Laminar Shear Stress Inhibits Vascular Endothelial
Cell Proliferation by Inducing Cyclin-Dependent
Kinase Inhibitor p21Sdi1/Cip1/Waf1
Shigeo Akimoto, Masako Mitsumata, Toshiyuki Sasaguri, Yoji Yoshida
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Abstract—Alterations in the functions of vascular endothelial cells (ECs) induced by fluid shear stress may play a pivotal
role in both the development and prevention of vascular diseases. We found that DNA synthesis of bovine aortic and
human umbilical vein ECs, determined by [3H]thymidine incorporation, was inhibited by steady laminar shear stress (5
and 30 dyne/cm2). This growth inhibition due to shear stress was associated with suppression of cell transition from the
G1 to S phase of the cell cycle. Therefore, we studied G1-phase events to find the molecules responsible for this cell cycle
arrest. Shear stress inhibited the phosphorylation of a retinoblastoma protein (pRb) and the activity of cyclin-dependent
kinase (cdk) 2 and cdk4, which phosphorylate pRb. The level of cdk inhibitor p21Sdi1/Cip1/Waf1 protein, but not that of
p27Kip1, increased as a result of shear stress, and the amount of p21 protein associated with cdk2 also increased, although
the protein level of cdk2 was unchanged. Shear stress markedly elevated the mRNA level of p21, and this elevation in
mRNA faded after the release of cells from shear stress, concomitant with a recovery of DNA synthesis. These results
suggest that steady laminar shear stress induces cell cycle arrest by upregulating p21. Derangement of the steady laminar
flow may release cells from this inhibition and induce cell proliferation, which, in turn, may cause atherosclerosis
through the induction of EC stability disruption. (Circ Res. 2000;86:185-190.)
Key Words: shear stress 䡲 cell cycle arrest 䡲 p21Sdi1/Cip1/Waf1 䡲 cyclin-dependent kinases 䡲 vascular endothelial cell
H
emodynamic forces have been implicated in the initiation
and localization of atherosclerosis.1 We found that 95% of
268 lipid depositions and 80% of 69 plaques in the aortas of
young subjects (aged ⬍39 years) in Japan were initiated within
a circle of 5-mm radius, with radii extending from the centers of
branch orifices, especially on the proximal side of each orifice.
In contrast, the development of atherosclerosis at flow dividers,
which are high shear stress regions, was restricted, consistent
with other data.2 We also found that disruption of atherosclerotic
plaques on the descending aorta was likely to occur at the distal
edges of plaques, where eddies and separation of the bloodstream from the vessel wall occur,3 resulting in the plaque wall
surfaces being exposed to low shear stress. These disruptions are
unlikely to occur at the top of plaques, which are exposed to high
laminar shear stress. There is evidence that the enhancement of
endothelial permeability and of lipid deposition preferentially
occurs at turbulent low mean shear stress regions in the arteries.4
In contrast, a thick glycocalyx5 and an increase in zonular-type
tight junctions between endothelial cells (ECs),6 which may
function as antiatherosclerotic factors, are more often observed
in high laminar shear stress regions of the arteries. These in vivo
observations were supported by evidence obtained in cultured
ECs; ie, high laminar shear stress promotes glycosaminoglycan
synthesis,7 tight junction formation, and the expression of
junction-related proteins.6 These observations suggest that shear
stress generated by blood flow may play a pivotal and antipodal
role in the induction and progression or prevention of atherosclerosis by changing EC functions.
Several investigations have indicated that the DNA synthesis of ECs preferentially occurs at branch orifices, where
atherosclerosis is initiated.8,9 Because increased cell division
may accelerate endothelial permeability,10 stabilization of
ECs may be achieved and maintained by restricting EC
proliferation. Consistent with other researchers,11 we found
that steady laminar shear stress inhibits the DNA synthesis of
ECs in vitro. However, the molecular mechanisms of this
inhibition are unknown. In the present study, we examined
the effects of steady laminar shear stress on cell cycle events
during the G1 and S phases, including the phosphorylation of
a retinoblastoma protein (pRb), the activation of cyclindependent kinases (cdks), and the expression of cdk inhibitors
in cultured vascular ECs.
Materials and Methods
Antibodies
The following antibodies were used: anti-human pRb (XZ-104,
G3–245), p21 (6B6), and p27 (G173–524) monoclonal antibodies
Received August 31, 1999; accepted November 3, 1999.
