© 1999 American Heart Association, Inc.
Cellular Biology |
Transient and Steady-State Effects of Shear Stress on Endothelial Cell Adherens Junctions
From the Vascular Research Laboratory, The Toronto Hospital Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. The current address for D.B.C. is Department of Anesthesia, Children's Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Dr B.L. Langille, Vascular Research Laboratory, Toronto General Hospital, 200 Elizabeth St, CCRW 1-856, Toronto, Ontario, Canada M5G 2C4. E-mail lowell.langille{at}utoronto.ca
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Abstract |
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Abstract—Endothelial cells exhibit profound changes in cell shape in response to altered shear stress that may require disassembly/reassembly of adherens junction protein complexes that mediate cell-cell adhesion. To test this hypothesis, we exposed confluent porcine aortic endothelial cells to 15 dyne/cm2 of shear stress for 0, 8.5, 24, or 48 hours, using a parallel plate flow chamber. Cells were fixed and stained with antibodies to vascular endothelial (VE) cadherin,
-catenin, ß-catenin, or
plakoglobin. Under static conditions, staining for all proteins was
intense and peripheral, forming a nearly continuous band
around the cells at cell-cell junctions. After 8.5 hours of shear
stress, staining was punctate and occurred only at sites of continuous
cell attachment. After 24 or 48 hours of shear, staining for
VE-cadherin, -catenin, and ß-catenin was intense and
peripheral, forming a band of "dashes" (adherens
plaques) that colocalized with the ends of stress fibers that inserted
along the lateral membranes of cells. Staining for plakoglobin was not
observed after 24 hours of shear stress, but returned after 48 hours.
Western blot analysis indicated that protein levels of
VE-cadherin, -catenin, and plakoglobin decreased, whereas
ß-catenin levels increased after 8.5 hours of shear stress. As cell
shape change reached completion (24 to 48 hours), all protein levels
were upregulated except for plakoglobin, which remained below control
levels. The partial disassembly of adherens junctions we have observed
during shear induced changes in endothelial cell shape
may have important implications for control of the
endothelial permeability barrier and other aspects of
endothelial cell function.
Key Words: shear stress • endothelium • vascular endothelial cadherin • -catenin • ß-catenin • plakoglobin
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Introduction |
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The structure and physiology of the endothelial cells that line the mammalian vasculature are greatly influenced by the shear stresses that are continuously imposed on them by blood flow. The most obvious structural responses of endothelium to shear stress are changes in cell shape and orientation; in areas of low or inconsistent shear stress in vivo, or when cells are maintained in static culture, endothelial cells assume a cuboidal, cobblestone morphology, whereas they elongate and align in the direction of flow when shear stress is moderate or high.1 2 These morphological changes are adaptive in that they reduce the spatial fluctuations in shear stress and the maximum shears to which the cells are exposed.3
Shape change of cells within monolayers probably necessitate changes in
the interaction of individual cells with their neighbors at cell-cell
junctions; however, changes in endothelial cell-cell
contacts during responses to altered shear stress have not been
examined. Endothelial cells form multiple junctional
complexes with their neighbors including adherens junctions, tight
junctions, and gap junctions, as well as adhesion mediated by
homophilic binding of the transmembrane protein,
platelet–endothelial cell adhesion
molecule-1.4 Of these, the adherens junction may be
particularly important, because its early formation after cell-cell
contact is thought to be a prerequisite for the assembly of other
junctional complexes.4 5 Adherens junctions are protein
complexes that mediate calcium-dependent adhesion through homophilic
binding of extracellular domains of transmembrane proteins called
cadherins.6 The cytoplasmic domains of cadherins are
linked to the actin cytoskeleton by the catenin
proteins.6 7 Within this complex, ß-catenin and
plakoglobin (
-catenin) are members of the armadillo family of
proteins that link cadherins to -catenin, a vinculin homologue that
binds directly or indirectly to filamentous
(F)–actin.8 9 10 In endothelial cells, the
predominant cadherin, vascular endothelial cadherin
(VE-cadherin/cadherin-5), is specific for this cell
type.11 12 VE-cadherin is a fully functional cadherin,
