HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Shear stress inhibits adhesion molecule expression in vascular endothelial cells
induced by coculture with smooth muscle cells
Jeng-Jiann Chiu, Li-Jing Chen, Pei-Ling Lee, Chih-I Lee, Leu-Wei Lo, Shunichi Usami, and Shu Chien
Vascular endothelial cells (ECs), which
exist in close proximity to vascular
smooth muscle cells (SMCs), are constantly subjected to blood flow–induced
shear stress. Although the effect of shear
stress on endothelial biology has been
extensively studied, the influence of SMCs
on endothelial response to shear stress
remains largely unexplored. We examined the potential role of SMCs in regulating the shear stress–induced gene expression in ECs, using a parallel-plate
coculture flow system in which these 2
types of cells were separated by a porous
membrane. In this coculture system,
SMCs tended to orient perpendicularly to
the flow direction, whereas the ECs were
elongated and aligned with the flow direction. Under static conditions, coculture
with SMCs induced EC gene expression
of intercellular adhesion molecule-1
(ICAM-1), vascular adhesion molecule-1
(VCAM-1), and E-selectin, while attenuating EC gene expression of endothelial
nitric oxide synthase (eNOS). Shear stress
significantly inhibited SMC-induced adhesion molecule gene expression. These
EC responses under static and shear
conditions were not observed in the absence of close communication between
ECs and SMCs, and they were also not
observed when ECs were cocultured with
fibroblasts instead of SMCs. Our findings
indicate that under static conditions, coculture with SMCs induces ICAM-1,
VCAM-1, and E-selectin gene expression
in ECs. These coculture effects are inhibited by shear stress and require specific
interaction between ECs and SMCs in
close contact. (Blood. 2003;101:2667-2674)
© 2003 by The American Society of Hematology
Introduction
Vascular endothelial cells (ECs), which provide an interface between the
blood and the vessel wall, are constantly subjected to hemodynamic
forces, including the shear stress imposed by blood flow. Shear stress
affects leukocyte–EC interaction and the subsequent leukocyte extravasation into inflamed tissues.1,2 The effects of fluid shear stress on
endothelial biology and gene expression have been extensively studied.3-7 In addition to its physical influence on leukocyte-EC adhesion,8,9
shear stress can alter the adhesive properties of ECs by modulating the
surface expression of adhesive proteins, for example, intercellular
adhesion molecule-1 (ICAM-1),10-15 vascular cell adhesion molecule-1
(VCAM-1),10,11,13,14,16,17 and E-selectin.13,18 Exposure of ECs to laminar
flow increases the gene and protein expression of ICAM-1.10-15 Our
recent study demonstrated that this shear flow–induced increase in
ICAM-1 expression is partially due to an elevation of the levels of
intracellular reactive oxygen species (ROS) in ECs.15 In contrast to
ICAM-1, shear stress treatment of ECs has only a minor effect on the
surface expression of VCAM-1, or may even cause a reduction.10,11,13,14,16,17 E-selectin has been reported to be less responsive to
laminar shear stress13 and more responsive to oscillatory flow condition.18
Most studies on how shear stress affects ECs have been
performed by using a cultured EC monolayer as an experimental
model. The vessel wall is composed of several types of cells,
including ECs, smooth muscle cells (SMCs), and fibroblasts. There
is increasing evidence that cell-to-cell interactions can control
cellular growth, migration, differentiation, and function.19 Physical
contact between ECs and SMCs via myoendothelial bridges has
been demonstrated in vivo20,21 and may play an important role in
cellular communication. Hence, in vitro models for studying ECs
need to simulate the in vivo environment by coculturing ECs in
close proximity to SMCs. Several coculture models have recently
been developed.22-26 Ziegler et al22 designed a coculture model that
includes SMCs, ECs, and a matrix of collagen gel. They demonstrated that ECs cocultured with SMCs aligned with the flow
direction more rapidly than ECs grown on plastic. Redmond et al23
developed a system in which ECs and SMCs were cocultured on
opposite sides of polypropylene capillary tubes. In this model, flow
was established through the lumen and the fluid medium diffused
through pores. A series of research studies have been conducted
using this coculture system, which represents a significant advance
over homogeneous culture.27-29 However, in this model the cells
cannot be observed directly until they are harvested, and direct
EC-SMC contact is prevented by the thickness of the capillary wall
(approximately 150 ␮m). Nackman et al24 constructed a system by
combining a parallel-plate flow chamber and a coculture module in
which ECs and SMCs are grown on opposite sides of a thin
permeable membrane containing 0.4-␮m pores. This system contains several valuable features, including a proper luminal/
abluminal orientation for ECs and SMCs, a short distance (10 ␮m)
between the cell layers, and the ability to separate different cell
types for assays. Moreover, EC-SMC contact has been demonstrated in this coculture module with the formation of the cytoplasmic projection from SMCs penetrating the entire length of the
From the Division of Medical Engineering Research, National Health Research
Institutes, Taipei, Taiwan, Republic of China; and the Department of
Bioengineering and Whitaker Institute of Biomedical Engineering, University of
California-San Diego, La Jolla.
Reprints: Jeng-Jiann Chiu, Division of Medical Engineering Research,
National Health Research Institutes, Taipei 114, Taiwan, Republic of China; email: [email protected].
Submitted August 20, 2002; accepted November 17, 2002. Prepublished online as
Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-08-2560.
Supported by grant ME-091-PP-I3 from the National Health Research
Institutes, Taiwan, Republic of China.
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
2667
2668
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
CHIU et al
pores in the membrane to reach the ECs.30,31 Using this system,
Nackman et al24 demonstrated that exposure of the EC side of the
coculture to shear flow attenuates the proliferation of the neighboring SMCs. More recently, Rainger et al25,26 developed a coculture
flow system, which is similar to the design of Nackman et al,24 to
investigate the adhesion of flowing leukocytes to ECs cocultured
with SMCs. They demonstrated that coculture of ECs with SMCs
markedly increased the adhesion of flowing leukocytes to ECs and
that the response of cocultured ECs to tumor necrosis factor-␣
(TNF-␣) stimulation was amplified.
Because close interaction between ECs and SMCs may play an
important role in regulating endothelial gene expression and hence
vascular biology, we established a parallel-plate coculture flow
system, based on the same principle as the systems of Nackman et
al24 and Rainger et al,25,26 to investigate the effects of shear stress
on the EC expression of ICAM-1, VCAM-1, and E-selectin. The
results of the present study demonstrate that ECs cocultured with
SMCs in a static condition had higher levels of gene expression of
ICAM-1, VCAM-1, and E-selectin and lower levels of endothelial
nitric oxide synthase (eNOS) than ECs in EC monoculture or
EC/EC culture. This SMC-induced modulation of gene expression
can be completely abolished by separating the ECs and SMCs by
using a cover slip instead of a porous membrane, while still
allowing humoral communication between these 2 types of cells
bathed in shared media. Shear stress applied to the EC side of the
coculture membrane significantly inhibited the induction of adhesion molecules in ECs. Our findings indicate that shear stress acts
as an important regulator of pathophysiologically relevant gene
expression in ECs and may thereby exert atheroprotective effects
on the vascular wall.
