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Am J Physiol Heart Circ Physiol 287: H2287-H2294, 2004. First published July 15, 2004; doi:10.1152/ajpheart.00808.2003
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Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

Akinori Ueda,1 Manabu Shimomura,2 Mariko Ikeda,3 Ryuhei Yamaguchi,5 and Kazuo Tanishita4

1School of Fundamental Science and Technology, 2Institute of Biomedical Engineering, 3Keio Leading-Edge Laboratory of Science and Technology, and 4Department of System Design Engineering Keio University, Yokohama 223-8522; and 5Department of Mechanical Engineering, Shibaura Institute of Technology, Tokyo 108-8548, Japan

Submitted 21 August 2003 ; accepted in final form 13 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

blood flow; shear stress; glycocalyx charge; glycocalyx thickness; in vitro model

ATHEROSCLEROTIC LESIONS appear in regions of low shear stress in relatively large arteries, such as the carotid bifurcation and the coronary artery (3). Atherosclerosis is initiated by the uptake of low-density lipoprotein (LDL) (24), which is highly associated with hemodynamic stress. Some studies demonstrate that the transport of macromolecules such as albumin across the cell membrane is strongly affected by the shear stress on endothelial cells (17, 27). Kudo et al. (11, 12) measured the effect of shear stress on albumin uptake into endothelial cells in vitro and reported an increased uptake at lower shear stress and a decreased uptake at higher shear stress. However, the mechanism of this biphasic response of uptake remains unclear.

The endothelial cell surface is characterized by various extracellular domains of membrane-bound molecules, the glycocalyx (20), which can sense the shear force of flowing blood (9, 15). Luft (14) visualized the endothelial glycocalyx layer in vitro using ruthenium red staining in an electron microscopic study and found that the glycocalyx layer is about 20 nm thick. Subsequent in vitro electron microscopic observations of the molecules revealed that the glycocalyx thickness is <100 nm (26). In vivo studies (19, 21) have revealed thicker glycocalyx layers ranging from 0.5 µm to over 1.0 µm. This difference in thickness is due to the preparation and staining techniques, which cause the collapse of the glycocalyx structures (14, 20, 32, 33).

The glycocalyx surface forms a complex three-dimensional array of soluble plasma components, including many proteins and solubilized glycosaminoglycans (GAG). Thus the glycocalyx layer restricts the flow of plasma and denies access of red blood cells and macromolecules to the vascular wall (4, 20, 33). Therefore, the glycocalyx layer is important to the interaction between blood and endothelium. Its properties, such as capillary barrier function and interaction with plasma components, have been studied (20). The glycocalyx consists of protein, glycolipid, and proteoglycans, including exposed charged groups, and also contains membrane-bound molecules, such as selectins and integrins (22, 25), involved in immune reactions and inflammatory processes (13, 18, 29).

The intracellular uptake of macromolecules is possibly regulated by glycocalyx properties, such as surface charge and morphological barriers. The glycocalyx surface has a negative charge because the glycocalyx has some acidic mucopolysaccharide sidechains (GAG), which contain many carboxyl and sulfate groups. Most plasma proteins, such as albumin, in the glycocalyx surface have a negative charge. Thus the glycocalyx layer repulses the anionic proteins electrostatically. Previous studies show increased permeability when glycocalyx or albumin is neutralized (30, 34).

The thickness of the glycocalyx layer might be associated with shear stress. Haldenby et al. (10) report that the glycocalyx thickness depends on the local region of the vessel and thus might be associated with shear stress because shear stress differs according to the type of blood vessel. In addition, Wang et al. (36) reported that the glycocalyx layer was thin in low-shear regions such as sidewalls of the bifurcation and thick in high-shear regions such as a divider of the bifurcation.

