Download pulsatile shear stress and high glucose concentrations induced

PULSATILE SHEAR STRESS AND HIGH GLUCOSE CONCENTRATIONS INDUCED
REACTIVE OXIGEN SPECIES PRODUCTION IN ENDOTHELIAL CELLS
J. Q. Yu1, L. K. Chin1, Y. Fu1, T. Yu2, K. Q. Luo2 and A. Q. Liu1
1
School of Electrical & Electronic Engineering,
School of Chemical & Biomedical Engineering,
Nanyang Technological University, Singapore 639798
2
Flow induced
shear stress
ABSTRACT
A hemodynamic Lab-on-a-chip system was
developed in this study. This system has two unique
features: (1) it consists of a microfluidic network with
an array of endothelial cell seeding sites for testing
them under multiple conditions, and (2) the flow rate
and the frequency of the culture medium in the
microchannel are controlled by a pulsation free pump
to mimic the flow profile of the blood in the blood
vessel under different physiological conditions. The
results demonstrate the advantage of utilizing this
system over the conventional non-pulsatile system in
the future shear stress related studies.
KEYWORDS
hemodynamic Lab-on-a-chip system, endothelial cell,
reactive oxygen species
INTRODUCTION
Reactive oxygen species (ROS) are natural byproducts of the normal aerobic metabolism in cells.
The main source of ROS production is the
mitochondrion during oxidative phosphorylation,
which involves the electron transport chain. In the
chain, electrons are passed through several proteins by
different oxidation–reduction reactions. However, a
small percentage of the electrons are leaked out from
the mitochondrion electron transport chain, and
subsequently superoxide anions are generated [1-2].
Hydrogen peroxide is a poorly reactive oxidizing
agent but it is cell membrane-permeable [3]. It is
generated by several enzymatic systems inside the cells.
Under normal metabolic condition, hydrogen peroxide
is involved in the regulation of the signal transduction
events. However, excess hydrogen peroxide will cause
cellular toxicity, causes the loss of homeostasis. The
level of oxidative stress of the cell can be determined
by monitoring the morphological change of the
mitochondria. Normally, mitochondria in endothelial
cells form a filamentous reticular network and
constantly undergo morphological changes through
fusion and fission. Fig. 1 shows the cellular responses
of endothelial cells interact with physical and
biochemical microenvironments. The shear stress is
imposed directly on the apical surfaces of the ECs and
Blood soluble
molecules
Endothelial cells
Nucleus
ROS↑
Basal lamina
Mitochondria
fission
Integrins
Figure 1: Endothelial cells interact with their physical and
biochemical microenvironments.
the blood soluble molecules send chemical cues to the
cells.
There are several reasons for the excess levels of ROS
production in cells [4]. First, short term elevation of
ROS level occurs after exhaustive exercise. Second, the
presence of exogenous ROS sources can also elevate the
ROS level, such as ultraviolet light, ionizing radiation,
inflammatory cytokines, environmental toxins, etc.
Third, diseases can also increase the ROS level, such as
hypertension, renal failure, diabetes, cardiac disease, etc
[5]. Therefore, researchers have put in efforts to
investigate the relationship between the elevated ROS
level and the ROS-related diseases, and further search
for potential drugs with antioxidant ability [6]. For
example, high glucose level in diabetes patients and
high sodium level14 in renal failure patients have been
proven to increase the ROS production level in cells. In
addition, different antioxidants or ROS scavengers were
studied and shown to reduce ROS production level.
However, these studies were conducted under either a
static condition in culture dishes or under a constant
shear stress, which were different from the
physiological condition with pulsatile shear stress [7].
In this paper, the designed Lab-on-a-chip system
mimics the flow rate of blood in the artery at the resting
condition or after an exhaustive exercise. Different
concentrations of glucose were added into the flow to
mimic hyperglycemic conditions in diabetes patients.
The shear induced cellular responses under different
extracellular condition were studied by investigating the
ROS production level in the endothelial cells and by
Medium
Medium with
20 mM glucose
(a)
(b)
(c)
(d)
(e)
(f)
Pneumatic
valve
90 min
60 min
30 min
15 min
Outlets
Figure 2: Schematic illustrations of the design of the
microfluidic chip.
monitoring
the
morphological
changes
of
mitochondrion using fluorescence imaging technique.
MATERIALS AND METHODS
The design of the hemodynamic Lab-on-a-chip
system is shown in Fig. 2. The chip consists of threelayer structures, namely bottom layer with a
microfluidic network for cell loading and liquid
injection, middle layer with pneumatic valves and top
layer with pneumatic connecting channels to control a
series of pneumatic valves simultaneously. Each
branch of the microchannel has 4 cell culture sites to
perform a series of time course experiments. Each cell
culture site has a width of 600 μm and a height of 150
μm. The downstream of each cell culture site is
connected to a common outlet and has a pneumatic
valve for flow switching. Three conditions with
different glucose concentration can be achieved with
the concentration gradient network. A pulsation free
precision pump (Nemesys, Cetoni) was used for cell
loading and media injection. The flow profile of the
pump was configured to mimic the blood flow in a
blood vessel.
