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INTRODUCTION |
The nature of blood flow patterns and shear forces within blood
vessels may be very variable depending upon vessel size, shape, branching, and partial obstructions (1). Biomechanical forces induced
within the cardiovascular system affect gene expression in cells of
blood vessel walls (2, 3) and functions of the cells in the vessel wall
and in the fluid phase (4-8). Changes of shear forces occur in
bifurcated or stenotic regions where atherosclerotic regions are prone
to develop.
According to the multistep theory in cell transmigration, monocytes
roll on the endothelial cells, interact with selectins, adhere to the
endothelial cells by firm adhesion to
ICAM-11 and vascular cell
adhesion molecule-1 (VCAM-1), and then migrate into the subendothelium
(9). Rolling of monocytes on endothelial cells is dependent on the
binding of E-selectin and sialyl Lewis X, and adhesion to the
endothelium is dependent on the interaction between integrins on
monocytes and adhesion molecules on the endothelial cells, such as
VCAM-1 and ICAM-1. Integrins consist of several subtypes, and each
subtype is specific for each ligand. For example,
4
1 integrin,
VLA-4, binds to VCAM-1, and
2 integrins bind to ICAM-1. Fibronectin,
one of the extracellular matrix proteins, is also known to bind to
1
integrins, mainly to
5
1 integrin. Thus activation of adhesion
molecules in endothelial cells and leukocytes is important for the cell
migration process.
In this study, we hypothesized that leukocyte adhesion might be
increased at bifurcations and in the downstream of the restricted vessels. In normal laminar flow, it has been reported that human leukocytes respond to fluid shear stress by retracting pseudopods and
down-regulation of integrins (10, 11), which is a requirement for
normal passage of circulating leukocytes through the microcirculation. In the downstream of the region where the vessel lumen is partially occluded, however, a backward vortex can be observed where cells in the
fluid phase are subjected to a vortex motion under low shear forces (1,
12-14). Although such a change of shear force on endothelial cells can
regulate the expression of adhesion molecules resulting in the
progression of atherosclerosis (15, 16), the effect of vortex-mediated
mechanical stress on leukocytes has not yet been determined. If
vortex-induced mechanical stress can induce cell adhesion in
leukocytes, leukocytes would be more prone to attach to the endothelial
lining in the turbulent flow because the residence time of leukocytes
in the regions with nonlaminar flow is longer than in those with
laminar flow (12, 13).
A variety of signaling systems are induced by a mechanosensor in
endothelial cells. As a mechanosensor, stretch-activated channels have
been reported to regulate Ca2+ influx induced by flow
stress in cells such as endothelial cells or smooth muscle cells (17).
There is much evidence that stretch increases intracellular
Ca2+ levels (4, 17). Thus the importance of
Ca2+ signaling in endothelial mechanotransduction has been
established. However, the role of Ca2+ in cell response to
the mechanical stress in leukocytes has not been examined so
far. Therefore, the aim of this study was to examine the effect
of mechanical stress on integrin-dependent cell adhesion in
human monocytic THP-1 cells and to elucidate the role of
Ca2+ signaling involved in this process.
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EXPERIMENTAL PROCEDURES |
Reagents--
RPMI medium was obtained from Nissui
Pharmaceuticals Co. Ltd. (Tokyo, Japan). Fetal calf serum was
purchased from Grand Cayman (British West Indies).
L-glutamine and penicillin/streptomycin were obtained from
Bio Whittaker (Walkersville, MD). Recombinant human soluble VCAM-1 and
ICAM-1 were from Genzyme/Techne (Minneapolis, MN). Fibronectin,
thapsigargin, W-7, ryanodine, U-73122, bovine serum albumin, RGDS
peptides, and RGES peptides were from Sigma. Anti-human
4 (VLA-4)
antibody was from Upstate Biotechnology (Lake Placid, NY).
GdCl3·6H2O and
NiCl2·6H2O were from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). BAPTA-AM was from Dojindo (Kumamoto, Japan).
Cell Lines--
The monocytic cell line THP-1 was a generous
gift from Dr. K. Nishida (Daiichi Pharmaceuticals Co. Ltd., Tokyo) and
was cultured in RPMI supplemented with L-glutamine and
penicillin/streptomycin plus 10% fetal calf serum in an atmosphere of
95% air and 5% CO2 at 37 °C.
