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1Department of Bioengineering, Proteins and Polymers at Interface Group, University of Utah, Salt Lake City; and 2Department of Anesthesiology, Lung Vascular Biology Laboratory, and 3Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah School of Medicine, Salt Lake City, Utah
Submitted 3 October 2006 ; accepted in final form 30 April 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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diffusion; permeability
Its role in capillary barrier function has been its most studied attribute, and the general consensus is that the glycocalyx forms a molecular filter overlying the intercellular junction, creating the primary determinant for both water and solute flux into the cell-cell junction (1, 2). Support for the molecular filter hypothesis developed from the observation that serum protein concentration influenced ferritin penetration into the glycocalyx and reduced the loading of luminal vesicles with ferritin (16). These observations were explained by serum proteins adsorbing onto the glycocalyx and forming a protein-matrix barrier to both diffusion and convection of macromolecules to the cell surface. Based on these findings, it was assumed that the glycocalyx could sieve macromolecules from entering the intercellular junction. Likewise, numerous studies have shown that capillary hydraulic conductivity is strongly dependent on perfusate protein concentration, suggesting that protein adsorption onto the glycocalyx decreases transendothelial fluid flux in vivo (15, 18, 21) and in vitro (8, 19). Interventions that directly alter the structure of the glycocalyx, such as enzymatic degradation, result in increased hydraulic conductivity, adding additional support to the fiber-matrix hypothesis (1, 2).
More recently, in vivo studies have examined the penetration of the glycocalyx by fluorescently labeled dextrans of varying sizes, charge, and fluorophore labels. Although generalized trends were observed such that neutral dextrans penetrated the glycocalyx in a size-dependent manner, inconsistencies in the apparent penetration rate were observed depending on the fluorophore label (32). Similarly, albumin and fibrinogen had similar entry rates despite markedly different molecular masses and overall shape. Collectively, these observations bring into question the notion that the glycocalyx behaves as a simple filter and highlights the need for high resolution evaluation of mass transport into and within the endothelial surface layer under highly defined conditions.
In this study, we obtained novel biophysical determinants of albumin behavior within the region believed to be occupied by the endothelial glycocalyx, using fluorescence correlation spectroscopy (FCS) (17, 28), which measured the diffusivity and local concentration of human serum albumin. Our measurements were made directly above intercellular junctions of cultured lung microvascular endothelial cells, since this area would be most relevant for paracellular transport. The methodologies presented provide the highest spatial and temporal resolution to date for examining macromolecular interactions with the glycocalyx in vitro and further our understanding of the role of the glycocalyx in lung endothelial cell function.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Bovine serum albumin (30% BSA solution), type XIV bacterial pronase from Streptomyces griseus (EC 3.4.24.31 [EC] ), heparin lyase III from Flavobacterium heparinum (heparinase III; EC 4.2.2.8 [EC] ), and hyaluronate lyase from Streptomyces hyalurolyticus (hyaluronidase; EC 4.2.2.1 [EC] ) were obtained from Sigma-Aldrich (St. Louis, MO). Other materials included fraction V fatty acid-free human serum albumin (HSA) from ICN Biomedicals (Mp Biomedicals, Irvine, CA) and AlexaFluor 532 carboxylic acid succinimidyl ester (AF532; Molecular Probes, Eugene, OR). HSA was labeled with AlexaFluor 532 carboxylic acid succinimidyl ester to a near 1:1 molar protein-fluorophore ratio.
Methods
Cell culture. Bovine lung microvascular endothelial cells (BLMVEC) and MCDB-131 complete cell medium were obtained from Vec Technologies (Rensselaer, NY). Clean round glass coverslips (1-in. diameter, 1.5-in. thickness) were coated with 0.2% type B bovine gelatin for 1 h at 37°C, followed by exposure to 30 µg/ml bovine fibronectin solution in serum-free MCDB-131 for another hour. Cells were used at 7 days postplating, and measurements were made from confluent monolayers.
