Figure 1.
Restoration of HS after 24 h of shear exposure.
(A) Top: Phase contrast micrographs of confluent RFPEC monolayers show that they fail to undergo change in cell shape under shear stress. Bottom: Representative immunofluorescent images of HS under static and shear stress conditions showing boundary clustering at 30 min and restoration of coverage at 24 h. (B) MFI (n = 16 images), (C) Coverage, and (D) Radial distribution of HS. The zero-radius represents the center of cell. The boundary of each cell was outlined by ImageJ. Then, the radial profile plug-in automatically changed the borders to the best-fit circles (D inset) for subsequent analyses. Scale bar: 20 µm. *P<0.05; **P<0.01.
Figure 2.
Validation of the clustering and subsequent restoration of HS under shear stress using bovine aortic endothelial cells.
(A) Top: Phase contrast micrographs of confluent BAEC monolayer reveal a typical dynamic change in cell morphology from cobblestone (static control) to the elongated (fusiform) and oriented in the direction of flow. Bottom: Representative immunofluorescent images of HS under static and shear stress conditions. (B) MFI, (C) Coverage, and (D) Scattering distribution displays the average intensity along lines from the centroid to the boundary of cells (n = 10 cells). For each cell, 16 lines (D left insert) were selected to analyze the scattering distribution. We also showed the radial distributions (D right insert, plotted by normalized radius on the horizontal axis and normalized intensity on the vertical; n = 50 cells). No significant differences were found between radial distribution and scattering distribution at both static and 30 min (P>0.05) indicating the radial distribution is appropriate for cobblestone ECs. The clustering of HS at 30 min and restoring of HS at 24 h on BAECs are similar to the results on RFPECs showed in our previous [7] and the present study, respectively. Scale bar: 20 µm. *P<0.05; **P<0.01.
Figure 3.
Redistribution of CS after 24 h of shear exposure.
(A) Immunofluorescent images of CS under static condition and shear exposure for 30 min and 24 h. (B) MFI, (C) Coverage, and (D) Radial distribution of CS. Scale bar: 20 µm. *P<0.05.
Figure 4.
Redistribution of glypican-1 under shear stress.
(A) Immunofluorescent images show that shear stress induces clustering of glypican-1 at 30 min, and enhances the glypican-1 intensity at 24 h. (B) MFI, (C) Coverage, and (D) Radial distribution of glypican-1 (n = 80 cells). Scale bar: 20 µm. *P<0.05; **P<0.01.
Figure 5.
Synthesis and distribution of Syndecan-1 under shear stress.
(A) Immunofluorescent images show that shear stress induces the syndecan-1 increase after 24 h of shear exposure. (B) MFI, (C) Coverage, and (D) Radial distribution of syndecan-1. Scale bar: 20 µm. **P<0.01.
Figure 6.
Redistribution of caveolae/caveolin under shear stress.
(A) Immunofluorescent images of caveolin-1 at time points. (B) MFI, (C) Coverage, and (D) Radial distribution of caveolin-1. After 24 h exposure to shear stress, the MFI of caveolin-1 was increased, and its localization at the center of cells was enhanced. Scale bar: 20 µm. *P<0.05.
Figure 7.
The vertical spatial distribution of caveolae/caveolin under shear stress.
(A) Z-projection of the apical and basal stack. The interface between two substacks is the surface (layer) crossing the center of the cell edges. (B) MFI, (C) Coverage, (D) radial distribution in the apical stack and (E) the basal stack. Much more caveolin-1 was distributed in the apical stack than in the basal stack under shear stress, especially for 24 h. In the apical stack, the caveolin-1 concentrated more near the cell boundary under the static condition, and moved to the cell interior after exposure to shear stress for 24 h; in the basal stack, the caveolin-1 was distributed nearly uniformly under static conditions and at 30 min; the caveolin-1 near the cell boundary decreased after 24 h. Scale bar: 20 µm. *P<0.05.