Here, we studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. We measured the albumin uptake into cultured endothelial cells with imposed shear stress stimulus and visualized the morphology of the glycocalyx layer, such as thickness. To determine the effect of surface charge of the glycocalyx layer on the uptake, we measured the albumin uptake for a neutralized glycocalyx layer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Cultured bovine aorta endothelial cells (BAECs; lot no. 32010, Cell Systems) were used in all experiments. Specimens for the albumin uptake measurements were prepared as follows. BAECs were seeded in 25-cm2 culture flasks (3014, Falcon) and cultured in DMEM (31600-34, GIBCO-BRL) containing 10% FBS (lot. 9K2087, JRH Biosciences), 1% antibiotic-antimycotic (15240-062 GIBCO-BRL), and 15 mM HEPES (346-D1373, DOJINDO). The culture flasks were maintained at pH 7.3 and 37°C in a CO2 incubator. BAECs of passages 5–9 were used for the uptake measurements. Specimens for the observation of the glycocalyx layer using electron microscopy were prepared as follows. BAECs were seeded on a glass dish (3910-035, IWAKI) or plastic-bottom dish (Falcon) coated with 2% collagen type IV (CELLMATRIX-4–20, Nitta Gelatin) and then used for observation when they reached confluency in 7–10 days. A glass-bottom dish was used because when BAECs are observed at high magnification the bottom of the dish must be thin with respect to the working distance. A plastic-bottom dish was used when BAECs were observed using an electron microscope.

Shear stress loading. The culture medium described above was used as the perfusate. A rectangular flow chamber (0.02 cm high, 2.0 cm wide, 1.20 cm long) (Fig. 1) was placed over the dish containing BAECs and then placed in the flow circuit (Fig. 1). The perfusate was circulated through the flow circuit by a roller pump (MP-3N, EYELA), thus exposing the BAECs to laminar flow (the pulsatile component was about ±5%). The total priming volume was 50 ml. The wall shear stress in the rectangular flow chamber, and thus the shear stress applied to the endothelial cells, was calculated as

(1)
where {tau} is wall shear stress (in Pa), µ is viscosity (8.5 x 10–4 Pa·s, measured by a rotational viscometer at 37°C), Q is flow rate (in cm3/s), h is flow channel height (0.02 cm), and b is flow channel width (2.0 cm). Table 1 lists the flow rate and calculated wall shear stress, which ranged between 0.5 and 3.0 Pa.



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  		Fig. 1. A: flow chamber installed inside a glass-base dish. B: cross section of assembled flow chamber showing endothelial cells (on coverglass) exposed to shear stress by a flow of perfusate.

 

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  		Table 1. Wall shear stress calculated from the measured flow rate

 
BAECs were exposed to the shear stress for 48 h. The perfusate was kept at 37°C using a water bath and was gassed with a mixture of 5% CO2-95% air to maintain pH 7.3 throughout the experiment.

Preparation of fluorescent albumin. The albumin uptake into the cells was determined by measuring the fluorescence of tetramethylrhodamin isothiocyanate-conjugated albumin (TRITC-albumin; A-847, Molecular Probes). To remove free TRITC dye, TRITC-albumin was centrifuged at 4°C eight times with an ultrafilter (CENTREX UFB-C30, IWAKI). The resulting TRITC-albumin was mixed with BSA (B-4287, Sigma) and dissolved in DMEM to reach a final concentration of 4.0 mg/ml (albumin concentration of 10% FBS).

Albumin uptake. Figure 2A summarizes the procedure for uptake measurements. After shear stress had been applied to the cells for 48 h, the flow chamber was switched to another flow circuit to determine the uptake of TRITC-albumin by perfusing TRITC-albumin for 1 h under the same shear stress. The flow chamber was then removed from the flow circuit and rinsed with fresh medium without TRITC-albumin for 5 min.



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  		Fig. 2. Schema of experimental procedures. A: procedure to measure albumin uptake into endothelial cells with and without applied shear stress. B: procedure to measure cell surface charge. TRITC, tetramethylrhodamin isothiocyanate.

