL.

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.03 to 0.37 +/- 0.05 and from 0.15 +/- 0.01 to 0.31 +/- 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 13.8 +/- 3 and from 10.7 +/- 2.1 to 16.3 +/- 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.

Glycocalyx modulation is a physiological means of regulating cell adhesion.

Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity. Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble &ggr; interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells. The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells. Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion. Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins.

Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM)

Large polysaccharide molecules composing the glycocalyx have been shown to prevent cell adhesion. However, this process was not observed microscopically. Terbium labeling, combined with a new quantitative imaging method based on electron energy loss spectroscopy, allowed specific glycocalyx staining with excellent contrast. Image analysis enabled us to compare glycocalyx structure in free membrane areas and contacts between monocytic cells and bound erythrocytes. Apparent glycocalyx thickness, in contact areas, was half of the sum of glycocalyx thicknesses in free areas without label density increase. Ultrastructural immunogold localization of CD43 molecules, a major component of glycocalyx, was also demonstrated to be excluded from contact areas during adhesion. Thus, both approaches strongly suggest that some glycocalyx elements must exit from contact to allow binding of adhesion molecules.

Microvascular blood flow resistance: role of endothelial surface layer.

Observations of blood flow in microvascular networks have shown that the resistance to blood flow is about twice that expected from studies using narrow glass tubes. The goal of the present study was to test the hypothesis that a macromolecular layer (glycocalyx) lining the endothelial surface contributes to blood flow resistance. Changes in flow resistance in microvascular networks of the rat mesentery were observed with microinfusion of enzymes targeted at oligosaccharide side chains in the glycocalyx. Infusion of heparinase resulted in a sustained decrease in estimated flow resistance of 14-21%, hydrodynamically equivalent to a uniform increase of vessel diameter by approximately 1 micron. Infusion of neuraminidase led to accumulation of platelets on the endothelium and doubled flow resistance. Additional experiments in untreated vascular networks in which microvascular blood flow was reduced by partial microocclusion of the feeding arteriole showed a substantial increase of flow resistance at low flow rates (average capillary flow velocities < 100 diameters/s). These observations indicate that the glycocalyx has significant hemodynamic relevance that may increase at low flow rates, possibly because of a shear-dependent variation in glycocalyx thickness.

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Morphological changes in rainbow trout (Oncorhynchus mykiss) gill epithelia following repeated intermittent exposure to chloramine-T

Chloramine-T is a widely used prophylactic and therapeutic agent in freshwater aquaculture. This study examined the effects of repeated intermittent exposure of healthy rainbow trout (Oncorhynchus mykiss) fingerlings to sublethal concentrations of chloramine-T (0, 5, 10, or 20 mg/L) twice weekly in 1-h pulses at 11 °C for 4 weeks in a replicate-tank facility. Gills were excised from subsamples of fish prior to exposure and at the end of the 4-week experimental period. Tissue was fixed and processed for light microscopy and transmission and scanning electron microscopy. The gill epithelium from fish treated with 10 and 20 mg/L chloramine-T appeared swollen and vacuolated, with extensive intercellular edema. There was a significant reduction in the number of lamellar mucous cells and an apparent increase in the numbers of chloride cells. Chloride cells from both the base of the lamella and the lamellar surface of gills exposed to chloramine-T had a significant increase in the area of the apical plasmalemma after treatment with 10 and 20 mg/L, and a reduction in the thickness of the apical plasmalemma-associated glycocalyx. These morphological changes are consistent with a compensatory mechanism for the remedial uptake of ions, suggesting that chloramine-T increased epithelial ion permeability coincident with a possible influx of water leading to intercellular edema. Chloride cell proliferation and intercellular edema may also have affected gas exchange across the branchial epithelium.