INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE ENDOTHELIAL CELL glycocalyx is a dynamic structure that envelopes the endothelial cells. The glycocalyx has been likened to a deep-pile carpet covering and enmeshing endothelial surface enzymes and receptors (24). On the luminal surface, it has recently been shown to be as much as 0.4-0.5 µm thick (29) and, in vivo, the glycocalyx binds lectins (10, 12). Recently, it has been shown to be capable of restricting access of cells and macromolecules to the endothelial cell plasma membrane (29). It excludes anionic macromolecules the size of 70-kDa dextran and larger, reduces the functional diameter of the vessel by excluding red blood cells, and can be removed by light-dye treatment (29).

Vascular endothelial cells produce integral membrane macromolecules such as heparan sulfate proteoglycan and thrombomodulin (13, 14) as constituents of the glycocalyx. The glycocalyx is apparently a dynamic component of endothelial cell function, since the distribution of endothelial surface glycoconjugates can change with altered states of permeability, for example, after cold-lesion injury to the brain (30). Thus knowledge of the structure of the glycocalyx may be key to understanding endothelial cell function. The structure of the glycocalyx is made more complex by binding plasma macromolecules such as fibrinogen (18) and albumin (2).

In cell systems and histological specimens, as well as intact animals, enzymatic tools have been used to explore the composition of the glycocalyx. It can be disrupted by heparinase (5), and perfusion with neuraminidase, heparinase, and pronase has been shown to disrupt the blood-retinal barrier (21) and to increase the permeability of frog mesenteric vessels (1). Less well recognized is the fact that vascular endothelial cells in culture can secrete and bind hyaluronan, a high-molecular-weight, unsulfated glycosaminoglycan on their apical surfaces (3, 6, 20). Hyaluronan contains no core protein and is synthesized in the plasma membrane rather than the Golgi. Synthesis is associated with continuous secretion directly into the pericellular matrix, and shedding from the cell surface is mediated by an undefined mechanism (22, 23). In rat vascular endothelium, hyaluronan has been shown scattered on the plasmalemma and concentrated in the caveolae (6).

To date, more is known of the role of hyaluronan in chondrocytes and extracellular matrix than in endothelial cells. In cultured cells, hyaluronan creates a matrix that is highly hydrated and excludes fixed erythrocytes from contact with the chondrocyte plasma membrane (16). Its structure is that of a highly hydrated random coil (17) that, in the presence of proteoglycans, extends to form complexes with a brushlike configuration (19, 27). The principal cell surface receptor of hyaluronan is CD44H (4), an integral glycoprotein with three major domains. Hyaluronan may also be cleaved by the enzyme hyaluronidase, and subsequently, the fragments can be bound by scavenger receptors termed hyaladherins located on the surface of liver endothelium (15, 26).

Few if any direct measurements have been made in vivo, but based on the foregoing, we hypothesized that the structural organization of the microvascular endothelial glycocalyx might be determined in part by hyaluronan, since it readily interacts with other surface macromolecules to create matrices with molecular-sieving properties. In the present study, we used enzymatic removal of hyaluronan from the endothelial apical surface to explore a role for this molecule in maintaining the permeation properties and structural integrity of the in vivo capillary glycocalyx. For the purposes of this study, we are using the word "permeation" to describe the depth of penetration of tracer molecules into the apical glycocalyx after a given period of time and to distinguish this phenomenon from "permeability," which describes the transvascular movement of molecules.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal preparation. All procedures and the care of animals were in accordance with institutional guidelines. Male Syrian golden hamsters weighing 110-160 g were anesthetized with intraperitoneal pentobarbital sodium in saline (70 mg/kg body wt). The trachea was cannulated to ensure a patent airway, and the animal spontaneously breathed room air. The left femoral vein was cannulated for fluid replacement, maintenance of anesthesia (0.49 ml/h), and infusions of dextran, enzyme, or glycosaminoglycan solutions (Sigma Chemical). During the surgical preparation, esophageal temperature was maintained at 37°C. In animals receiving continuous infusion of glycosaminoglycans via the femoral cannula, anesthesia was maintained by supplemental doses of intraperitoneal pentobarbital sodium (70 mg/kg) given via a secured catheter. After placing the animal on a Plexiglas platter, the right cremaster muscle was prepared for visualization according to standard methodology (29). The tissue was continually superfused at 5 ml/min with a bicarbonate-buffered salt solution consisting of (in mM) 131.9 NaCl, 4.6 KCl, 2 CaCl₂, 1.2 MgSO₄, and 20 NaHCO₃. To this solution, succinylcholine (10⁵ M, Sigma Chemical) was added to reduce spontaneous muscle contractions. The pH of the solution was maintained at 7.35-7.45 by bubbling 5% CO₂-95% N₂ gas, and the temperature was maintained at 34°C. The body temperature of the hamster was maintained at 37-38°C with conducted heat. After at least 30 min of stabilization, during which time arteriolar tone was evaluated, a bolus of 0.2-0.3 ml FITC-dextran (70, 145, 580, or 2,000 kDa; 20 mg/ml in saline; Sigma Chemical) was given via the femoral cannula. Systemic hematocrit was measured at the start and end of each experiment in blood samples taken from a toe clip and collected in heparinized microhematocrit tubes.