because its expression in Chinese hamster ovary cells leads to
association with catenins, adherens junctions formation, contact
inhibition of cell growth, and restriction of monolayer
permeability.13 14 15 N-Cadherin has also has been detected
but is not localized to cell junctions16 ; apparently
N-cadherin is displaced from the adherens junction by
VE-cadherin.17 Recently, an additional
endothelial cell cadherin has been described, but its
functional significance is unclear.18
The assembly of adherens junctions in epithelia is not fully understood, but recent research has provided important insights. ß-Catenin contributes to early junction formation, and tyrosine phosphorylation of ß-catenin, and probably other members of the adherens junction complex, regulates the disassembly of adherens junctions.19 20 21 In endothelium, such disassembly occurs during the complex processes of shape change, reorientation, migration, and proliferation that are associated with repair of wounds to the monolayer, and with the achievement of confluence in sparsely plated cultures.22 23 Whether disassembly also occurs during the more subtle events that accompany shear-induced shape change is unclear. This issue is important, because the status of the adherens junctions is important to the physiology of the endothelium. For example, adherens junctions influence the permeability of the endothelial monolayer, either directly or through influences on tight junctions,5 14 24 and vascular sites that are exposed to extreme or fluctuating shear stresses display abnormally high permeability.25 These sites are prone to the development of atherosclerotic vascular disease,26 27 and increased permeability is thought to contribute to its pathogenesis.28 Atherogenesis and many other pathological processes are also associated with leukocyte diapedesis across endothelium, a process that involves complex interplay between endothelial-endothelial and endothelial-leukocyte adhesion complexes.29 30 Consequently, changes in adherens junctions may contribute to shear sensitivity of diapedesis.31
Finally, changes to adherens junctions may occur when shear stress
changes chronically. Such changes could reflect adaptations that
enhance adhesion between cells that are exposed to increased mechanical
loads. Alternatively, shear-induced reorganization of the actin
cytoskeleton, to which the adherens junctional proteins are linked by
-catenin, may influence the distribution of these proteins. Thus,
shear stress induces redistribution of F-actin from cell-cell junctions
(the dense peripheral band of microfilaments), where it is
prominent in static cultures, to centrally distributed stress fibers,
which are sparse in the absence of shear but prominent when it is
present.32 33
In this study, we have examined transient and steady-state effects of shear stress on the cadherin-catenin complex at endothelial adherens junctions. We report that shear stress causes partial disassembly of the adherens junction complex, followed by a reassembly that reflected shear-induced reorganization of F-actin distribution. After adaptation to shear stress, adherens junction proteins were localized in adhesion plaques (adherens plaques) that were distinct from the linear, beltlike distribution that predominated in static cultures. The extent of the redistribution of the catenins, and the temporal relationships of this redistribution, was consistent with the putative role for plakoglobin in the establishment of stable, long-term junctional complexes.22
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Antibodies
The following antibodies were used: mouse monoclonal antibody to VE-cadherin (clone 30Q8A-1), a gift from ICOS Corp, and goat polyclonal antibodies to human
E-catenin, ß-catenin, and -catenin (Santa
Cruz Biotechnology, Inc). Secondary antibodies used included
FITC-conjugated donkey anti-mouse and donkey anti-goat antibody
(Jackson ImmunoResearch Laboratories), respectively, in the presence of
rhodamine-labeled phalloidin (Molecular Probes), which labels
F-actin.
Cell Culture
Porcine thoracic aortas were obtained from a local
slaughterhouse, and endothelial cells were isolated and
purified as previously described.34 Cultures were
maintained at 37°C in complete medium 199 that was supplemented with
5% FBS (GIBCO), 1% amphotericin B (Fungizone), and 2%
penicillin/streptomycin, and equilibrated with humidified 95% air, 5%
CO2. Experiments were performed on confluent
cultures that had been transferred to sterile, 75x38x1–mm type 2947
glass microslides (Corning) that were prescored into 6 equal segments
for fluorescence staining (see below) or left unscored for
Western blot analysis. The cells used in this study were from
passages 4 to 6.
