Real-time
Detection of Nitric Oxide in Cultured Rat Aorta Endothelial Cells Induced by
Shear Stress
YE Jian-Feng^1,3, ZHENG Xiao-Xiang³, XU Lisa Xueming ^1,2*

( ¹Department of Biomedical Engineering,
Purdue University, West Lafayette, IN 47906, USA;

²School
of Mechanical Engineering, Purdue University, West Lafayette, IN 47906, USA;

 ³Department of Biomedical Engineering, Zhejiang University, Hangzhou
310027, China )

Abstract       To
establish a stable and real-time method to detect the production of
intracellular nitric oxide (NO) of endothelial cells under different shear
stresses. After the cultured endothelial cells were loaded with DAF-FM, the
relative NO production was determined by fluorescence intensity, which was
detected using Zeiss Axioskop 2 fluorescence microscope and ICCD-camera. The
fluorescence of DAF-FM can reflect NO production. Shear stress can induce a
dose-dependent increase of NO production, and the increase can be totally
inhibited by L-NAME and partly inhibited by Ca²⁺-free buffer. The
method can be used to detect the change of NO production in real time, and it
can also be used to study the mechanism related to NO increase induced by shear
stress.

Key words   endothelial
cells; nitric oxide; shear stress; DAF-FM; calcium

Shear stress affects endothelial cells in many ways,
such as cytoskeletal rearrangement, decrease of intracellular pH, release of
PGI2 and some growth factors (PDGF, FGF, ECGF,TGF-b, etc), activation of IP3
and mitogen-activated protein kinases, and significant increase of nitric
oxide(NO) production[1–4]. Therefore, hemodynamic forces play a key role in
many pathological processes. Atherosclerotic lesions tend to develop in regions
where there are separations from unidirectional laminar blood flow, typically
near branches, bifurcations, regions of arterial narrowing, and curvatures in
the arteries[1–2], where the shear stress is not stable. Studies also indicate
that vascular endothelium function disturbance, especially impairment of
endothelium dependent vasodilation, is involved in the development of
atherosclerosis[5]. As an important function factor of vascular endothelial
cells, NO is closely related to the endothelial dysfunction, atherosclerosis
and many other diseases[6]. Endothelial-derived NO involves in many events in
the vasculature, including vasodilation, inhibition of platelet aggregation,
adhesion molecule expression, and vascular smooth muscle proliferation, which
are directly or indirectly related to many cardiovascular pathological
processes. Endothelial cells release NO more potently in response to increased
shear stress than to agonists that raise intracellular free calcium
concentration [Ca2+]i. However, it is not clear how the shear stress induces
the synthesis of NO. Studies have indicated that NO production increases with a
calcium/CaM dependent manner in the first few minutes after endothelial cells
were exposed to shear stress, followed by a sustained NO production that
occurred more than 30 min, which was Ca²⁺ independent[7]. The
activation of eNOS(endothelial nitric oxide synthase) by shear stress, which
modulated by Ca/CaM, G protein, tyrosine kinase phosphorylation and eNOS gene
expression, is responsible for the increase of NO production[8]. However, the
contribution of extracellular calcium to the production of NO is somewhat
contradictory.

As a short half-life (several seconds) molecule, NO
is not stable. It can rapidly react with molecule oxygen, superoxide anion, and
transition metal ions found in metalloproteins[9]. These properties make
real-time measurements of NO production extremely difficult. Different methods
have been used to detect NO, such as chemiluminescence assays, bioassays,
Griess reaction, oxy-hemoglobin assays, ESR, HPLC and electrochemical
electrodes. These methods have different advantages and disadvantages[10]. Each
of them has limitation in its ability to continuously detect NO production in
real-time in living cells because of poor specificity, low sensitivity, or
experimental difficulty. However,
4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), a
new cell-permeable fluorescent probe, can react rapidly with NO and produce
high fluorescence. Fluorescence indicators can be useful for the quantitation
of NO at low concentration in real time[7,11]. Therefore, the application of
this technique would be helpful to study the process of NO synthesis induced by
shear stress, and the mechanism involved.

In this research, a method was developed to detect
the NO production in real time in cultured rat aorta endothelial cells (RAECs)
under different shear stresses using DAF-FM diacetate. Moreover, the effects of
extracellular calcium to the synthesis of NO were also studied.