Fluorescence correlation spectroscopy. All fluorescence fluctuation measurements were performed using a custom-built FCS instrument (34). A 532-nm laser beam (Nd:YAG 75 mW laser; CrystaLaser, Reno, NV) was collimated and directed into the epi-port of a Nikon Diaphot 200 inverted microscope with a Nikon x100, 1.4 NA oil Plan Apochromat objective and a dichroic filter (Chroma Q565LP; Chroma, Rockingham, VT). Emitted fluorescence was collected by the same objective, passed through a 532-nm notch filter (Kaiser Optical, Ann Arbor, MI), and imaged onto a pinhole positioned at the objective conjugate plane. A fiber optic (OZ Optics, Carp, Ontario, Canada) collected the photons passing through the pinhole. The fiber optic split the collected light evenly and sent it to two avalanche photodiodes (PerkinElmer, Wellesley, MA), thus enabling cross-correlation analysis. Single photons created transistor-transistor logic pulses that were registered by a Flex99/160 hardware correlator (http://correlator.com). FCS samples were placed on an MFC-2000 xyz microscope stage (ASI, Eugene, OR) with a fine focus controlled by a computer.
Cell monolayers were washed in phenol red-free DMEM, covered with AF532-HSA solution (1.24 µg/ml), and sealed into a custom-made perfusion chamber. A z-axis FCS scan was performed by stepping the focus in 100-nm increments: a typical z-axis profile was carried out over a cell-cell junction starting from the coverslip, through the cell, and into the solution above the cell (n = 6–9 different cells for each run).
Three separate sets of FCS data were acquired: 1) from control cells, 2) by measuring competitive displacement of AF532-HSA using excess unlabeled bovine serum albumin (BSA), and 3) by measuring interactions of AF532-HSA within the glycocalyx after three different enzymatic degradations of the glycocalyx. For excess BSA experiments, 5 mg/ml BSA solution in DMEM was added to the cells 30 min before FCS data collection. In the enzyme experiments, cell monolayers were first treated with the enzyme solution before DMEM wash, addition of AF532-HAS, and FCS data collection. Pronase, a broad-spectrum protease, was used at 0.01 mg/ml in MCDB-131 for 5 min, because longer exposure times caused the cells to detach from the glass coverslip. Heparanase III was diluted to 15 mU/ml (Sigma units), and hyaluronidase was diluted to 50 IU/ml, both in MCDB-131. Each enzyme solution was placed on the cells for 1 h. In controls, z-axis profiling was performed on cells with no AF532-HSA and on coverslips without cells in the presence of AF532-HSA.
Fluorescent fluctuations in time, 
F(t), are defined as
 | (1) |
F
is the average fluorescence intensity. The normalized autocorrelation function G(
) results from correlating a signal against itself:
 | (2) |
), for a molecule diffusing through a Gaussian shaped 3-D volume illuminated with excitation intensity, is given by
 | (3) |
D is the characteristic time of their diffusion, G
is equal to one (9, 29), and S represents the ratio of axial and radial distances at which the intensity of the Gaussian excitation falls to 1/e2 of its maximum. S was determined experimentally to be 
10 by using diffusion of rhodamine-6G (D = 2.8 x 10–6 cm2/s) (29). Experimental FCS data were fitted to Eq. 1 using Levenberg-Marquardt nonlinear least-squares optimization in IGOR Pro (WaveMetrics, Lake Oswego, OR). Each fit resulted in a pair of parameters, N and D. These were averaged for every z-distance, and the standard error of the mean was computed. Confocal microscopy. Cells were cultured on glass coverslips as described above. Monolayers were washed three times with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde for 30 min at room temperature. Heparan sulfates were immunostained with HSS-1 (US Biologicals, Swampscott, MA) at a concentration of 9.5 µg/ml for 1 h at room temperature, washed with PBS, and incubated with labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA). Hyaluronan was localized using the combination of biotinylated hyaluronan-binding protein (Associates of Cape Cod, East Falmouth, MA) and FITC-labeled anti-biotin antibody (Jackson ImmunoResearch). Images were obtained using a Fluoview 300 confocal microscope (Olympus, Melville, NY) equipped with a PLAPON x60 oil objective. Three-dimensional rendering and image analysis were performed using Volocity (Improvision, Lexington, MA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The z-axis profile for AF523-HSA concentration in an aqueous protein solution with no cells, N(z)aqsol, was subtracted from the AF523-HSA concentration profile in the presence of the cell monolayer, N(z)cells, and is given by 
N = N(z)cells – N(z)aqsol (Fig. 1B). Note the fivefold increase in N occurring between 1.5- and 2.5-µm distances measured above the glass surface. The characteristic diffusion time of AF523-HSA, D(z), is shown in Fig. 1A. The D(z) in an aqueous solution corresponded to the free albumin diffusion coefficient, DHSA = 6 x 10–7 cm2/s. In the presence of the cell monolayer, D(z) values increased by 50% over the same distance range, where N increased (i.e., from 1.5 to 2.5 µm above the glass interface) (Fig. 1B). This suggests the presence of a structure that both concentrates albumin molecules and slows their diffusion (albumin diffusion coefficient dropped to 4 x 10–7 cm2/s). Below the 1-µm distance, N and D increased for both the control (coverslip without cells) and cell monolayer sample because of the nonspecific accumulation of albumin to the glass-cell interface.
