J. Biomedical Science and Engineering, 2010, 3, 206-212
doi:10.4236/jbise.2010.32027 Published Online February 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online February 2010 in SciRes. http://www.scirp.org/journal/jbise
Pressure shift mediated anoikis of endothelial cells in the flow
field in vitro
Jia Hu1, Er-Yong Zhang1, Jiang Wu2, Wei-Lin Xu3, Huai-Qinq Chen2, Ying-Kang Shi1,
Ying-Qiang Guo1,3*
1Dept. Thoracic & Cardiovascular surgery, West China Hospital of Sichuan University, Chengdu, China;
2Institute of Biomedical Engineering, West China Medical Center, Sichuan University, Chengdu, China;
3The State Key Lab of Hydraulics on High Speed Flow, Sichuan University, Chengdu, China.
Received 16 November 2008; revised 10 December 2009; accepted 13 December 2009.
ABSTRACT
Dramatic changes of pressure in the local circulation
flow field would lead to alterations in biorheological
characteristics of Endothelial cells(ECs), and futher
resulted in the apoptosis induced by loss of anchorage,
a form of cell death known as anoikis. In this study,
we set levels of pressure(negative and positive pres-
sure) loaded ECs groups and non-activated cultured
ECs ,single shear stress loaded ECs as control group
to demonstrate the effects of pressure shift on cell
morphogenesis and adhesion. Furthermore, we in-
vestigate the effects of pressure shift on ECs proli-
feration and apoptosis to elucidate the influences of
pressure shift on vitality of ECs. We present these
data here to suggest that the negative pressure might
be another important factor beyond velocity and shear
stress in biomechanical impairment on ECs, then to
trigger the apoptosis with the extracellular matrix
(ECM) detachment (anoikis). As the negative pressure
is thought to play a role in the anoikis process, these
results have implications for both the path- ogenesis
and therapeutics investigations of stenostic vessel dis-
eases and the future vascular tissue engineering.
Keywords: Endothelial Cells; Anoikis Cell Adhesion;
Pressure; Flow Field
1. INTRODUCTION
The role of the Extracellular matrix (ECM) goes beyond
providing the physical scaffold on which the Endothelial
cells (ECs) adhere, it also provides ECs with information
for proliferation, migration, differentiation and survival
through the structural and functional links. Loss of these
links with ECM could induce apoptosis which has been
termed as anoikis, a Greek ancient word meaning
“homelessness”. Previous Hydrodynamics investigations
on the mechanisms of the cardiovascular wall damage,
in vitro assays and in vivo models, focused on the rela-
tionship between the velocity, shear stress and the ECs,
while investigations on the pressure shift (especially the
negative pressure) mediated anchorage-related apoptosis
(anoikis) of ECs in vitro were rarely described. Based on
the engineering hydrodynamics advance, the dilated
downstream of the stenosis could lead to a decrease of
wall pressure and an increase of pressure pulsation, fur-
ther resulted in a low pressure environment to generate
the cavitation phenomenon which would damage the
wall structure severely [1,2]. We therefore suggest that
the distribution and variations of pressure located down-
stream of the stenostic vessel may be another factor for
biomechanical impairment on ECs, then may contribute
to the pathogenesis of cardiovascular stenostic diseases.
In the present study, we developed an efficient assay
for the effects of pressure shift on the expression of cy-
toskeleton(F-actin), Vascular adhesion molecule (VCAM)
and one of the important transmembrane heterodimeric
receptors(IntegrinαVβ3). Combined with our analysis of
Ecs proliferation, apoptosis and the expression of apop-
tosis-associated protein (Caspase-3, P53, Bcl-2 and Fas),
we also presented correlative evidence that the negative
pressure played a certain role in the genesis and progress
of anoikis in the flow field in vitro.
2. MATERIALS AND METHODS
2.1. Cell Cultures and Maintenance
Human umbilical vein ECs EA.Hy926 obtained from
Jiangsu Institute of Hematology were cultured in a 5%
CO2 atmosphere at 37³C, in RPMI medium (Gibco BRL,
USA) supplemented with 10% fetal bovine serum(FBS).
Cells were detached by D-Hank’s solution with 0.25%
trypsin 1ml and then repelleted to suspend in the RPMI
medium. The supernatants were collected and centri-
fuged at 1000rpm for 5min in a MIKRO12–24 centri-
*The study supported by NSF of China (Grant NO. 30700149, 30670515
and Youth Scientific Fund of Sichuan University (Grant NO.06062).