Initially, endothelial cells were loaded, cultured
and stained prior to the application of shear stress and
chemical treatments as shown in Figure 3a. Harvested
cells were concentrated by centrifugation and then
resuspended in the growth medium at the concentration
of about 1×10-6 cells ml-1. The chips were then
incubated in the cell culture incubator with medium
replenishment every 24 hours until the cells in the
microchannel reached almost full confluence for the
experiments as shown in Figure 3b.
The intracellular ROS level was measured using a
cell-permeable
fluorescent
dye
of
2’,7’dichlorodihydrofluorescein diacetate (H2DCFDA).
Prior to the chemical and shear stress experiments, the
cultured endothelial cells in the microfluidic chips
Figure 3: Microphotos of (a) seeding ECs in the
microchannel; (b) culture for 48 hours; (c-d) measurement
of intracellular ROS level using H2DCFDA; (e) filamentous
reticular mitochondrial networks; and (f) mitochondrial
fragmentation.
were incubated in culture medium with 10 mM
H2DCFDA for 45 min to establish a stable intracellular
level. The cells were then being exposed under several
chemical and shear conditions with the same
concentration level of H2DCFDA dissolved in culture
medium. With the presence of ROS in the cells, the
H2DCFDA retained in the cytoplasm was oxidized and
became fluorescent as illustrated in Figure 3(c-d). For
each condition, 300 cells were measured to obtain the
average fluorescence intensity which is related to the
ROS concentration.
Mitochondrial morphology can be visualized by
staining the mitochondria inside the cells using a green
fluorescent dye (MitoTracker Green FM, Invitrogen) as
shown in Figure 3(e-f). Prior to the chemical and shear
stress experiments, the endothelial cells grown in the
microfluidic chips were incubated in culture medium
with a working concentration of 200 nM of MitoTracker
dye for 45 min. For each chemical and shear treatment
condition, 300 cells were examined to determine the
morphological changes of the mitochondria. Each data
point was expressed as a mitochondrial fission ratio,
which is the ratio of the number of cells with diffused
mitochondria to the total number of cells evaluated.
RESULTS AND DISCUSSIONS
Three different shear treatment conditions were
selected to investigate the shear-induced responses on
1.0
1.0
Normalized fluorescent Intensity
Normalized fluorescent intensity
Negative control (Static)
Shear stress 30 (Constant)
Shear stress 15 (Pulsatile)
0.8
Shear stress 30 (Pulsatile)
0.6
0.4
0.2
0
15
30
60
90
120
Figure 4: Time-dependent responses of the ROS level in
ECs under different shear stress profiles.
cultured endothelial cells in the microchannel: (1) a
constant shear stress of 30 dyne cm-2 with a steady
flow rate of 8.46 mL s-1, (2) a normal physiological
pulsatile shear stress of 15 dyne cm-2 (mean) with a
frequency of 70 bpm and (3) a fast pulsatile shear
stress of 30 dyne cm-2 (mean) with a frequency of
140bpm to mimic the condition under exhaustive
exercise.
The temporal responses of the endothelial cells in
ROS production level under different shear treatment
conditions are shown in Fig. 4. The static condition
without a fluidic flow serves as a negative control of
the experiment and it can be seen that the fluorescence
intensity increased when the dye molecules stained the
cells for longer than 15 min. To exclude this intensity
increment, the normalized fluorescence intensities for
the other three shear treatment conditions were
subtracted with that of the negative control. The
intracellular ROS level when the endothelial cells were
being exposed to pulsatile shear stress of 30 dyne cm-2
for different time periods. The ROS level was
increased at the first 60 min by nearly 4-fold.
Prolonged exposure of the endothelial cells to the shear
flow up to 2 hours resulted in a sustained elevation in
ROS levels, which is similar to the one reported
previously. The ROS levels of endothelial cells under
different shear treatment conditions for up to 120 min.
By comparing the ROS levels in various shear
treatment conditions at the 60 min time point, there is
no significant increase of ROS level between the
constant shear stress of 30 dyne cm-2 and the negative
control, which has no shear stress as the culture
medium was maintained in a static condition in the
microchannel (0.36/0.34).
