Relative shedding of glycosaminoglycans from the endothelial glycocalyx during inflammation and their contribution to stiffness of the glycocalyx

Abstract

BACKGROUND:

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Details of shedding of specific constituents of the glycocalyx remain to be determined.

OBJECTIVES:

To review the contributions of the primary glycosaminoglycans that comprise the glycocalyx, heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA), as barrier to WBC-EC adhesion, and elucidate the rates of shedding of each component in response to an inflammatory stimulus. Assess the potential role that stiffness of the glycocalyx plays in resisting infiltration by WBCs during the adhesion process.

METHODS:

Quantitate shedding of the glycocalyx in post-capillary venules of rat mesentery in response to superfusion of the tissue with 10⁻⁶ M fMLP. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS-1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP).

RESULTS:

Following a 30 min exposure of the mesentery to fMLP about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The time constant for HBP shedding (5.8 min) was one-third that for BS-1 labelled GAGs (14.3 min). An attempt was made to assess stiffness of the glycocalyx by observing the motion of adhered lectin coated fluorescently labelled microspheres (FLM) under oscillatory flow conditions. Estimates of the elastic modulus of the glycocalyx revealed a value of 26 mPa, which was orders of magnitude below published data obtained by atomic force microscopy.

CONCLUSIONS:

The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC-EC adhesion. Prior observations that HA lies closer to and parallel to the endothelial membrane, compared to HS suggests that the compact layer of HA near the EC membrane surrounds WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. The relatively low elastic modulus of the glycocalyx under shear is consistent with the hypothesis that the FLMs adhered to strands of HS normal to the EC surface that extended above the relatively more compact and stiffer HA layer below. Gradients of stiffness within the glycocalyx may not be detected by compressive indentation tests published to date.

Keywords

Glycocalyx shedding inflammation stiffness heparan sulfate chondroitin sulfate hyaluronan glycosaminoglycans

1. Introduction

1.1. Historical background

This thematic issue of Biorheology that focuses on the endothelial glycocalyx is a fitting recognition of the foresight of the journal’s founding editor, Alfred L. Copley. As noted in his historical account of the endo-endothelial fibrin lining (EEFL) [1], in the 1940’s the endothelial cell (EC) surface was becoming recognized as a crucial site for maintaining hemostasis, mitigating thrombosis, and as a barrier to cell interactions with the wall and vascular permeability. Initial studies of Chambers and Zweifach explored the intercellular cement [2] and basement membrane that is continuously secreted by the EC. They drew attention to the surface of the endothelium as an extension of the basement membrane that formed the so-called “hematoparenchymal barrier” [3]. Based upon early observations that introduction of plasmin into small blood vessels resulted in extensive petechial hemorrhages [4], Copley hypothesized that a fibrin lining on the surface of the EC was responsible for maintenance of small blood vessel integrity, as schematized in Fig. 1.

Copley theorized that the cell-free layer, observed in microvessels by Poiseuille [6] and others, was comprised of an immobilized portion that bounded a fibrin layer on the EC [7]. The concept of an immobilized layer of plasma has been suggested as the cause of the anomalous low capillary hematocrit on the order of 10–20% of systemic values that fell below the hypothetical maximum reduction of 50%, based on red cell velocity profiles in small tubes. Klitzman and Duling [8] hypothesized that the low capillary hematocrits arose from retardation of fluid within a 1.2 μm thick layer on the EC surface. To validate this hypothesis and explore the role of the glycocalyx in contributing to the anomalous low capillary hematocrits, Desjardin and Duling [9] inserted finely drawn micropipettes into feeding vessels and perfused individual capillaries with heparinase to strip off the glycocalyx. Their results showed a two-fold rise in capillary hematocrit, presumably because of the resultant increase in the effective capillary diameter with degradation of the glycocalyx. Subsequent studies have shown increases in capillary hematocrit in response to glycocalyx removal by perfusion with hyaluronidase [10], or degradation due to the presence of reactive oxygen species [11].

1.2. Composition of the glycocalyx

The fibrin layer hypothesis, however, has been supplanted by direct observations that the endothelial glycocalyx may serve as a barrier to blood cell adhesion [12,13] to the EC and extravasation of macromolecules [14,15], as schematize in Fig. 2.