Intravital microscopy. After FITC-dextran infusion, microvessels (capillaries, arterioles, and venules) were observed at ×55 with a Leitz water-immersion objective (numerical aperture = 0.84) on a Zeiss intravital microscope equipped with a SIT 66 MTI video camera. Transillumination for bright-field measurements was obtained with a 150-W xenon lamp, and measurements were made as described previously (10). Light from a 75-W xenon lamp and epi-illuminator equipped with a 450- to 490-nm excitation filter, a dichroic beam splitter (FT 510), and a barrier filter (LP 520) was used for epi-illumination of the fluorescent tracer (29). Viewing of the fluorescent tracer was limited to a total time of 10 s or less per vessel to prevent light-dye injury to the endothelium (29). Images of vessels were displayed on a Dage MTI video monitor and recorded on S-VHS video tapes for subsequent image analysis.

Enzyme treatment and controls. For each data set, at least three animals were used to yield from 5 to 10 of each type of vessel, i.e., arterioles, capillaries, and venules, per animal. Streptomyces hyaluronidase (70 kDa; pH optimum 5-6.0; Sigma Chemical) was given in bolus doses via the femoral cannula. At 15, 30, 60, and 120 min after enzyme infusion, microvessels were observed and recorded as described above. To yield a time course of enzyme action, selected microvessels were also recorded at 5-min intervals starting 30 min after hyaluronidase infusion and continuing up to 100 min. Control experiments were performed by analyzing vessels before enzyme treatment and also 60 min after systemic infusion of a bolus dose of either 0.2 ml saline alone or 0.2 ml heat-inactivated hyaluronidase (95°C for 10 min).

Intravenous glycosaminoglycans for reconstitution of the matrix. To deepen our understanding of the effects of enzymatic treatment, we conducted experiments designed to restore the enzymatically modified glycocalyx using defined macromolecular systems. For hamsters of this age (2-3 mo), the normal plasma concentration of circulating hyaluronan is ~80 ng/ml (31). On the basis of a 3 ml plasma volume of distribution in the hamster and a half-life of 5 min (8), clearance of circulating hyaluronan is 33.6 ng/min. To quickly raise the plasma concentration to at least 8 mg/ml (100 times the baseline plasma concentration), a bolus loading dose of 24 mg hyaluronan in 0.1 ml saline was given intravenously after 1 h of enzyme treatment. This was immediately followed by constant infusion of a hyaluronan solution of 0.4 mg/ml at a rate of 0.49 ml/h to maintain an estimated plasma steady-state concentration of ~8 mg/ml. We chose this large dose to counteract the short half-life of hyaluronan in plasma and to saturate circulating hyaluronidase. The infused glycosaminoglycan was allowed to equilibrate for 2 min before observations were made. For an additional 30 min, microvessels were viewed and images were recorded as described above.

Quantitative determination of plasma hyaluronidase levels. About 5 ml blood from control, hyaluronidase-treated, and glycosaminoglycan-treated animals were obtained by cardiac puncture after injection of saline containing heparin sodium. The samples were centrifuged at 3,500 g for 10 min at 25°C to remove the cellular components. Triton X-100 (1%) was added to the plasma samples which were then frozen at 70°C until further use. Hyaluronidase levels were assayed by ELISA in the laboratory of Dr. Robert Stern at University of California, San Francisco, by a previously described method (9).

