HUVEC ICAM-1 and VCAM-1 Synthesis in Response to Potentially
Athero-Prone and Athero-Protective Mechanical and Nicotine
LIAM T. BREEN,1,2,3PETER E. MCHUGH,1,2and BRUCE P. MURPHY1
1National Centre for Biomedical Engineering Science, National University of Ireland Galway, University Road, Galway,
Ireland;2Department of Mechanical and Biomedical Engineering, National University of Ireland, University Road, Galway,
Ireland; and3Department of Mechanical and Manufacturing Engineering, Parsons Building, Trinity College Dublin, College
Green, Dublin 2, Ireland
(Received 23 June 2009; accepted 3 February 2010; published online 17 February 2010)
Associate Editor Scott I. Simon oversaw the review of this article.
investigated the endothelial cell (EC) morphological response
to mechanical stimuli; generally consisting of a wall shear
stress (WSS) and a cyclic tensile hoop strain (THS). More
recent studies have investigated the EC biochemical response
(intercellular adhesion molecule, ICAM-1, and vascular
cellular adhesion molecule, VCAM-1, expression) to idealized
mechanical stimuli. However, current literature is lacking in
the area of EC biochemical response to combinations of
physiological WSS and THS mechanical stimuli. The objec-
tive of this study is to investigate the EC response to
physiological WSS and THS stimuli and to compare this
response to that of ECs exposed to idealized steady WSS and
cyclic THS of the same magnitudes. This study also investi-
gated the EC response to a nicotine chemical stimulus
combined with a suspected athero-prone physiological
mechanical stimulus. A bioreactor was designed to apply a
range of combinations of physiological WSS and THS
waveforms. The bioreactor was calibrated and validated
using computational fluid dynamics and video extensometry
techniques. The bioreactor was used to investigated the
biochemical response exhibited by human umbilical vein
endothelial cells (HUVECs) exposed to physiological athero-
protective (first bioreactor test case, pulsatile WSS combined
with pulsatile THS) and athero-prone (second bioreactor test
case, oscillating WSS combined with pulsatile THS) mechan-
ical environments. The final testing environment (third
bioreactor test case) combined a nicotine chemical stimulus
with the mechanical stimuli of the second bioreactor test case.
In first and second bioreactor test cases, the addition of a
pulsatile THS to the WSS resulted in opposite trends of
ICAM-1 down-regulation and up-regulation, respectively.
This outcome suggests that the effect of the additional
pulsatile THS depends on the state of the applied WSS
waveform. Similarly, in first and second bioreactor test cases,
the addition of a pulsatile THS to the WSS resulted in a
VCAM-1 up-regulation. However, it has been previously
shown that the addition of a cyclic THS to a high- or low-
steady WSS resulted in a VCAM-1 down-regulation, indicat-
ing that the EC response to idealized mechanical stimuli
(steady WSS and cyclic THS) is not comparable to physio-
logical mechanical stimuli (unsteady WSS and pulsatile THS),
even though in both situations the average magnitude of WSS
and THS applied were similar. In third bioreactor test case, a
nicotine chemical stimulus induced a substantial VCAM-1
up-regulation and a moderate ICAM-1 up-regulation. The
addition of the mechanical stimuli of the second bioreactors
test case resulted in a greater VCAM-1 up-regulation than
what was expected, considering the observations of the
previous second bioreactor test case alone. This study found
that the EC biochemical response to physiological mechanical
stimuli is not comparable to the previously observed EC
response to idealized mechanical stimuli, even though in both
environments the mechanical stimuli were of a similar
magnitude. Also, the level of VCAM-1 expressed by the
nicotine stimulated ECs showedanelevated levelof sensitivity
to the athero-prone mechanical stimuli.
Keywords—Cell mechanotransduction, Wall shear stress,
Tensile hoop stretch, ICAM-1, VCAM-1, Nicotine.
The human vascular system is continuously exposed
tensile hoop strain (THS), and pressure. The magni-
tudes of these forces can vary greatly throughout the
arterial system, this can be caused by changes in arterial
wall mechanical properties, anatomical location, and
arterial geometry. Pathological studies show that arte-
rial disease is frequently found in localized regions that
are exposed to very specific hemodynamic WSS.23,28,50
Address correspondence to Liam T. Breen, Department of
Mechanical and Manufacturing Engineering, Parsons Building,
Trinity College Dublin, College Green, Dublin 2, Ireland. Electronic
Annals of Biomedical Engineering, Vol. 38, No. 5, May 2010 (? 2010) pp. 1880–1892
0090-6964/10/0500-1880/0 ? 2010 Biomedical Engineering Society
In the majority of previous in vitro mechano-
transduction studies, isolated mechanical forces have
examined the endothelial cell (EC) response to either
Additional studies have investigated the morphological
and cytoskeletal response of ECs to combinations of
pulsatile WSS and THS.30,52These studies have indi-
cated that cell reorientation and alignment and F-actin
filament reorganization are a result of a WSS stimulus,
and that this cellular response is greatly enhanced with
the addition of a THS. The latter study52alluded to the
hypothesis that the addition of the THS might enhance
the sensitivity of the ECs toward a WSS stimulus, i.e.,
a situation where the combination of the WSS and
THS resulted in having a synergetic effect on the ECs
morphological response. This experimental result
tends to imply that a simultaneous mechanical force
environment will result in a better understanding of the
in vivo function of ECs.