Cell Adhesion Assay--
Cell adhesion assays were carried out
essentially as described (18). Briefly, polystyrene 96-well
flat-bottomed microtiter plates (Costar 3595, Corning Inc., Corning,
NY) were coated with 50 µl of soluble VCAM-1 (2.5 µg/ml), soluble
ICAM-1 (2.5 µg/ml), or fibronectin (10 µg/ml) for 1 h at room
temperature. After incubation, wells were blocked by incubation with
200 µl of 10 mg/ml heat-denatured bovine serum albumin for 30 min at
room temperature. Control wells were filled with 10 mg/ml
heat-denatured bovine serum albumin. One hundred µl of THP-1 cells
suspended at a concentration of 106/ml in 10% fetal calf
serum-RPMI were incubated for the indicated times in a CO2
incubator at 37 °C after exposure to vortex flow by vortex machine
(MS1 minishaker from IKA Works, Wilmington, NC). After
incubation, nonadherent cells were removed by centrifugation (top side
down) at 48 × g for 5 min. The plates were then
centrifuged inversely at 80 × g for 5 min. Attached
cells were fixed with 5% glutaraldehyde for 30 min at room
temperature. Cells were washed three times with water, and 100 µl of
0.1% crystal violet in 200 mM MES (pH 6.0) was added to
each well and incubated at room temperature for 20 min. Excess dye was
removed by washing with water three times, and the bound dye was
solubilized with 100 µl of 10% acetic acid. The absorbance of each
well at 595 nm was then measured using a multiscan enzyme-linked
immunosorbent assay reader (SPECTRA classic, Tecan, Maennedorf,
Austria). Each sample was assayed in triplicate. The absorbance
was linear to the cell number up to OD of 1.9 (data not shown). For
example, 0.05 of OD. represents adhesion of about 2,000 cells, and 0.5 of OD represents adhesion of about 25,000 cells.
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RESULTS |
Vortex-mediated Mechanical Stress Increased Adhesion of THP-1 Cells
to VCAM-1 and Fibronectin--
To determine the regulation of integrin
avidity or affinity by mechanical stress mediated by vortex flow, we
studied adhesion of THP-1 cells to purified adhesion molecules. Cell
adhesion to soluble VCAM-1, soluble ICAM-1, and fibronectin was
determined after cells were exposed to vortex flow for 5 s at
1,500 rpm to mimic vortices that may occur in the cardiovascular system
(12, 13, 19). Vortex-mediated mechanical stress increased adhesion of
THP-1 cells to VCAM-1 and fibronectin by approximately five-fold but
not to ICAM-1 (Fig. 1).

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Fig. 1.
Vortex flow stimulates cell adhesion to
VCAM-1 and fibronectin, but not ICAM-1, in THP-1 cells. THP-1
cells were subjected to adhesion assays on ICAM-1, VCAM-1, or
fibronectin for 5 min (VCAM-1, fibronectin) or 10 min (ICAM-1) with (open bar) or without
(filled bar) vortexing at 1,500 rpm for 5 s. Data
represent the mean ± S.D. of triplicate measurements from three
independent experiments.
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Vortex-mediated cell adhesion to VCAM-1 and fibronectin increased in a
speed-dependent manner (Fig.
2). To show that this cell adhesion is
dependent on
4
1 and
5
1 integrins, we preincubated the cells
with anti-
4 antibody and RGDS peptides. Preincubation of the cells
with anti-
4 antibody inhibited vortex-mediated cell adhesion to
VCAM-1 by about 80%, but not with control IgG (Fig. 3A). Preincubation with RGDS,
but not with REDS peptides, inhibited vortex-mediated cell adhesion to
fibronectin (Fig. 3B). We also studied the change of
1
integrin expression on THP-1 cells induced by vortex-mediated
mechanical stress, but we could not find any change of the expression
by flow cytometry (data not shown). These data indicate that cell
adhesion in our assay depends on the interaction between integrins and
their ligands and that vortex-mediated mechanical stress increased the
avidity or affinity of both
4
1 and
5
1 integrins in THP-1
cells.

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Fig. 2.
