Lateral Zone of Cell-Cell Adhesion as the Major Fluid
Shear Stress–Related Signal Transduction Site
Yumiko Kano, Kazuo Katoh, Keigi Fujiwara
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Abstract—It has been proposed previously that actin filaments and cell adhesion sites are involved in mechanosignal
transduction. In this study, we present certain morphological evidence that supports this hypothesis. The 3D disposition
of actin filaments and phosphotyrosine-containing proteins in endothelial cells in situ was analyzed by using confocal
microscopy and image reconstruction techniques. Surgical coarctations were made in guinea pig aortas, and the same
3D studies were conducted on such areas 1 week later. Stress fibers (SFs) were present at both basal and apical regions
of endothelial cells regardless of coarctation, and several phosphotyrosine-containing proteins were associated with SF
ends. Apical SFs had one end attached to the apical cell membrane and the other attached to either the basal membrane
or the lateral cell border. Within the coarctation area, the actin filament– containing and vinculin-containing structures
became prominent, especially at the apical and the lateral regions. Substantially higher levels of anti-phosphotyrosine
and anti-Src staining were detected in the constricted area, particularly at the cell-cell apposition, whereas the anti–focal
adhesion kinase, anti–CT10-related kinase, anti–platelet endothelial cell adhesion molecule-l, anti-vinculin, and
phalloidin staining intensities increased only slightly after coarctation. We propose that apical SFs directly transmit the
mechanical force of flow from the cell apex to the lateral and/or basal SF anchoring sites and that the SF ends associated
with signaling molecules are sites of signal transduction. Our results support the idea that the cell apposition area is the
major fluid shear stress– dependent mechanosignal transduction site in endothelial cells. (Circ Res. 2000;86:425-433.)
Key Words: endothelium 䡲 mechanotransduction 䡲 stress fibers 䡲 phosphotyrosine 䡲 coarctation
N
umerous studies have indicated that endothelial cells
(ECs) have some mechanism for responding to fluid
flow.1 When cultured ECs are exposed to the flow of culture
medium for a short period of time, a number of so-called
early flow responses occur, including NO release,1 G-protein
activation,2– 4 Ca2⫹ mobilization,5– 8 protein kinase C activation,9 –11 increased levels of tyrosine phosphorylation,1,12
platelet endothelial cell adhesion molecule-l (PECAM-l)
tyrosine phosphorylation,13,14 mitogen-activated protein kinase activation,11,12 and directed cell migration.15,16 These
events are followed by later responses that become detectable
typically after several hours of flow application, including
changes in various gene expressions,1 changes in cell
shape,17–22 and cytoskeletal reorganization.1,23–27 It is not
certain whether and how any of the early types of responses
relate to any of the later events. It is also uncertain whether
these flow-induced responses are expressed by in situ ECs
that are constantly exposed to blood flow.
Although these observations demonstrate the ability of ECs
to respond to fluid flow, the mechanism for flow sensing and
signal transduction remains largely unknown. Fluid shear
stress is the frictional force that acts on the apical cell surface.
If the cell surface is easily deformable, the force will mostly
be dissipated by deformation and may not be able to elicit
flow responses in cells. Thus, shear stress– dependent signal
transduction is expected to begin at sites where cell surface
deformation is less likely to occur. One possible place is the
apical stress fiber (SF)–plasma membrane attachment site
(the apical plaque),28 although there is no evidence that the
apical plaque is a site for mechanosignal transduction. There
are other rigid structures associated with the plasma membrane, such as the cell-substrate adhesion site (focal adhesion)
and the cell-cell adhesion site. Bundled actin filaments are
associated with all of these sites, and via the actin bundle
network, the force of shear stress acting on the apical plaque
may be transmitted to the other fixed parts of the cell, where
activation of specific signaling molecules may occur. This
scheme suggests the possibility that some flow-dependent
signaling events are initiated at the focal adhesion site, which
is not directly exposed to flow. Indeed, in cultured cells,
flow-dependent and cell adhesion– dependent mitogen-activated protein kinase activation has been reported,11,12 and
there are apical SFs that appear to span the distance between
the apical and the basal cell surfaces.28 This scheme also
predicts signal transduction at the cell-cell attachment site.