In Vitro Shear Stress Experiments
Porcine aortic endothelial cells were subjected
to laminar fluid shear stress in a parallel plate flow chamber that was
perfused by gravity feed from a glass reservoir system.35
Cells were subjected to a constant shear stress of 15
dyne/cm2 for 8.5, 24, and 48 hours. After each
experiment, cells were either fixed for fluorescence staining
or collected for Western blot analysis.
Fluorescence Staining and Confocal Microscopy
Cells were fixed with 3% paraformaldehyde in
PBS for 20 minutes at room temperature, and then the glass slides were
broken into 6 equal pieces along the prescored lines. Permeabilization
by topical application of 0.2% Triton X-100 (TX-100) was preceded and
followed by three 5-minute washes with PBS. Incubation with the primary
antibody was followed by FITC-conjugated secondary antibody in the
presence of rhodamine-labeled phalloidin, which labels F-actin. In some
0-hour and 48-hour experiments, adherent endothelial
cells were stained with the fluorescent membrane label
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate (DiI; Molecular Probes, Inc), an inert, nontoxic
hydrophobic probe. A stock solution of 2.5 mg/mL in ethanol was diluted
to a final concentration of 20 µmol/L in culture medium. Cells
were incubated with DiI-containing medium for 10 minutes at 37°C,
washed with PBS, and fixed for 15 minutes with 3%
paraformaldehyde in PBS. Endothelial
cells were examined using a Bio-Rad MRC 600 laser scanning confocal
microscope (Nikon x60 oil-immersion objective; numerical aperture,
160/0.17). FITC was excited at a wavelength of 488 nm, and a band-pass
filter (506 to 538 nm) was used to detect fluorescence.
Rhodamine and DiI were excited at 568 nm, and fluorescence was
detected between 589 and 621 nm.
Western Blot Analysis
Protein extractions were performed as described by Lampugnani et
al,22 23 with minor modifications. To separate the
cellular extracts into TX-100–soluble and –insoluble fractions,
endothelial cells on glass microslides were placed in a
100-mm plastic dish and washed 4 times with ice-cold
Ca2+- and Mg2+-containing
PBS. Cells were then extracted at 0°C with TX-100 extraction buffer
(1x Tris-buffered saline [TBS; pH 7.5], 1% NP-40, 1% TX-100, and
1x complete protease inhibitor cocktail
[Boehringer Mannheim], 100 µmol/L sodium
orthovanadate, and 1 µmol/L phenylmethylsulfonyl
fluoride [PMSF], 500 µL for every 25
cm2) for 30 minutes on ice with gentle agitation.
The extraction buffer was collected and centrifuged at
14 000g for 5 minutes at 4°C. The supernatant was defined
as the TX-100–soluble fraction. After this extraction, cells were
examined with phase-contrast microscopy and appeared adherent to the
glass slide with preserved nuclei and cytoskeletal fibers. The cells
were then washed gently 3 times with ice-cold buffered saline (TBS
containing protease inhibitors (1x complete protease
inhibitor cocktail, 100 µmol/L sodium orthovanadate,
and 1 µmol/L PMSF) and extracted with SDS extraction buffer (1x
TBS [pH 7.5], 1% NP-40, 1% SDS, 1x complete protease
inhibitor cocktail, 100 µmol/L sodium orthovanadate,
and 1 µmol/L PMSF, 500 µL for every 25
cm2) for 20 minutes on ice. The cells were then
scraped from the glass slide with a Costar cell lifter, and both cells
and extract were collected and vigorously pipetted through a 30-gauge
needle. The extract was centrifuged at 14 000g for
5 minutes at 4°C. The supernatant was defined as the
(TX-100)–insoluble fraction. Supernatants were stored at -20°C.
Total protein extractions were obtained by placing
endothelial cell–covered glass microslides in 100-mm
Petri dishes and washing them 3 times with ice-cold PBS without
Ca2+ or Mg2+. Cells were
then scraped from the slide in 1 mL of PBS with a Costar cell lifter
and collected into a 1.5-mL Eppendorf tube. After a brief spin
(3000g) to pellet the cells, the PBS was removed and 100
µL of lysis buffer was added (20 mmol/L Tris HCl [pH 7.5],
150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% sodium deoxycholate,
1% NP-40, 1% SDS, 100 µmol/L sodium orthovanadate, 1
µmol/L PMSF, and 1x complete protease inhibitor
cocktail). After a brief vortex, the cells were left on ice for 10
minutes to lyse. The lysate was centrifuged
(14 000g) for 3 minutes, revortexed, and spun again. The
supernatant was collected and stored at -80°C. A Lowry assay was
used to assess protein content for both extraction studies and total
protein studies, using BSA as the standard.