Fig. 1.

Copley’s hypothesis of the blood-endothelial interface as a cell-free layer with mobile and immobile portions and a molecular coat on the EC surface comprised of fibrin. From Copley [5].

Fig. 2.

The endothelial glycocalyx is comprised of transmembrane and membrane-bound proteoglycans that are decorated with the glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate. These are encased in a meshwork of hyaluronan that is linked by hyaluronan binding proteins (HBP) to the endothelium. The glycocalyx serves as a barrier to leukocyte (WBC) adhesion to the endothelium, which follows the initial radial migration of WBCs to the EC surface with subsequent rolling on the EC, mediated by selectins on the EC surface, and followed by firm adhesion mediated by integrin receptors on the EC. The composition of this barrier results from the continued synthesis of new glycans by the EC, and shear dependent removal by dissolution of the proteoglycan core protein by proteases and cleavage of GAGs by lyases secreted by the EC.

It is now recognized that the vascular endothelium is coated with a layer of polysaccharides and transmembrane proteins that serves as a barrier to extravasation of solutes and leukocyte (WBC) adhesion, and plays a role in the transmission of shearing forces to the EC surface and the process of mechanotransduction [16]. With the addition of an adsorbed layer of proteins [2] the resultant endothelial surface layer (ESL) has been shown to extend into the lumen of microvessels 400–500 nm [17], which significantly exceeds dimensions obtained in either fixed specimens or cultured cells [18]. Copley’s observations that the serine protease plasmin affects the blood-EC interface arises not from dissolution of fibrin, but rather the ability of plasmin to either directly cleave EC surface proteoglycans [19] or activate other proteases [20], such as matrix metalloproteases [21] that degrade proteoglycans on the EC surface.

The molecular composition and structure of the ESL has been summarized in numerous reviews [18,22–25]. The fine structure of the glycocalyx has been described as a network of glycoproteins on the order of 50 to 100 nm thick, with a characteristic spacing of 20 nm that accounts for the resistance to filtration of molecules the size of plasma proteins [26]. It is now recognized that these glycoproteins are decorated with glycosaminoglycans (GAGs, principally heparan sulfate, HS, and chondroitin sulfate, CS) and that both the core proteins and GAGs can be enzymatically cleaved by either matrix metalloproteases [27] or endoglycosidases such as heparanase [28], respectively. Heparan sulfate (HS), a glucosaminoglycan and the most common GAG on the EC glycocalyx, is associated with 50–90% of endothelial proteoglycans [29] and is typically present in a ratio of 4:1 with the second most common GAG, the galactosaminoglycan chondroitin sulfate (CS) [30]. The GAGs dermatan and keratin sulfate may also be present on the EC to a lesser extent than CS and HS [31]. The principal proteins on the EC surface that bind HS and CS to form the proteoglycans are the transmembrane syndecans and the membrane-bound glypicans [23]. The most prevalent proteoglycan, syndecan-1, is one of four members of this family of proteoglycans and is a type I integral membrane protein composed of an intracellular domain, a transmembrane domain, and an extracellular core [32]. Syndecan-1 typically has one to three HS and one to three CS GAG molecules attached to its core protein. The number of GAGs attached to the core protein is variable and may depend on the physiological state and location of the tissue. It has been found that HS and CS are preferentially distributed along the syndecan protein core, with most of the HS bound near the N-terminus, distal to the plasma membrane and most of the CS near the C-terminus near the plasma membrane [33]. Studies of syndecan-1 knockout mice have suggested that its absence leads to enhanced WBC-EC adhesion, although the extent to which the WBCs or the ECs contribute to this response remains to be determined [34]. It has also been suggested that the GAG hyaluronan (HA), which is not bound to a proteoglycan core, but held in place by hyaluronan binding proteins (HBP) anchored to the EC membrane, may play a role in permeation of the glycocalyx by macromolecules [15] and contribute to the structural integrity of the GAG protein matrix [35]. However, the proportion of HA relative to HS and CS remains to be determined.