 
The albumin uptake was measured using confocal laser scanning microscopy (MRC 600 system, Bio-Rad Laboratories), which involved an inverted microscope (TMD 300, Nikon) with an objective lens [x63 (numerical aperture 1.4) Plan-apo, Nikon]. The fluorescence of TRITC excited by the 488-nm spectral line of a 25-mW argon ion laser was detected at a wavelength longer than 515 nm. The scan rate was 512 lines/s, and each individual image was an average of three successive images. The fluorescence and phase-contrast images were acquired simultaneously as follows. The flow chamber was mounted on the stage of the invert microscope, and the fluorescent and phase-contrast images were acquired at five random locations in the specimen. Each set of image data consisted of 11 serial tomographic images in a vertical direction (height direction of the cell) at intervals of 1 µm. Simultaneously, the albumin uptake into the same cells (same lot and generation of cells) was measured without shear stress stimulus as control data.

Image analysis. The acquired fluorescence images were analyzed using image analysis software (NIH Image) on a personal computer (Macintosh G4, Apple computer). The fluorescence intensity of TRITC-albumin was determined using an integrated image from the 11 tomographic images (see Albumin uptake). The edges of BAECs were determined using phase-contrast images and then applied to the corresponding fluorescence image. The average fluorescent intensity for each individual cell was determined. Finally, the normalized uptake was calculated as the ratio of average intensity of an individual cell under shear stress conditions to that under static conditions.

Measurement of the thickness of stained glycocalyx layer structures using transmission electron microscopy. After shear stress loading, the structure of the glycocalyx layer was observed using transmission electron microscopy (TEM). After the loading, the BAECs on the plastic dish were washed in PBS(+) (05913, Nissui) and 0.2 M sodium cacodylate buffer (29810, TAAB) and then prefixed with 3.6% glutaraldehyde (EM Grade, 35330, TAAB) including 1,500 ppm ruthenium red and 0.2 M sodium cacodylate buffer for 24 h at room temperature. The BAECs were then rinsed with 0.2 M sodium cacodylate buffer with sucrose and were postfixed by 1.0% osmium tetroxide including 1,000 ppm ruthenium red and 0.2 M sodium cacodylate buffer for 3 h at room temperature. The BAECs were dehydrated using acetone and then embedded in Epon. The Epon samples were sectioned into ultrathin specimens (about 80 nm thick), which were then observed using TEM. Acquired images were analyzed using image analyzing software (NIH Image) on a computer (Macintosh G4, Apple computer) to determine the thickness of the glycocalyx layer. The thickness of the stained glycocalyx layer was estimated based on TEM images to determine the brightness along the line normal to the glycocalyx layer direction. In this estimation, we defined the edge of the stained glycocalyx layer as the position of middle brightness along the line as illustrated in Fig. 8. The thickness of the stained structure was measured at 30 points for each individual cell.



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  		Fig. 8. Determination of the thickness of the glycocalyx layer. The edge of the glycocalyx layer was defined by the position of middle brightness along the line shown in A and the thickness was determined by the distance between the points of middle brightness as shown in B. The brightness is expressed by 256 gradations.

 
Measurement of cell surface charge. After the shear stress loading, the charge of the glycocalyx layer was determined by measuring the amount of toluidine blue (Sigma) on the glycocalyx layer. Toluidine blue was used because it was cationic ions that combined with the negative charge on the glycocalyx layer; thus the charge on the cell surface is directly related to the amount of toluidine blue in the layer (31, 32).

After being loaded, the BAECs were washed in PBS(+) and 0.25 M sucrose (31365–1201, Junsei Chemical) and then stained by a mixture of 0.25 M sucrose and 0.001% toluidine blue for 1 h at 4°C. BAECs were washed in 0.25 M sucrose five times and then soaked for 30 min at 4°C in 0.1 mg/ml protamine sulfate (type X, P-4020, Sigma) to extract the toluidine blue, which combined with the glycocalyx. The toluidine blue was displaced by protamine sulfate because protamine sulfate has higher affinity for carboxyl groups and sulfate groups in the glycocalyx than toluidine blue.

To measure the amount of charge on the cells, we used Van Damme's method (32), and the amount of toluidine blue on the glycocalyx was estimated based on the absorption rate, which depends of the concentration of toluidine blue. First, the extract was transferred to quartz glass cuvettes to measure the absorbance of the extract by using a double beam spectrophotometer (U-3400, Hitachi) at an extinction wavelength of 640 nm. Next, the average absorbance for each individual cell was determined (average by measuring the fluorescence of the same sample 10 times).