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To test whether the changes in N and D(z) shown in Fig. 1 were due to interactions of AF523-HSA with the glycocalyx, we applied an excess (5.0 mg/ml) of unlabeled BSA to the cells before adding AF532-HSA. The effects of excess unlabeled albumin are shown in Fig. 2B. Two N curves are shown: the difference between control N(z)cell and N(z)aqsol and the difference between AF532-HSA concentration from cells bathed in saturating unlabeled BSA, N(z)cell+BSA, and AF532-HSA in an aqueous solution, N(z)aqsol. The N profile without excess albumin (open squares) practically retraces the control N profile shown in Fig. 1B. The saturation of glycocalyx with unlabeled BSA caused N to drop slightly below zero from 1.5 to 2.5 µm above the glass interface, indicating that the concentration of AF523-HSA in this region was smaller than in the control sample (with no cells and no excess of BSA added).
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D(z), within the glycocalyx (Fig. 2A). AF532-HSA diffusion was somewhat faster when the glycocalyx was saturated with unlabeled BSA compared with control cells, indicating that BSA has possibly displaced glycocalyx-associated AF532-HSA. However, the shallow maximum of D(z) shifted by 0.5 µm into the region between 2.5 and 3.0 µm, so it is also possible that excess BSA concentrations might have swelled the glycocalyx and thus caused an outward shift. The actual AF532-HSA fluorescence counts were 16% lower in the systems saturated with BSA relative to the respective values for AF532-HSA in controls with cell monolayer but no BSA (data not shown). Pronase Treatment
Enzymatic digestion of the glycocalyx with pronase resulted in moderate changes in the AF532-HSA concentration z-profile; N(z) was below the level seen in controls (no pronase) and was also reduced to a level lower than N(z) of the aqueous control (Fig. 3B). The fluorescence counts from AF532-HSA in the pronase-treated glycocalyx were 16% lower than in control experiments (untreated glycocalyx; data not shown). The changes in N(z) suggest that protein accumulation with glycocalyx has been reduced with pronase treatment. Variation of the characteristic diffusion times for AF532-HSA within the pronase-treated glycocalyx (Fig. 3A) displayed a slightly shorter D(z) than in controls and a flatter D(z) profile.
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Enzymatic digestion of the glycocalyx with heparanase III resulted in an increase of N at smaller distances compared with the untreated cells and also diminished the N maximum found for untreated cells at 2.0- to 2.5-µm distances (Fig. 4B). The concentration of AF532-HSA in the heparanase-treated glycocalyx was 25% higher than that of AF532-HSA in solution between 0.5 and 2.0 µm and 15% lower than that of HSA in an untreated glycocalyx between 2.0 and 2.5 µm. The characteristic diffusion times for AF532-HSA in the heparanase-treated glycocalyx (Fig. 4A) were slightly longer than in the solution control, but heparanase reduced the maximum in D, making the D(z) profile flatter and similar to that for pronase treatment.
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Digestion of the glycocalyx with hyaluronidase resulted in large changes in both N(z) and D(z). Hyaluronidase treatment completely eliminated any variation in N(z) and D(z) over the entire range of z-distances above the cell surface. N was reduced to levels lower than those found in the protein solution controls at all distances measured (N < 0, Fig. 5B). The characteristic diffusion times after hyaluronidase treatment, D(z), were no different than the D values found for the aqueous solution of AF532-HSA with no cells (Fig. 5A).