It is well recognized that the structure and composition of the ESL results from a dynamic balance between continued biosynthesis of glycans and shear-dependent alterations [36,37]. Shedding of the glycocalyx occurs in response to hyperglycemia [38], endotoxemia and septic shock [39], oxidized LDL [40], TNF-𝛼 [41], atrial natriuretic peptide [42], abnormal blood shear stress [43,44], ischemia–reperfusion injury [45], light-induced production of free radicals [17], and during by-pass surgery [46,47]. Dramatic structural changes in the glycocalyx occur during inflammation that leads to shedding of proteoglycans and GAGs and alterations in permeability. Topical stimulation of the endothelium for prolonged periods (20–120 min) with the cytokine TNF-𝛼 results in an increased porosity of the glycocalyx in the absence of WBC–EC adhesion [48]. Acute activation of the endothelium with the chemoattractant fMLP results in a rapid (<5 min) shedding of glycans from the EC surface of arterioles, capillaries and venules [49]. Chemokine induced shedding of the glycocalyx has been shown to increase exposure of leukocyte integrin receptors on the EC (ICAM-1) which may exacerbate WBC-EC adhesion [12]. Although it has been suggested that reductions in ESL thickness during inflammation are less than that required to expose adhesion receptors [50], i.e. only 200 out of the 500 nm total thickness, the analysis therein does not address the process of leukocyte penetration of the surface layer and ESL structural integrity. It has been suggested that penetration of the ESL by microvilli on the WBC surface is an important determinant of WBC-EC adhesion [51]. Thus, reductions in enzymatic degradation of the ESL by either inhibiting heparanase or its secretion [52], may lessen reductions in ESL stiffness that limit penetration of the layer [53]. While the precise enzymatic processes responsible for shedding of the glycocalyx are intertwined between metalloproteinases that directly cleave proteoglycans on the EC surface [27], and heparanase which may cleave heparan sulfate [52], it is apparent that stabilizing the ESL to resist cytokine induced changes may mitigate numerous pathological conditions.

1.3. Mechanical properties of the glycocalyx

The mechanical properties of the glycocalyx has been recognized as a significant determinant of cell adhesion and rolling on the EC. It has been shown that stiffness of the ESL may affect penetration of cells [51] as well as affecting the resistance to fluid movement within the glycocalyx [54]. Deformation of the ESL may depend primarily on the resistance to fluid movement within the ESL, and to a lesser extent the rigidity of the proteoglycan structure [54]. Transmission of shear stresses to the EC surface is also dependent upon the rigidity of the molecular structure and the resistance to permeation of fluid within the layer [55]. Modeling studies have examined the structural rigidity of the glycocalyx in light of its molecular structure. Based upon the ultrastructural observations of Squire et al. [26], Han et al. [56] have hypothesized that proteoglycans on the EC surface are matted down by prevailing shear stresses and as wall shear stresses are diminished recoil further into the vessel lumen. Compression of the glycocalyx by the passage of blood cells is countered by lubrication pressures generated as fluid is squeezed through the molecular matrix of the ESL and increases as the resistance to fluid movement in the layer increases [54,56]. Experimental evidence for deformation of the ESL with varying wall shear stress has been obtained in post-capillary venules of the mesentery (rat) [57]. With a constant circulating concentration of fluorescently labelled microspheres (FLMs) coated with the lectin BS-1, the adhesion of FLMs to the EC surface was invariant with basal levels of shear stress (characterized by measured red cell velocity and calculated wall shear rate) Fig. 3A. In contrast, with reductions in shear rate imposed by reducing red cell velocity with hemorrhagic hypotension, the adherence of microspheres within an individual vessel increased significantly, Fig. 3B. It was hypothesized this the increased adhesion resulted from unfurling of the proteoglycans on the EC surface that gave rise to an increase in binding sites for the microspheres, which could not be discerned amongst the randomly distributed shear rates and adhesion in the resting normal circulation.

Fig. 3.