J. Hu et al. / J. Biomedical Science and Engineering 3 (2010) 206-212 207
fuge at 4℃. The purified cells were collected, tested as
previously described [3,4] and viability was determined
by trypan blue exclusion. After been attached to the fi-
bronectin plates (plastic plates coated for 30min at 37℃
in CO2 incubator with 50ug/ml of fibronectin, washed
twice with D-Hanks solution before use), the cells were
grown at 37℃ in 5% CO2 incubator (Heraeus, Germany)
with RPMI medium to confluence.
2.2. Levels of Pressure Loading in the Flow
Experiment
The flow system was remanufactured from the system
which was previously described by H.Q. Chen [5] (Fig-
ure 1). The plastic slides containing the endothelial
monolayer were inserted into the parallel-plate flow
chamber that was installed between the upper and
lower reservoir connected by tubing. Continuous flow
in this system was maintained by circulating cell cul-
ture medium (as arrow shown in Figure 1) by a peri-
staltic pump installed between the upper and lower
reservoir, while altitude difference provided constant
flow through the chamber to expose the bottom of the
inserts to laminar flow at a consistent levels of pressure.
As the periodical fluctuation of the pressure that caused
by the peristaltic pump and fluid flow could interfere
the experiment results, we set two reservoirs as a feed-
back regulator to maintain consistent and steady pres-
sure. The numerical analysis of the chamber flow per-
formed by Fluent 6.0 indicated the flow in chamber
was 1) laminar flow; 2) two-dimensional flow; 3) suf-
ficient developed steady flow; 4) the pressure distrib-
uted in chamber averagely; 5) maintained steady flow
in negative pressure environment. We set nonactivated
cultured ECs(Con), single shear stress (1.85 dyn/cm2)
loaded ECs (S-con)as control groups and levels of
pressure loaded groups in the context of low shear
stress (1.85dyn/cm2): –10cmH2O(N10), –20cmH2O(N20),
–40cmH2O(N40), +20cmH2O(P20) and +40cmH2O(P40).
Figure 1. The schematic diagram of the improved paral
alized by using BODIPY FL phalloidin (Molecularlel
plate flow chamber: 1) upper reservoir; 2) peristaltic
pump; 3) lower reservoir and; 4) flow chamber.
2.3. F-actin and Adhesion Molecule Analysis
Loaded by levels of pressure for 2h and fixed by 4%
paraformaldehyde at 4℃ for 15min, ECs were perme-
abilized in 1% Triton X-100 and then F-actin was visu
Probes, USA). The cell membranes were imaged at an
excitation of 505nm and emission of 512nm by the laser
confocal scanning microscope (Bio-Rad Mre-1024ES)
and fluorescence density value analysis of F-actin were
tested by IMAGEPRO plus 5.0(Media Cybernetics Inc.).
The collected cells were incubated with specific mono-
clonal antibody (PE-VCAM monoclonal antibody in
1:40 dilution, FITC-IntegrinαVβ3 monoclonal antibody
LM609 in 1:100 dilution) respectively for 20 minutes.
Cells were then resuspended in PBS to a density of 2～
3×105cells/ml. The levels of cell surface fluorescently
labeled protein were quantified by immunofluorescent
flow cytometry (ELITE ESP, Coulter, USA): we set the
fluorescent density value of non-activated cultured
ECs(Con) as 100% and the fluorescent density value of
other groups as Related fluorescent density value
(RF%).
2.4. Proliferation and Apoptosis Assay
To label chromosomal DNA with propidium iodide (PI),
the pressure loaded cells were washed twice with PBS,
0.25％ trypsin and the resulting cell pellet was resus-
pended at 1×105 cells/ml in PBS containing 100ug/ml of
PI and 500mg/ml RNase (Sigma; St. Louis, MO) for a
30min incubation at 4℃. The stained cells were then
analyzed by flow cytometry and quantification was per-
formed by using Cell Quest (Becton–Dickinson, Moun-
tain View, CA). We use PI [Proliferation Index, PI=(S +
G2M) / (G0/1+ S + G2M ) × 100%] and AI (Apoptosis
Index, AI=number of cells displaying red fluorescence
lower than the G0-G1 diploid peak / total number of
cells × 100%) to assay ECs proliferation and apoptosis
changes in cell cycle. RT-PCR technology as previously
described [6,7,8] and Western blot analysis were applied
to caspase–3, p53, Bcl–2 and Fas protein expression
assay.
2.5. Statistics
All experiments were repeated three times. Statistic in-
formation was analyzed by one sample T-test with
SPSS11.5 and differences at P<0.05 were considered
statistically significant.