Speed-dependence of vortex-induced adhesion
of THP-1 cells to VCAM-1 and fibronectin. THP-1 cells were
subjected to adhesion assays on VCAM-1 or fibronectin for 5 min after
vortexing at the indicated speeds for 5 s. Data represent the
mean ± S.D. of triplicate measurements from three independent
experiments.
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Fig. 3.
Time-dependent increase of
vortex-induced adhesion of THP-1 cells to VCAM-1 and fibronectin.
THP-1 cells were subjected to adhesion assays on VCAM-1 or fibronectin
for 5 min after vortexing at 1,500 rpm for the indicated seconds. Data
represent the mean ± S.D. of triplicate measurements from three
independent experiments.
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Transient Integrin Activation after Vortex-mediated Mechanical
Stress--
Next, we studied the time-dependent effect of
vortex flow on cell adhesion to VCAM-1 and fibronectin. We found that
vortex-mediated mechanical stress increased cell adhesion to both
VCAM-1 and fibronectin quite rapidly, reaching a peak at 2-5 s of
stimulation, indicating that such a brief vortex stimulation is enough
to activate
1 integrin (Fig. 4). To
examine reversibility of this integrin activation, cells were vortexed
at 1,500 rpm for 5 s and left static for the indicated minutes.
Cell adhesion to VCAM-1 or fibronectin was then determined. After the
cells were left static for only 4 min, the cell adhesion induced by
vortex flow was rapidly reduced to ~50% (Fig.
5), showing that this integrin activation
induced by vortex-mediated mechanical stress is quite transient and
reversible.

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Fig. 4.
Cell adhesion depends on
1 integrin. THP-1 cells were preincubated with
10 µg/ml anti- 4 antibody or control IgG (for VCAM-1) and 2 mM RGDS or RGES peptide (for fibronectin) for 1 h in
an atmosphere of 95% air and 5% CO2 at 37 °C. After
the incubation, cells were subjected to adhesion assays on VCAM-1 or
fibronectin with (open bar) or without (filled
bar) vortexing at 1,500 rpm for 5 s. Data represent the
mean ± S.D. of triplicate measurements from three independent
experiments.
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Fig. 5.
Vortex-induced integrin activation is
transient and reversible. THP-1 cells were subjected to adhesion
assays on VCAM-1 or fibronectin for 5 min after left static for the
indicated minutes after vortexing at 1,500 rpm for 5 s. Data
represent the mean ± S.D. of triplicate measurements from three
experiments. The value at the baseline was expressed as 100%.
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Integrin Activation Induced by Vortex-mediated Mechanical Stress
Depends on IP3-sensitive Ca2+ Release from
Intracellular Stores--
Calcium signals are reported to be important
for various cell responses such as integrin activation leading to cell
adhesion (20). To determine whether Ca2+ is involved in
integrin activation induced by vortex-mediated mechanical stress, we
next pretreated the cells with BAPTA-AM, an intracellular
Ca2+chelator. Pretreatment of the cells with BAPTA-AM
inhibited vortex-mediated cell adhesion to fibronectin (Fig.
6A) and VCAM-1 (data not
shown), indicating that intracellular Ca2+ is necessary for
this integrin activation. To determine whether a stretch-activated
Ca2+ channel, a well known sensing system for mechanical
stress (17), or Ca2+ influx from the extracellular space is
involved in integrin activation induced by vortex-mediated mechanical
stress, we next pretreated the cells with
GdCl3·6H2O, a specific stretch-activated
channel inhibitor, or NiCl2·6H2O, a
nonspecific Ca2+ influx inhibitor. Pretreatment of cells
with these inhibitors did not affect vortex-mediated cell adhesion to
fibronectin (Fig. 6B) or VCAM-1 (data not shown), indicating
that this integrin activation does not depend on stretch-activated
channels or Ca2+ influx from outside of the cells. These
data indicate that Ca2+ release from intracellular
Ca2+ stores such as endoplasmic reticulum may play a key
role for this phenomenon.

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Fig. 6.
