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Introduction
Exposure to acute or chronic hypoxia
leads to the development of pulmonary arterial hypertension (PAH).
The cardinal features of PAH are persistent vasoconstriction and
structural remodeling of the pulmonary vessels[1].
Hyperplasia of pulmonary artery smooth muscle cells (PASMCs) is the
main component of pulmonary vascular remodeling which is associated
with progressive elevation in pulmonary arterial pressure[2].
However, the exact mechanism of the proliferation of PASMCs was
unclear.
Several studies have shown that
5-hydroxytryptamine (5-HT), endothelin-1 (ET), platelet derived
growth factors (PDGF), angiotensin II (Ang II), and epidermal growth
factor (EGF) participate in the regulation of proliferation in
PASMCs[3]. Among these mediators, 5-HT plays an important
role in the pathogenesis of PAH. 5-HT exerts potent mitogenic and
co-mitogenic effects on PASMCs, and these effects are associated
with cellular internalization of 5-HT mediated by the
5-hydroxytryptamine transporter (5-HTT)[4,5]. Yet, it is
not clear if inhibition of 5-HTT may abolish the proliferation of
PASMCs to 5-HT.
Furthermore, mitogen activated
protein kinases (MAPKs) are a superfamily of serine/threonine
protein kinases distributed extensively in cytoplasm. Many stimuli
resulting in cell growth, differentiation, and vascular contraction
may activate MAP kinase-dependent signaling pathways.
Among this family, extracelluar
signal-regulated kinases (ERKs) are activated in response to growth
and differentiation factors[6-8]. Previous studies have
shown that 5-HT induced ERK1/2 activation in rat aortic smooth
muscle cells and rabbit isolated renal artery smooth muscle cells[9,10].
However, whether the intracelluar signal pathway of 5-HT in PASMCs
is dependent on the ERKs activation is largely unconcerned.
Therefore, the present study was designed to observe the effect of
fluoxetine, a selective inhibitor of 5-HTT, on the proliferation of
PASMCs in response to 5-HT, and then to study the effect of
downregulation of ERK1/2, using antisense oligodeoxynucleotides (ODNs),
on the proliferation of 5-HT-stimulated PASMCs in vitro.
Materials and methods
Drugs and reagents
Dulbecco's modified Eagle's medium (DMEM),
fetal bovine serum (FBS), and trypsin were purchased from Gibco Co
(Grand Island, New York, USA). 5-HT and propidium iodide (PI)
were purchased from Sigma Co (St Louis, USA). Lipofectin reagent was
from Life Technologies Inc, Ltd (Rockville, USA). Fluoxetine
hydrochloride was from Eli Lilly Co (Indianapolis, USA). ODNs were
synthesized by Sangon (Shanghai, China).
Pulmonary artery smooth muscle
cell culture Lungs were removed from male Wistar rats weighing
250-300 g (supplied by the Animal Center of China Medical
University, Grade II, Certificate No LN 03-0009). Under
aseptic conditions, proximal pulmonary arteries were isolated. After
removing the tissue around the arteries, the pulmonary arteries were
cut into small pieces about 1 mm2 and then seeded into
30-mm Petri dishes and cultured in DMEM (containing 20% FBS, 100 kU/L
of penicillin and 100 mg/L of streptomycin, pH 7.2). Then these
explants were incubated at the atmosphere of 37 ¡ãC and 95% O2/5%
CO2[11]. When the cells had grown out from the
explants and reached the confluence of more than 70%, cells were
passaged to T25 flasks. Cells used in experiments were from passages
3 to 9.
Oligodeoxynucleotides On
account of GCG package locating in the translation initiation region
of rat ERK1/2 mRNA, the sequences of antisense ODN were designed as
follows: 5'-GCC GCC GCC GCC GCC AT. This ODN has been used
successfully to downregulte ERK1/2 in VSMCs, rat cardiac myocytes
and rat cardiac fibroblasts[12]. Sense ODN (5'-AT GGC GGC
GGC GGC GGC), random ODN (5'-CGC GCG CTC GCG CAC CC) were used as
controls. All bases were protected by phosphorothioation. One batch
of antisense ODN was labeled with fluorecein isothiocyanate (FITC)
used in fluorescence microscopy experiments.