3. RESULTS
3.1. The Effects of Pressure Shift on Cells
Morphological Changes
Stained green-fluorescence, the F-actin filament of
non-activated cultured ECs(Con) showed no oriented

208 J. Hu et al. / J. Biomedical Science and Engineering 3 (2010) 206-212
Figure 2. The effects of pressure shift (loaded for 2h) on
F-actin A. The variations of the distribution and organi-
zation of F-actin (A: con; B: S-con; C: N10; D: N20; E:
N40; F: P20; G: P40) B. The quantitative analysis of the
effects of pressure shift on F-actin expression (n=6,
**compared with S-Con group p<0.01).
shear stress for 2h (S-con), the F-actin filament showed a
tendency to orient parallel to the long axis of the cells
(Figure 2 B). As the negative pressure increasing, the
redistribution and organization of F-actin were more
obviously oriented parallel to the long axis of the cells
and flow vector (Figure 2 c, d, e). However, the F-actin
filament presented no orientation propensity to the posi-
tive pressure changes (Figure 2 f, g). As the fluores-
cence value density of BODIPY FL phalloidin was con-
sistent with the F-actin content, we analyzed the expres-
sion of F-actin by testing the green-fluorescence value
density with Image Pro Plus 5.0. The expression of
F-actin was generally enhanced by the increasing nega-
tive pressure (n=6, compared with S-con p<0.01), while
no significant change of F-actin expression was ob-
served in the positive pressure loaded group.
3.2. The Effects of Pressure Shift on Cell Adhe-
sion Molecules
When exposed to levels of pressure and loaded for 2h,
the expression of VCAM and IntegrinαVβ3 demon-
strated significant changes with different tendency:
1) VCAM expression up-regulated with increasing
positive pressure and down-regulated with gradually
increasing negative pressure;
Table 1. The effects of pressure shift (loaded for 2h) on the
expression of VCAM and IntegrinαVβ3 (n=6,**compared with
S-Con group p<0.01).
Assay Items (relative fluorenscence values)
Groups
VCAM Integrin αVβ3
con 100±0 100±0
S-con 110.7±31.2 151.5±16.9
N10 113.8±11.3 152.5±29.7
N20 108.7±11.8 217.4±50.2**
N40 78.3±4.9** 328.7±14.5**
P20 144.7±22.6** 233.2±31.7**
P40 155.8±24.5** 270.3±23.5**
Figure 3. The duration-dependent effects of –40cmH2O
pressure on the expression of VCAM and IntegrinαVβ3
A. The variations of VCAM expression B. The varia-
tions of IntegrinαVβ3 expression (n=6**compared with
S-Con group p<0.01).
2) The increasing negative and positive pressure both
resulted in the intensified expression of the IntegrinαVβ3
(Table 1).
As –40cmH2O pressure caused a significant changes
in the expression of VCAM and IntegrinαVβ3, we fur-
ther studied the duration-dependent effects of the –40cm
H2O pressure on the expression of VCAM and Integri-

J. Hu et al. / J. Biomedical Science and Engineering 3 (2010) 206-212 209
nαVβ3: VCAM expression down-regulated with the
pressure loaded duration while the expression of In-
tegrinαVβ3 up-regulated initially and started to decrease
gradually after pressure loaded for 2h (Electronic sup-
plementary Material).
JBiSE
3.3. The Effects of Pressure Shift on ECs
Proliferation and Apoptosis
As Loaded by levels of pressure for 2h, groups of ECs
show different viability changes: 1) certain proliferative
activity could be observed in the low negative pressure
loaded groups (N10, N20) and significant apoptosis only
occurred in the –40cm H2O pressure loaded group (N40);
2) significant changes of proliferation and apoptosis
were not observed in the positive pressure loaded
groups(P20, P40). We therefore analyzed the time course
of the –40cmH2O pressure-induced proliferation and
apoptosis changes in ECs (Table 2).
As the RT-PCR results showed, the expression of cas-
pase-3 was only significantly up-regulated in N40 group.
We therefore investigated the duration dependent effects
of –40cmH2O pressure on caspase-3 and apoptosis asso-
ciated protein: P53、Bcl-2 and Fas by Western blotting
analysis (Figure 4): the expression of Caspase-3, Bcl-2
and Fas protein up-regulated with duration-dependence
in –40cmH2O pressure loading, while P53 protein
showed a fluctuated increasing expression during the
first 4h negative pressure (–40cmH2O) loading and
sharply decreased back to the baseline in the next 2h.
Table 2. The duration-dependent effects of –40cmH2O on
the cell cycle, proliferation and apoptosis index (
s
x

, n=6).