Denaturing discontinuous SDS-PAGE was performed on 8% Laemmli gels as
described by Gallagher.36 Electrophoresis and transfer
onto polyvinylidene difluoride (PVDF) membranes were carried
out using a Bio-Rad Mini-Protean II apparatus according to
Gallagher.36 For immunoblotting, PVDF
membranes were incubated for 1 hour with blocking buffer (5% BSA and
0.1% Tween 20 in TBS), followed by overnight incubation with
anti–VE-cadherin (dilution, 1:500) or 1-hour incubation with
anti–E-catenin (dilution, 1:500), anti–ß-catenin (dilution,
1:1000), or anti-plakoglobin (dilution 1:500). Membranes were then
incubated with either horseradish peroxidase–conjugated anti-mouse
peroxidase (dilution 1:2500) or anti-goat peroxidase (dilution
1:25 000) for 1 hour. Between the various incubation steps, the PVDF
membrane was washed several times with blocking buffer. Proteins were
detected with the enhanced chemiluminescence kit (Amersham), and film
was exposed for up to 5 minutes. Band intensity (mean optical density
integrated for the band area) was quantified on unsaturated x-ray film
by a digital image analyzer (Molecular Analyst Software,
Version 1.5; Bio-Rad Laboratories). All comparisons were made relative
to control conditions (0 hours shear stress).
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Results |
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Localization of VE-Cadherin and the Catenins in Static Endothelial Cell Cultures
Antibodies that recognized VE-cadherin,
-catenin, ß-catenin,
and plakoglobin all yielded continuous, linear staining around the
periphery of cultured porcine aortic endothelial cells
maintained in static cultures (Figures 1a
and 5a through 5c), as previously
described for other vascular endothelia. Optical sectioning, using the
confocal microscope, revealed that staining was localized to the
midplane of the cell along the lateral junctions (Figure 2e through
2h). This finding is consistent with current models that depict
extracellular homophilic binding of VE-cadherin at the junctions of
adjacent cells and intracellular linkage of cadherins to the
cytoskeleton by the catenins.22 In addition to linear
junctional staining, some broader regions at the margins of cells
showed punctate staining (see Figure 5a through 5c, arrows).
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Distribution of VE-Cadherin During Shear Stress–Induced Shape
Change and Reorientation
On exposure to 15 dyne/cm2 shear stress,
endothelial cells first display morphological changes
at 
8.5 hours (Figure 1c
and 1d). At this time, cells were no
longer continuously apposed to their neighbors; instead, gaps between
cells were frequently observed (Figure 1c and 1d, arrows).
Fluorescence staining revealed that the dense
peripheral band of junctional F-actin seen in static
cultures was less prominent, and F-actin stress fibers were more
numerous and distributed randomly throughout the cells (compare Figure 1b and 1d). After 24 and 48 hours of shear stress, the cells
were elongated and aligned with their major axis in the direction of
flow, and cells were continuously apposed to each other (Figure 1f and 1h). At these times, F-actin formed long, thick stress
fibers that ran the length of the cells. A continuous dense
peripheral band of F-actin was not observed; however, many
stress fibers terminated at the cell-cell junctions.
At no time after the initiation of shear stress was the junctional
staining for VE-cadherin lost (Figure 1a, 1c, 1e, and 1g);
however, the junctional staining was discontinuous after 8.5 hours; ie,
no staining was observed at free margins of cells that had separated
from their neighbors (Figure 1c, arrow). After 24 and 48 hours
of shear stress, staining was re-established around the complete
periphery of the cells (Figure 1e and 1g), but predominantly as
small, distinct "dashes" rather than as the continuous linear
staining that is observed in endothelium without shear
or in other epithelial cells.