1   Methods

1.1  Culture of RAECs

The rat aorta endothelial cells were cultured in
MCDB-131 media (VEC Technology, NY) and maintained in a 95% air – 5%CO₂
humidified incubator at 37 °C. Cells were grown to confluence on glass cover
slips coated with 10 mg/L fibronectin. RAECs were subcultured (1:2) after
confluence. Mainly, cells were washed twice by PBS and 2 ml 0.025% trypsin-2%
EDTA solution was added. After cells were rounding, MCDB-131 media was used to
terminate digestion. Cells were detached by gently shaking and blowing, then
devided into two parts. The medium was changed every two days. RAECs of passage
7–10 were used in experiments.

1.2  NO and [Ca^2+]_i
imaging

RAECs grown on fibronectin-coated cover slips were
loaded with 5 μmol/L DAF-FM diacetate (Molecular Probe, OR), 5 μmol/L Fura-2 AM
(Molecular Probe, OR) individually for 30 min in the Krebs Ringer solution at
room temperature in dark. Then they were subsequently washed five times using
the Krebs Ringer solution. Then, the cells were loaded in a parallel plate flow
chamber and excited with filtered light from a mercury lamp. Under the
excitation light of wavelength 488 nm, DAF emits fluorescence at the wavelength
of 515 nm upon binding NO. The light of wavelengths of 340 nm and 380 nm were
used to excite Fura-2/Ca²⁺, and the induced fluorescence intensity
ratio was detected at 535 nm[12]. The pictures and fluorescence were detected
by Zeiss Axioskop2 fluorescence microscope and Attofluor ICCD camera (Atto
Bioscience, USA), then the fluorescence intensity was analysed by the software
attofluor ratiovision. The resolution of each picture is 256 pixels, and the
spatial resolution is 0.4 pixel/μm (100× oil objective lens). The pictures were
captured every 2.5 s.

1.3  Fluid flow

Fluid flow was applied to cells in a parallel plate
flow chamber using a closed flow loop (Fig.1). This system uses a constant
hydrostatic pressure to drive fluid through the flow chamber, subjecting the
monolayer to steady laminar flow produces a well-defined fluid shear stress.
All the apparatus were maintained at 25 ℃.
Krebs Ringer solution with 1.3 mmol/L CaCl₂ or Ca²⁺-free
was used as normal circulation fluid. No fluid was applied when the reaction of
RAECs to sodium nitroprusside (SNP, 100 μmol/L, Sigma), the donor of NO, was
studied. NG-nitro-L-arginine methyl ester (L-NAME 100 μmol/L Sigma) was
added into circulation solution to inhibit the synthesis of NO. In another
group of experiments, Krebs Ringer solution without calcium was used to study
the influence of extracellular calcium on the process of NO synthesis when
RAECs exposed to shear stress.

Fig.1      Flow
system schematic

This system uses a constant hydrostatic
pressure to drive fluid through the flow chamber, subjecting the monolayer to
steady laminar flow produces a well-defined fluid shear stress.

1.4  Statistical analysis

Data are reported as the 2     Results and Discussion

Using Zeiss Axioskop 2 fluorescence
microscope and ICCD-camera, the intracellular fluorescence intensity was
detected in individual cells. The average of signals from five cells was used.
Fig.2 shows the effect of SNP on the change in NO production in static RAECs.
SNP is a donor of NO, which can release NO to react with DAF-FM and produce
fluorescence. The addition of SNP resulted in a step increase of intracellular
fluorescence intensity. It indicated that DAF-FM would allow highly sensitive,
real-time detection and continuous measurement of NO production in living
cells. However, caution should be taken to reduce the power and the exposure
time of excitation light to prevent photobleaching.

Fig.2 Effect of SNP (100 μmol/L) on NO production in
static RAECs without flow (n=4)

Percent of
Fluorescence intensity increase=current intensity/baseline intensity.

When cultured RAECs were exposed to laminar flows at
different shear stresses (4, 8 and 16 dyne/cm²), there was an
increase of NO in the first few minutes (Fig.3). After 5 min, significant
increase of NO production was observed when cells were under 8 and 16 dyne/cm²
compared to the baseline (P<0.01). However, there was no significant increase in NO production until 20 minutes after the cells were exposed to 4 dyne/cm2 flow. It increased slowly with time and reached a peak after 60 min. Moreover, the synthesis of NO was shear stress dependent. The time course of NO production in RAECs subjected to flow was somewhat different from other observations using different methods. It might be caused by reactive oxygen species, which
can affect the kinetics of reaction between NO and DAF-FM.