One known EC response to mechanical stimulation
is the up- or down-regulation of adhesion molecules.
The adhesion molecules ICAM-1 and VCAM-1 when
expressed in abnormal quantities have been shown to
be associated with the initiation and progression of
atherosclerosis.3,10,16,17,29,32,33,36,43It has been demon-
strated that a steady WSS can down regulate the level
of EC VCAM-1 expression,3this down-regulation was
shown to be time and shear stress dependent and
reversible. Similarly Nagel et al.33investigated the
response of HUVECs to a range of steady WSS, and
showed that ECs expressed a time-dependent, but
force-independent ICAM-1 up-regulation in response
to the WSS. Additionally, other studies have focused
on EC ICAM-1 and VCAM-1 expression in response
to a cyclic THS. Cheng et al.15and Yun et al.49
exposed ECs to a cyclic THS for up to 24 h and
up-regulation. Ali et al.2found that a 25% cyclic THS,
applied over 6 h, resulted in a 5.5-fold increase in the
level of EC ICAM-1 expression.
The effect of combined chemical and mechanical
stimulation has received limited coverage in the litera-
ture. For example, the EC response to a nicotine
chemical stimulus has been investigated previously.1,45
However, the effect on adhesion molecule expression of
a combined nicotine and mechanical stimulus has not
been observed in the literature to date. Zhang et al.1
showed that a nicotine chemical stimulus (10?5M and
10?7M) resulted in significant increased mRNA levels
of endothelial nitric oxide synthase and VCAM-1 after
24 h. Albaugh et al.1also found that a nicotine stimulus
(10?8M) up-regulated EC VCAM-1 expression after
3 h exposure. Additionally, this study also showed that
an ICAM-1 up-regulation occurred, but to a lesser
degree. Neither of these studies assessed a simultaneous
mechanical and chemical environment, which would
more accurately reflect the in vivo situation.
In this study, we propose to investigate the response
of ECs to a multiaxial physiological mechanical force
environment, in combination with a chemical stimulus.
The study used a custom bioreactor which was vali-
dated for the application of simultaneous physiological
mechanical loading patterns. The specific force pat-
terns applied to the cells relate to physiological con-
ditions found at the carotid sinus, an athero-prone
condition similar to the conditions found at the outer
wall of the carotid bulb, and an athero-protective
condition relating to the force environment at the distal
region at the carotid bifurcation. The test environment
was designed to test the hypothesis that atherosclerosis
initiation (i.e., inappropriate expression of adhesion
molecules) could be identified when different simulta-
neous WSS and THS patterns are applied to ECs. The
study also provides information on the compounding
effect that two significant risk factors have on the
function ofECs, via an
mechanical stimulus and a nicotine chemical stimulus.
AND WAVEFORM VALIDATION
The bioreactor was designed to simultaneously
apply a range of physiological WSS and THS
mechanical stimuli to a variety of tissues and cell types.
The bioreactor achieves this goal by combining two
previous bioreactor techniques: a cone and plate rhe-
ometer6and multiple flexible silicone substrates upon
which the cells are seeded.45The cone and plate rhe-
ometer generates the desired cellular WSS, while the
strain applied to the cellular substrates replicates the
THS applied to the arterial wall, similar to the situa-
tion if an arterial wall was cut longitudinally and rolled
out flat and stretched laterally. The WSS and the
substrate strain are applied in perpendicular directions,
as would normally be the case in the in vivo arterial
system. A cone diameter and cone angle of 120 mm
and 1.0?, respectively, were used. The tip of the cone
was machined flat (20 mm diameter) to prevent rub-
bing between the cone and plate surface. The cone to
plate separation at the center point was 175 lm. Eight
square windows were cut from the plate at a mean
radial position of 35 mm. Eight corresponding flexible
cellular substrates were placed beneath the plate at
each of the window locations, as shown in Fig. 1. For
each experiment 300 mL of cell media (EGM-2 Cell
Media) was used. Both the WSS and THS are sepa-
rately controlled by two stepper motors, which are
individually programed using LabView (version 6.1i).
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