Adhesion of lectin (BS-1) coated fluorescently labelled microspheres (FLMs) to the endothelium of post capillary venules in rat mesentery. Redrawn from Lipowsky et al. [57]. (A) Number of FLMs adhered per 100 μm length of venule vs wall shear rate, randomly sampled in 20 venules ranging in diameter from 17 to 50 μm. Shown is a linear regression bracketed (dashed lines) by the confidence interval of the slope. No significant correlation with shear rate was discernible. (B) Number of FLMs adhered, normalized with respect to initial values at normal flow, during reductions in shear rate caused by hemorrhagic hypotension. As shear rate is reduced, the number of adhered FLMs increases significantly, P < 0.05. The rise is attributed to unfurling of glycoproteins on the EC surface and increased availability of binding sites for BS-1 on the EC surface.

Thus, within this framework, the present study was undertaken to: (1) Shed additional light on the relative amounts of shedding of the main components of the ESL in response to inflammatory stimuli, and (2) to explore the mechanical properties of the ESL. To that end, studies were conducted to delineate the shedding of hyaluronan relative to other glycans, mainly heparan sulfate, and to provide a more quantitative measure of glycocalyx stiffness from observations of the movement of microspheres adhered to the EC under conditions of pure oscillatory shear.

2. Methods

2.1. Animal preparation

All animal studies conformed to the Guiding Principles in the Care and Use of Animals established by the American Physiological Society, and all protocols have been approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University.

Male Wistar rats, weighing 250–400 g, were anesthetized with Inactin (120 mg/kg, i.p.), tracheostomized, and allowed to breathe under spontaneous respiration. The right jugular vein and its paired carotid artery were cannulated with polyethylene tubing. Supplemental anesthetic was administered via the jugular catheter, as needed, to maintain a surgical plane of anesthesia. The carotid catheter was connected to a strain-gage pressure transducer to monitor central arterial pressure, which averaged a nominal 125 mmHg. Core temperature was monitored by a rectal probe and was maintained between 36 and 37 °C with the aid of a heating pad.

2.2. Intravital microscopy

The intestinal mesentery was exteriorized through a midline abdominal incision, placed on a glass pedestal to permit viewing under either epi- or trans-illumination and superfused with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered Ringer’s solution (pH = 7.4) at a temperature of 37.0 °C. Visual recordings of the mesentery were made with a Yokogawa CSU-10, spinning disk confocal microscope, (Solamere Technology, Salt Lake City, UT) using an XR/MEGA-10 intensified CCD camera (Stanford Photonics, Palo Alto, CA). Output of the camera was digitized and saved to computer disk in tagged image format (TIF) with each image being 1024 × 1024 pixels in area with a depth in intensity of 10 bits (1024). To measure glycan concentration on the microvessel wall, confocal epi-fluorescence microscopy images were acquired using a Zeiss 20x/0.50NA water immersion objective and spanned 170 × 170 μm in the focal plane, with an effective pixel size of 0.166 μm (170/1024). Venule diameters were measured from the digitized images by scaling with reference to a stage micrometer. Measurement of the spatial oscillations of fluorescently labelled microspheres (100 nm diameter) adhered to the EC were made at a higher magnification using a Zeiss 63x/1.2 water immersion objective that gave an interpixel spatial resolution of 0.036 μm/pixel.