Materials and methods
Cell cultures
ECs were isolated from fresh human umbilical cords by means of the
collagenase perfusion technique as previously described.32 The cell pellet
was resuspended in a culture medium consisting of medium 199 (M199;
Gibco, Grand Island, NY) supplemented with 20% fetal bovine serum
(FBS; Gibco) and 1% penicillin/streptomycin (Gibco). ECs were grown in
Petri dishes to reach confluence and then treated with trypsin and harvested.
Third-passage human umbilical cord SMCs were obtained commercially
(Clonetics, Palo Alto, CA) and maintained in F12K medium (Gibco)
supplemented with 10% FBS. Identity of SMCs was verified by immunostaining with mouse monoclonal antibody to ␣-smooth muscle specific
actin (Sigma, St Louis, MO). Human skin fibroblasts were purchased from
American Type Culture Collection (Rockville, MD) and grown in a culture
medium consisting of minimal essential medium (MEM; Gibco) supplemented with 10% FBS, L-glutamine, and penicillin. Cells between passages
4 and 7 were used in the experiments.
Coculture modules
Coculture was established by plating cells on the 2 sides of a 10-␮m-thick
porous polyethylene terephthalate (PET) membrane (Falcon cell culture
inserts; Becton Dickinson, Lincoln Park, NJ). The membrane has a diameter
of 2.3 cm with 0.4-␮m pores configured at a density of 1.6 ⫻ 106
pores/cm2. ECs were first seeded onto the outer side of the membrane at a
density of 5 ⫻ 105 cells/cm2. After allowing 2 hours for adherence, the
membrane was placed with the EC side down into a 6-well plate containing
the culture medium, and the opposite side of the membrane (ie, the inner
side) was either kept unseeded (EC/␾) or seeded with identical ECs
(EC/EC) or SMCs (EC/SMC) at a density of 2 ⫻ 105 cells/cm2. ECs and
SMCs were maintained in their respective media until confluence. The
culture medium was then exchanged with a medium that was identical to the
previous medium except that it contained only 2% FBS. The cells were
further incubated for 24 hours prior to the experiment.
Parallel-plate coculture flow system
We built a parallel-plate coculture flow chamber similar to the designs of
Nackman et al24 and Rainger et al.25,26 This coculture flow chamber permits
the on-line microscopic visualization of cells during and after the flow
experiment. The design and construction of the chamber are illustrated in
Figure 1. Figure 1A shows an exploded view of the component parts of the
chamber. Two sets of glass slides (parts 1 and 5; 80 ⫻ 10 mm, 0.7 mm
thick) and silicone gaskets (parts 2 and 4; 80 ⫻ 10 mm, 0.25 mm thick)
were fastened by vacuum suction through ports v and vi with a polycarbonate insert holder (part 3; 80 ⫻ 10 mm, 17 mm thick), which was machined
precisely to allow the incorporation of the coculture module (viii) into a
channel cut in the lower silicone gasket (part 4). The compartment of the
SMC side was fully filled with the medium. Since the medium is a
noncompressible fluid, this treatment permits the reduction of membrane
vibration induced by transmural flow across the EC monolayer under shear
flow conditions. The fluid medium entered at port i through slit iii into the
channel, and exited through slit iv and port ii. The resulting flow channel is
shown schematically in Figure 1B. In the test section, the channel width (w)
and height (h) were 38 mm and 0.25 mm, respectively. The wall shear stress
(␶) produced by the flow chamber in the present study was estimated to be
12 dyne/cm2 using the formula ␶ ⫽ 6␮Q/wh2, where ␮ is the viscosity of
the perfusate and Q is the flow rate. The chamber was connected to a
perfusion loop system, kept in a constant-temperature controlled enclosure,
and maintained at pH 7.4 by continuous gassing with a mixture of 5%
carbon dioxide (CO2) in air. The EC side of the coculture was subsequently
subjected to flow with the designated shear stress, while the opposite side
was maintained in a static condition. The chamber was placed on the stage
of an inverted microscope (Axiovert 200M; Zeiss, Göttingen, Germany). A
charge-coupled device (CCD) video camera (CCD-72; Dage-MTI, Michigan City, IN) was attached to the microscope and the video image was
Figure 1. Design and construction of the parallel-plate
coculture flow chamber. (A) Components of the chamber.
Two sets of glass slides (1 and 5) and silicone gaskets (2 and
4) and the polycarbonate insert holder (3) are held together
by a vacuum suction applied at the perimeter of the side via
ports v and vi, forming a channel inside the lower silicone
gasket (4). The insert holder is machined precisely to allow
the introduction of the EC/SMC coculture module (viii) into
the channel. Cultured medium enters at port i through slit iii
into the channel, and exits through slit iv and port ii. The
chamber allows on-line microscopic visualization of the cells
during the flow experiment via the observation window (vii)
opened in the upper silicone gasket (2). (B) Schematic
diagram of the parallel-plate coculture flow channel. ECs
were seeded onto the inverted side of the membrane containing 0.4-␮m pores configured at a density of 1.6 ⫻ 106
pores/cm2 and then subjected to flow, while SMCs cultured in
the opposite side of the membrane were maintained in a
static condition.
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
transmitted to a video monitor (HR-1000; Dage-MTI) and recorder
(SR9090U; JVC, Tokyo, Japan), enabling the recording of all results in the
video field.
Experimental procedure
To validate the coculture flow system, the EC side of the coculture was
subjected to flow for 24 hours, and the subsequent morphological alterations and cytoskeletal reorganization in the cocultured cells were investigated. Immunostaining of platelet endothelial cell adhesion molecule-1
(PECAM-1), which is an interendothelial junctional protein, under static
and flow conditions allows an assessment of the integrity of the cocultured
ECs. Various sets of coculture modules (EC/␾, EC/EC, and EC/SMC) were
inserted into a parallel-plate flow chamber, and then the EC side was
subjected to flow for 6 hours to investigate the regulatory effect of shear
stress on the expression of various adhesion molecule genes (ICAM-1,
VCAM-1, and E-selectin) in the cocultured ECs. Porous PET membranes
containing 3-␮m and 8-␮m pores configured at a density of more than
5 ⫻ 105 pores/cm2 were used to further confirm this regulatory effect. In
additional experiments, ECs were cocultured with another type of vascular
cells (ie, fibroblasts) to test the specificity of the influence of SMCs on
endothelial responses to shear stress. Moreover, to elucidate the effect of
close interaction between ECs and SMCs in the coculture, SMCs growing
on a cover slip were placed in a compartment opposite the EC compartment.
This eliminated the possibility of direct contact between the cocultured ECs
and SMCs, while maintaining the interaction between these cells via
communication of medium that contains substances released from the cells
on both sides of the cover slip. The role of nitric oxide (NO) in modulating
the SMC-induced gene expression in cocultured ECs under the static
condition was elucidated by pretreating the ECs with an NO donor,
S-nitroso-N-acetylpenicillamine (SNAP, 100 ␮M; Calbiochem, San Diego,
CA) or DETA/NO (100 ␮M; Calbiochem), for 30 minutes before coculture
with SMCs in the presence of the same reagent. In addition, the eNOS gene
expression in ECs cocultured with SMCs was examined.