In contrast, the increment was more substantial
when the endothelial cells were being exposed to
10 mM glucose (Static)
Shear stress 15 (Pulsatile)
10 mM glucose + shear stress
0.8
0.6
0.4
0.2
0
30
60
90
Time (min)
Figure 5: The ROS level in ECs being exposed to 10 mM
glucose with pulsatile shear stress of 15 dyne cm-2
physiological pulsatile shear stress conditions. The ROS
level was increased by 1.7 fold (0.56/0.34) and 2.2 fold
(0.76/0.34) at pulsatile shear stress of 15 and 30 dyne
cm-2, respectively. This results show that it is necessary
to investigate the effect of shear stress on the
endothelial cells under a physiological condition, which
can truly reflect the pulsatile pattern of the blood flow in
the artery. In addition, reports stating that pulsatile shear
stress are critical to maintain the functionalities of
endothelial cells in the artery have been presented.
Therefore, it is more realistic to expose the endothelial
cells using pulsatile shear stress under different
chemical conditions in the experiments.
For normal people, the concentration of glucose in
the blood is ranging from 4 to 6 mM. For diabetes
patients, however, the concentration of glucose in the
blood is above 6 mM and may be up to 20 mM. In these
experiments, 10 mM of glucose was added to mimic the
plasma condition of a diabetes patient and the ROS
level of the endothelial cells was monitored under
normal physiological pulsatile shear stress condition or
under exhaustive exercise. The experimental results for
the normal physiological pulsatile shear stress condition
are shown in Fig. 5. When the endothelial cells were
being exposed to 10 mM glucose under the normal
pulsatile shear stress of 15 dyne cm-2, the ROS level was
quickly increased for over 4-fold in the first 60 min and
stabilized afterward. By comparing the ROS level at the
60 min time point between the cells treated with 10 mM
glucose in a static state and that under a normal shear
stress of 15 dyne cm-2, the ROS level was increased 1.7
fold (0.81/0.49). It can be concluded that although high
concentration of glucose can elevate the level of
intracellular ROS, a much higher level of ROS can be
detected if the glucose solution is delivered under a
normal pulsatile shear stress condition.
Mitochondrial fission ratio (%)
40
studied by mimicking the physiological pulsatile flow
profiles in the blood vessel, i.e. during resting and
exhaustive exercising. The results show that ROS level
was elevated during exhaustive exercise (shear stress of
30 dyne cm_2) and with high glucose concentration
(diabetes patient). The results also show that pulsatile
shear stress is an essential element to mimic the
physiological conditions in the blood vessel, which also
highlights the potential of using the developed Lab-ona-chip system in future hemodynamic studies as
compared to the conventional experimental setup.
20 mM glucose
20 mM glucose + shear stress 15
20 mM glucose + shear stress 30
30
20
10
0
0
1
2
3
Time (h)
Figure 6: Mitochondrial morphology of endothelial cells
with 20 mM glucose under different shear treatment
conditions.
To investigate the mitochondrial morphological
changes of endothelial cells, 20 mM of glucose was
added and the endothelial cells were being exposed
under different shear treatment conditions for 4 hours.
The fluorescent images of the endothelial cells after
being exposed for 4 hours and the mitochondrial
fission ratios monitored for the first 4 hours are shown
in Fig. 6. For endothelial cells treated with 20 mM
glucose for 4 hours, mitochondrial fragmentation was
prevalent with a mitochondrial fission ratio of 20%.
When the endothelial cells were being exposed with 20
mM glucose plus the pulsatile shear stress of 15 or 30
dyne cm-2, the mitochondrial fission ratio was further
elevated from 20% to 25% and 26%, respectively. The
results show that the chemical treatment (i.e. high
concentration of glucose) produced a more dominant
effect in promoting mitochondrial fission than the
physical treatment (i.e. pulsatile shear stress),
especially in such an extreme case of 20 mM of
glucose.
CONCLUSIONS
In this paper, the intracellular ROS level and the
mitochondrial morphology of endothelial cells under
physiological pulsatile shear stresses and different
glucose concentrations were investigated using a
hemodynamic Lab-on-a-chip system. The intracellular
ROS level was studied using real-time fluorescence
microscopy with the measurement of the oxidation of
H2DCFDA by hydroxyl radicals or hydrogen peroxide
molecules. The elevated cellular ROS level led to
morphological changes of mitochondria from
filamentous reticular networks to diffused and short
fragments. The mitochondrial morphology was studied
via fluorescent MitoTracker staining. The shearinduced
cellular responses of endothelial cells under glucose
concentration of 10 mM or 20 mM were realized and
ACKNOWLEDGEMENT
This work was supported by the Environmental and
Water Industry Development Council of Singapore
(Grant No. MEWR C651/06/171).
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CONTACT
A. Q. Liu
Email: [email protected]
Tel: (65) 6790-4336
Fax: (65) 6793-3318