Data and statistical analyses. Recorded video images of the microvessels were captured using Image-1 software (Universal Imaging). Visualization of still frames was enhanced by using a digital, time-based corrector. For each microvessel, the anatomic diameter, width of the dextran column, and width of the red blood cell column were measured using on-screen calipers that were calibrated with a vertical and horizontal ×55 image of a stage micrometer (Graticules). Positioning of the calipers for anatomic measurements was done according to a previously designed method (10), and its accuracy in measuring the width of the fluorescent tracer column has been demonstrated by Vink and Duling (29) with a comparison of in vivo and in vitro measurements. Briefly, the vessel midplane was brought into focus during transillumination, and the vessel was then viewed under epi-illumination. One of the calipers was positioned outside the dextran column with its luminal surface against the edge of the dextran column. Prior measurement established the accuracy of the endothelial boundary by comparing the bright-field diameter measurement to peaks of an intensity profile across a vessel labeled with a fluorescent membrane dye in the endothelium.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic illustration of region on capillary apical surface from which red blood cells and Dextran 70 are excluded. Diagram is not drawn to scale.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Enzyme dosage and activity. Streptomyces hyaluronidase is specific for hyaluronan. This enzyme was initially tested at doses of 35, 70, 140, and 280 U in saline, equivalent to at least 11.7, 23.3, 46.7, and 93.3 U/ml plasma, respectively (based on a 3 ml plasma volume in a 110- to 160-g hamster). A dose of 140 U hyaluronidase produced the greatest penetration of the Dextran 70 into the apical glycocalyx (Fig. 2A). This dose was used in subsequent experiments. Compared with control animals, enzyme activity in animals that were treated with 140 U hyaluronidase increased two- to threefold (Table 1). An evaluation of the time course of enzyme action showed that the maximum effect of 140 U occurred ~1 h after treatment (Fig. 2B). After 120 min, the dextran did not appear to penetrate further. Therefore, in subsequent experiments, unless otherwise stated, data were collected starting 1 h after enzyme treatment and continuing for an additional 30 min.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of Streptomyces hyaluronidase on penetration of FITC-Dextran 70 into apical glycocalyx of hamster cremaster capillaries. A: effect of increasing doses of enzyme. B: time course of effect of 140 U of enzyme. Data represent means ± SE; total number of vessels per treatment is given in parentheses on bars. Measurement is difference between endothelial cell membrane and point of dye penetration. Three animals were used in A, and two animals were used in B. * P < 0.05 compared with 0, 30, 70, and 280 U. ** P < 0.05 compared with 0-30 min.

Effect on penetration of macromolecules into the glycocalyx. In the control capillaries (5-7 µm) as well as small arterioles and venules 7-10 µm in diameter, FITC-Dextran 70 had no access to a region extending ~0.4 µm from the membrane of the endothelium (Fig. 3). In larger arterioles and venules (diameters of 10-15 µm), this region was ~0.5 µm in width. Red blood cells had no access to a region extending ~0.7 µm into the lumen of the capillaries and averaging ~0.8-1 µm in arterioles and venules of different sizes. The difference in size between the regions that red blood cells and Dextran 70 did not penetrate was similar to that observed by Vink and Duling (29) in which a fluid layer 0.1-0.2 µm thick was present between the surfaces of flowing red blood cells and the boundary of the region from which Dextran 70 is excluded. The different size of the region that Dextran 70 did not penetrate within the capillaries, arterioles, and venules may be due to heterogeneity in the composition of the apical glycocalyx among vessels. During normal angiogenesis, endothelial cells from different segments of a microvascular unit display heterogeneity in their lectin-binding ability, reflecting differences in the composition of their surface glycoconjugates (12). Hence, it is likely that the dextran molecules may not penetrate the glycocalyx of capillaries, arterioles, and venules to the same degree.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of 140 U Streptomyces hyaluronidase on penetration of FITC-Dextran 70 into apical glycocalyx of different microvessels after 1 h of treatment. Measurement is difference between endothelial cell membrane and point of dye penetration at 90 min. Difference between open and shaded bars represents enzyme-induced dye penetration. Data represent means ± SE; total number of vessels per treatment is indicated in parentheses on bars. Three animals were used per treatment. * P < 0.05 compared with control.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of 140 units Streptomyces hyaluronidase on penetration of different sizes of dextrans into capillary endothelial glycocalyx. Measurement is difference between endothelial cell membrane and point of dye penetration at 90 min. Difference between open and shaded bars represents enzyme-induced dye penetration. Data represent means ± SE; total number of vessels per treatment is indicated in parentheses on bars. Three animals were used per treatment. * P < 0.05 compared with control. # P < 0.05 compared with FITC-Dextrans 580 and 2,000.