From the Department of Pathology (S.A., M.M., Y.Y.), School of Medicine, Yamanashi Medical University, Nakakoma, Yamanashi, Japan, and the
Department of Bioscience (T.S.), National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Correspondence to Masako Mitsumata MD, PhD, Department of Pathology, School of Medicine, Nihon University, 30-1 Kamicho, Oyaguchi, Itabashi,
Tokyo 173-8610, Japan.
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
185
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Circulation Research
February 4, 2000
from PharMingen; anti-human cdk2 polyclonal antibody from
Upstate Biotechnology; anti-human cdk4 (H-22) and p21 (C-1)
polyclonal antibodies from Santa Cruz Biotechnology; and
anti-human p27 (57) monoclonal antibodies from Transduction
Laboratories.
Exposure to Shear Stress
Bovine aortic ECs (BAECs) and human umbilical vein ECs
(HUVECs) were plated on plastic slides or polyester sheets, as
described previously.12 Flow experiments were performed according
to a method similar to that described previously.13 A confluent
monolayer of ECs in a parallel-plate flow chamber was exposed to
steady laminar shear stress. Control ECs were grown in the same
manner as the sheared ECs and were placed into fresh medium
before being maintained in an incubator.
DNA Synthesis
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After being exposed to shear stress, ECs were incubated in culture
medium containing 2 ␮Ci/mL [3H]thymidine for 2 hours. After the
cells were treated with trichloroacetic acid, they were dissolved with
0.1 mol/L NaOH, and the incorporated radioactivity was counted and
normalized by cellular protein, which was measured in parallel
samples according to the method of Lowry.
Flow Cytometry
BAECs (1⫻106) were pelleted and fixed with 70% ethanol. After the
cells were digested with RNase, the DNA was stained with propidium iodide and then analyzed with a flow cytometer.
Immunoprecipitation and Western Blotting
HUVECs were lysed in a lysis buffer containing 10 mmol/L
Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
Na3VO4, 50 mmol/L NaF, 20 ␮g/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40, followed by centrifugation. The supernatant was reacted with an antibody for 1 hour
at 4°C. Immunoprecipitates recovered by protein A– or protein
G– conjugated Sepharose beads were separated by SDS-PAGE,
transferred onto a polyvinylidene difluoride membrane, and then
incubated with an antibody for 2 hours. Immunoblots developed with
a peroxidase-conjugated secondary antibody were visualized with
enhanced chemiluminescence reaction reagents (Amersham) and
analyzed with the public domain NIH image program.
cdk Assay
The immunoprecipitates, which were isolated from cells by reaction
with an antibody for cdk2 or cdk4, were reacted with glutathione
S-transferase–fused murine pRb carboxyl terminal (GST-Rb, Santa
Cruz Biotechnology) in a kinase reaction buffer in the presence of
50 ␮mol/L cold ATP and 3.7 MBq/mL [␥-32P]ATP. The radiolabeled
pRb, fractionated with SDS-PAGE, was quantified with a BAS-2000
Bio Image analyzer.
Northern Blotting
The cDNA for p21 was obtained from human vascular smooth
muscle cells as described previously.14 Extraction of total RNA,
electrophoresis, and transfer onto nylon membranes were performed
as previously described.13 Blots were hybridized with cDNA probes
for 16 hours at 50°C and were visualized and quantified with a
BAS-2000 Bio Image Analyzer.
Statistical Analysis
The results are expressed as mean⫾SD of the number of observations (n⫽3 to 5 at each point) and analyzed with a 1-factor ANOVA,
after confirming variance homogeneity by use of the Bartlett test.
An expanded Materials and Methods section is available online at
http://www.circresaha.org.
Figure 1. Time course of the shear stress–induced inhibition of
EC DNA synthesis. Immediately after confluent BAECs and
HUVECs were exposed to 30-dyne/cm2 (b), 5-dyne/cm2 (c), or
1-dyne/cm2 (d) laminar steady shear stress for the indicated
times, the cells were exposed to 2 ␮Ci/mL [3H]thymidine for 2
hours. The radioactivity incorporated in trichloroacetic acid–insoluble materials was counted with a liquid scintillation counter
and normalized with a cellular protein that was measured in parallel samples. The data are presented as values relative to the
static control, which was set at 100%. There was no obvious
detachment of BAECs subjected to 30-dyne/cm2 shear stress
for 24 hours (a). The flow was from left to right. *P⬍0.01 vs
static control.