SHEAR STRESS REGULATES EC/SMC INTERACTION
2669
triphosphate (dNTP) mix, 10 mM dithiothreitol (DTT) and oligo-dT12-18
(0.5 ␮g/mL) for 50 minutes at 42°C. Reactions were terminated with
Escherichia coli RNase H (2 U/␮L). The cDNA was diluted 1:20 before the
performance of polymerase chain reaction (PCR) by using 1 ␮L cDNA in
20 mM Tris-HCl, pH 8.4, 3 mM MgCl2, 0.2 mM dNTP mix, 0.5 ␮M sense
and antisense primers, and Taq polymerase (2 U/␮L; Takara Shuzo, Shiga,
Japan). Primer sequences were designed as ICAM-1 (sense: 5⬘CGA-CTGGAC-GAC-AGG-GAT-TGT3⬘; antisense: 5⬘ATT-ATG-ACT-GCG-GCTGCT-ACC3⬘; product length, 290 base pair [bp])35; VCAM-1 (sense:
5⬘GGA-AGT-GGA-ATT-AAT-TAT-CCA-A3⬘; antisense: 5⬘CTA-CAC-TTTTGA-TTT-CTG-TG3⬘; product length, 441 bp)36; E-selectin (sense: 5⬘AGTAAT-AGT-CCT-CCT-CAT-CAT-G3⬘; antisense: 5⬘ACC-ATC-TCA-AGTGAA-GAA-AGA-G3⬘; product length, 185 bp)37; eNOS (sense: 5⬘AGATCA-CCG-AGC-TCT-GCA-TT3⬘; antisense: 5⬘ATT-TCC-ACT-GCT-GCCTTG-TC3⬘; product length, 400 bp). Amplification of the cDNA was
performed in parallel samples using human GAPDH (glyceraldehyde-3phosphate dehydrogenase) primers (sense: 5⬘CCA-CCC-ATG-GCA-AATTCC-ATG-GCA3⬘; antisense: 5⬘TCT-AGA-CGG-CAG-GTC-AGG-TCCACC3⬘; product length, 599 bp).37 The samples were amplified using
primers for 25 cycles of denaturation at 94°C for 1 minute, annealing at
60°C for 1 minute, and extension at 72°C for 2 minutes, using a GeneAmp
System 9700 (PE Biosystems, Foster City, CA). Amplification was linear
with respect to the cDNA concentration by optimizing the primer concentration, amplification cycles, and MgCl2 concentration for each PCR reaction.
The amplified cDNAs were analyzed by 1% agarose gel electrophoresis and
ethidium bromide staining. Band intensities were quantified directly from
the stained agarose gels using video imaging and a densitometry software
system (GDS-8000 Imaging Workstation; UVP, Upland, CA). To verify the
identity of the PCR product, aliquots of the PCR product were separated by
gel electrophoresis and transferred to nylon membranes. The Southern blots
were probed with 32P-labeled full-length cDNAs of ICAM-1, VCAM-1,
E-selectin, and eNOS (gifts from Dr D. L. Wang, Academia Sinica, Taiwan).
Statistical analysis
Morphological study and fluorescence immunostaining
A ⫻ 10 objective with a 13-␮m depth of field was used to directly examine
the morphology of the cocultured ECs and SMCs, which were separated by
the membrane with a thickness of 10 ␮m. After 24 hours of flow application
to the EC side, cocultured ECs and SMCs were rinsed with phosphatebuffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 30
minutes. The cytoskeletal reorganization in the cocultured cells and the EC
border were examined by immunostaining the cells with antibodies against
actin and PECAM-1, respectively, as described previously.33,34 To determine the SMC orientation in the coculture, the cell boundaries were traced
manually and roughly 200 cells in the images before and after 24 hours of
flow application were analyzed quantitatively using image software written
by Wayne Rasband of the National Institutes of Health. Basically, each of
the individual cells was represented by an ellipse that best fits the boundary
of the cells. The direction of the major axis of the ellipse, which represents
the direction of the cells relative to the flow direction, was measured by
using the angle tool in the software. The ratio of the cell numbers at
10-degree intervals to the total cell number in the images was then
determined. This method, as used in this study, was verified to have less
than 1% error by calculating specific shapes with known theoretical angles.
RNA isolation and reverse transcriptase–polymerase
chain reaction (RT-PCR)
After shear stress treatment, 5 to 7 ␮g total RNA of the EC monolayer
(approximately 2.5 ⫻ 106 cells) in coculture was isolated by the guanidium
isothiocyanate/phenochloroform method as previously described15 and then
converted to cDNA using the Superscript II reverse transcriptase system
and oligo-dT primers (Life Technologies, Rockville, MD). Briefly, total
RNA (2 ␮g, in diethyl pyrocarbonate [DEPC] water) was incubated with 50
U/␮L Superscript II RNase H reverse transcriptase (RT) in buffer containing 20 mM Tris-HCl, pH 8.4, 2.5 mM MgCl2, 0.5 mM deoxynucleoside
Results are expressed as mean ⫾ SEM (n ⫽ number of individual coculture
modules from which cells were harvested). Statistical analysis was
performed by using an independent Student t test for 2 groups of data and
analysis of variance (ANOVA) for multiple comparisons. A P value less
than .05 was considered significant.
Results
Morphological findings
Under the static condition (0 hours), the cocultured SMCs were
uniformly distributed and the hill-and-valley pattern was absent
(Figure 2A). This result is consistent with that found by Powell et
al.38 After 24 hours of shear stress (12 dyne/cm2) application to the
EC side, the SMCs in coculture tended to orient perpendicularly to
the flow direction. This result is comparable to the in vivo
orientation of SMCs in blood vessels39 and to observations on
homogeneous SMC culture directly exposed to flow.40,41 A typical
polar plot of the quantitative analysis of SMC reorientation is
shown in Figure 2B. A relatively random distribution of cell angle
was found in the static condition. In contrast, 24 hours of shearing
caused the cells to be aligned perpendicularly to the flow direction
(2.08 ⫾ 5.34 degrees in the static condition vs 62.73 ⫾ 3.28
degrees in the flow condition, P ⬍ .05; Figure 2B). The elongation
and alignment of ECs with the flow direction were not clearly
observable under the microscope owing to interference by the
porous membrane and the cocultured SMCs. However, this alignment of cocultured ECs with the flow direction was demonstrable
in immunostained preparations, which showed the reorganization
2670
CHIU et al
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
ing for PECAM-1 in cocultured ECs showed uniformly circumferential bands in the cell borders under static and flow conditions
(data not shown). These observations of flow-induced morphological changes and cytoskeletal reorganization and the uniform
distribution of interendothelial junctional protein supported the
utility of our cocultured flow system for studying endothelial
responses to mechanical forces in the presence of
neighboring SMCs.