Effect of exogenous macromolecules on the glycocalyx. Attempts to increase the size of the intact apical glycocalyx by exogenous glycosaminoglycans before enzyme treatment and to reconstitute the matrix after degradation were made. Hyaluronan alone or chondroitin sulfate alone proved unsuccessful in increasing the thickness of the glycocalyx above baseline. Similarly, when hyaluronan and chondroitin sulfate were premixed and infused into the vasculature, there was no significant increase in the size of the intact glycocalyx. However, when the hyaluronan and chondroitin sulfate complex was infused after enzyme treatment, there was a significant decrease in access of FITC-Dextran 70 to the space bounded by the glycocalyx (Fig. 5). Treatment with each glycosaminoglycan separately had no effect on Dextran 70 access after enzyme treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effect of combined hyaluronan (HA) and chondroitin sulfate (CS) on penetration of FITC-Dextran 70 into endothelial apical glycocalyx in microvessels after 1 h of Streptomyces hyaluronidase treatment. Measurement is difference between endothelial cell membrane and point of dye penetration at 90 min. Difference between open and shaded bars represents enzyme-induced dye penetration. Data represent means ± SE; total number of vessels per treatment is indicated in parentheses on bars. Three animals were used per treatment. * P < 0.05 compared with control. ** P < 0.05 compared with enzyme alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, Streptomyces hyaluronidase treatment increased access of FITC-Dextrans 70 and 145 to the glycocalyx but altered neither the width of the red blood cell column nor increased access of FITC-Dextrans 580 and 2,000. We interpret these data as showing that hyaluronan plays a significant role in the maintenance of the permeation properties of the apical glycocalyx. We conclude that its removal causes an upward shift in the molecular weight cutoff beyond which macromolecules are normally restricted from penetrating the glycocalyx.

Although the permeation of the apical glycocalyx increased, there was no effect of enzyme treatment on red blood cell column width. We therefore conclude that molecules other than hyaluronan can stabilize the rheological properties of the glycocalyx. We hypothesize that the matrix-forming properties of hyaluronan and its affinity for binding other glycoproteins and proteoglycans may create a meshlike matrix spanning other molecules (e.g., heparan sulfate, chondroitin sulfate, fibronectin, and thrombomodulin) on the endothelial apical surface. We further hypothesize that since hyaluronidase is similar in size to albumin, hyaluronidase enters the matrix and creates a more open structure, enabling the smaller dextran molecules to gradually diffuse deeper into the glycocalyx (Figs. 4 and 6). However, it appears that some component of the glycocalyx still retains extended configuration and restricts red blood cell access to the endothelial membrane in the presence of an increased dextran permeation within the glycocalyx. Earlier work had shown that heparinase and chondroitinase can disrupt the rheological properties of the endothelial surface layer (5). Taken together, the enzyme data suggest that the structure of the glycocalyx is the aggregate of linking properties of hyaluronan that stabilize properties of the heparan sulfate and chondroitin sulfate proteoglycans.

View larger version (73K): [in this window] [in a new window]   Fig. 6.   Schematic illustration of hypothesized effect of hyaluronidase on access of red blood cells and dextrans to apical glycocalyx in a capillary. A: intact glycocalyx. B: after 1 h of enzyme treatment. Diagram is not drawn to scale.