This laboratory has shown that PECAM-1, a cell-cell adhesion molecule, is tyrosine-phosphorylated when ECs are
exposed to flow.13,14 This PECAM-l phosphorylation could
Received August 19, 1999; accepted December 3, 1999.
From the Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Correspondence to Dr Yumiko Kano, Department of Structural Analysis, National Cardiovascular Center Research Institute, 5 Fujishiro-dai, Suita,
Osaka, 565-8565, Japan.
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
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Figure 1. Stereo pairs of in situ ECs in a control guinea pig
aorta doubly stained by phalloidin (A) and anti-phosphotyrosine
(PY-20) (B). A, 3D distribution of actin filaments. Thick SFs are
observed at the base, and thin SFs are observed at the apical
portion. Some apical SFs run into the actin circumferential ring
(arrowheads), and some attach to the basal part of cells
(arrows). B, PY-20 staining associated with the plasma membrane, especially the lateral portion. Anti-phosphotyrosine staining without corresponding actin filament staining was rarely
observed.
be triggered by the apical SF system that links the apical
plaque and the cell adhesion site. Indeed, we have shown that
PECAM-1 tyrosine phosphorylation does not occur in ECs
treated with cytochalasin D.14 Whether apical SFs terminate
on the cell-cell adhesion site requires investigation.
This signal transduction involving actin filaments predicts
a certain SF organization in the cell. More specifically, it
predicts the presence of strategically placed SF–plasma membrane attachment sites on the apical, lateral, and basal parts of
ECs and the association of signaling molecules with the
lateral and basal sites. Previously, we have provided evidence
for the presence of apical SFs and plaques in ECs in situ.29 In
the present study, by using guinea pig aortas and confocal
microscopy together with image reconstruction techniques,
we show the 3D organization of actin filament– containing
structures in ECs in situ. This overall actin cytoskeletal
organization is consistent with the proposed model for mechanosignal transduction. We found that the apical SFs run
between the apical cell surface and the lateral and the basal
part of the cell but that the basal SFs stay on the basal part of
Figure 2. Stereo pairs of in situ ECs in the coarctation zone
doubly stained with phalloidin (A and C) and anti-vinculin (B). A
and C, Compared with Figure 1A, the actin cytoskeleton is more
developed. Arrows indicate apical SFs connecting to the basal
portions of ECs. At high magnification (C), an apical plaque is
shown by an arrowhead. B, Vinculin localization is shown. Bright
and large staining spots are observed at both the cell apex and
base. Bar⫽20 ␮m.
the cell, presumably providing tight adhesion of ECs to the
basement membrane. When the flow rate (as well as fluid
shear stress) in the vessel is increased by making a region of
coarctation, SF expression is increased, especially the apical
type. Also increased in the coarctation area is the expression
of Src and tyrosine-phosphorylated proteins, especially in the
cell-cell overlap area. These results are consistent with the
idea that the lateral cell apposition zone is a major fluid
flow–related mechanosignal transduction site.
Materials and Methods
En Face Aortic Sample Preparation
Guinea pig abdominal aortas were used. A small silicon tube was
placed over some of the aortas for a week to analyze the effect of
increased fluid shear stress on various morphological parameters.
Aortas were fixed, longitudinally cut, and spread flat to expose the
endothelium.29
Kano et al
Cell-Cell Adhesion and Signal Transduction
427
Figure 3. A stereo pair of ECs stained with anti-vinculin in a
control vessel. Although dotty staining was present in the specimen at both the apical and the basal portions, weak apical
staining was lost during the image reconstruction procedure.
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Microscopy and 3D Image Reconstruction
En face aortic samples were stained with various antibodies and
observed under a laser scanning microscope. To make 3D images,
serial optical sections were made and, with the use of computer
software, reconstructed into stereo images. Each 3D image is
presented as a pair of sized-matched appropriately mounted micrographs that should be viewed through a special stereo viewer.