Shear stress inhibits the induction of ICAM-1, VCAM-1, and
E-selectin in ECs by coculture with SMCs
Figure 2. Morphologic changes induced in the cocultured cells by exposing the
EC side of the coculture to a shear stress of 12 dyne/cm2 for 24 hours. (A) SMCs
in coculture tend to orient perpendicularly to the flow direction. The photographs
labeled 0 hours and 24 hours show the SMCs before and after shear flow,
respectively. (B) A typical polar representation of the quantitative analysis of SMC
reorientation. Cocultured cells were photographed in the preshear condition and after
24 hours of shear stress (12 dyne/cm2) application to the EC side, and the SMC
orientation was analyzed quantitatively as described in “Materials and methods.” Cell
orientation is defined as the angle between the direction of the major axis of the
ellipse that best fits the boundary of the cells and the flow direction. Data represent
the cell numbers at 10-degree intervals as percentages of total cell number in the
images. Inverted open triangles (ƒ) connected by dashed lines represent the data
before shear flow. Solid circles (F) connected by solid lines represent the data
obtained after 24 hours of shear flow. Note the relatively random distribution of cell
angle before flow and the increase in cell alignment perpendicular to flow direction
after 24 hours of shearing. The percentages of cells (%) are shown in the radial axes.
of actin filaments and circumferential distribution of the interendothelial junctional protein (ie, PECAM-1) in response to flow
application. In the static condition, very long, parallel actin bundles
were observed in the central regions of the cocultured SMCs,
whereas the actin filaments in the cocultured ECs mainly localized
at the cell periphery (data not shown). After 24 hours of flow
application to the EC side, the cocultured ECs displayed very long,
well-organized, parallel actin stress fibers aligned with the flow
direction in the central regions of the cells. There was no noticeable
change of actin organization in the cocultured SMCs. Immunostain-
The effect of shear stress on the expression of various adhesion
molecules (ie, ICAM-1, VCAM-1, and E-selectin) was determined
in ECs that had been cultured either in the absence (EC/␾) or
presence of ECs (EC/EC) or SMCs (EC/SMC). The goal was to
assess the role, if any, of the cocultured SMCs in mediating the
shear-induced changes in the expressions of these adhesion molecules in ECs. As shown in Figure 3, ECs cocultured with SMCs
(EC/SMC) in a static condition showed markedly higher ICAM-1,
VCAM-1, and E-selectin mRNA levels than did the ECs cultured
alone (EC/␾) and the ECs cocultured with identical ECs (EC/EC).
These results indicate that coculture with SMCs induced the EC
expression of these adhesion molecules. Application of shear stress
(12 dyne/cm2) for 6 hours to EC/␾ or EC/EC significantly
increased their ICAM-1 mRNA level as compared with the static
control (Figure 3A), without altering the mRNA levels of VCAM-1
(Figure 3B) and E-selectin (Figure 3C). These results are consistent
with those found using the monocultured EC monolayer as an
experimental model.11-17 Shear stress to the EC side of the EC/SMC
coculture completely abolished the SMC-induced ICAM-1,
VCAM-1, and E-selectin mRNA expression found in cocultured
ECs under the static condition. These results indicate that the
application of shear stress to EC/SMC coculture inhibits the SMC
induction of ICAM-1, VCAM-1, and E-selectin gene expression in
the cocultured ECs in the static condition. Our findings thus
implicate shear stress as an inhibitor of pathophysiologically
relevant gene expression in ECs that are located in close proximity
to SMCs.
To further confirm the regulatory effect of shear stress on
SMC-induced adhesion molecule gene expressions, various coculture membranes containing 3-␮m and 8-␮m pores were used. As
shown in Figure 4, ECs cocultured with SMCs using either type of
membrane yielded results for the adhesion molecule gene expression under static and flow conditions similar to those obtained
using 0.4 ␮m-pore membranes (Figure 3). This result indicates that
SMCs modulate adhesion molecule gene expression in cocultured
ECs under static and flow conditions through the membrane with
pore sizes of 0.4, 3, and 8 ␮m.
Figure 3. Shear stress inhibits the gene expression of
ICAM-1, VCAM-1, and E-selectin in ECs induced by
coculture with SMCs. ECs cultured alone (EC/␾) or cocultured with identical ECs (EC/EC) or SMCs (EC/SMC) were
maintained in a static condition or exposed to a shear stress of
12 dyne/cm2 for 6 hours and their mRNA levels were determined by RT-PCR analysis, as described in “Materials and
methods.” Amplification of cDNA was performed in parallel
samples using human GAPDH primers. Data are presented
for expression of ICAM-1 (A), VCAM-1 (B), and E-selectin (C)
as a percentage change in band density normalized to GAPDH
samples (ie, differences from densities in static control ECs
cultured alone [Control EC/␾]), and are shown as mean ⫾
SEM from 4 independent experiments. *P ⬍ .05 vs control
EC/␾. #P ⬍ .05 vs control EC/SMC.
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
Figure 4. Effect of membrane pore size on the expression of ICAM-1, VCAM-1,
and E-selectin in ECs cocultured with SMCs. ECs were cultured alone (EC/␾) or
cocultured with identical ECs (EC/EC) or SMCs (EC/SMC), using membranes
containing (A) 3-␮m or (B) 8-␮m pores. Cocultured ECs were maintained in a static
condition or exposed to shear stress of 12 dyne/cm2 for 6 hours and their mRNA
expression was determined by RT-PCR analysis, as described in “Materials and
methods.” Amplification of cDNA was performed in parallel samples using human
GAPDH primers. Data represent duplicate experiments with similar results.
ECs cocultured with fibroblasts did not alter their gene
expressions of ICAM-1, VCAM-1, and E-selectin in
response to static and flow conditions
In addition to SMCs, fibroblasts also exist in close proximity to
ECs in the vascular wall. To determine whether the fibroblasts also
exert a regulatory effect on the expression of ICAM-1, VCAM-1,
and E-selectin in ECs, the gene expressions of these adhesion
molecules were examined in ECs cocultured with fibroblasts in the
static condition or subjected to shear stress. In the static condition,
unlike the results in EC/SMC coculture (Figure 3), the adhesion
molecule mRNAs in ECs cocultured with fibroblasts (EC/FB) did
not change relative to the corresponding levels in ECs cultured
alone (EC/␾; Figure 5). The exposure of EC/FB to a shear stress of
12 dyne/cm2 for 6 hours increased the ICAM-1 mRNA expression
in ECs but did not change the expression of VCAM-1 and
E-selectin mRNA. Thus, the adhesion molecule gene expressions
in EC/FB under both static and flow conditions are the same as
those of ECs cultured alone. These results, which are fully
consistent with the results from the study using cultured EC
monolayers,11-17 indicate that the findings on EC/SMC coculture
resulted from a specific action of the neighboring SMCs in
modulating the EC responses to shear stress.