Neutralization of glycocalyx charge. The glycocalyx was neutralized by adding protamine sulfate (type X, P-4020, Sigma) to the perfusate to a final concentration of 0.001 mg/ml. Protamine sulfate was used as a charge neutralizer because its isoelectric point is 10–12 (30).

After the shear stress was applied for 48 h, BAECs were exposed to the perfusate with protamine sulfate for 30 min to neutralize the glycocalyx. The flow circuit was then rinsed with fresh DMEM without protamine sulfate. BAECs were then exposed to the perfusate with TRITC-albumin, and finally the fluorescence intensity of TRITC was measured to determine the amount of albumin uptake.

Statistical analysis. Data are presented as means ± SD; n is the number of replicated experiments performed. Student's t-test was used to test for differences, which were considered significant at an error level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of shear stress on albumin uptake. Figure 3 shows the fluorescent and phase-contrast images of endothelial cells under applied shear stress up to 3.0 Pa for 48 h. Each fluorescent image is the integration of 11 serial tomographic images of endothelial cells. The phase-contrast images reveal the morphology changes of endothelial cells due to the applied shear stress. The polygonal shape of the cells under static conditions (no applied shear stress) became elongated in the flow direction, similar to previously reported results (6–8, 16).



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  		Fig. 3. Phase-contrast and confocal images of albumin uptake into endothelial cells with and without applied shear stress.

 
Figure 3 reveals the dependency of the albumin uptake on the applied shear stress. The distribution of fluorescent spots in individual cells at a shear stress of 1.0 Pa appeared to be denser than under static conditions, revealing enhanced albumin uptake by the applied shear stress. In contrast, the distribution of fluorescent spots at a shear stress of 3.0 Pa was clearly sparser compared with that under static conditions. Thus the albumin uptake was restricted when the shear stress exceeded 2.0 Pa.

To quantify the albumin uptake into the cells, we measured the brightness intensity of the fluorescent images of Fig. 3 using NIH Image analysis. Figure 4 shows the average brightness intensity of each image in Fig. 3. The relative light intensity (y-axis) is an indicator of the ratio of the albumin uptake with applied shear stress to that without shear stress. The albumin uptake (i.e., relative light intensity) increased by 16% at a shear stress of 1.0 Pa between shear stress conditions and static conditions, a statistically significant difference (P < 0.05). In contrast, at a shear stress of 3.0 Pa, the uptake decreased to 27% between shear stress conditions and static conditions, a statistically significant difference (P < 0.001).



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  		Fig. 4. Effect of shear stress on albumin uptake. The relative light intensity (y-axis) was used as an indicator of the normalized albumin uptake (ratio of the uptake with applied shear stress to that without shear stress). Five fluorescent images per dish were taken randomly, and the average fluorescent intensity for an individual cell was determined. Data are means ± SD; n = 6. Significant differences were evident between static conditions and applied shear stress of 1.0 Pa (*P < 0.05) and 3.0 Pa (**P < 0.001).

 
Effect of shear stress on morphology of the glycocalyx layer. Figure 5 shows a TEM image of a cross section of a representative endothelial cell under static conditions. The glycocalyx layer stained by ruthenium red appears on the surface edge of the endothelial cell. The brightness in the TEM image corresponds to the atomic weight of the detected substance, namely, the brighter the area, the higher the atomic weight. Therefore, the glycocalyx layer labeled with ruthenium red is dimmer. The image reveals that endothelial cells were uniformly covered by the glycocalyx layer. Figure 6 is a magnified view of the gap junction site between endothelial cells as indicated by a white arrow. The thickness of the glycocalyx layer slightly varies along the surface edge of each cell.



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  		Fig. 5. Transmission electron microscopy (TEM) image of the cross section of a representative endothelial cell covered with the glycocalyx under static conditions.

 


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  		Fig. 6. TEM image of the gap junction (white arrow) between representative endothelial cells covered with the glycocalyx under static conditions.