An expanded Materials and Methods section is available online at
http://www.circresaha.org.
Results
Actin Filament Organization
Whole-mount preparations of guinea pig abdominal aortas
were stained with fluorescein-labeled phalloidin, and 3D
images were reconstructed from confocal serial optical sections (Figure 1A). Each EC was delineated by a ring of strong
staining near the lumenal surface, presumably representing
actin filaments of the adherens junction. However, there were
also actin rings near the base of the cell. Whether adherens
junctions are present near the base of ECs requires investigation. SFs were generally aligned in the direction of blood
flow. Thick SFs were present at the base, and some of them
were quite short. Thin SFs were found in the apical portion of
the cell. These apical SFs tended to span the whole length of
the cell and appeared to run into the actin ring of the apical
location (Figure 1A, arrowheads). Some SFs ran vertically,
connecting the apical and the basal surfaces (Figure 1A,
arrows).
Weakly fluorescent structures were lost during 3D
image processing of confocal optical serial sections. Thus,
structures consisting of a small number of actin filaments
are not represented in the reconstructed images. It is
known that in situ SF expression is high in the region of
increased fluid shear stress,22,27,30,31 and indeed, in the
guinea pig aorta, the actin cytoskeletal architecture was
more exaggerated near bifurcations (data not shown). We
had attempted to take advantage of this exaggerated actin
filament organization for our analyses, but it was difficult
to obtain flat whole-mount preparations of branching
areas. Thus, to create an area with increased fluid shear
stress that could be easily made into a whole-mount
Figure 4. FAK and Crk localization in ECs. Optical sections
were made near the cell base. A and B, Anti-FAK staining in the
control area (A) and the coarctation zone (B), with an increased
size and number of staining spots in panel B. C, Anti-Crk
stained focal adhesions (arrowheads) and the general cytoplasm
(seen as smooth staining).
preparation, we made a coarctation in the straight region of
the abdominal aorta. A stereo pair of the constricted area
showed ECs with increased staining with rhodaminelabeled phalloidin. Apical SFs terminating on the circumferential actin ring were clearly demonstrated. Also clearly
seen were the SFs that ran between the apical and the basal
surfaces (Figure 2A, arrows). Stereo pairs showed that the
actin cytoskeleton was membrane-associated, forming a
cagelike structure underneath the plasma membrane and
surrounding the entire body of cytoplasm. Increased shear
stress upregulated the expression of apical SFs. Figure 2C,
a more magnified stereo pair, shows the centrally located
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Vinculin
Anti-vinculin staining associated with both basal and apical
plaques of in situ ECs has been shown.28,29 Stereo pairs of
normal aortic ECs showed basal anti-vinculin spots but not
the apical spots, presumably because of their low level of
labeling (Figure 3). The extent of cell delineation by antivinculin was minimal. On the other hand, the level of
anti-vinculin labeling in the coarctation area was considerably higher, and each staining spot in Figure 2B was
significantly larger than the spots shown in Figure 3. The
lateral and the apical vinculin spots also became noticeably
prominent, indicating that heightened shear stress increased
the size of vinculin-containing structures.
FAK and Other Focal
Adhesion-Associated Proteins
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Focal adhesion kinase (FAK) association with focal adhesions but not with apical plaques was reported in cultured
cells.28 In ECs in vivo, this kinase was localized to discrete
spots at the base of cells both in control (Figure 4A) and
constricted (Figure 4B) vessels. They are presumably focal
adhesions that are arranged linearly along the direction of
blood flow. Under increased shear stress, the number and size
of anti-FAK spots increased, suggesting that ECs reenforced
their adhesion to the basement membrane by making more
and larger focal adhesions.
CT10-related kinase (Crk), an adopter protein for both
paxillin and pp130cas, also binds to FAK. Anti-Crk stained the
basal portion of ECs, most notably focal adhesions (Figure
4C). Paxillin and pp130cas were also immunolocalized to focal
adhesions (data not shown). Our data indicate colocalization
of FAK, Crk, paxillin, and pp130cas in the focal adhesion of
ECs in situ.