Regulatory effect of SMCs on the adhesion molecule gene
expressions in cocultured ECs is via the close
interaction between these cocultured cells
With the same type of coculture membrane, it has been
demonstrated that the close communication, or even cell-to-cell
contact, between the ECs and SMCs in the coculture results
from the formation of the cytoplasmic projection from SMCs
penetrating the entire length of the membrane pores to reach
ECs.30,31 Because the close interaction or heterotypic contact
between the cocultured cells has been shown to be crucial for
regulating endothelial function,42-44 we postulated that the
regulatory effect of SMCs on the expressions of ICAM-1,
VCAM-1, and E-selectin in cocultured ECs is attributable to the
close interaction between these cocultured cells. To test this
hypothesis, a strategy was used to eliminate the potential direct
contact between cocultured ECs and SMCs by first seeding the
SMCs onto cover slips and then placing the cover slips in the
compartments opposite those of ECs. Although close communication or contact between ECs and SMCs in coculture was
prevented, these cells could interact with each other by crosstransference of substances released from opposite sides of the
SHEAR STRESS REGULATES EC/SMC INTERACTION
2671
intervening membrane into the shared medium. In the static
condition, separation of the cocultured ECs and SMCs (EC/slide/
SMC) prevented the SMC induction of ICAM-1, VCAM-1, and
E-selectin mRNA levels when PET membrane was used in the
EC/SMC cocultures (P’s ⬍ .01; Table 1). Incubation of ECs
with culture medium collected from the EC/SMC cocultures
under the static condition (EC/␾ ⫹ med) did not significantly
stimulate their ICAM-1, VCAM-1, and E-selectin mRNA
expression (P’s ⬎ .5 vs control EC/␾). This finding further rules
out a secondary effect of the medium due to substances released
from the cocultured SMCs. Consistent with the effect of shear
stress on EC monocultures,11-17 the application of shear stress to
ECs in cocultures separated from SMCs by cover slips caused an
increase of the EC expression of ICAM-1, but not VCAM-1 and
E-selectin, as compared with the static controls. This is to be
contrasted with the decrease in EC ICAM-1 expression in the
EC/SMC module. Our results thus clearly indicate that the
effects of SMCs on the expression of ICAM-1, VCAM-1, and
E-selectin in cocultured ECs under static and flow conditions are
exerted through the close interaction or heterotypic contact
between these cocultured cells.
NO modulates SMC-induced ICAM-1, VCAM-1, and E-selectin
gene expression in cocultured ECs in the static condition
NO has been shown to inhibit the expression of a number of
pathophysiologically relevant genes, including ICAM-1 and
VCAM-1,45 induced by various stimuli. To explore whether NO
modulates the SMC induction of ICAM-1, VCAM-1, and
E-selectin in EC/SMC coculture, ECs were preincubated with an
NO donor (SNAP or DETA/NO) for 30 minutes before coculture
with SMCs in the presence of the NO donor. As shown in Figure
6A, SNAP or DETA/NO treatment of ECs at a concentration of
100 ␮M significantly suppressed SMC-induced ICAM-1,
VCAM-1, and E-selectin mRNA expression. The same treatments with NO donors had no effect on the mRNA expressions
of these adhesion molecules in control EC/␾ (data not shown).
To elucidate whether the induction of these adhesion molecules
in ECs by coculture is associated with the attenuation of NO
generation, eNOS gene expression in these cocultured ECs was
examined by RT-PCR analysis, as described in “Materials and
methods.” As shown in Figure 6B, ECs cocultured with SMCs
(EC/SMC) in the static condition significantly reduced their
eNOS mRNA expression as compared with the EC/␾. This
reduction of eNOS gene expression was not found in ECs
cocultured with identical ECs (EC/EC). Separation of the
Figure 5. ICAM-1, VCAM-1, and E-selectin gene expression in ECs cocultured
with fibroblasts was not altered in response to static and flow condition. ECs
cultured alone (EC/␾) or cocultured with fibroblasts (EC/FB) were maintained in the
static condition or exposed to shear stress of 12 dyne/cm2 for 6 hours and their mRNA
expression was determined by RT-PCR analysis, as described in “Materials and
methods.” Amplification of cDNA was performed in parallel samples using human
GAPDH primers. Data shown are for a representative experiment. Similar results
were obtained in a duplicate experiment.
2672
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
CHIU et al
Table 1. SMCs regulate ICAM-1, VCAM-1, and E-selectin gene expression in cocultured ECs via close interaction between these cocultured cells
Adhesion
molecule
Control
Shear
EC/SMC
EC/slide/SMC
EC/␾ ⫹ med
EC/␾
EC/SMC
EC/slide/SMC
ICAM-1
561 ⫾ 86*
98 ⫾ 16†
102 ⫾ 16†
339 ⫾ 40*
88 ⫾ 17†
296 ⫾ 32*
VCAM-1
1153 ⫾ 264*
103 ⫾ 19†
95 ⫾ 8†
92 ⫾ 11
42 ⫾ 11*†
95 ⫾ 14
967 ⫾ 213*
108 ⫾ 12†
97 ⫾ 13†
124 ⫾ 31
93 ⫾ 16†
109 ⫾ 18
E-selectin
ECs cultured alone (EC/␾) or cocultured with SMCs grown on a cover slip (EC/slide/SMC) or without a cover slip (EC/SMC) were maintained in static condition or exposed
to shear stress of 12 dyne/cm2 for 6 hours and their mRNA expression was determined by RT-PCR analysis, as described in “Materials and methods.” Amplification of the cDNA
was performed in parallel samples using human GAPDH primers. ECs that were incubated in a static condition with culture medium collected from the EC/SMC coculture
(EC/␾ ⫹ med) did not significantly increase their expression of ICAM-1, VCAM-1, or E-selectin mRNA. Data on band density normalized to GAPDH RNA levels are presented
as percentages of the results for the control EC/␾ (taken as 100%). The results shown are mean ⫾ SEM from 4 independent experiments in each group.
* P ⬍ .05 vs control EC/␾.
† P ⬍ .05 vs control EC/SMC.
cocultured cells by insertion of a cover slip completely abrogated the SMC modulation of eNOS gene expression (data not
shown). The results from these studies suggest that NO may
serve as a negative regulator for the adhesion molecule gene
expressions in ECs cocultured with SMCs.
Discussion
Vascular ECs are constantly exposed to hemodynamic forces,
including the shear stress due to blood flow. Recent studies have
demonstrated that a number of pathophysiologically relevant
genes, such as the platelet-derived growth factor (PDGF)–B
chain,46,47 monocyte chemotactic protein-1 (MCP-1),48,49 early
growth response-1 (Egr-1),50-52 and ICAM-1,11-15 are activated
by shear stress. Virtually all of these studies used EC monolayers on slides as an experimental model, which may not reflect
the in vivo environment of ECs, which exist in close proximity
to SMCs. In vivo, physical contact (via myoendothelial bridges)
between ECs and SMCs has been demonstrated,20,21 and it is
conceivable that bidirectional cross-talk between ECs and
SMCs may influence EC responses to hemodynamic forces and
affect vascular biology.
Through the use of our parallel-plate coculture flow system,
the current study provides several lines of evidence to demonstrate that the neighboring SMCs play a pivotal role in
mediating the endothelial expression of adhesion molecules
Figure 6. NO modulates SMC-induced ICAM-1, VCAM-1, and E-selectin gene
expression in cocultured ECs under the static condition. (A) NO donor attenuates SMC-induced ICAM-1, VCAM-1, and E-selectin mRNA levels in cocultured ECs.