Under high power, the dashes of cadherin staining colocalized with the
ends of stress fibers that inserted into cell junctions. Most of these
structures were junctional and not associated with the basal surface of
the cells, given that careful optical sectioning by confocal microscopy
revealed more basal stress fibers (Figure 2a through 2d). Thus,
these findings reveal an adhesion plaque of adherens junctional
proteins, which we refer to as the adherens plaque, that is distinct
from those in static endothelial cultures or in
confluent cultures of other epithelia.
Optical sectioning revealed another interesting feature of stress fiber
distribution in shear-adapted endothelium; stress
fibers passed both above and below the nucleus. Nuclear location was
readily identifiable as ovoid regions devoid of fluorescence
after staining for F-actin and junctional proteins (Figure 2).
In static cultures, many stress fibers were basal to the nucleus, but
they were not observed apical to the nucleus (compare Figure 2e and 2h). In contrast, many stress fibers passed both over (Figure 2a) and under (Figure 2d) the nuclei after 48 hours of
shear stress.
To assess levels of VE-cadherin protein and its association with the
cytoskeleton, Western blots were performed using total protein, or
using TX-100–soluble and –insoluble cell extracts. Total VE-cadherin
protein levels were increased after 24 hours and were maximal after 48
hours of applied shear stress (Figure 3a, lanes 3 and 4); thus, one adaptation to elevated shear stress may
include increased cadherin-mediated cell-cell adhesion. Increased
levels of VE-cadherin associated with the cytoskeleton were confirmed
by Western blots using TX-100–insoluble protein fractions. Between 0
and 48 hours of shear stress, cytoskeleton-associated VE-cadherin
levels doubled (mean of 3 experiments; Figure 4a, lanes 2 and 8); however, we did not
detect a preferential redistribution to the cytoskeleton, because
VE-cadherin levels were also elevated in the TX-100–soluble fraction
(Figure 4a, lanes 1 and 7). The TX-100–insoluble fraction of
protein decreased slightly (mean decrease of 25% in 3 experiments) but
consistently from 0 to 8.5 hours (Figure 4a, lanes 2 and
4) and then increased for up to 48 hours of applied shear stress.
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Effects of Shear Stress on the Distribution of - and ß-Catenin
in Endothelial Cells
After 8.5 hours of shear stress, the continuous, junctional
localization of -catenin seen in static cultures (Figure 5a) became punctate and less intense
(Figure 5d). After 24 and 48 hours, staining became more intense
and was localized to the adherens plaques at the junctional ends of
stress fibers (Figure 5g and 5j), as observed for
VE-cadherin.
Although junctional staining often clearly defined the position of
cell-cell junctions, even with the discontinuous staining seen after
shear stress, there were frequently more broadly distributed regions of
punctate staining at the cell-cell interface both before and after
imposition of shear stress (Figure 5). We hypothesized that
these were regions of substantial overlap of cells at their junctions.
Such regions of overlap, in both static and shear-stressed cultures,
were demonstrated when the cell membranes were stained with
fluorescent probe, DiI (Figure 6a and 6b). It was not possible to double stain with DiI and junctional
protein labels, because even light detergent treatment, which was
necessary to give antibodies access to the intracellular proteins,
disrupted DiI staining of the membrane. Nonetheless, the pattern of
immunostaining for adherens junction proteins resembled
that of cell overlap detected by DiI staining (Figure 6c and 6d).
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Western immunoblotting using total protein extracts
revealed that -catenin levels decreased after 8.5 hours and then
increased to control levels by 48 hours (Figure 3b, lanes 2 and
4, respectively). Total -catenin levels correlated with the amount
of -catenin associated with the cytoskeleton, which decreased
slightly (average decrease by densitometry of 25% in 5 experiments)
after 8.5 hours of shear stress and then steadily returned to control
levels by 48 hours (Figure 4b, lanes 4, 6, and 8). Within the
TX-100–soluble fraction, protein levels increased steadily to maximum
levels after 24 hours (50% greater than static cultures) that were
sustained after 48 hours of shear stress (Figure 4b, lanes 1, 3,
5, and 7). These results suggest that when F-actin is dissociated from
cell junctions (ie, 8.5 hours), the amount of -catenin associated
with the cytoskeleton is at its lowest; however, -catenin
reassociates with F-actin once cytoskeletal reorganization is
completed.