Phosphotyrosine
Figure 5. Phosphotyrosine localization in the coarctation zone. A,
Low-magnification micrograph showing the downstream edge of a
coarctation (arrows). In the coarctation area (left), PY-20 staining is
significantly increased. Coarctations formed folds in the vessel wall
in the unconstricted area, and the top of folds showed increased
labeling. B, Confocal image of the coarctation zone optically sectioned at the middle level of ECs. Staining is at the cell-cell junction. C, Side view of the endothelium. A confocal image obtained
from a folded area shows strong staining at the cell-cell border
(arrows) and weak linear staining along the apical plasma membrane (arrowheads). Bar⫽20 ␮m.
apical plaque (arrowhead)28 and SFs running between the
apical plaque and the base of the cell (arrows). These
studies demonstrate that apical SFs are connected to either
the cell-cell adhesion site or the basal plasma membrane.
Because protein tyrosine phosphorylation occurs during intracellular signaling, we investigated phosphotyrosine levels
in ECs in the constricted region (Figure 5A). There was a
clear demarcation in anti-phosphotyrosine staining at the
edge of coarctation. Although immunostaining in the low
fluid shear stress area was low (Figure 5A, right), the area of
increased shear stress exhibited a high level of staining
(Figure 5A, left), which was closely associated with the
lateral plasma membrane (Figure 5B). The similar sharp
staining demarcation was observed at the proximal edge of
coarctations. The lateral staining did not always form a sharp
line, which might be expected if the adherens junction were
labeled (Figure 5B). Instead, sheetlike staining patterns were
observed, suggesting that the lateral cell membrane overlap
area was labeled. A side view of ECs clearly showed staining
along the entire cell-cell apposition (Figure 5C, arrows).
Dotty staining was associated with the base of the cell and the
apical cell membrane (Figure 5C, arrowheads).
A stereo pair of anti-phosphotyrosine–stained endothelium
showed immunoreactivity being spread throughout the lateral
plasma membrane overlap (Figure 1B). The specimen was
also stained for actin filaments and showed colocalization of
phosphotyrosine with actin filaments (Figure 1A). Although
spotty staining corresponding to focal adhesions and apical
Kano et al
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Figure 6. Localization of Src family
kinases. A to D, Cryosections (A and B)
and whole-mount preparations (C and D)
stained with 1 of the 2 types of Src antibodies, SRC-2 (A and C) and N-16 (B
and D). Src staining was mainly
observed at cell-cell junctions but also
along the top and base of cells in a dotted manner. IE indicates internal elastic
lamina; SM, smooth muscle cells. E and
F, ECs doubly stained with anti-vinculin
(green) and anti-Src (red). Superimposed
images show yellow spots indicating
colocalization of the 2 antigens in focal
adhesions and apical plaques (arrows).
An en face view of the basal portion (E)
and a side view (F) are shown.
Bar⫽10 ␮m.
plaques was present, SFs were not labeled. These results
suggest that the cell-cell border is an active site of signaling
events and that increased shear stress upregulates signaling
activities.
Src
Src family proteins are tyrosine-phosphorylated at the time
of signal transduction and are tyrosine kinases. Localization of Src was investigated by using 2 antibodies with
different specificity: SRC-2 (recognizing the Src family
kinases) and N-16 (recognizing only c-Src). Cryosections
of the aortic wall stained with SRC-2 showed labeling of
the cell border and some basal spots, presumably focal
adhesions (Figure 6A). The cell apex was also labeled in a
dotted manner (Figure 6A). Cryosections stained with
N-16 exhibited similar staining patterns, but anti– c-Src
images were better defined than SRC-2 images (Figure
6B). Like anti-phosphotyrosine staining, the anti-Src signal was spread along the entire length of cell-cell apposition. When these 2 antibodies were treated with appropriate synthetic Src peptides and then used to stain specimens,
no labeling was observed (data not shown).