ECs were pretreated with SNAP or DETA/NO for 30 minutes at a concentration of 100
␮M and then cocultured with SMCs in the presence of the respective NO donors.
Data represent duplicate experiments with similar results. (B) ECs cocultured with
SMCs reduced their eNOS mRNA expression. ECs cultured alone (EC/␾) or
cocultured with identical ECs (EC/EC) or SMCs (EC/SMC) were maintained in the
static condition and their eNOS mRNA levels were determined by RT-PCR analysis,
as described in “Materials and methods.” Amplification of cDNA was performed in
parallel samples using human GAPDH primers. Data are presented as percentages
of control EC/␾ in band density normalized to GAPDH RNA levels, and are shown as
mean ⫾ SEM from 4 independent experiments. *P ⬍ .05 vs control EC/␾.
(ICAM-1, VCAM-1, and E-selectin) in response to static and
flow conditions. First, ICAM-1, VCAM-1, and E-selectin mRNA
expression was augmented in ECs cocultured with SMCs in the
static condition. Second, the application of shear stress to the
cocultured EC/SMC inhibited these increases. Third, the responses of ECs cocultured with SMCs under static and flow
conditions were similar regardless of the pore size (0.4, 3, or 8
␮m) of the membrane placed between these cocultured cells.
Fourth, another type of vascular cells, fibroblasts, cocultured
with ECs could not substitute for SMCs in modulating the EC
expression of these adhesion molecules, suggesting that SMCs
have a specific regulatory effect on EC responses to shear stress.
Fifth, SMC modulation of the EC expression of these adhesion
molecules under static and flow conditions could be completely
abrogated when the cocultured cells were separated by insertion
of a cover slip, indicating that the contact or close proximity
between these cocultured cells is essential for the SMC modulation of the EC expression of these adhesion molecules. Finally,
incubation of ECs in a static condition with culture medium
collected from the coculture did not induce expression of these
adhesion molecules. These last 2 findings indicate that the effect
of SMC coculture did not result from a secondary effect via the
shared medium.
By introducing a coculture module into a parallel-plate flow
chamber, we have created a system that permits on-line direct
visualization of the cocultured cells during and after the flow
experiment. This model, in which the EC side of the coculture
can be subjected to shear flow while the opposite side, with
SMCs, remains in a static condition, mimics more closely the
physical environment of these vascular cells in vivo. The present
study on cell morphology in coculture has demonstrated, for the
first time, that SMCs in coculture tend to orient perpendicularly
to the flow direction after flow exposure of the cocultured ECs
for 24 hours. This result is consistent with the in vivo orientation
of SMCs in blood vessels39 and with observations on homogeneous SMC culture directly exposed to flow.40,41 The mechanism
underlying the perpendicular orientation of cocultured SMCs to
flow direction remains unclear. Several signaling molecules,
including calcium, have been suggested to be involved.40 Since
the SMCs in our model were not in direct contact with the shear
flow, it is likely that this flow-induced reorientation was caused
by the transmission of signals from ECs stimulated by the shear
flow. Although the morphological changes of ECs under coculture conditions were not clearly visible by phase microscopy,
owing to interference by the porous membrane and the overlying
SMCs, immonostainings for cytoskeletal reorganization and
junctional protein (ie, PECAM-1) distribution in these cocultured ECs provide evidence for actin fiber elongation and EC
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
alignment with the flow direction (data not shown). These
observations have validated the usefulness of our coculture flow
system for investigating EC-SMC interaction and their responses to hemodynamic forces.
The present result on increased ICAM-1, VCAM-1, and
E-selectin mRNA expression in cocultured ECs in a static
condition may explain the findings by Rainger et al25,26 and
Kinard et al53 that ECs cocultured with SMCs greatly increased
the adhesion of leukocytes. The mechanisms by which SMCs in
coculture augment these adhesion molecule expressions in ECs
under the static condition remain unclear. It has been shown that
ECs and SMCs regulate each other’s quiescent phenotype under
static conditions.30,31,38,42-44 Several growth factors (eg, activated transforming growth factor–␤1 [TGF-␤1]) have been
shown to be produced by the ECs and SMCs in coculture and
may be involved in some of the effects exerted by SMCs on
ECs.43 TGF-␤1 activation of ICAM-1, VCAM-1, and E-selectin
in ECs has been reported.54 The recent study by Rainger and
Nash26 demonstrated that increased adhesion of flowing leukocytes to ECs cocultured with SMCs could be abolished by a
neutralizing monoclonal antibody against TGF-␤1. It is likely
that these growth factors produced by ECs or SMCs in static
coculture exert autocrine or paracrine effects on ECs to contribute to their elevated expression of the adhesion molecules.
An additional factor that may be involved in regulating
endothelial function is NO, a relaxing factor released from ECs
via the activation of eNOS. EC-derived NO modulation of
collagen production by SMCs has been reported.55 We52 and
others45 have demonstrated that NO inhibits the EC expression
of a number of pathophysiologically relevant genes, including
Egr-1, ICAM-1, and VCAM-1, induced by chemical or mechanical stimulation. It was not clear whether a higher level of these
adhesion molecule expressions in cocultured ECs under the
static condition resulted from a lower level of NO production by
the cells. By RT-PCR analysis, the present study demonstrated
that the mRNA expression of eNOS in ECs cocultured with
SMCs under static conditions was significantly attenuated in
comparison with the EC monoculture or EC/EC culture. Moreover, exogenous addition of NO donors SNAP and DETA/NO
inhibited the mRNA expression of these adhesion molecules in
the EC/SMC coculture to an extent similar to that in the EC
monoculture or EC/EC culture. The precise molecular mechanism by which ECs and SMCs in the coculture communicate and
subsequently regulate endothelial function warrants further
investigation.
Another pathway by which SMCs transmit signals that
ultimately affect endothelial function is via the direct physical
contact between ECs and SMCs.20,21,42-44,56 Using the same type
of coculture membrane, Fillinger et al30,31 demonstrated that
physical contact between ECs and SMCs in the coculture may
exist, as evidenced by the extension of cytoplasmic projections
from SMCs across the entire length of the membrane pores to
reach ECs. It is very likely that this potential contact between
ECs and SMCs was responsible for the SMC modulation of
ICAM-1, VCAM-1, and E-selectin gene expressions in the
cocultured ECs. Despite the potential contribution of the
EC-SMC contact, the possible effect of close communication
between the cocultured cells via humoral transmission through
the tiny distance between these cells cannot be totally excluded.
Since the cells were separated by a very small volume formed in
the tiny slender pores (0.4 ␮m ⫻ 10 ␮m), it is also likely that the
humoral substances released from the cells on either side of the
membrane may reach those on the other side through a limited
SHEAR STRESS REGULATES EC/SMC INTERACTION
2673
distance at a very high concentration, which consequently exerts
significant effects. In the current study, the importance of cell
contact or close interaction between the cocultured ECs and
SMCs in regulating EC gene expression was indicated by the
lack of induction of ICAM-1, VCAM-1, and E-selectin in ECs
cocultured with SMCs grown on a cover slip, which eliminates
the possibility of EC-SMC contact. Moreover, ECs incubated in
the static condition with culture medium collected from the
EC/SMC coculture did not stimulate the mRNA expression of
these adhesion molecules. These findings rule out a secondary
effect of the medium, which contains a much lower concentration of substances released from the cells as compared with that
in the tiny pores. Thus, although the precise mechanism by
which SMCs regulate the adhesion molecule expressions in
cocultured ECs is not well understood, our data clearly indicate
that contact or close communication between these cocultured
cells is essential for the SMCs to exert their regulatory effects on
the EC expression of adhesion molecules.