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Immunofluorescent imaging of heparan sulfates revealed a dense heparan sulfite layer across the surface of the cell (Fig. 6A). The thickness of the heparan sulfate layer was 2.8 ± 0.5 µm (n = 10). Hyaluronan localization also revealed a dense layer of staining across the surface of the cell (Fig. 6B). The thickness of the hyaluronan layer was 3.1 ± 0.4 µm (n = 10). Confocally derived glycocalyx thickness was nearly identical to FCS measurements of the glycocalyx.
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DISCUSSION |
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FCS Limitations
FCS spatial resolution is defined by the dimensions of the observation volume, which in turn is defined by the microscope optics. The observation volume, with an ellipsoidal shape extending 1.0 µm in the z-axis and 0.2 µm in the x- and y-axes, was stepped incrementally in the z-direction through the cell and into the aqueous solution above the cell. Since the glass coverslip surface is the only fixed reference in the z-direction, one has to establish where along the z-axis the cell membrane exists and where the glycocalyx begins. Pesen and Hoh (24) used atomic force microscopy and confocal fluorescence microscopy to study live bovine pulmonary artery endothelial cells in vitro and found that the thickness of these cells at the cell-cell junctions was 0.5 µm. Accordingly, we assigned the fluorescence collected below this z-level to both the glass-cell interface and to the cell body. At distances greater than 1.5 µm from the glass coverslip surface (i.e., 0.5 µm of cell thickness + 1 µm of optical resolution in z-direction), any changes in AF532-HSA concentration (N) and characteristic diffusion time (D) were attributed to structures that are above the cell membrane.
FCS z-profiles demonstrated that albumin diffusion, D(z), and its concentration, N, both increased within the same region located at a distance of 1.5 to 2.5 µm above the glass-cell interface. We attribute these effects to the glycocalyx structure; it reduced albumin's characteristic diffusion times by 30% and increased local concentrations of AF532-HSA fivefold. The variations of N(z) and D(z) were wide and symmetrical as expected from the broadening caused by 1-µm optical z-resolution. This optical broadening prevented us from deriving a precise thickness measurement of the glycocalyx structure; however, its location well above the cell surface was not affected by optics. Assuming the cell thickness was 0.5 µm, the structure responsible for albumin interactions was located between 1.0 and 2.0 µm above the endothelial cell membrane. The experimental results (Fig. 1A) also indicate that albumin diffusion was largely unhindered between the cell surface (at 0.5 µm from the glass-cell interface) up to a distance of 1.5 µm. These results rule out the possibility that the observed effect was due to albumin diffusion inside cytoplasmic vesicles (35).
The presence of excess unlabeled BSA (5 mg/ml) reduced AF532-HSA concentrations within the region attributed to the glycocalyx to a level below that found in the aqueous solution and reduced D(z) over the range of 1.5 to 2.5 µm above the glass surface. Between 2.5 and 3.0 µm, however, D(z) increased slightly. There appears to be two distinct mechanisms that explain the dynamic behavior of labeled albumin as assessed using FCS. In the initial experiments, cell monolayers were washed with protein-free medium and the FCS experiments were performed with tracer albumin alone, where the tracer albumin concentration was 1.24 µg/ml. The goal of these experiments was to assess the dynamics of albumin within the glycocalyx without the confounding influence of other proteins (including albumin itself). Tracer albumin dynamics were then reevaluated in the presence of excess unlabeled albumin, with the hope of distinguishing the role of binding versus hindered diffusion. Clearly, in the absence of unlabeled albumin, tracer albumin dynamics suggest that albumin interacts with components of the glycocalyx.