2.3. Measurement of endothelial surface glycan concentration

To obtain an index of the glycan concentration on the endothelial cell (EC) surface, the lectin Bandeira Simplicifolia (BS-1, Sigma, St. Louis, MO) was labeled with Alexa Fluor 488 (Invitrogen, Inc., Carlsbad, CA) and infused i.v. via the jugular indwelling catheter, as described previously [49]. Previous studies have shown that 60% of BS-1 bound to the venular surface could be removed by direct perfusion with 50 U/ml heparinase III [58]. A single bolus of labeled BS-1 was administered i.v. at a dose of 1 ml/kg, and allowed to equilibrate for 10 min prior to intensity measurements on the EC surface. All acquired fluorescence images were corrected for the non-uniformity of the laser illumination by field-flattening and scaling to a reference standard, as described previously [49]. A representative post-capillary venule that is stained with BS-1 is shown in Fig. 4. Glycan concentration was taken as proportional to the average peak fluorescence intensity along the length of the venular wall. A radial profile of intensity, averaged along the length of a venule, is shown in the inset of Fig. 4B. The apparent thickness of the lectin-stained layer is much larger than that estimated by exclusion of 70 kDa Dextran of glycocalyx layer thickness [49]. As shown therein, thickness of the venular glycocalyx thickness averaged 600 nm, taken as the distance between the edge of the exclusion zone and the surface of the endothelium. In contrast, the width of the lectin stain averaged about 1800 nm, as indicated by the width of the peak intensity at 50% of maximum along one wall. As a frame of reference, also shown in this image are 100 nm diameter lectin-coated fluorescently labelled microspheres (FLM). Diameter of the FLMs appear to be about 600 nm, as indicated by the width of the peak intensity along a line through its center. The substantially larger thickness of the glycocalyx compared to that obtained by dextran exclusion is likely due to the combined effects of the finite depth of field of the objective (∼1 μm) and the point-spread function of the optical train. The latter is clearly evidenced by the apparent five-fold larger diameter of the FLMs compared to that obtained by electron microscopy (100 nm, manufacturers measurements).

Fig. 4.

Staining of the glycocalyx with fluorescently labelled lectin BS-1. (A) Brightfield image of a 25 μm diameter post-capillary venule. (B) Fluorescence image obtained by confocal microscopy after labelling with BS-1. The average peak intensity of the fluorophore along the length of a venule was obtained from an average radial intensity profile (inset) and used as a measure of the concentration of glycans on the vessel wall. Following staining of the glycocalyx, fluorescently labelled microspheres, 100 nm in diameter, were coated with BS-1 and infused into the circulation. The number of FLMs adhered per length of venule was also used as a measure of the concentration of glycans on the EC surface. Images acquired with a 50x/1.0 NA water immersion objective.

Quantitation of specific components of the glycocalyx was obtained using a combination of these techniques. To determine the change in composition of hyaluronan on the EC surface, the glycocalyx was stained with fluorescently labelled (Alexa 488) hyaluronan binding protein (HBP, Sigma, St. Louis, MO). The shedding of BS-1 and HBP following activation of the EC was obtained by superfusion of the mesenteric tissue with the peptide f-Met-Leu-Phe (fMLP) at a concentration of 10⁻⁶ M in Ringer’s solution. For comparison, the shedding of heparan sulfate from the EC surface was obtained by first infusing 100 nm FLMs that were covalently linked to an antibody of HS (F58-10E4, Associates of Cape Cod Inc., Falmouth, MA). These data were reported, in part, previously [45]. With a steady infusion (via jugular vein) of FLMs a constant circulating concentration of FLMs was maintained and the number of FLMs adhered per 100 μm length of venule was taken as a measure of HS concentration [45].

3. Results

Shedding of components of the glycocalyx was initiated by superfusion with fMLP and the concentration of fluorophores was monitored over a 30 min period, as shown in Fig. 5. During stabilization of the tissue, fluorescently labelled BS-1 (for GAGs), hyaluronan binding protein (HBP) or antibodies for heparan sulfate (HS-Mab) were introduced and the mesenteric tissue was superfused with Ringer’s solution. In separate experiments bound fluorescent HBP did not vary significantly over a 30 min control period with Ringer’s solution lone (dotted line, ○). Following onset of fMLP, a rapid fall in fluorescently labelled HBP was observed for 14 venules (diameter = 31.4 ± 13.8 SD μm). For comparison, also shown is the previously reported shedding of BS-1 label and HS-Mab during superfusion with fMLP. A 25% reduction in BS-1 fluorescence intensity following onset of exposure to fMLP signifies a general loss of glycans over the 30 min period, as reported previously for 17 venules (diameter = 28.8 ± 14.8 SD μm) [49] and indicated by the dashed line of Fig. 5 and symbol ( $\blacktriangle$ ). This decrease in glycans was matched closely by a previously shown decrease in HS-Mab ( $\blacksquare$ ) obtained in 18 venules of similar size [45]. To quantitate differences in the time course of shedding, the fraction shed over the 30 min observation period was calculated as 1 − I∕I₀, which was then fit to an exponential function, $1-\frac{I}{I_{0}}=A(1-e^{-t/{\tau}})$ , where 𝜏 is the time constant of the shedding response and A is the asymptotic value of intensity. The time constant for an exponential decrease in HBP was about one-third that of the loss of either HS-Mab or BS-1, i.e. 5.8 vs 14.3 min, p < 0.04 (t-test), suggesting that the HBP was shed more rapidly than the BS-1 labelled glycans. The asymptote of the HBP was also significantly lower than that of the BS-1 label, p < 0.001.