Results
Effects of Shear Stress on DNA Synthesis
There was no obvious detachment of ECs subjected to shear
stress (Figure 1a, 24-hour exposure). The DNA synthesis of
BAECs significantly decreased after exposure to 30-dyne/cm2
shear stress (Figure 1b). This decrease was first evident 2
hours after the start of cell exposure to shear stress, reached
40.4% of the static control level after 4 hours, and remained
at this low level for at least 24 hours. An inhibitory effect on
DNA synthesis was also observed with 5-dyne/cm2 shear
stress (4 and 12 hours) in BAECs (Figure 1c). However, no
significant inhibition was observed at 1-dyne/cm2 shear stress
(Figure 1d), suggesting that there is a threshold of growth
inhibition due to steady laminar flow between 1- and 5-dyne/
cm2 shear stress in certain populations of BAECs. The DNA
synthesis of HUVECs also decreased significantly, to 38.6%,
Akimoto et al
Shear Stress–Induced Cell Cycle Arrest in ECs
187
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Figure 3. Inhibition of pRb phosphorylation by shear stress.
HUVECs were exposed to 30-dyne/cm2 shear stress for the
indicated times. ECs were lysed, and immunoprecipitates were
size-fractionated in a 6% polyacrylamide gel and reacted with
an anti-Rb monoclonal antibody for Western blotting. ppRb indicates hyperphosphorylated form of pRb. The left lane shows the
molecular marker.
Figure 2. Effect of shear stress on the cell cycle. After BAECs
were subjected to 30-dyne/cm2 shear stress for the indicated
times, sheared and control ECs were harvested by trypsin, fixed
with ethanol, and treated with RNase and EDTA. After DNA was
stained with propidium iodide, we evaluated the fluorescence by
flow cytometry. The data are the percentages of cells in each
phase, as determined by EPICS Profile Analyzer.
17.9%, and 7.9% of the static control level, after exposure to
30-dyne/cm2 shear stress for 6 hours and 5-dyne/cm2 shear
stress for 4 and 12 hours, respectively (Figures 1b and 1c).
However, unlike BAECs, 1-dyne/cm2 shear stress also significantly inhibited the DNA synthesis of HUVECs with 4 and
12 hours of exposure (Figure 1d), suggesting that the threshold in HUVECs may be set at a lower level than that in
BAECs. This diversity in response to flow may represent a
species difference (bovine versus human) or a vascular
difference (artery versus vein or aorta versus umbilical
vessel).
system, which regulates the G1/S transition, participated in
this mechanism.
As shown in Figure 3, a hyperphosphorylated form of pRb,
which moves more slowly than the hypophosphorylated form
in SDS-polyacryamide gels, decreased 12 hours after the start
of exposure to shear stress (30 dyne/cm2) in HUVECs.
Therefore, shear stress was considered to inhibit a cell cycle
event upstream from pRb phosphorylation. Thus, we analyzed the effects of shear stress on the activity of cdk2 and
cdk4, which are responsible for the phosphorylation of pRb.
The phosphorylation of GST-Rb by both cdk2 (Figure 4a)
and cdk4 (Figure 4b) in HUVECs was greatly decreased by
shear stress (30 dyne/cm2) within 2 and 4 hours, respectively.
Cell Cycle Arrest by Shear Stress
Flow cytometric analysis for the DNA content in BAECs
indicated that the cell populations in the S and G2/M phases
progressively decreased after the cells were subjected to shear
stress (30 dyne/cm2). The percentage of cells in the S phase
was reduced to 10.4% compared with the static control value
of 13.1% after 6 hours and was reduced to 9.7% compared
with the static control value of 12.3% after 12 hours.
Similarly, the G2/M population was 15.7% versus 21.8%,
14.0% versus 20.7%, and 9.1% versus 17.9% in sheared and
control cells after 2, 6, and 12 hours, respectively. This
reduction was accompanied by an increase in the percentage
of cells in the G0/G1 phase (Figure 2). Similar results were
obtained after 24 hours (n⫽5, data not shown). These data
indicate that shear stress inhibits the DNA synthesis of ECs
via inhibition of the cell transition from the G0/G1 to the S
phase of the cell cycle.
Inhibition of cdk Activities by Shear Stress
To elucidate the mechanism underlying cell cycle arrest
induced by shear stress, we examined whether the cdk-pRb
Figure 4. Effect of shear stress on cdk activity. The activity of
cdk2 (a) and cdk4 (b) was assayed by using a pRb fragment as
the substrate. After HUVECs were subjected to 30-dyne/cm2
shear stress, immunoprecipitates isolated from sheared and
static control cells via the reaction with a polyclonal antibody for
human cdk2 or cdk4 were incubated with [␥-32P]ATP and GSTRb. The reaction products were fractionated with 8%
SDS-PAGE.