In the presence of saturating unlabeled albumin, very different tracer dynamics can be resolved with the FCS. Excess unlabeled albumin reduced tracer concentration within the region of the glycocalyx to levels below that of the aqueous media bathing the cells. The only biophysical interpretation of these data is that the saturating unlabeled albumin altered the structure of the glycocalyx in a manner that allowed it to exclude tracer entry. These observations closely parallel the effects of albumin on capillary permeability, as reported by Huxley and Curry (13). Briefly, they examined the effect of a stepwise reduction in albumin concentration on capillary permeability as albumin was reduced over a range from 0.1 to 0.001 g/dl. They observed that capillary hydraulic conductivity (Lp) remained constant until perfusate albumin levels were very low (below 0.01 g/dl), after which Lp increased. Therefore, even very low concentrations of albumin can maintain glycocalyx barrier function at concentrations that would be the equivalent of clinical hypoproteinemia. The authors provided two interpretations for these data: 1) albumin "may occupy sites within a network of the fiberous molecules within the water pathway" (meaning within the glycocalyx), and 2) albumin cross-links the fibrous glycoproteins within the glycocalyx to reduce the overall free space (i.e., reduces porosity), as hypothesized by Curry and Michel (4, 20).
Ultrastructural evidence for the effects of plasma proteins, including albumin, on glycocalyx function can be found in the work of Adamson and Clough (2). They examined the effects of plasma proteins and albumin on the binding of cationized ferritin (CF) to the endothelial surface layer. In vessels perfused with serum, the CF layer was widely separated from the plasma membrane and no CF was observed within the region attributed to the glycocalyx. When the vessels were perfused with protein-free Ringer solution, the CF layer appeared to reside on the plasma membrane and CF was seen in vesicles. Finally, when the vessels were perfused with a solution of albumin, the CF layer was located at an intermediate distance between the plasma membrane and the outer region observed when full serum was used to penetrate the vessel. The authors concluded that plasma proteins, including albumin, modify the configuration of the glycocalyx to prevent CF penetration.
We can suggest a comparison between FCS-derived albumin dynamics with observations of Huxley and Curry (13) and Adamson and Clough (2). In our experiments, the tracer concentration of labeled albumin (AF532-HSA) was 1.24 µg/ml (essentially zero) and clearly well below the albumin concentration required to maintain barrier properties (e.g., 0.01 g/dl). Thus we expect that this negligible concentration of tracer albumin 1) promotes little, if any, cross-linking of matrix elements and that 2) the tracer albumin occupies very little of the diffusion space within the glycocalyx matrix. However, 5 mg/ml (i.e., 0.5 g/dl) of excess unlabeled albumin is more than enough to both occupy weak binding sites and alter the glycocalyx structure such that it increases barrier function, as evidenced by the ability to exclude tracer albumin to a concentration less than that found in the aqueous media above the cell. An albumin concentration of 5 mg/ml is consistent with hypoproteinemia in the clinical setting, yet this scant concentration of unlabeled albumin was able to modify the glycocalyx to such an extent that it completely excluded tracer albumin from gaining entry. In fact, we reported that albumin concentration markedly influences Lp of cultured endothelial cells by showing a 10-fold decrease in Lp between 0 and 1% BSA (8). These data support the hypothesis that cultured endothelial cells possess a functional glycocalyx and that monolayer permeability is protein dependent and therefore very similar to the in vivo capillary.
Enzyme Studies
The primary goal of enzymatic degradation experiments was to identify components of the glycocalyx that contributed to the dynamic behavior of albumin. Relatively little is known about the structural determinants of the glycocalyx, although heparan sulfate and hyaluronan are believed to be important components. We have previously shown that syndecan, a major heparan sulfate proteoglycan on endothelial cells, is localized to the cell periphery (7) and could spatially contribute to barrier properties of the glycocalyx over the cell-cell junction. Hyaluronan has been shown to significantly influence permeation of the glycocalyx to fluorescently labeled dextrans (11), suggesting that it may also play a major structural role. Therefore, we examined the effects of three enzymes, pronase, heparanase III, and hyaluronidase, on FCS-observed glycocalyx-albumin interactions.
Pronase (protease E), a broad-spectrum protease, has been used to digest the capillary glycocalyx in vivo, where it significantly increased Lp, but without altering the dimensions of the intercellular junction (1), suggesting that the change in permeability was due solely to structural alterations of the glycocalyx. Chang (3) tested the effect of various concentrations of pronase on cultured endothelial cell permeability and reported that Lp increased threefold with low concentrations of pronase (0.10–0.125 mg/ml), whereas albumin diffusion was unaffected; at higher concentrations (0.20 mg/ml), both Lp and albumin diffusion increased. Collectively, these data suggest that a graded removal of the glycocalyx can be accomplished and that this cell surface layer provides significant resistance for both water and protein entrance into the cell-cell junction.