Cell cycle distribution (%)
Group
G0/G1 S G2/ M
PI (%) AI (%)
0 min 59.1±4.9 19.8±5.8 22.7±1.5 41.4±7.90.1±0.1
15 min57.8±2.5 22.9±7.8 22.2±5.6 44.8±7.50.4±0.0
30 min67.2±4.4 22.5±1.2 10.9±4.1 33.2±6.66.5±0.6
1 h 82.6±3.6 7.6±0.5 11.9±4.6 18.4±5.110.2±0.3
2 h 81.6±7.5 6.8±0.4 11.2±6.7 17.4±2.018.4±0.6
4 h 83.6±11.2 7.0±0.2 9.1±0.5 15.2±7.319.4±024
6 h 90.6±13.3 5.3±0.3 5.2±1.6 10.1±5.927.6±1.1
Figure 4. Western blotting results of anoikis-associated proteins A Caspase-3; B P53; C Bcl-2; D Fas protein.

210 J. Hu et al. / J. Biomedical Science and Engineering 3 (2010) 206-212
4. DISCUSSION
Anchorage-related apoptosis appeared in ECs which
were experimentally detached from their extracellular
matrix had suggested the role of ECM as a suppressor of
apoptosis induced by biomechanical forces [9]. Since the
discovery of adhesion-activated tyrosine kinase pp125
FAK [10,11], a web of signaling networks spread out
and revealed the multiple pathways that could regulate
the adhesion-related apoptosis(anoikis). Through these
pathways, variety of biomechanical factors in flow field
was capable of triggering the anoikis process. Many
studies demonstrated [12,13] that observable changes in
cytoskeleton and viability of ECs only occurred under
the condition of loaded shear stress>8 dyn/cm2 and du-
ration>24h. To differ the focus from previous reports in
the literature, we therefore adopted extremely low shear
stress(1.85dyn/cm2) and short pressure loading duration
(≤6h) to evaluate the pressure shift effects specifically.
The cytoskeleton, particularly the content and distri-
bution of F-actin, is a strong determinant in the me-
chanical properties of ECs, such as cell shape, stiffness
and cytoplasm viscosity. We had identified that the sig-
nificant changes in F-actin expression and cell shape
were only observed after certain negative pressure ex-
posure for 2h(N20 and N40 group). Interestingly, up-
regulated expression of F-actin and streamlined cell
shape resulted in down-regulated expression of VCAM、
up-regulated expression of apoptosis-associated pro-
tein(especially Fas protein) and increased Apoptosis
Index(AI%). However, these findings were different
from previous studies that streched cells were found to
be susceptible to rescuing from anoikis [14,15]. As our
previous data demonstrated [5], when exposed to high
shear stress, the adhesion ability of cells enhanced with
the concentrated distribution of F-actin from the cortex
to the perinuclear area of cells. We therefore considered
that the increasing negative pressure may disassembly
the F-actin filaments to monomers but inhibited its reor-
ganizaton in the perinuclear area of cells, further resulted
in the increased amount of F-actin presenting only in the
cortex but sharply decreased the adhesion ability. Some
evidence demonstrated that the cytoskeletal disruption
regulated the FasL expression and were associated with
anoikis [16], which was coincident with our findings in
the consistent up-regulation of F-actin and Fas expres-
sion with negative pressure(–40cmH2O) loaded for 2h.
However, the detailed mechanism of this process re-
mains to be determined by further investigations. In the
experiment, we demonstrated the readily apparent dif-
ferences between F-actin and ECs viability of negative
versus positive pressure loaded ECs, which implied that
the negative pressure could regulate both signaling
molecules and apoptosis-related protein that were asso-
ciated with the cytoskeleton, and as such may together
regulate anoikis by serving as sensors of cytoskeletal
integrity.
Sufficient and appropriate adhesion to the ECM, rep-
resented by the VCAM, is critical for proper ECs func-
tion and signaling. With the organization of the cy-
toskeleton and transduction of biochemical signals, the
external (ECM) to internal (ECs) signaling transduction
are mediated by the integrins [17]. As the member of the
integrin family, IntegrinαVβ3 is capable of recognizing
and bind many ECM proteins and induce intracellular
biochemical responses to regulate anoikis process[18].