Fig. 5.

Shedding of the venular glycocalyx following superfusion of tissue with 10⁻⁶ M fMLP. Shown are the normalized fluorescence intensities of hyaluronan binding protein (HBP, ●) following fMLP for 14 venules, and from separate experiments without fMLP for 10 venules during a 30 min control period (○). As a frame of reference, the general shedding of glycans is indicated by EC-bound BS-1 ( $\blacktriangle$ ), previously reported [49], and shedding of 0.1 μm FLMs coated with Mab (■) for heparan sulfate (HS), shown previously [45]. Data are mean ± SE for 14, 17 and 18 vessels, labelled with fluorescently labelled HBP, BS-1 and HS Mab, respectively.

To examine the mechanical properties of the glycocalyx, the movement of lectin coated fluorescently labelled microspheres (FLMs) was examined under oscillatory flow conditions in capillaries. Capillaries were used for this experiment to preclude obstruction of the video field by red cells. It was thus assumed that the structure of the capillary glycocalyx would be similar to that in venules. Following a steady infusion of lectin (BS-1) coated FLMs into the circulation the movement of adhered FLMs was tracked during pure oscillatory motion of the free stream. Oscillatory flow in capillaries was induced by gentle compression of a capillary with a blunted micropipette at the downstream end of the capillary, as schematized in Fig. 6A. With occlusion of the downstream end, the upstream pressure was rapidly transmitted downstream, which caused an inflation of the capillary. The total volume of fluid movement in and out of the capillary during a pulse was estimated from the peak to peak axial displacement of red cells. Because of the capillary volume between upstream and downstream locations, the axial movements of free FLMs and red cells were amplified at the upstream end, increasing from zero at the occlusion site to 10 μm at 500 μm upstream. An example of peak-peak oscillations of RBCs is shown in Fig. 6A. Given a 6 μm average capillary diameter, the radial increase in microvessel diameter was found to be on the order of 0.06 μm.

Fig. 6.

Characterization of the deformation of the glycocalyx induced by oscillatory wall shear stress. Oscillatory wall shear stresses were generated by occluding a capillary at its downstream end. Due to inflation of the microvessel by the propagating pulse-pressure wave, RBCs oscillated (inset) and the radius of the vessel varied accordingly on the order of 0.06 μm. Deformation of the glycocalyx was measured by monitoring the position of an adhered FLM (BS-1 coated 100 nm bead) along a measuring line (panel B). The shift in peak fluorescence intensity was monitored by frame-by-frame analysis of the video. A maximum oscillatory displacement of the FLM was about 0.36 μm, which was much less than that of an FLM in the free stream of 1.27 μm.

Axial movement of FLMs in the free stream or at the wall were measured by tracking changes in peak fluorescent intensity along a measuring line (Fig. 6B). The overall excursion of the peak intensity was measured during the sinusoidal oscillation of the FLM. A rough approximation of the shear forces acting on an adherent FLM was made assuming a linear shear field in this pure oscillatory flow. For the illustrative example shown in Fig. 6C, the bead at the wall underwent a 0.36 μm excursion during the oscillations. In contrast, excursion of an FLM in the center of the capillary equaled 1.27 μm. Assuming that the glycocalyx deforms in simple shear one may roughly estimate its shear modulus. Given that the adherent FLM moves parallel to the vessel wall and that it is situated at a distance of 0.5 μm from the endothelial membrane, one may calculate the shear strain (𝛾) as 0.36∕0.5 = 0.72. For a linear shear field on the glycocalyx surface, the shear stress (𝜏) on the glycocalyx surface was estimated from the velocity of the centerline FLM, and for a plasma viscosity of 1.2 cP, equaled 0.06 dyn∕cm². Thus, for this representative measurement, the shear modulus (G) was estimated from G = 𝜏∕𝛾, and equaled 8.3 mPa. For eight measurements in two capillaries, G averaged 26 ± 16 SD mPa. This value is an order of magnitude below estimates obtained by other means, as noted in the Discussion.