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Figure 5. Shear stress–induced p21 protein expression. After
HUVECs were exposed to 30-dyne/cm2 shear stress for the
indicated times, protein extracts from sheared or static ECs
were immunoprecipitated with a polyclonal anti-p21 antibody,
size-fractionated with 14% SDS-PAGE, and reacted with a
monoclonal anti-p21 antibody (a). To measure the protein level
of cdk2-associated p21 and cdk2, cell extracts from sheared
and control ECs were immunoprecipitated with an anti-cdk2
polyclonal antibody, separated by SDS-PAGE, and then immunoblotted with an anti-p21 monoclonal antibody (b) or anti-cdk2
polyclonal antibody (c). The p27 protein level was also measured by immunoprecipitation and immunoblotting by use of
antibodies for p27 (d).
Induction of cdk Inhibitor p21 by Shear Stress
The activities of cdks are controlled by the cdk inhibitor
proteins, which include Cip/Kip family members, such as
p21Sdi1/Cip1/Waf1 and p27Kip1, and Ink4 family members, such as
p15Ink4b and p16Ink4a.15 The Cip/Kip family universally inhibits
several cdks, whereas the Ink4 family specifically inhibits
cdk4 and cdk6. Because shear stress inhibits both cdk2 and
cdk4, we wondered whether shear stress stimulates the
expression of the Cip/Kip family inhibitors.
As shown in Figure 5a, the p21 protein level increased 2
hours after the start of exposure to shear stress (30 dyne/cm2)
and maintained a high level for 12 hours, with an almost
2.2-fold increase over that of the control. To measure the
amount of cdk2-associated p21, immunoprecipitates from an
anti-cdk2 antibody were blotted with an anti-p21 antibody.
The protein level of the cdk2-associated p21 clearly increased
after the exposure of HUVECs to shear stress (30 dyne/cm2)
for 4 and 12 hours (Figure 5b), although the total amount of
cdk2 itself was unchanged by shear stress (Figure 5c). In
contrast to p21, the p27 protein did not increase as a result of
shear stress (30 dyne/cm2) (Figure 5d).
Shear stress (30 dyne/cm2) increased the level of p21
mRNA quite quickly in HUVECs (Figure 6a). This expression was first evident after 15 minutes and became maximal
4 hours after the start of exposure to shear stress. It was
maintained at a high level for 12 hours, similar to the protein
level, with an almost 2.3-fold increase over that of the control
Figure 6. Shear stress–induced p21 mRNA expression. After
HUVECs were exposed to 30-dyne/cm2 (a) or 5-dyne/cm2 (c)
shear stress, total RNA from sheared or static ECs was sizefractionated and hybridized with a labeled p21 cDNA probe. For
the analysis of mRNA expression, the same blot was rehybridized with ␤-actin cDNA. Radioactivities were quantified with an
image analyzer, and the p21/␤-actin ratios were calculated (b).
(Figure 6b). The induction of p21 mRNA expression was also
observed with lower shear stress. With 5-dyne/cm2 shear
stress, HUVECs expressed p21 mRNA within 30 minutes and
maintained a high level for 12 hours (Figure 6c).
Akimoto et al
Shear Stress–Induced Cell Cycle Arrest in ECs
189
Figure 7. Effect of the release of ECs from laminar shear stress on DNA synthesis and p21
mRNA expression. After HUVECs were subjected
to 30-dyne/cm2 shear stress for 6 hours, the cells
were released from shear stress, placed in culture dishes, and incubated in a static condition
for 1, 6, or 12 hours. The [3H]thymidine uptake
into the cells and the p21 mRNA expression were
measured at each time point, as described in the
legends of Figures 1 and 6, respectively.
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For further confirmation of the linkage between shear
stress–induced growth inhibition and p21 expression, we
measured the DNA synthesis and p21 mRNA expression
after releasing the cells from shear stress. When we
released the cells from shear stress (30 dyne/cm2) after 6
hours of exposure and incubated them in a static condition
for various periods up to 12 hours, the DNA synthesis of
the cells recovered to almost the control level within 6
hours after the cessation of shear stress. Simultaneously,
the p21 mRNA level, which had increased after 6 hours of
exposure to shear stress, decreased during the same period
of incubation (Figure 7).
Discussion
In the adult aorta, the average turnover rate of ECs is very low
(⬍0.8% per day).8,16 An exception was observed in the area
surrounding the branch orifices, where flow separation and a
vortex occur, and 1.3% of these ECs were found to be in the
S phase of the cell cycle.8 In tissue cultures, consistent with
our data, it has been shown that the exposure of ECs to steady
laminar flow decreases proliferation,11 whereas turbulent
flow stimulates proliferation.17 When a flow-disturbing bar
was placed in a parallel-plate flow chamber and cell division
was monitored, cell division increased in the restricted area of
flow separation with a high shear stress gradient (just behind
the obstacle) but not in the area where shear stress recovered
to its nondisturbed value with uniform laminar flow (1.5 mm
downstream from the obstacle).18 Together with our other
results, these data suggest that blood flow has antipodal
effects on EC growth, namely, inhibition of growth with
steady laminar flow and a stimulation of growth with disturbed flow.