The linear, junctional localization of ß-catenin seen in static
cultures (Figure 5b) became punctate and sparse after 8.5 hours
of shear stress and occurred only at sites where cells were still in
contact (Figure 5e, arrowhead). After 24 and 48 hours of shear
stress, ß-catenin staining was intense, junctional, and
discontinuous; ie, it was associated with the adherens plaque defined
by VE-cadherin and -catenin staining (Figure 5h and 5k).
Western immunoblots using total protein extracts revealed
that ß-catenin levels increased over the time of applied shear stress
and were maximal at 48 hours (Figure 3c). Bands detected from
the TX-100–soluble fraction were always substantially stronger than
those detected from the insoluble fraction. Within the
TX-100–insoluble fraction, ß-catenin protein levels increased from 0
to 8.5 hours (Figure 4c, lanes 2 and 4) before rising to 3 times
the levels seen in static cultures (mean of 3 experiments) after 48
hours of applied shear stress (Figure 4c, lane 8). Within the
TX-100–soluble fraction, ß-catenin levels steadily increased (0.6-
to 1.1-fold) to peak levels at 48 hours (Figure 4c, lanes 1, 3,
5, and 7). These results indicate that ß-catenin levels are
upregulated during adaptation to shear.
Plakoglobin Redistribution Under the Influence of Shear
Stress
Within 8.5 hours of initiation of shear stress, continuous,
junctional immunostaining of plakoglobin became sparse
and localized, sporadically, to sites where lateral cell membranes were
still in contact (Figure 5f). Nuclear staining of the protein
was consistently observed at this time. After 24 hours, no
plakoglobin was detectable by immunostaining (Figure 5i); however, a junctional staining pattern was again observed
after 48 hours of shear stress (Figure 5l). At this time,
plakoglobin distribution resembled that of VE-cadherin, -catenin,
and ß-catenin; ie, it localized to the adherens plaques at the ends
of stress fibers that inserted into cell junctional regions.
Levels of plakoglobin detected by immunoblots of total
protein extracts decreased from 0 to 8.5 hours of shear application and
then increased up to 48 hours, although levels remained below controls
(Figure 3d). Western immunoblots using the
TX-100–insoluble fraction revealed that the amount of plakoglobin
associated with the cytoskeleton decreased 40% (mean of 3 experiments)
after 8.5 hours of shear stress (Figure 4d, lane 4). Levels
increased up to 48 hours (Figure 4d, lane 8) but remained below
control conditions by one third. The TX-100–soluble fraction
consistently yielded more plakoglobin than the
TX-100–insoluble fraction, but again, levels never reached control
conditions.
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The intimate interactions of the protein complex that forms adherens junctions with the adjacent actin cytoskeleton probably constrain the mobility of cell-cell junctions. This inference is consistent with the dispersal of adherens junctions during the profound morphological changes that occur when endothelial cells grow to confluence or when they repair wounds to the cell monolayer.22 These changes include extension of lamellipodia, gross alterations of cell shape, cell migration, and cell proliferation. The absence of adherens junctions is associated with compromised function of these cells, particularly with respect to the formation of permeability barriers,13 14 24 so it is not surprising that these structures are rapidly assembled when monolayers establish confluence.
VE-cadherin is the predominant cadherin in endothelial cells.11 12 Unlike cadherins in epithelia, VE-cadherin remains localized to sites of cell-cell contact in subconfluent cultures.11 22 It is thought that "diffusion trapping" through homophilic binding is adequate to maintain peripheral distribution of this protein.14 In contrast, the catenins redistribute away from junctions in subconfluent endothelial cell cultures,22 as in epithelia. Thus, homophilic binding of VE-cadherin may contribute to cell-cell adhesion, whereas dispersal of catenins, and therefore dissociation of the adherens junction from the cytoskeleton, may allow a more fluid cell-cell interface that accommodates morphological changes in subconfluent monolayers. It does not appear, however, that homophilic binding of VE-cadherin can fully substitute for complete adherens junctions, because mutant VE-cadherin that lacks cytoplasmic domains, and cannot interact with catenins, localizes to cell-cell junctions but cannot form a normal permeability barrier.14
Endothelial cells undergo marked shape change in response to altered shear stress that undoubtedly requires some reorganization of junctional structures. This shape change involves elongation of the cells and their alignment in the direction of shear stress,1 2 but it does not require formation of lamellipodia, cell migration, or cell proliferation. Therefore, we asked how the changes to adherens junctions that occur during shear stress-induced cell shape change compared with those previously reported for endothelial wound repair or growth to confluence.