A confocal optical section at the mid level of ECs showed
dotty staining by SRC-2 at the cell-cell border (Figure 6C).
N-16 gave a similar but better defined staining pattern (Figure
6D). An en face preparation stained doubly with anti-vinculin
and SRC-2 was optically sectioned at the base of cells, where
anti-vinculin stained focal adhesions. Figure 6E is a merged
image of anti-vinculin (red) and anti-Src (green) signals and
shows colocalization (yellow) of the 2 antigens at focal
adhesions. A double-labeled confocal side view is similarly
illustrated in Figure 6F, demonstrating localization of Src
kinases at focal adhesions. This micrograph also demonstrates the localization of Src kinases at apical plaques
(arrows).
Src expression was investigated in the coarctation zone.
A low-magnification photograph showed increased antiSrc staining in the area of coarctation (Figure 7A). The
border between low and high levels of Src expression was
sharp and corresponded well with the edge of coarctation
(Figure 7B). Increased staining occurred at all localization
sites, including the general cytoplasm, but staining was
particularly strong at the cell border (Figure 7C). The
localization of Src kinases was remarkably similar to the
distribution of phosphotyrosine-containing proteins.
Csk
C-terminal Src kinase (Csk) is a nonreceptor-type tyrosine
kinase that phosphorylates a tyrosine residue in the
C-terminal region of Src family kinases and inactivates them.
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Figure 7. Localization of Src in the
coarctation zone. A, Low-magnification
view of the entire coarctation area. The
boundary between the low and the high
shear stress areas is clearly observed. B,
Upstream edge of a coarctation showing
a sharp staining boundary. C, Confocal
optical section at the middle level of
ECs, showing staining at the cell-cell
boundary.
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Strong anti-Csk staining was present at the cell-cell apposition (Figure 8). This staining was broad, inasmuch as it was
seen with anti-Src kinases and anti-phosphotyrosine.
Discussion
The response of ECs to fluid flow has mostly been studied
by using in vitro systems. Although this approach has
provided information regarding the basic property of this
cellular response, it is commonly believed that in vitro data
may not totally reflect the situation in vivo. Therefore, it is
of paramount importance that ECs in situ be closely
studied. Understandably, however, performing experiments on cells in vivo is difficult, and available data on in
situ ECs are severely limited. In the present study, using
guinea pig abdominal aortic ECs in situ, we obtained
certain morphological information pertinent to mechanosignal transduction by these cells. Although these
immunolocalization studies do not provide definitive solutions to the mechanism of flow-induced signaling by
ECs, we suggest that they provide insights into the way
these cells respond to flow.
plaque, and the other end appears to terminate at the basal
plasma membrane.28 Likewise, one end of the apical SF in
ECs in situ is attached to the apical plaque, whose molecular
makeup is similar to that of the apical plaque of cultured
cells.29 However, it was not clear where the other end of the
apical SF was located. In the present study, we demonstrated
that this end was attached to either the basal focal adhesion or
the cell-cell apposition site. The apical SF attachment to the
focal adhesion site is presumably mediated by the integrin
system. The lateral termination sites were clearly above the
base of the cell, indicating that they were not the cellsubstrate attachment sites. This brings up a new set of
questions regarding the structure and the molecular makeup
of this lateral SF–plasma membrane binding site, which may
Actin Bundle Organization and
Signal Transduction
A role of the cytoskeleton in mechanosignal transduction by
ECs has been proposed.1,32–36 We have suggested that the
actin cytoskeleton might transmit mechanical forces directly
from one part of the cell to another. Such a scheme predicts
a certain pattern of actin bundle disposition. In the present
study, we have demonstrated the presence of SFs that run in
the specific ways that have been predicted by our mechanosignal transduction hypothesis.
Of particular interest are the SFs located in the apical part
of ECs. In cultured fibroblasts, one of the ends of the apical
SF is attached to the apical plasma membrane via the apical
Figure 8. Anti-Csk staining at the cell-cell border. A confocal
optical section at the middle height of cells is shown.