Integrin activation induced by
vortex-mediated mechanical stress depends on IP3-sensitive
Ca2+ release from intracellular stores. A,
THP-1 cells were preincubated with 50 µM BAPTA-AM for
1 h in an atmosphere of 95% air and 5% CO2 at
37 °C. After incubation, cells were subjected to adhesion assays on
fibronectin for 5 min with (open bar) or without
(filled bar) vortexing at 1,500 rpm for 5 s. Data
represent the mean ± S.D. of triplicate measurements from three
independent experiments. B, THP-1 cells were preincubated
with 50 µM GdCl3·6H2O for
1 h or 1 mM NiCl2·6H2O for
1 h followed by treatment with or without 1 µM
thapsigargin (THG) for 3 h in an atmosphere of 95% air
and 5% CO2 at 37 °C. After incubation, cells were
subjected to adhesion assays on fibronectin for 5 min with (open
bar) or without (filled bar) vortexing at 1,500 rpm for
5 s. Data represent the mean ± S.D. of triplicate
measurements from three independent experiments. C and
D, THP-1 cells were preincubated with the indicated
concentrations of U-73122 (C) or ryanodine (D)
for 1 h in an atmosphere of 95% air and 5% CO2 at
37 °C. After incubation, cells were subjected to adhesion assays on
VCAM-1 for 5 min with (open bar) or without (filled
bar) vortexing at 1,500 rpm for 5 s. Data represent the
mean ± S.D. of triplicate measurements from three independent
experiments.
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Ca2+ is released from the intracellular Ca2+
stores via two known channels, one sensitive to inositol
1,4,5-trisphosphate (IP3) and the other sensitive to
ryanodine. Therefore, to determine the mechanism of Ca2+
release from intracellular Ca2+ stores, we pretreated the
cells with thapsigargin, an inhibitor of Ca2+-ATPase that
inhibits IP3-dependent Ca2+ release
from intracellular stores (21, 22). Because thapsigargin itself induces
sustained elevation of intracellular calcium mediated by capacitative
Ca2+ influx (23, 24), we added NiCl2 to block
this Ca2+ influx. Pretreatment of THP-1 cells with
thapsigargin and NiCl2 inhibited vortex-mediated mechanical
stress-induced cell adhesion to fibronectin (Fig. 6B) and
VCAM-1 (data not shown). We also pretreated the cells with U-73122, a
specific PLC inhibitor, because mechanical stimulation of a
single cell can activate PLC to elevate IP3 (25).
Pretreatment of the cells with U-73122 inhibited vortex-mediated cell
adhesion to fibronectin and VCAM-1 (data not shown) in a dose-dependent manner (Fig. 6C).
To examine the role of ryanodine-sensitive Ca2+ release
from intracellular Ca2+ stores, we next pretreated the
cells with ryanodine, which can inhibit ryanodine-sensitive
Ca2+ release (26). Pretreatment of THP-1 cells with
ryanodine up to 10 µM did not affect vortex-mediated cell
adhesion to fibronectin (Fig. 6D). These data indicate that
IP3-dependent Ca2+ release from
intracellular Ca2+ stores plays a key role in this phenomenon.
Calmodulin Is Also Necessary for Integrin Activation Induced by
Vortex-mediated Mechanical Stress--
We also examined the potential
role of Ca2+-calmodulin in integrin activation induced by
vortex-mediated mechanical stress. To determine the involvement of
calmodulin in integrin activation induced by vortex-mediated mechanical
stress, we pretreated the cells with W-7, a calmodulin inhibitor,
before vortexing the cells. Pretreatment of cells with W-7 inhibited vortex-mediated cell adhesion
to VCAM-1 and fibronectin in a dose-dependent manner (Fig.
7), indicating that calmodulin is also involved in integrin activation
induced by vortex-mediated mechanical stress.

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Fig. 7.
Calmodulin inhibitor inhibits vortex-induced
adhesion to VCAM-1 and fibronectin in a dose-dependent
manner. THP-1 cells were preincubated with W-7 (calmodulin
inhibitor) at indicated concentrations for 2 h in an atmosphere of
95% air and 5% CO2 at 37 °C. After incubation, cells
were subjected to adhesion assays on VCAM-1 or fibronectin for 5 min
with (open bar) or without (filled bar) vortexing
at 1,500 rpm for 5 s. Data represent the mean ± S.D. of
triplicate measurements from three independent experiments.