Fig.3 Effect of different laminar flow (n=4–5)
on NO production in RAECs (n=5)

Percent
of fluorescence intensity increase=fluorescence intensity with flow/
fluorescence intensity before the flow. *P<0.05, **P<0.01 (vs baseline).

The upregulation of NO production was totally
inhibited by 100 μmol/L L-NAME, a NOS (nitric-oxide synthase) inhibitor
(Fig.4). When Ca²⁺-free Krebs Ringer solution was used in the circulation,
much less NO production was induced by flow at 16 dyne/cm2 (Fig.5). After RAECs
exposed to flow stress, an increase of [Ca²⁺]_i was
observed in the first 15 s (about 25%-30%), then [Ca²⁺]_i
was decreased to baseline (Fig.6). Further research will be performed to study
if the increase of [Ca²⁺]_i is due to the influx of
excelluar Ca²⁺ or the release from intracellular storage of Ca²⁺.

Fig.4 L-NAME inhibits NO synthesis induced by shear
stress of 16 dyne/cm2 (n=4)

Percent of fluorescence intensity
increase=fluorescence intensity with flow/ fluorescence intensity before the
flow. *P<0.05, **P<0.01 (vs baseline). ^#P<0.05, ^##P<0.01 (vs L-NAME group).

Fig.5 Shear
stress (16 dyne/cm²) induced NO synthesis was partly inhibited when
Ca²⁺-free Krebs Ringer solution was used as the circulation fluid

Percent of fluorescence intensity
increase=fluorescence intensity with flow/ fluorescence intensity before the
flow (n=5). *P<0.05, **P<0.01 (vs baseline). ^#P<0.05, ^##P<0.01 (vs Ca²⁺ free group).

Fig.6
Effect of laminar flow (shear stress 8 dyne/cm2) on [Ca²⁺]i in RAECs
(n=4)

[Ca²⁺]_i
ratio=current ratio signal/baseline ratio under excitations of 334 nm and 380
nm.

Although it is well known that exposure of
endothelial cells to shear stress stimulates production of NO, the molecular
mechanisms by which shear stress regulates NO production have not been clearly
elucidated. Studies indicated that the increase of intracellular calcium[13],
eNOS phosphorylation[14] and translocation[15] plays key role in the process.
Studies indicated shear stress can induce the phosphorylation of eNOS on serine
and threonine residues by different kinases, such as PKA, PKC, calmodulin
kinase II, Akt, etc.[8,16,17]. The phosphorylation can change the activity of
eNOS and thus regulate NO production. After several hours exposed to shear
stress, the expression of and synthesis of eNOS was increased by kinase signal
and transcription factors. eNOS activity is regulated by Ca²⁺
through its interaction with calmodulin, with the binding of the Ca²⁺-calmodulin
complex to displace specific region binded by caveolin-1[18]. Therefore, the
increase of [Ca²⁺]_i can induce the synthesis of NO.
However, it is confusing if shear stress can induce the increase of [Ca²⁺]_i
and the importance of [Ca²⁺]_i related to NO
production induced by shear stress. The contradictory results may be caused by
different circulation medium, the rate of onset of the flow-force, and
different cells[1]. The results above indicated that [Ca²⁺]i was
increased when endothelial cells exposed to laminar shear stress. Moreover, the
remove of Ca²⁺ from buffer significantly inhibited NO production,
which indicated the important roles of Ca²⁺ influx in the initial
and the process of NO synthesis induced by shear stress. It is reasonable to
hypothesis that the initial production of NO is Ca²⁺-calmodulin
dependent, whereas the sustained release phase is more dependent on eNOS
phosphorylation and eNOS expression.

Clearly, the method is very useful to study NO
release of endothelial cells toward different shear stresses and the mechanisms
involved. Based on the results obtained in the present study, future experiments
can be conducted to study the relationship of NO, [Ca²⁺]_i,
shear stress and different chemical and mechanical stimuli after endothelial
cells are treated with OxLDL, cholesterol, ATP, bradykinin, etc, which are very
important factors affecting some physiological and pathological processes, such
as atherosclerosis, thrombosis, inflammation. Therefore, the method would help
partly explain mechanisms of endothelial dysfunction, atherosclerosis and other
related pathological processes.

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Received: October
14, 2002     Accepted:
November 15, 2002

This work was
supported by grants from the Showalter Fund (USA) and the National Natural
Science Foundation of China (No. 30170275)

*Corresponding author: Tel, 001-765-494-6637; Fax, 001-765-494-0539;
e-mail, [email protected]