Figure 8.
Redistribution of CTx-B labeled GM1 under shear stress.
(A) The ganglioside GM1 was labeled with fluorescent CTx-B after shear stress exposure. The arrows indicate the clustering of GM1. (B) MFI, (C) Coverage, and (D) Radial distribution of CTx-B. The zero-radius represents the center of cell. GM1 was found to be clustered and recruited after 30 min of shear exposure [7]. The distribution of GM1 recovered close to the static level after 24 h, although much of GM1 was still clustered. Scale bar: 20 µm. *P<0.05.
Figure 9.
Redistribution of actin cytoskeleton under shear stress.
(A) After flow application, the actin cytoskeleton was visualized with fluorescent phallotoxin. Blue arrows indicate the dense peripheral actin bands; white arrows indicate the stress fibers; and yellow arrows and red arrowheads denote the filopodia and lamellipodia, respectively. (B) MFI, (C) Coverage, and (D) Radial distribution of F-actin. F-actin was found most densely distributed along the edges of EC under static conditions, while fluid shear stress induced the polymerization of actin, the polarization of actin filaments (30 min), and the formation of stress fibers (24 h). Scale bar: 20 µm. *P<0.05; **P<0.01.
Figure 10.
The vertical spatial distribution of actin cytoskeleton under shear stress.
(A) F-actin in the apical and basal stack. The interface between two substacks is the plane crossing the center of the cell edges. Blue arrows indicate the dense peripheral actin bands; white arrows indicate the stress fibers; and yellow arrows and red arrowheads denote the filopodia and lamellipodia, respectively. (B) MFI, (C) Coverage, (D) radial distributions in the apical stack and (E) the basal stack. The polarized actin filaments were distributed slightly more in the basal stack than in the apical stack initially and after shear for 30 min. After exposure to shear stress for 24 h, the stress fibers were well assembled in the apical stack, but not the basal stack. In the apical stack, the actin distribution was still concentrated near the cell boundary after 30 min of shear exposure, and became more uniform after 24 h, compared to the static conditions; in the basal stack, the distribution was more uniform at all times. Scale bar: 20 µm. *P<0.05.
Figure 11.
Redistribution of HS in the presence of cytochalasin D (CD).
Cells were treated with CD, an inhibitor of actin polymerization, 1(A) Confocal images, (B) MFI, (C) Coverage, and (D) Radial profile of HS in the presence of CD. Disruption of the actin cytoskeleton by CD did not influence HS under static conditions and after shear exposure for 30 min, but attenuated the shear stress-induced recovery of HS at 24 h. The distribution of HS at 24 h in the presence of CD was close to the level at 30 min. Scale bar: 20 µm. **P<0.01.
Figure 12.
Adaptive remodeling of glycocalyx with membrane rafts and actin cytoskeleton.
Under static conditions, glypican-1carrying only HS is localized on the dispersed lipid rafts and caveolae on the membrane. The actin cytoskeleton interacts with the transmembrane protein syndecan-1 and the caveolar structural protein caveolin-1 for stabilization. After 30 min of shear exposure, lipid rafts have carried glypican-1 with anchored HS to the cell boundary (clustering), while syndecan-1 carrying HS and CS, and caveolae with localized glypican-1 and anchored HS, do not move. Actin microfilaments increase in both apical and basal aspects of the cell. After 24 h of exposure, new caveolae are assembled on the apical surface, which may associate with newly synthesized glypican-1. Syndecan-1 (HS/CS), and glypican-1(HS) that is bound to anchored caveolae, and mobile lipid rafts are synthesized and result in nearly uniform distributions of HS and CS. Numerous long stress fibers form and most distribute in the apical part of the cell, where they stabilize new caveolae and syndecan-1. In the basal part of the cell, actin microfilaments increase, scatter and arrange in a disorderly fashion. Our findings portray a dynamic reorganization of the EC glycocalyx.