Microculture tetrazolium (MTT) assay
Cells were seeded into 96-well plates at a density of 1´104
cells/well. The cells were then incubated in medium containing
vehicle (5% FCS DMEM) and 5-HT (1 µmol/L) for 24 h with or without
fluoxetine (10 µmol/L, 1 µmol/L, and 100 nmol/L) added 30 min before
5-HT. The effect of different ODNs (0.2 µmol/L) on the proliferation
induced by 5-HT was also observed after transfection. The group with
the cells incubated in serum-free medium was used as the control. At
the end of this period, MTT (5 g/L) was added to each well, and
incubation proceeded at 37 ¡ãC for 4 h. Thereafter, the medium was
removed and the cells were solubilized in 150 µL Me2SO.
Optical density (OD) of each well was determined by
enzyme-linked ELISA at 490 nm of wavelength. Then the proliferation
rates (PR) of each group were calculated.
PR=(ODexperiment-OD
control)/ODcontrol
Flow cytometry (FCM) PASMCs
grown in T25 plates were treated with fluoxetine or antisense ODN
(0.2 µmol/L) before 5-HT was added. The cells were harvested by
trypsinization, washed twice with PBS, and the pellets
were resuspended in 0.5 mL of PBS containing 100 mg/L RNase for
incubation at 37 ¡ãC for 30 min. Then, 0.5 mL of PI
solution (100 mg/L in PBS) was added, and the mixture was incubated
in the dark at 4 ¡ãC for 30 min. The cells were analyzed with a
FACScan flow cytometer. Then S-phase cell fractions (SPF)
and proliferation index (PI) of each group were calculated. SPF=S/(G0G1+S+G2M);
PI=(G2M+S)/(G0G1+S+G 2M).
Liposomal transfection Prior
to transfection, PASMCs were cultured in serum-free medium for 24 h.
ODNs were mixed with antibiotic- and serum-free medium to a
concentration of 0.8 µmol/L, then mixed with equal volume of medium
containing 80 mg/L of lipofectin and incubated at room temperature
for 15 min. ODN/lipofectin mix 20 µL was added to each well of a
96-well plate, 200 µL to each well of a 12-well plate, and 1000 µL
to the T25 plates, with equal volume of antibiotic- and serum-free
medium. The cells were incubated for a further 6 h with gentle
agitation every 2 h. The medium was then replaced with the same
volume of liposome-free medium containing the same concentration of
ODN and 5% FBS. Cells were incubated for another 24 h before MTT
assay and FCM.
Fluorescence microscopy
PASMCs were seeded into 12-well plate and transfected using FITC-labeled
antisense ODN. After 24 h and 48 h of incubation, the cells were
viewed by fluorescence microscopy.
Statistical analysis All the
data are presented as mean¡ÀSD, and assessed by ANOVA and t-test.
P<0.05 was considered significant.
Results
Uptake of ODNs by PASMCs
Twenty four hours after liposomal
transfection, FITC-labeled antisense ODN were observed in both
cytoplasm and the nuclei of PASMCs (Figure 1). More than 90% of the
cells exhibited fluorescence.
Effect of fluoxetine and ODNs on
the proliferation of PASMCs induced by 5-HT MTT assay showed
that 5-HT (1 µmol/L) induced increased proliferation of PASMCs and
the PR in response to 5-HT increased from 172%¡À5% to 217%¡À4%
compared with the vehicle. Pretreatment of the cells with fluoxetine
(10 µmol/L, 1 µmol/L, and 100 nmol/L) could produce a
concentration-dependent reduction in PR (Figure 2). According to
these results, 1 µmol/L was chosen as the concentration of
fluoxetine used in following experiments.
Pretreatment of PASMCs with ERK1/2
antisense ODN resulted in a significant inhibition of 5-HT-induced
PASMCs proliferation (Figure 3) and this inhibitory effect is rather
more potent than fluoxetine. In contrast, sense ODN and random ODN
did not have such effect.