Pronase treatment reduced AF532-HSA concentration at distances of 1.25 to 2.0 µm to a level slightly below that found in protein solution. Pronase also slightly affected D(z); however, these effects were barely outside the measurement errors (Fig. 3). The uncertainty about the effects of pronase treatment is most likely due to the inability to use higher enzyme concentrations or longer treatment times. Attempts to use higher concentrations of pronase caused the cells to detach from the glass coverslip, even at relatively short incubation times. Therefore, we selected a lower enzyme concentration (0.01 mg/ml) and an exposure shorter than that for other enzymes (5 min), and the minimal alterations in FCS parameters likely reflect limited removal of glycocalyx structure.
Heparanase III digestion of the glycocalyx reduced the maximum in N and shifted the N profile closer to the cell membrane (Fig. 4). The D(z) profile was flattened and also shifted inward. There was a notable increase in D(z) from distances of 1.0 to 1.5 µm, indicating that albumin diffusion was slower at distances closer to the cell surface compared with controls. It appears that heparanase III digestion reduced the ability of the glycocalyx to exclude albumin above the cell surface and allowed more AF532-HSA into lower regions of the glycocalyx. The heparanase-induced increase of D(z) closer to the cell surface suggests that the glycocalyx may have partially collapsed into this region or that the loss of heparan sulfates unmasked previously inaccessible binding sites for albumin.
Digestion of the glycocalyx with hyaluronidase had the most marked effect on FCS-derived parameters. Hyaluronidase reduced N to values below that of the protein solution (Fig. 5), suggesting a complete abolition of albumin accumulation over the entire z-axis. In addition, hyaluronidase treatment reduced D(z) to the characteristic diffusion times found in aqueous solution, suggesting that hyaluronidase treatment disrupted all albumin binding and/or eliminated hindrance to albumin diffusion at all distances above the cell membrane. In fact, the data suggest that hyaluronidase completely eliminated albumin-glycocalyx interactions; as far as albumin is concerned, hyaluronan appeared to be a major structural component of the lung capillary glycocalyx in vitro.
To validate the biophysical measurements of the glycocalyx thickness derived by FCS/albumin profiling, we obtained 3-D confocal images by immunostaining for heparan sulfate and hyaluronan on the endothelial surface. Immunostaining for both glycosaminoglycans revealed a dense surface layer that was 3.0 µm in thickness, consistent with our FCS measurements. There has been considerable debate about the existence, structure, and function of the glycocalyx found on cultured endothelial cells and whether it is comparable to the glycocalyx found in vivo. Our results, utilizing FCS and confocal microscopy, demonstrate that 1) cultured endothelial cells possess a significant glycocalyx layer; 2) the glycocalyx is at least as thick as the endothelial surface layer measured in vivo (32, 33); 3) functionally, the in vitro glycocalyx can alter albumin dynamics to increase albumin concentration fivefold; and 4) heparan sulfates and hyaluronan are present as a 2- to 3-µm-thick layer covering much of the cell surface.
Summary
In this study we have used albumin as a dynamic molecular probe of glycocalyx function; hence, our model of the glycocalyx is mechanistically based on those structural attributes that affect albumin diffusion and local concentration. Since albumin is the major serum protein and is responsible for 70% of the oncotic pressure of plasma, we believe that the choice of albumin as a probe is important: any characterization of the structure-function relationship of endothelial glycocalyx must account for its ability to interact with albumin. FCS provided the highest spatial and temporal resolution possible to make detailed measurements of fluorescently labeled analytes using live cells. We acknowledge that there is likely a fine structure to glycocalyx that exists between the cell surface and the region where FCS effects were observed, a region that does not hinder albumin diffusion and, therefore, was not measurable in our experiments. We are currently conducting experiments to examine dynamic behavior of a wider range (in terms of molecular sizes) of fluorescently labeled proteins within the glycocalyx region.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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increases entry of macromolecules into luminal endothelial cell glycocalyx. Am J Physiol Heart Circ Physiol 279: H2815–H2823, 2000.
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