 
Figure 7 shows TEM images of the glycocalyx layer under various levels of applied shear stress. Although the glycocalyx layer showed a slightly uneven surface, the overall nature of the glycocalyx layer geometries illustrate the influence of applied shear stress. The glycocalyx layer under an applied shear stress up to 1.0 Pa was similar to that under static conditions. However, the glycocalyx layer under an applied shear stress above 2.0 Pa was 26% thicker than that under static conditions. The glycocalyx layer under an applied shear stress of 3.0 Pa was significantly thicker, 74%. Figure 8 shows the edge of the stained glycocalyx layer as the position of middle brightness along the line. Figure 9 shows the estimated layer thickness. The glycocalyx thickness is relatively constant for shear stress up to 2.0 Pa but 74% thicker at a shear stress of 3.0 Pa compared with that under static conditions (P < 0.05).



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  		Fig. 7. TEM images of glycocalyx layers (A) without shear stress (static conditions) and with shear stress of 0.5 Pa (B), 1.0 Pa (C), 2.0 Pa (D), and 3.0 Pa (E). Magnification was x20,000.

 


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  		Fig. 9. Effect of applied shear stress on glycocalyx thickness. Data are means ± SD; n = 5. A significant difference was evident between the static conditions and applied shear stress conditions only at 3.0 Pa (*P < 0.05).

 
Effect of shear stress on endothelial surface charge. Figure 10 shows the effect of applied shear stress on the endothelial surface charge determined from the amount of toluidine blue. The relative absorbance (y-axis) is an indicator of the surface charge of each individual cell and is defined as the measured absorbance divided by the number of cells (31). Similar to results for the glycocalyx thickness (see Effect of shear stress on morphology of the glycocalyx layer), the charge level (i.e., relative absorbance) remained relatively constant for shear stress up to 2.0 Pa and was 84% higher at a shear stress of 3.0 Pa compared with that under static conditions (P < 0.05).



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  		Fig. 10. Effect of shear stress on surface charge. Relative absorbance (y-axis) was used as an indicator of the surface charge of an individual cell and was defined as the measured absorbance divided by the number of cells. Data are means ± SD; n = 4. A significant difference was evident between static conditions and applied shear stress conditions only at 3.0 Pa (*P < 0.05).

 
Effect of neutralized surface charge on shear-dependent albumin uptake. Figure 11 shows the effect of applied shear stress on albumin uptake with the charge of the glycocalyx neutralized by protamine sulfate. Again, the same as in Fig. 4, the relative light intensity (y-axis) is used as an indicator of the normalized albumin uptake. The solid and open symbols indicate the albumin uptake with and without neutralized charge, respectively. The albumin uptake without neutralized charge was the same as in Fig. 4. The albumin uptake with the neutralized charge increased with applied shear stress except for a shear stress of 2.0 Pa and increased by 75% at 3.0 Pa compared with that without a neutralized charge.



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  		Fig. 11. Effect of shear stress on albumin uptake with and without the charge of the glycocalyx being neutralized. Data are means ± SD; n = 6 (without neutralized charge) and 3 (with neutralized charge). Significant differences were evident between conditions not neutralized and neutralized conditions at 0.5, 1.0 Pa (*P < 0.05), and 3.0 Pa (**P < 0.001). The relative intensity was defined as the ratio of albumin uptake with shear stress to that without shear stress.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Shear dependence of albumin uptake. This study identifies the dual response of albumin uptake to applied shear stress (Fig. 3). Kudo et al. (11) also mentioned the dual response of albumin uptake to applied shear stress. In their study, the albumin uptake increased by 30% at lower shear stress (1.0 Pa) compared with that under static conditions and decreased by 76% at higher shear stress (6.0 Pa). Our results are similar, with the only difference being in the experimental procedure. In the study by Kudo et al., albumin was transported into the cells under static conditions for 1 h after shear stress was applied for 48 h. They assumed that the cells have memories of previous experiences of imposed shear stress stimulus. In our study, the albumin was transported into the cells after shear stress was applied for 48 h, but the shear stress was still applied to the cells during the uptake process for 1 h. Our results therefore confirmed the reproducibility of the dual response of albumin uptake to shear stress stimulus and we provide additional information on the role of the glycocalyx in albumin transfer.