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DISCUSSION |
In this study we have examined the effect of vortex-mediated
mechanical stress on integrin-dependent cell adhesion in
human monocytic THP-1 cells and have clearly shown that a brief period of vortex-mediated mechanical stress activated
1 integrin, resulting in cell adhesion to VCAM-1 and fibronectin in a transient and reversible manner. We have also shown that
IP3-dependent Ca2+ release from
intracellular Ca2+ stores and calmodulin are involved in
this integrin activation. This mechanism might explain why
atherosclerosis is prone to progress in bifurcated or stenotic regions,
and this may be a novel aspect of atherosclerosis and inflammation.
Most of the studies on mechanotransduction in the cardiovascular field
have been done in endothelial cells and smooth muscle cells. The
endothelial cells are normally subjected to mechanical stimuli from
shear stress and from strain associated with stretch of the vessel
wall. These stimuli can be detected by a mechanosensor that initiates a
variety of signal transduction cascades (17, 27). For example, in
response to the change in shear stress the endothelium can change the
gene expression of various cytokines and adhesion molecules (15, 16,
28) that would be related to the promotion of atherosclerosis,
thrombosis, and inflammation. Few studies, however, have been conducted
to elucidate the changes in the adhesive property of leukocytes in the
vortex flow, which might be also related to the induction of
atherosclerosis. Fukuda et al. (11) have reported that human
leukocytes respond to fluid shear stress by retracting pseudopods and
down-regulate the integrin expression under the laminar flow condition,
which would help leukocytes to run in the vessel wall. However, in the
tortuous cardiovascular system, such as branching of the vessels and
downstream of partially occluded vessels, leukocytes and platelets can
be subjected to differing shear forces under nonlaminar flow patterns (1, 12, 13). In this study, therefore, we exposed cells to vortex flow
in order to mimic vortices that may occur in the cardiovascular system.
In the study of platelet aggregation, a stirring bar has been used to
expose platelets to vortex flow (19). Because it is important to expose
whole cells to vortex flow instantaneously to mimic the in
vivo situation, vortexing the cells in a vortex machine would be
more reasonable to stimulate the cells in vitro.
Establishing an in vivo model would be more important to
show the relevance of this data to in vivo situations.
In previous studies, the endothelial intracellular Ca2+
concentration in response to mechanical stress is biphasic, consisting of an initial transient rise that depends on Ca2+ release
from IP3-sensitive stores, followed by a sustained
elevation mediated by Ca2+ influx (22, 29, 30). However, in
this report we have shown that Ca2+ influx from the
extracellular space is not necessary for integrin activation induced by
vortex-mediated mechanical stress on THP-1 cells. Our data also clearly
indicate that IP3-dependent Ca2+
release from intracellular Ca2+ stores plays a key role in
this mechanism. Although the reason why only Ca2+ release
from intracellular stores is required for vortex-mediated integrin
activation remains unclear, it might be because of the shortness of
vortex stimulation and integrin activation.
Calmodulin is a Ca2+ binding protein and is reported to be
important for various cell responses, such as integrin activation leading to T cell adhesion (20) and aggregation (31). Our study clearly
demonstrates that calmodulin also plays an essential role in regulating
integrin activation induced by vortex-mediated mechanical stress as
shown in various cell responses (32, 33). However, at present it is not
clear how Ca2+ release from intracellular stores can be
linked to the activation of calmodulin and integrin activation in THP-1
cells. Further studies, therefore, are required to clarify this mechanism.
In this study we have not been able to identify the sensing mechanism
for vortex-induced mechanical stress in THP-1 cells. There is a
possibility that a mechanosensor itself is not involved in this
process. The forces applied at the cell surface might be transmitted to
other locations via cytoskeleton. This kind of mechanotransduction is
shown in the area of mechanical stretch (34). Therefore, an
explanation of the sensing mechanism would be required to understand
this process. Further understanding of how leukocyte adhesion functions
in the tortuous cardiovascular system would enhance our knowledge of
the nuances of the atherosclerotic and inflammatory process and should
facilitate the development of drugs to regulate the process.
In summary, we have provided clear evidence that vortex-mediated
mechanical stress on THP-1 cells quickly induces Ca2+- and
calmodulin-dependent integrin activation, and
IP3-dependent Ca2+ release from intracellular
Ca2+ stores is involved in its mechanism. These findings
might enlighten another aspect of increased atherosclerosis at stenotic
or bifurcated regions.