An important finding of the current study is that shear stress
applied to the EC side of the coculture totally abolished
SMC-induced gene expressions of ICAM-1, VCAM-1, and
E-selectin in the cocultured ECs. The way in which shear stress
inhibits these EC gene expressions in coculture is unclear but is
likely to be multifactorial. Biologic mediators responsible for
augmented adhesion molecule expression in cocultured ECs
under the static condition may be suppressed or dominated by
another mediator or signal after flow application. Moreover, a
small transmural flow across the EC monolayer may exist owing
to the pressure difference between the opposite sides of the
porous membrane under the shear flow condition. This could
significantly alter local concentrations in the membrane pores
for substances released from the cocultured cells, thereby
modulating EC gene expressions. In addition to these adhesion
molecule expressions, our recent study further demonstrated
that the shear stress inducibility of various monocyte attractant
chemokines, including MCP-1 and growth-related oncogene
(GRO)–␣, could also be reduced in ECs by coculture with SMCs
(data not shown). These results suggest that this shear stress
inhibition of adhesion molecule expressions in ECs cocultured with
SMCs may reflect the regulatory roles of shear stress in gene
expressions in ECs. The present data suggest that shear stress may
play a protective role in vascular homeostasis by inhibiting the
expression of a number of pathophysiologically relevant genes in
ECs that are located in close proximity to SMCs.
In summary, the present study demonstrates that coculture with
SMCs induces the expression of ICAM-1, VCAM-1, and E-selectin
genes in ECs in the static condition, whereas the application of shear
stress to ECs inhibits these coculture-induced gene expressions. SMC
modulation of the EC expression of these adhesion molecules under
static and flow conditions is mediated via close interaction, or even
direct contact, between these cocultured cells. Our findings indicate that
shear stress acts as a protective regulator of pathophysiologically
relevant gene expression in vascular ECs, which normally exist in close
proximity to SMCs.
Acknowledgment
The authors are indebted to Dr Ned H. C. Hwang for helpful advice
and discussion.
2674
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
CHIU et al
References
1. Harlan JM. Leukocyte-endothelial interaction.
Blood. 1985;65:513-525.
2. Osborn L. Leukocyte adhesion to endothelium in
inflammation. Cell. 1990;62:3-6.
3. Nerem RM. Hemodynamics and the vascular endothelium. J Biomech Eng. 1993;115:510-514.
4. Frangos JA. Physical Forces and the Mammalian
Cell. San Diego, CA: Academic Press; 1993.
5. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519-590.
6. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transducer an
atheroprotective force. Arterioscler Thromb Vasc
Biol. 1998;18:677-685.
7. Chien.S, Li S, Shyy YJ. Effects of mechanical
forces on signal transduction and gene expression in endothelial cells. Hypertension. 1998:3(pt
2):162-169.
8. Gallik S, Usami S, Jan KM, Chien S. Shear
stress-induced detachment of human polymorphonuclear leukocytes from endothelial cell
monolayers. Biorheology. 1984;26:823-834.
9. Lawrence MB, Smith CW, Eskin SG, McIntire LV.
Effect of venous shear stress on CD18-mediated
neutrophils adhesion to cultured endothelium.
Blood. 1990;75:227-237.
10. Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL.
Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered
shear stress. Arterioscler Thromb Vasc Biol.
1995;15:2-10.
11. Nagel T, Resnick N, Atkinson WJ, Dewey CF Jr,
Gimbrone MA Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells.
J Clin Invest. 1994;94:885-891.
12. Tsuboi H, Ando J, Korenaga R, Takada Y, Kamiya
A. Flow stimulates ICAM-1 expression time and
shear stress dependently in cultured human endothelial cells. Biochem Biophys Res Commun.
1995;206:988-996.
13. Morigi M, Zoja C, Figliuzzi M, et al. Fluid shear
stress modulates surface expression of adhesion
molecules by endothelial cells. Blood. 1995;85:
1696-1703.
14. Sampath R, Kukielka GL, Smith CW, Eskin SG,
McIntire LV. Shear stress-mediated changes in
the expression of leukocyte adhesion receptors
on human umbilical vein endothelial cells in vitro.
Ann Biomed Eng. 1995;23:247-256.
lial cell-smooth muscle cell coculture model for
use in the investigation of flow effects on vascular
biology. Ann Biomed Eng. 1995;23:216-225.
23. Redmond EM, Cahill PA, Sitzmann JV. Perfused
transcapillary smooth muscle and endothelial cell
coculture—a novel in vitro model. In Vitro Cell
Dev Biol Anim. 1995;31:601-609.
24. Nackman GB, Fillinger MF, Shafritz R, Wei T,
Graham AM. Flow modulates endothelial regulation of smooth muscle cell proliferation: a new
model. Surgery. 1998;124:353-361.
25. Rainger GE, Stone P, Morland CM, Nash GB. A
novel system for investigating the ability of
smooth muscle cells and fibroblasts to regulate
adhesion of flowing leukocytes to endothelial
cells. J Immunol Methods, 2001;255:73-82.
26. Rainger GE, Nash GB. Cellular pathology of atherosclerosis: smooth muscle cells prime cocultured endothelial cells for enhanced leukocyte
adhesion. Circ Res. 2001;88:615-622.
27. Redmond EM, Cahill PA, Sitzmann JV. Flow-mediated regulation of endothelial receptors in
cocultured vascular smooth muscle cells:an endothelium-dependent effect. J Vasc Res. 1997;
34:425-435.
28. Redmond EM, Cahill PA, Sitzmann. Flow-mediated
regulation of G-protein expression in cocultured vascular smooth muscle and endothelial cells. Arterioscler Thromb Vasc Biol. 1998;18:75-83.
29. Hendrickson RJ, Cappadona C, Yankah EN, Sitzmann JV, Cahill PA, Redmond EM. Sustained pulsatile flow regulates endothelial nitric oxide synthase
and cyclooxygenase expression in cocultured vascular endothelial and smooth muscle cells. J Mol Cell
Cardiol. 1999;31:619-629.
30. Fillinger MF, O’Connor SE, Wagner RJ, Cronenwett JL. The effect of endothelial cell coculture on
smooth muscle cell proliferation. J Vasc Surg.
1993;17:1058-1068.
31. Fillinger MF, Sampson LN, Cronenwett JL, Powell
RJ, Wagner RJ. Coculture of endothelial cells and
smooth muscle cells in bilayer and conditioned
media models. J Surg Res. 1997;67:169-178.
32. Gimbrone MA Jr. Culture of vascular endothelium. In: Spact TH, eds. Progress in Hemostasis
and Thrombosis. Vol 3. New York, NY: Grune &
Stratton; 1976:1-28.