Effect of fluoxetine and
antisense ODN on cell cycle Flow cytometric analysis of cell
cycle distribution showed that the cells treated with 5-HT had
larger values of S-phase cell fraction (SPF) and PI than the
vehicle. But pretreatment with fluoxetine or antisense ODN decreased
these values (Figures 4, 5). These results indicated that 5-HT
promoted the PASMCs from the G0/G1 phase of
the cell cycle into S phase and this effect was inhibited by
fluoxetine and antisense ODN.
Discussion
Results from the present study
demonstrated that 5-HTT played a key role in the mitogenic effect of
5-HT on PASMCs. Fluoxetine, a highly selective inhibitor of 5-HTT
inhibited the proliferation of PASMCs induced by 5-HT in vitro.
Meanwhile, antisense ODN to ERK1/2 inhibited 5-HT-induced
proliferation of PASMCs. These findings suggest that
5-HT-induced proliferation of PASMCs is 5-HTT and ERK pathway
dependent.
In response to hypoxia, 5-HT is
released from pulmonary neuroendocrine cells and neuroepithelial
bodies distributed throughout the airways. An increase in 5-HT may
contribute to secondary pulmonary artery hypertension[13].
The proliferation of PASMCs induced by 5-HT is an important
component of pulmonary arterial remodeling. RT-PCR analyses of
PASMCs indicated the presence of 5-HT1B/1D, 5-HT2
receptors and 5-HTT mRNA[14,15]. The present results show
that the proliferation of PASMCs induced by 5-HT is inhibited by
fluoxetine in a manner that is concentration-dependent. In contrast,
the 5-HT1B/1D receptor antagonist GR127935, the 5-HT2A
receptor antagonist ketanserin, or the 5-HT2B/2C receptor
antagonist SB206553 had no this effect[16]. It has been
recently reported that exposure of PASMCs to hypoxia increased 5-HTT
expression and activity, and this effect was associated with
potentiation of the mitogenic action of 5-HT[5,17]. Some
scholars found that mice deficient in 5-HTT or treated with
selective inhibitors of 5-HTT developed less PAH than controls when
exposed to hypoxia[18,19]. These evidences suggest that
5-HTT in PASMCs may be a key determinant of pulmonary arterial
remodeling and the development of PAH. Therefore, our result means
that 5-HTT is one of the important mechanisms of PAH and this may
provide a novel therapeutic target for PAH.
The uptake of FITC-labeled antisense
ODN by PASMCs with liposomes proved the high efficiency of
transfection. In the present study, the antisense ODN to ERK1/2
delivered by lipofectin resulted in an effective suppression of the
proliferative response to 5-HT in PASMCs. Meanwhile, the effect of
antisense ODN stronger than fluoxetine suggests that antisense ODN
to ERK1/2 could inhibit the proliferation of PASMCs not only induced
by 5-HT, but also by the
serum[20]. The present study proves that 5-HT induced the
proliferation of PASMCs and is dependent on the activation of
ERK1/2. Therefore, it is reasonable to consider that 5-HT induced
activation of ERKs through 5-HTT in PASMCs. Previous studies showed
that 5-HT induced the Tyr phosphorylation of GTPase-activating
protein (GAP) and the effect was mediated by 5-HTT, which was the
upstream of the ERK pathway, not by 5-HT receptors[21].
The mechanism of 5-HT signaling for PASMCs through 5-HTT has also
been shown to involve the production of reactive oxygen species (ROS)
such as superoxide and H2O2 via the
activation of NAD(P)H oxidase, and the activation of the ERK pathway
occurs secondary to ROS formation[22,23]. Therefore, the
activation of ERKs induced by 5-HT is mediated by 5-HTT.
The present study showed that
fluoxetine concentration-dependently inhibited 5-HT-induced
proliferation of PASMCs in vitro, and demonstrated that
antisense ODN to ERK1/2 significantly inhibited mitogenesis of
PASMCs. Therefore, we concluded that 5-HT-induced mitogenesis of
PASMCs was mediated by 5-HTT, in which the signal transduction for
5-HT was dependent on ERKs signal pathway.
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