This dual response of albumin uptake is consistent with the lower-shear hypothesis proposed by Caro et al. (3), who postulated that increased transport of LDL under low shear stress initiates the formation of atherosclerotic lesions. The shear dependence of macromolecule uptake in the vessel wall has been reported (5, 28, 36, 37).

Change in stained glycocalyx structures. In our in vitro experiment, the glycocalyx layer labeled with ruthenium red appeared "fluffy" (Figs. 6 and 7) and covered the entire endothelial surface membrane at a thickness ranging from 20 to 40 nm.

The glycocalyx thickness tended to increase at a high shear stress of 3.0 Pa but remained relatively constant at a shear stress up to 2.0 Pa, indicating that the glycocalyx is sensitive to shear stress above 3.0 Pa. This is consistent with results reported by Wang et al. (36), in which the glycocalyx layer thickness at rabbit aortic bifurcation was thicker at high shear stress regions. Our results are also consistent with those by Baldwin et al. (2), in which the glycocalyx layer thickness of rabbit aortic endothelium was 20 nm, and those by Rostgaard and Qvortrup (23), in which the glycocalyx layer thickness of rat capillary of intestinal villus was 50 nm.

Arisaka et al. (1) reported that the application of shear stress to endothelial cells of thoracic aortas of pigs for 24 h increased both GAG and protein synthesis. Because proteoglycan consists of GAG and core protein, the increased thickness of the glycocalyx layer in our study is associated with the enlarged proteoglycan.

Change in surface charge. At a shear stress of 3.0 Pa, the glycocalyx thickness increased by 74% compared with that under static conditions (Fig. 8) and the surface charge level increased by 84% (Fig. 9). The similarity in these increases indicates that the shear dependency of glycocalyx thickness is associated with that of the charge levels. We measured the charge of glycocalyx layer by using Van Damme's method (32) and toluidine blue, which stained the glycocalyx and was transported into the intracellular space. The transport of toluidine blue hinders the identification of the charge distribution in glycocalyx. Such detrimental transport was avoided by doing the staining of glycocalyx at 4°C, because a low temperature restricts the metabolic and diffusion activities in cells.

Effect of neutralized charge on albumin uptake. In this study, protamine sulfate was used as an anionic neutralizer. Cytotoxic aspects of protamine sulfate had to be considered. We confirmed the viability of endothelial cells stained by protamine sulfate by doing a dye exclusion test with 1.0% trypan blue. Another problem of using protamine sulfate is that it might induce albumin permeability across the membrane. Swanson and Kern (30) showed that native albumin permeability across pulmonary endothelial cells increased with the addition of protamine sulfate, whereas cationic albumin permeability did not. Their findings indicate that protamine sulfate does not destroy cellular functions but binds to the anionic charges on the endothelium.

Conclusive summary. The albumin uptake into endothelial cells at a shear stress of 1.0 Pa increased by 16% and that at 3.0 Pa decreased by 27% compared with the uptake in cells under static conditions (no shear stress). The shear dependence of albumin uptake is highly associated with glycocalyx. At a shear stress of 3.0 Pa, the thickness of stained glycocalyx layer structures increased by 70% and the glycocalyx charge increased by 80% compared with those under static conditions. The albumin uptake at a shear stress of 3.0 Pa for a neutralized (no charge) glycocalyx layer was almost twice that of cells with a charged layer. The findings in our study reveal that glycocalyx influences the albumin uptake in endothelial cells at higher shear stress (3.0 Pa).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by Japanese Ministry of Education, Science, Sports and Culture Grant-in-Aid for 2002 Research Project of Academic Frontier "Biofluid and Biological Research for Generation and Development of Cardio-Vascular Disease and Biomedical Treatment."


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Ueda, Tanishita Laboratory, Keio Univ., 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan (E-mail: akinori{at}tani.sd.keio.ac.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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