Therefore as a signaling transductant, the expression of
IntegrinαVβ3 were observed significantly up-regulated
in both positive and negative pressure groups with
enough loaded duration and intensity (Table 1). Fur-
thermore, we demonstrated the up-regulated expression
of VCAM with low AI% in positive pressure loaded
groups(P20, P40) and significant down-regulated expr-
ession of VCAM accompanied by high AI% in N40
group with duration-dependence, which implied that the
negative pressure (–40cmH2O) induced apoptosis could
be adhesion-dependant. However, in our time course
experiment on IntegrinαVβ3 and apoptosis-associated
proteins (Figure 4), the up-regulated expression of In-
tegrinαVβ3 in ECs were consistent with the intensified
expression of Caspase-3、Bcl-2 and Fas protein when
exposed to –40cmH2O pressure loaded for 2h, during
which the expression of VCAM showed insignificantly
changing. The findings give evidence of the Integri-
nαVβ3 mediating some proapoptotic responses that
could contribute to the integrated mechanism of negative
pressure induced anoikis process with adhesion-inde-
pendence. In support of our findings, previous studies
[19] suggest the apoptotic effects observed with Integri-
nαVβ3 antagonist (echistatin) in ECs were not due to
detachment but rather due to activation of intracellular
signals.
Multiple pathways could initiate the caspase activa-
tion to induce anoikis process converging at the level of
effector caspases-3 [20]. As two main pathways in which
caspases cascade are initially activated, the death recep-
tor pathway and mitochondrial pathway are regulated by
a host of key effector proteins, such as P53、Bcl-2 and
Fas proteins which have been previously confirmed as
important regulators in different pathways [21,22]. As
we noted, the expression of P53 showed a fluctuated
increasing within 4h negative pressure (–40cmH2O)
loading (Figure 4), which might imply the multiple
functions of P53 protein in regulating the early stage of
anoikis process. During the first 2h, P53 expression
up-regulated initially and then down-regulated with the
up-regulation of Bcl-2 expression. Meanwhile, the de-
creasing PI% that still exceeded the gradually increasing
AI% (Table 2). Published studies [23] demonstrated that
P53 could encode a transcription factor to activate genes

J. Hu et al. / J. Biomedical Science and Engineering 3 (2010) 206-212 211
involved in growth arrest (p21, GADD45) and also
could control the anti-apoptotic protein Bcl-2 in mito-
chondrial pathway. Therefore, we considered the main
function of P53 protein during the first 2h was inducing
the growth arrest to reduce the sensitivity of ECs to
apoptosis. While the slight down-regulated P53 expres-
sion on the 2h checkpoint could be explained by some
evidence that P53 and Bcl-2 may combined as p53-Bcl2
complexes in contributing to the direct mitochondrial
p53 pathway of apoptosis [24]. Although much of p53-
mediated apoptosis signals were through mitochondrial
pathways, p53 inducible genes could alter the localiza-
tion of death receptors normally found in the cytoplasm
to the cell surface to enhance the sensitivity to death
receptor-mediated apoptosis (Fas) [25]. Therefore, at the
end of the first 2h, the homeostasis in the proliferation
and apoptosis of ECs broke down (AI% started to ex-
ceed PI %) with the significantly up-regulated Fas ex-
pression and P53 expression. Meanwhile, the expression
of VCAM showed a significant down-regulation, which
further supported the notion that the negative pressure
induced apoptosis could be adhesion-dependent (anoi-
kis). As we noted, the P53 expression surprisingly de-
creased back to the baseline with up-regulated Bcl-2
expression after being loaded by negative pressure
(–40cmH2O) for 4h. During the same period, consistent
with the up-regulated Fas expression, the expression of
Caspase-3 still up-regulated with increasing AI%, which
indicated the predominating function of Fas protein in
the latter stage(after 4h) of negative pressure induced
anoikis process.
We have confirmed a certain role of negative pressure,
particularly –40cmH2O pressure, in biomechanical im-
pairment on ECs with adhesion-dependence. While our
preliminary investigations on the mechanism of negative
pressure induced anoikis demonstrated that P53 acts dual
function in regulating the early stage of anoikis process
and Fas protein (death receptor pathway) predominated
the end stage of the negative pressure induced anoikis
process. These data give us insights into integrated in-
vestigations on mechanisms of downstream vascular
impairment in stenostic vessel diseases (eg. atheroscle-
rosis, post-stenostic aneurysm formation) and Integri-
nαVβ3, P53, Fas represent attractive targets for protec-
tive therapeutics aiming at downstream vessels for a
better long-term results in the patients with stenostic
vessel dieases. However, the key to anoikis regulation
depends on the sum of intrinsic and extrinsic input, It
will be of interest for us to sort out the precise manner
by which the architectural state of the cytoskeleton, in-
tegrin signal transduction events and posttranslational
apoptotic factors are interrelated.
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