Kano et al
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be different from the binding sites involved in the integrinbased anchoring of SFs to the membrane. Integrin is not
concentrated at the cell-cell adhesion site. Cadherin and
PECAM-l are the major transmembrane proteins at the lateral
cell adhesion site, and both proteins have a ␤-catenin binding
activity.37– 40 Actin filament bundles running into the lateral
portion of cultured cells has been reported, and ␤-catenin is
localized to such sites.41 There is a circumferential ring of
actin filaments associated with the lateral plasma membrane
of ECs forming a monolayer. In many instances, apical SFs
appeared to run into this structure, at the level of resolution of
a light microscope. Whether these 2 actin filament bundles
are physically linked is an intriguing question, which we are
now investigating.
As we have outlined in the introductory section of the
present study, fluid shear stress acts on the rigid part of cell.
The apical plaque is such a part because it is anchored by the
SF system. Thus, it is a candidate site for shear stress sensing
and initial signal transduction. However, it is also possible
that through the SF system, the force is transmitted to focal
adhesions and cell-cell apposition sites, where appropriate
signaling pathways can be activated. Thus, any individual
region or combinations of these 3 regions are good candidates
for fluid shear stress sensing and signal initiation sites. The
present study has provided a structural basis for this
hypothesis.
Localization of Signaling Molecules
Tyrosine residues of many proteins involved in signal transduction are phosphorylated at the time of their activation or
inactivation.1,42 Thus, the level of tyrosine phosphorylation at
a given area in a cell can be a reflection of local signal
transduction activities. Anti-phosphotyrosine staining was
detected at apical plaques, regions of cell-cell apposition, and
focal adhesions. The focal adhesion has been identified as a
site of signal transduction,43– 45 and several tyrosine-phosphorylated polypeptides, such as FAK, vinculin, paxillin,
Crk, pp130cas, and Csk, have been localized.46 –52 Vinculin and
paxillin are also concentrated at apical plaques.28,29 These
proteins may be responsible for anti-phosphotyrosine staining
of focal adhesions and apical plaques, but other proteins,
perhaps specific for shear stress signal transduction, may also
contribute to this staining.
Anti-phosphotyrosine staining was much stronger at the
cell-cell contact site than at the other 2 locations, suggesting
more active signaling events at the lateral cell overlap. Because
increased shear stress significantly increased this staining, the
region of cell-cell apposition may be the most active signal
transduction site of shear stress. We have reported previously
that when a confluent monolayer of cultured ECs is exposed to
⬎5 dyne/cm2 of shear stress or osmotic changes, PECAM-1 is
tyrosine-phosphorylated within 1 minute.13,53 PECAM-1 is an
EC adhesion molecule localized at the membrane overlap region
between neighboring cells. Thus, a portion of the phosphotyrosine localized at the cell-cell border may be in PECAM-l.
Unlike vascular endothelial (VE)-cadherin localization, which
appears as a line at the cell-cell overlap, anti–PECAM-l staining
was seen as a belt.14,54 As shown in the present study, antiphosphotyrosine staining was broad along the cell-cell border.
Cell-Cell Adhesion and Signal Transduction
431
Src family kinases and Csk were also localized to the entire
cell-cell overlap. Thus, PECAM-1, Src, and Csk, all of which are
phosphotyrosine-containing molecules, are localized in a nearidentical pattern to the cell-cell border. These results suggest that
all or some of these molecules work together for mechanosignal
transduction at the cell-cell border. Indeed, our in vitro study
shows that c-Src is an effective PECAM-1 kinase.13,53 Immunolocalization also suggests that the activity of Src kinases is
tightly controlled at the cell-cell border by Csk.
Effect of Coarctation
A coarctation was made in the straight region of the abdominal aorta to create a region of increased fluid shear stress that
can be easily made into an en face specimen for microscopy.