33. Chiu JJ, Wang DL, Chien S, Skalak R, Usami S.
Effects of disturbed flow on endothelial cells.
J Biomech Eng. 1998;120:2-8
15. Chiu JJ, Wung BS, Shyy YJ, Hsieh HJ, Wang DL.
Reactive oxygen species are involved in shear
stress-induced intercellular adhesion molecule-1
expression in endothelial cells. Arterioscler
Thromb Vasc Biol. 1997;17:3570-3577.
34. Osawa M, Masuda M, Kusano K, Fujiwara K. Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal
transduction: is it a mechanoresponsive molecule. J Cell Biol. 2002;158:773-785.
16. Ohtsuka A, Ando J, Korenaga R, Kamiya A,
Toyama-Sorimachi N, Miyasaka M. The effect of
flow on the expression of vascular adhesion molecule-1 by cultured mouse endothelial cells. Biochem Biophys Res Commun. 1993;193:303-310.
35. Lucchiari N, Panajotopoulos N, Xu C, et al. Antobidies eluted from acutely rejected renal allografts
bind to and activate human endothelial cells.
Hum Immunol. 2000;61:518-527.
17. Korenaga R, Ando J, Kosaki K, Isshiki M, Takada
Y, Kamiya A. Negative transcriptional regulation
of the VCAM-1 gene by fluid shear stress in murine endothelial cells. Am J Physiol. 1997;273:
C1506–1515.
18. Chappell DC, Varner SE, Nerem RM, Medford RM,
Alexander RW. Oscillatory shear stress stimulates
adhesion molecule expression in cultured human
endothelium. Circ Res. 1998;82:532-539.
19. Robenek H, Severs NJ. Cell interactions in atherosclerosis. Boca Raton, FL: CRC Press Inc; 1992.
20. Spagnoli LG, Villaschi S, Neri L, Palmieri G. Gap
junctions in myoendothelial bridges of rabbit carotid arteries. Experientia. 1982;38:124.
21. Little TL, Xia J, Duling BR. Dye tracers define differential endothelial and smooth muscle coupling
patterns within the arteriolar wall. Circ Res. 1995;
76:498-504.
22. Ziegler T, Alexander RW, Nerem RM. An endothe-
36. Birdsall HH, Lane C, Ramser MN, Anderson DC.
Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence. J Immunol. 1992;148:27172723.
37. Weber KSC, Draude G, Eri W, Martin R, Weber
C. Monocyte arrest and transmigration on inflamed endothelium in shear flow is inhibited by
adenovirus-mediated gene transfer of I␬B-␣.
Blood. 1999;93:3685-3693.
38. Powell RJ, Cronenwett JL, Fillinger MF, Wagner
RJ, Sampson LN. Endothelial cell modulation of
smooth muscle cell morphology and organization
growth pattern. Ann Vasc Surg. 1996;10:4-10.
39. Liu SQ. Influence of tensile strain on smooth
muscle cell orientation in rat blood vessels. J Biomech Eng. 1998;120:313-320.
40. Liu SQ, Goldman J. Role of blood shear stress in the
regulation of vascular smooth muscle cell migration.
IEEE Trans Biomed Eng. 2001;48:474-483.
41. Lee AA, Graham DA, Dela CS, Ratcliffe A, Karlon
WJ. Fluid shear stress-induced alignment of cultured vascular smooth muscle cells. J Biomech
Eng. 2002;124:37-43.
42. Orlidge A, D’Amore PA. Inhibition of capillary endothelial cell growth by pericytes and smooth
muscle cells. J Cell Biol. 1987;105:1455-1462.
43. Hirschi KK, Rohovsky SA, D’Amore PA. PDGF,
TGF-␤, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of
10T1/2 cells and their differentiation to a smooth
muscle fate. J Cell Biol. 1998;141:805-814.
44. Korff T, Kimmina S, Martiny-Baron G, Augustin
HG. Blood vessel maturation in a 3-dimensional
spheroidal coculture model: direct contact with
smooth muscle cells regulates endothelial cell
quiescence and abrogates VEGF responsiveness. FASEB J. 2001;15:447-457.
45. Khan BV, Harrison DG, Olbrych MT, Alexander RW.
Nitric oxide regulates vascular cell adhesion molecule-1 gene expression and redox-sensitive transcriptional events in human vascular endothelial
cells. Proc Natl Acad Sci U S A. 1996;93:9114-9119.
46. Resnick N, Collins T, Atkinson W, Bonthron DT,
Dewey CF Jr, Gimbrone MA Jr. Platelet-derived
growth factor B chain promoter contains a cisacting fluid shear stress-responsive element.
Proc Natl Acad Sci U S A. 1993;90:4591-4595.
47. Khachigian LM, Resnick N, Gimbrone MA Jr, Collins T. Nuclear factor-kappa B interacts functionally with the platelet-derived growth factor
B-chain shear stress-responsive element in vascular endothelial cells exposed to fluid shear
stress. J Clin Invest. 1995;96:1169-1175.
48. Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid
shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad
Sci U S A. 1994;91:4678-4682.
49. Shyy YJ, Lin MC, Han J, Lu Y, Petrime M, Chien
S. The cis-acting phorbol ester “12-O-tetradecanoylphorbol 13-acetate”-responsive element is
involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl
Acad Sci U S A. 1995;92:8069-8073.
50. Khachigian LM, Anderson KR, Halnon NJ, Gimbrone
MA Jr, Resnick N, Collins T. Egr-1 is activated in endothelial cells exposed to fluid shear stress and interacts with a novel shear stress-response element in
the PDGF-A chain promoter. Arterioscler Thromb
Vasc Biol. 1997;17:2280-2286.
51. Schwachtgen JL, Houston P, Campbell C,
Sukhatme V, Braddock M. Fluid shear stress activation of egr-1 transcription in cultured human
endothelial and epithelial cells is mediated via the
extracellular signal-related kinase 1/2 mitigenactivated protein kinase pathway. J. Clin Invest.
1998;101:2540-2549.
52. Chiu JJ, Wung BS, Hsieh HJ, Lo LW, Wang DL.
Nitric oxide regulates shear stress-induced early
growth response-1 expression via the extracellular signal-regulated kinase pathway in endothelial
cells. Circ Res. 1999;85:238-246.
53. Kinard F, Jaworski K, Sergent-Engelen T, et al.
Smooth muscle cells influence monocyte response to LDL as well as their adhesion and
transmigration in a coculture model of the arterial
wall. J Vasc Res. 2001;38:479-491.
54. Kang YH, Brummel SE, Lee CH. Differential effects of transforming growth factor-beta 1 on lipopolysaccharide induction of endothelial adhesion
molecules. Shock. 1996;6:118-125.
55. Paul RM, Tanner MA. Vascular endothelial cell
regulation of extracellular matrix collagen: role of
nitric oxide. Arterioscler Thromb Vasc Biol. 1998;
18:717-722.
56. Dora KA, Doyle MP, Duling BR. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc
Natl Acad Sci U S A. 1997;94:6529-6534.