4. Discussion

The aim of the current study has been to establish a framework for the role of the endothelial glycocalyx as a barrier to leukocyte-endothelial adhesion in post-capillary venules of the microcirculation. It is well established that the composition and structure of the venular glycocalyx may dramatically change during the inflammatory process. The seminal studies of Henry and Duling [48] in which permeation of high molecular weight dextrans into the microvascular endothelial glycocalyx was increased by bathing the cremaster muscle with TNF-𝛼 suggested that proinflammatory cytokines disrupt the glycocalyx, increase its porosity and increase the volume occupied by RBCs in a microvessel. Upon comparison with their earlier work in which dextran permeation into the glycocalyx was increased following perfusion with hyaluronidase [15], it was concluded that TNF-𝛼 degraded the glycocalyx to a greater extent than hyaluronidase [48], due to degradation of other components of the glycocalyx, presumably by activation of endogenous proteases. Subsequent studies from this laboratory have modelled the inflammatory process by topical application of the chemoattractant fMLP and revealed a shedding of HS and CS that results from either activation of matrix metalloproteases on the EC surface [59] to cleave proteoglycans, or secretion of heparanase from the endothelium [60] that cleaves HS from its protein core. Measurements of the exclusion of 70 kDa dextran from the EC surface following perfusion of venules with either heparinase, chondroitinase, hyaluronidase or a mixture of all three GAG cleaving enzymes were made to determine the relative contributions of each GAG to the apparent thickness of the glycocalyx [58]. It was suggested that HS, CS and HA contributed 43%, 34% and 12% respectively to the glycocalyx thickness, and collectively, the three GAGs account for 90% of its thickness.

4.1. Shedding of the glycocalyx

Previous studies of fMLP induced shedding of the glycocalyx revealed the rapid loss of HS [45] as shown in Fig. 5. The shedding of HS was consistent with the loss of HS and CS indicated by reduction of the bound lectins [45]. Following a prolonged 30 min exposure to fMLP, HS and CS labels fell about 30%. In contrast, HA decreased over 50% during a similar period (Fig. 5) and exhibited a much greater fall during the initial phases (<5 min) of EC exposure to fMLP. Assuming that the apparent concentration of HA was much less than either HS or CS (12 vs 43 and 34%, respectively) if the density of HBPs is less than or equal to that of the other GAG bearing proteoglycans, then similar rates of HBP and PG cleavage, by for example MMP activation, would likely result in a greater percentage decrease in HA density on the surface. This disparity could be amplified by a greater fraction of the total HA bound per HBP molecule on the EC surface. Thus, speculation of the relative contributions of HS, CS and HA as a barrier to WBC-EC adhesion would suggest that HS and CS bearing proteoglycans may constitute the main structural and steric hindrance to WBC infiltration to reach EC bound adhesion molecules. Direct observation of the structure and density of glycans on the surface of culture ECs using stochastic optical reconstruction microscopy [61] suggest otherwise. These studies suggest that HA lies in a plane parallel to the cell surface with a coverage greater than that of HS which is much shorter and lies perpendicularly to the EC membrane. However, with regard to WBC-EC adhesion, this static picture may not accurately represent the dynamics of the adhesion process. For example, the time constant of WBC-EC adhesion in response to fMLP appears to be quite variable amongst species and tissues. In situ observations in rat mesentery of fMLP induced transient adhesion of circulating beads coated with antibody to ICAM-1 (1A29) reveal a time constant for adhesion of 10.9 min [12]. This value lies midway between the time constant of 5.8 min observed here for HA shedding, and the 14.3 min shown previously for HS shedding [45]. Thus. more detailed studies of alterations in the relative concentration of GAGs on the EC surface need to be performed.