In the present study, steady laminar shear stress blocked a
cell cycle event that occurred before entry into the S phase,
because the accumulation of the cell population in the G0/G1
phase after exposure to shear stress was accompanied by a
decrease in the number of cells in the S phase as well as a
decrease in the G2/M population. These results suggest that
laminar shear stress plays a key regulatory role in the
inhibition of proliferation, in association with maintaining
cells in the quiescent G0/G1 phase of the cell cycle. Further-
more, the derangement of blood flow (such as at branch
orifices) may release cells from this inhibition and induce cell
proliferation, which may in turn disturb the stability of ECs.
How is the shear stress signal transmitted to the EC nuclei,
leading to G0/G1 arrest in the cell cycle? Many mRNAs,
second messengers (for a review, see References 19 and 20),
and transcription factors,21 which could be involved in the
signal transduction of EC proliferation, have been shown to
be induced by shear stress. However, almost none of them has
been shown to be directly responsible for the mechanisms by
which shear stress– dependent growth arrest is induced. The
importance of cdk2 and cdk4 activation and the subsequent
phosphorylation of pRb in the G1 to S transition has been
emphasized in a variety of cells (for a review, see Reference
22). The phosphorylation of pRb is required to release E2F
transcription factors from the pRb/E2F complex; these transcription factors then activate the expression of genes required for initiating DNA synthesis.23 The activities of cdk
are negatively regulated by several cdk inhibitor proteins.
p21Sdi1/Cip1/Waf1 binds to several cdks, including cdk2 and cdk4,
to inhibit their activities.24 In the present study, we have
demonstrated for the first time that steady laminar shear stress
induces p21 expression and enhances cdk2-p21 binding,
concomitant with the suppression of both cdk2 activation and
pRb phosphorylation, causing cell cycle arrest in ECs. Because cdk4 activity was also suppressed by shear stress, p21
may also inhibit cdk4-mediated pRb phosphorylation.
On the other hand, the level of p27 was not altered by shear
stress. Therefore, the contribution of p27 to the shear stress–
induced inhibition of cdks appeared to be small. However,
this does not rule out the possibility of the involvement of
p27, because the manner in which p27 inhibits cdks may be
different from that of p21. In epithelial cells, transforming
growth factor-␤ (TGF-␤) elevates the expression of the
cdk4/cdk6-specific inhibitor p15Ink4B and induces the release
of p27 from cdk4 and cdk6. This release increases the binding
of p27 to cdk2, which leads to cell cycle arrest.25 TGF-␤ is
also induced by shear stress in ECs.26 Therefore, the possibility remains that TGF-␤ induced by shear stress shifts p27
from cdk4 and cdk6 to cdk2 and inhibits cdk2 activity. To
study this possibility, we exposed HUVECs to shear stress
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February 4, 2000
(30 dyne/cm2) for 8 hours in the presence of either a
neutralizing antibody for TGF-␤ 1 (10 ng/mL) or tetraethylammonium (3 mmol/L), a K⫹ channel blocker that inhibits
shear stress–induced TGF-␤ expression.26 Neither of these
affected the inhibition of DNA synthesis by shear stress (data
not shown), suggesting that TGF-␤ may not be involved in
the mechanotransduction of shear stress–mediated growth
inhibition. The upstream signal transduction of p21 expression, involved in the growth inhibition of ECs induced by
shear stress, remains to be determined.
Acknowledgments
This study was supported in part by a grant-in-aid from the Ministry
of Education, Science, and Culture, the Ministry of Science and
Technology Agency, and the Ministry of Health and Welfare of
Japan. The authors are grateful to Drs Akihiko Hashi (Yamanashi
Medical University) and Katsuhiro Teramoto (Yamanashi Prefectural Central Hospital) for providing the human umbilical cords.
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Laminar Shear Stress Inhibits Vascular Endothelial Cell Proliferation by Inducing
Cyclin-Dependent Kinase Inhibitor p21 Sdi1/Cip1/Waf1
Shigeo Akimoto, Masako Mitsumata, Toshiyuki Sasaguri and Yoji Yoshida
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Circ Res. 2000;86:185-190
doi: 10.1161/01.RES.86.2.185
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