Our experiments suggest a role for hyaluronan in maintaining the apical glycocalyx as a barrier to large macromolecules. However, although Streptomyces hyaluronidase is specific for hyaluronan, removal of hyaluronan from the glycocalyx could also result in the removal of other molecules associated with the hyaluronan chains. Thus the contribution of other molecules cannot be ruled out. Underhill and Toole (28) reported that when exogenous hyaluronan was added to a suspension of SV-3T3 cells, most binding occurred within the first 2 min of incubation and persisted for >1 h. Although treatment with hyaluronidase was sufficient to cause changes in dextran penetration of the apical glycocalyx, our failure to reconstitute the matrix with additional hyaluronan alone suggests that exogenous hyaluronan is unable to incorporate into the glycocalyx unless another glycosaminoglycan is present. This supports earlier observations of the behavior of chondrocytes in culture (15, 19) and may be due to the fact that, once released from the cell, circulating hyaluronan molecules assume random coiled meshworks that become highly hydrated (17). This hydrated coil conformation may limit interaction with molecules already present on the cell surface or recognition by binding sites on hyaluronan receptors (4). However, in the presence of chondroitin sulfate, this random coil extends to form complexes (19, 27). Although the accepted mechanism by which chondroitin sulfate interacts with hyaluronan is via the core protein in chondroitin sulfate proteoglycans, interaction between the carbohydrate side chains is also possible, although both glycosaminoglycans are highly negatively charged (27). Scott (25) has proposed that the driving force behind such an interaction in the absence of a core protein results from the presence of hydrophobic patches in the secondary structure of these glycosaminoglycans. Mutual attraction of these domains leads to an ordered aggregation that opposes the electrostatic repulsion of each molecule. Thus hyaluronan and chondroitin sulfate aggregates may incorporate into the glycocalyx more readily than coiled hyaluronan molecules alone. The reconstitution of the surface matrix that we observed using hyaluronan premixed with chondroitin sulfate chains supports this hypothesis.

Our data are also consistent with the idea that newly synthesized hyaluronan is constantly extruded into the glycocalyx (22) and replaces what is degraded by circulating hyaluronidase. Heldin and Pertoft (11) showed that the coats around human mesothelial cells pretreated with Streptomyces hyaluronidase regained full size after 5 h in culture. We hypothesize that in the presence of excess circulating hyaluronan, a type of competitive binding of hyaluronidase molecules will occur and nascent, cell-surface hyaluronan will be spared from degradation, resulting in a net resynthesis of the layer. In this case, the apparent reconstitution that we observed could be the result of ongoing extrusion of hyaluronan from the basal layer of the glycocalyx toward the lumen rather than binding of circulating hyaluronan/chondroitin sulfate complexes. The fact that we did not observe reconstitution with exogenous hyaluronan alone could be attributed to its removal by scavenger receptors in the liver (7, 15, 26) so that competitive binding of circulating hyaluronidase molecules could not occur. However, the infused hyaluronan/chondroitin sulfate complexes may have prevented recognition and endocytosis by scavenger receptors and allowed the complexes to circulate long enough to saturate the hyaluronidase and spare nascent hyaluronan chains.

We also found that addition of the hyaluronan/chondroitin sulfate complexes to the intact glycocalyx did not increase the size of the matrix above the control. This might suggest that there is a size limit (~0.4-0.5 µm) to the glycocalyx. A mechanism responsible for this size limit is unknown, but given the deformation of the glycocalyx that occurs each time a white blood cell passes, the surface layer of the glycocalyx could be constantly sheared off while resynthesis of matrix from the basal region occurs. Alternatively, synthetic rate by the endothelium may limit the matrix size and thus prevent an increase in vascular resistance to blood flow.

Taken together, these findings suggest that the apical endothelial cell glycocalyx is a highly complex structure composed of molecules with distinct functional roles and that it cannot be treated as a single entity. Hence, to comprehend the significance of such a structure at the endothelial-blood interface, one must first decipher the functions of its constituent parts. This is by no means an easy task, since in vivo visualization of the glycocalyx with conventional light microscopy is extremely difficult because of its small size (<1 µm) and low ratio of organic material to water. Attempts to visualize its ultrastructure via electron microscopy are usually defeated by dissolution of the glycoconjugates during aqueous fixation, followed by dehydration and collapse during routine tissue processing. However, if the glycocalyx can be labeled and stabilized in situ before fixation for electron microscopy, the structural relationships between its various components will be more readily clarified.