4.2. Structural properties of the glycocalyx

In view of the aforementioned importance of the stiffness of the glycocalyx as a factor that limits WBC infiltration to reach EC bound adhesion molecules, the modulus of elasticity of the glycocalyx was roughly estimated from the oscillatory movement of surface bound lectin-laden microspheres (Fig. 6). As noted in the RESULTS, a shear modulus of 26 mPa was estimated. If the glycocalyx were an isotropic elastic solid with a Poison’s ratio on the order of 0.5, the Young’s modulus (E) would be about 3-fold greater, i.e. on the order of 78 mPa. Several studies have attempted to measure the elastic modulus of the glycocalyx by a variety of indentation techniques using atomic force microscopy (AFM). Estimates of E vary considerably, in part because of methodological differences, such as size of the AFM probe, and analysis methods, as well as sample differences. For example, based upon the AFM data in Oberleithner et al. [53], obtained with a 1 μm diameter AFM probe on the EC of a split human umbilical artery mounted on a slide, a value of E equal to about 350 Pa may be calculated. Using cultured bovine lung microvascular ECs, O’Callaghan et al. [62], with an 18 μm AFM tip, calculated E equal to 250 Pa. For cultured human umbilical vein ECs, with a 40 nm and 2.4 μm tips, Bai and Wang [63] and Marsh and Waugh [64], determined values of E equal to 390 and 700 Pa, respectively. In contrast, Nijenhaus et al. [65] used a microrheology based method based upon measuring oscillations of micron sized particles in an optical trap to calculate the viscoelastic shear modulus of mixtures of HA that mimicked physiological glycocalyx concentrations estimated for cells in culture. They determined a range in elastic modulus from 0.1 Pa at a frequency of 1 Hz, to 10 Pa at 10⁴ Hz. Thus, the shear modulus of 26 mPa found here is orders of magnitude below these published values. This disparity may represent a failure of the assumption that the glycocalyx can be characterized in terms of an isotropic elastic continuum.

It is likely that the oscillating spheres on the surface of the EC (Fig. 6) represent the movement of a microsphere that is tethered to a strand of HS that rises normal to the EC membrane and pivots around its attachment point under oscillatory surface shear stresses. This behavior would be consistent with the structural observations of Fan et al. [61] and the concept that the density of the glycocalyx increases from the outer edge of the surface layer to the EC plasma membrane [58]. As shown therein, the transient diffusion of 70 kDa dextran through the glycocalyx was greatly reduced near the EC surface suggestive of a more compact layer near the EC membrane. Analysis of solute transport through a porous bi-layer by Curry and Michel [66] clearly supports a more compact layer near the EC membrane, that could manifest increasing stiffness of the glycocalyx near the EC membrane. A compact layer of HA could very well cover WBC adhesion receptors, such as ICAM-1, which is only about 20 nm in length [67]. The outer portion of the glycocalyx may be much more deformable, as observed here when comparing shear to compressive loadings imposed by indentation techniques. The unfurling of the outer strands of the glycocalyx with reductions in shear stress, as hypothesized for the increased exposure of binding sites for FLMs noted in Fig. 4, could be mostly HS bearing proteoglycans, that are easily bent in shear. It is thus conceivable that the glycocalyx offers little resistance to penetration by microvilli on the surface of rolling WBCs until they penetrate deeper into a layer of hyaluronan. Enzymatic shedding HA could thus greatly enhance firm WBC-EC adhesion.

5. Conclusions

In summary then, there appears to be strong evidence that heterogeneity of the glycocalyx structure is a significant determinant of WBC adhesion. WBC may easily penetrate the outermost layer comprised mainly of HS proteoglycans. When the endothelium is activated by inflammatory mediators, the first component to be shed may be HA that resides in a compact layer near the surface of the EC. Increases in porosity of that layer could contribute to the onset of rapid WBC-EC firm adhesion as adhesion receptors become more accessible. Such increases could be mediated by either proteolysis of HBP or secretion of hyaluronidase from the EC or surrounding cells.

Footnotes

Acknowledgements

This work was supported by NIH HL382986. The author is grateful to Ann Lescanic for her technical assistance in performing the animal studies.

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