VE-Cadherin, -Catenin, and ß-Catenin Protein Levels Are
Altered During Shear-Induced Shape Change, but the Proteins Remain
Localized at Endothelial Cell Junctions
The persistence of junctional localization of most adherens
junction proteins during shear-induced shape change strongly suggests
that homophilic binding between cadherin molecules on adjacent cells is
maintained during these morphological adaptations. This inference is
consistent with findings that junctional localization of
VE-cadherin is maintained during shape change associated with wound
repair.22
Persistent localization of -catenin and ß-catenin at cell-cell
junctions throughout adaptations to shear contrasts with the dispersal
of the catenins from junctional sites during
endothelial cell wound repair or during growth to
confluence.22 This difference probably reflects more
subtle morphological changes induced by shear stress that can be
completed with only partial disassembly of the adherens junction and
only partial dissociation of the adherens junction protein complex from
the F-actin cytoskeleton. These inferences are consistent with
our observations that cytoskeleton-associated (Triton-inextractable)
VE-cadherin, -catenin, and ß-catenin were only slightly diminished
after 8.5 hours of shear stress. Thus, reorganization of the
cytoskeleton and these junctional proteins, without their dissociation,
may achieve shape change and cell reorientation that are driven by
exposure to shear stress.
Plakoglobin Redistributes Away From Cell Junctions During
Shear-Induced Cell Shape Change, and Its Reassociation With Junctions
Is a Late Adaptation to Shear Stress
In contrast to VE-cadherin, -catenin, and ß-catenin,
the complete loss of plakoglobin from the endothelial
cell-cell junction 24 hours after initiation of shear stress, and its
reassociation with these junctions after 48 hours, was
consistent with previous reports that plakoglobin contributes
only to long-term, very stable adherens junctions.22 Its
absence from junctions at 24 hours indicates that the cells had not
reached steady state at this time, even though no subsequent changes to
cell morphology were detectable. The basis for association of
plakoglobin with only stable, steady-state cell-cell junctions is
poorly understood. Possibly the protein forms more stable associations
than ß-catenin with VE-cadherin and -catenin, so that turnover of
the complex is slow. Alternatively, plakoglobin associates with
desmoplakin at endothelial cell-cell
junctions,37 and it is possible that these complexes are
important in stabilizing cell-cell adhesion.
Surprisingly, the loss of immunostaining at 24 hours was not reflected in large changes in total protein levels; however, immunostaining detects local concentration of protein, not content. We infer that failure to detect staining reflects redistribution of protein from high concentrations at junctions to a diffuse distribution over the cell surface and/or within the cytoplasm.
An additional interesting feature of plakoglobin redistribution was its nuclear localization after 8.5 hours, when the cells first displayed morphological responses to initiation of shear stress. This observation raises the possibility that plakoglobin is acting as a signaling molecule during morphological adaptations to shear stress. Plakoglobin, like ß-catenin, is a member of the armadillo family of proteins that are involved in regulation of epithelial cell phenotype and growth control.8 9 These proteins bind to members of the Tcf family of proteins, and this complex translocates to the nucleus to regulate gene transcription after the Tcf moiety binds to high-mobility-group DNA domains. Most studies have focused on ß-catenin signaling, which regulates many aspects of epithelial cell phenotype,38 and similar functions may be performed by plakoglobin.