The same approach has been taken by other investigators.55
Precise fluid mechanical analyses on the blood flow pattern
and assessment of the magnitude of the increase of fluid shear
stress within the constricted region were not performed.
Because we have used a straight region of the blood vessels,
it is expected that the flow pattern within the coarctation is
generally laminar (as is assumed for a nonbranching straight
blood vessel) and that the level of fluid shear stress in the
constricted zone is significantly heightened. The flow pattern
in the region immediately distal to the coarctation is expected
to be nonlaminar, and accordingly, the fluid shear stress level
there is expected to be lower than that of the normal flow
area. Although these local differences in fluid shear stress
levels are likely, ECs are expected to be under higher shear
stress in the constricted region than in the areas immediately
proximal and distal to a coarctation. Thus, we expect that the
expression of any shear stress– dependent events is heightened in the area of coarctation. The basic localization patterns
of various proteins were the same between regions with and
without a coarctation. However, the actin cytoskeleton and
the structures labeled with anti-vinculin, anti-paxillin, and
anti-FAK were enlarged in ECs in the coarctation area.
Inasmuch as most of these latter structures are focal adhesions, our results suggest that cells under increased shear
stress reenforce their adhesion to the substrate by making
larger and possibly more numerous adhesion sites.
All these changes, however, were detectable only at a high
magnification. When en face specimens stained with antiFAK, anti-vinculin, anti-paxillin, or fluorescent phalloidin
were observed at low magnifications, there was no fluorescence boundary between the low and the high shear stress
areas. This indicates that the extent of increased expression of
these proteins in the coarctation area is not incredibly high.
However, in the specimens stained with anti-phosphotyrosine
or anti-Src, a clear demarcation was detected between the low
and the high shear stress regions, indicating that Src expression is substantially upregulated by shear stress and that the
amount of proteins with phosphorylated tyrosines is also
highly concentrated in the high shear stress area. Observations at high magnifications revealed that increased staining
was mostly associated with the lateral cell overlap region. It
is not clear at this time what types of signaling pathways are
activated at the cell border, but they are presumably fluid
shear stress– dependent signaling events perhaps involving
PECAM-1, Src, and Csk. These data suggest the cell-cell
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apposition to be an important site for mechanosignal transduction in ECs.
Many types of EC flow responses, especially the early
ones, are transient. The present study indicates that although
the ECs in the coarctation zone have been exposed to
increased shear stress for 1 week, certain types of signaling
events are still occurring. Consistent with this interpretation is
our observation that both anti-Src and anti-phosphotyrosine
heavily stain ECs at arterial branching, where the average
fluid shear stress is always higher than the straight region of
the same vessel. Some of the long-lasting responses are
related to cell morphology, including cell-shape changes and
SF alignment, and these responses are known to be reversible.
To maintain the expression of a stimulus-dependent, reversible response, cells may need to continuously monitor the
level of the stimulus and to generate a positive signal for the
response. Thus, we suggest that the increased expression of
SFs and focal adhesions and the elongated cell shape are
maintained in ECs in the region of high shear stress because
the fluid shear stress– dependent signal transduction machinery of these cells is continuously active and keeps generating
positive signals. This may be the reason for the increased
expression of phosphotyrosine in the zone of high shear
stress. We propose that the expression and organization of the
SF system, which includes the SF–plasma membrane attachment site and possibly of other morphological parameters in
ECs in situ, are dynamically maintained by fluid shear
stress– dependent signals transmitted continuously from the
area of cell-cell adhesion.
Acknowledgments
This study was supported in part by grants-in-aid for Scientific
Research from the Ministry of Education, Science, and Culture of
Japan, grants from the Ministry of Health and Welfare of Japan, and
a grant from the Organization for Pharmaceutical Safety
and Research.
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Lateral Zone of Cell-Cell Adhesion as the Major Fluid Shear Stress−Related Signal
Transduction Site
Yumiko Kano, Kazuo Katoh and Keigi Fujiwara
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Circ Res. 2000;86:425-433
doi: 10.1161/01.RES.86.4.425
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