Cellular Distribution of Adherens Junction Proteins Parallels
Shear-Induced Reorganization of the Cytoskeleton: Formation of the
Adherens Plaque
In static cultures, both F-actin and adherens junction proteins
are distributed continuously around the cell-cell junction, as they are
in all other confluent epithelioid monolayers. Steady-state adaptation
to shear stress resulted in loss of the dense peripheral
band of F-actin; in addition, VE-cadherin and the catenins became
localized to discontinuous plaques, which we define as adherens
plaques, at the ends of stress fibers that inserted into cell-cell
junctions. These structures are distinct from adherens junctions of
stable epithelial monolayers and may be structurally analogous to the
focal adhesion plaques that link the F-actin cytoskeleton to
extracellular matrix via transmembrane protein complexes. Similar
structures have been observed in fibroblasts39 40 and
smooth muscle (M. Jones, B.L. Langille, unpublished observations,
1999). Stress fibers projected from these plaques in
opposite directions into both of the neighboring cells, possibly
because these adhesion plaques act as nucleation sites for stress fiber
formation during adaptation of the cells to shear. We previously
reported that paired stress fibers project into adjacent cells from
a junctional site in regions of rabbit arteries that are exposed to
high shear stresses in situ.41 To the extent that adherens
junctions contribute to regulation of permeability of the
endothelial cell monolayer,14 24 the
discontinuous distribution of the protein complex may contribute to the
chronically elevated permeability of the endothelium at
sites of unusual shear stress in vivo.42
Only adherens plaques were observed after adaptation to shear stress, and linear, beltlike staining predominated in static cultures; however, regions of punctate staining for adherens proteins were observed in some areas of cell overlap both with and without shear stress. Thus, both linear adherens junctions and some adherens plaques characterized cell-cell adhesion under static conditions.
Steady-State Expression of Adherens Junction Proteins Depends on
Shear Stress
Although cellular levels of VE-cadherin and the catenins declined
slightly as the cells initiated shear-induced shape change, levels of
VE-cadherin, and - and ß-catenin were elevated after 48 hours of
shear stress. It is probable that increased expression of these
proteins is an adaptation to the mechanical load that is imposed on
cell-cell junctions by shear stress. Surprisingly, however, levels of
plakoglobin, the catenin most associated with stable
junctions,22 were depressed after 48 hours of shear
stress. Although the cells may not have adapted fully to
physiological levels of shear stress by 48 hours,
it is also possible that substantially altered protein-protein
interactions in the punctate, plaquelike adherens junctions that
characterize endothelium under shear may limit
participation of plakoglobin. The level of regulation of adherens
junction proteins was not pursued in this study, because changes in
total protein were modest and occurred over extended times; therefore,
discriminating transcriptional control versus mRNA or protein stability
was not practical.
Implications for In Vivo Endothelial Cell
Biology
In vivo shear stresses vary over the cardiac cycle and also over
longer time scales that reflect the physiology of the organism. Changes
in mean blood flow rate that take hours or longer, eg, circadian
rhythms that are displayed by many arterial blood flow
rates, occur over time intervals that can elicit the junctional
reorganization we have observed. In particular, local flow direction
depends on mean flow rate near arterial bends and branch
sites; therefore, cells at these sites may continually receive signals
to change their orientation. These sites may be particularly vulnerable
to perturbations in endothelial cell physiology that
are influenced by the integrity of adherens junctions.
In summary, we have shown that initiation of shear stress on endothelium causes partial disassembly of adherens junctions that involves complete, but temporary, loss of junctional plakoglobin and partial, transient dispersal of other catenins and VE-cadherin. Adherens junctions, which form a beltlike structure contiguous with the dense peripheral band in static cultures, are reassembled into adherens plaques that localize at the ends of stress fibers that insert into cell-cell junctions. Thus, adherens junctions in endothelium exposed to physiological levels of shear stress are structurally distinct from such junctions in static endothelial cell cultures or in other epithelial monolayers.
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Acknowledgments |
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This work was supported by Grant MT-15044 from the Medical Research Council of Canada. B.L.L. is a Career Investigator of the Heart and Stroke Foundation of Ontario, and S.N. is the recipient of a studentship from the Heart and Stroke Foundation of Canada. D.B.C. was a Medical Research Council of Canada postdoctoral fellow at the time of this work.
Received May 17, 1999; accepted July 7, 1999.
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References |
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