© 1999 American Heart Association, Inc.
Original Contribution |
Induction of Serotonin Transporter by Hypoxia in Pulmonary Vascular Smooth Muscle Cells
Relationship With the Mitogenic Action of Serotonin
From the Département de Physiologie (S.E., B.R., S.A.), INSERM U492, Créteil, France, and INSERM U288, NeuroPsychoPharmacologie Moléculaire, Cellulaire et Fonctionnelle (V.F., C.B., M.P.M., M.H.), Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Correspondence to S. Eddahibi, INSERM U492 et Département de Physiologie Centre Hospitalier Universitaire Henri Mondor, 94010 Créteil, France. E-mail eddahibi{at}im3.inserm.fr
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Abstract—The increased delivery of serotonin (5-hydroxytryptamine, 5-HT) to the lung aggravates the development of hypoxia-induced pulmonary hypertension in rats, possibly through stimulation of the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). In cultured rat PA-SMCs, 5-HT (10–8 to 10–6 mol/L) induced DNA synthesis and potentiated the mitogenic effect of platelet-derived growth factor-BB (10 ng/mL). This effect was dependent on the 5-HT transporter (5-HTT), since it was prevented by the 5-HTT inhibitors fluoxetine (10–6 mol/L) and paroxetine (10–7 mol/L), but it was unaltered by ketanserin (10–6 mol/L), a 5-HT2A receptor antagonist. In PA-SMCs exposed to hypoxia, the levels of 5-HTT mRNA (measured by competitive reverse transcriptase–polymerase chain reaction) increased by 240% within 2 hours, followed by a 3-fold increase in the uptake of [3H]5-HT at 24 hours. Cotransfection of the cells with a construct of human 5-HTT promoter-luciferase gene reporter and of pCMV-ß-galactosidase gene allowed the demonstration that exposure of cells to hypoxia produced a 5.5-fold increase in luciferase activity, with no change in ß-galactosidase activity. The increased expression of 5-HTT in hypoxic cells was associated with a greater mitogenic response to 5-HT (10–8 to 10–6 mol/L) in the absence as well as in the presence of platelet-derived growth factor-BB. 5-HTT expression assessed by quantitative reverse transcriptase–polymerase chain reaction and in situ hybridization in the lungs was found to predominate in the media of pulmonary artery, in which a marked increase was noted in rats that had been exposed to hypoxia for 15 days. These data show that in vitro and in vivo exposure to hypoxia induces, via a transcriptional mechanism, 5-HTT expression in PA-SMCs, and that this effect contributes to the stimulatory action of 5-HT on PA-SMC proliferation. In vivo expression of 5-HTT by PA-SMC may play a key role in serotonin-mediated pulmonary vascular remodeling.
Key Words: 5-hydroxytryptamine transporter • hypoxia • pulmonary arterial smooth muscle cell • pulmonary hypertension
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Hypoxia is well known to affect markedly both the tone and the structure of blood vessels. In the lung, acute hypoxia causes arteriolar vasoconstriction with a subsequent rise in pulmonary artery pressure. Moreover, chronic hypoxia results in smooth muscle cell proliferation and sustained elevation in pulmonary artery pressure. One possible mechanism that may account for the latter effect is a direct action of hypoxia on the expression of specific genes involved in smooth muscle cell proliferation.1
That serotonin (5-hydroxytryptamine, 5-HT) plays an important role in the remodeling of the pulmonary circulation, notably during exposure to hypoxia, is suggested by several studies. Fawn-Hooded rats, which have a genetic deficit in 5-HT platelet storage, are characterized by high plasma levels of the indolamine. Interestingly, these animals, but not Sprague-Dawley rats (which have markedly lower levels of plasma 5-HT), develop pulmonary hypertension when they are exposed to mild hypoxia.2 Similarly, primary pulmonary hypertension has been reported in patients with increased plasma 5-HT levels associated with platelet storage deficit.3 The possibility that 5-HT may favor the development of pulmonary hypertension is further supported by recent data showing that continuous intravenous infusion of the indolamine during a 2-week exposure to hypoxia aggravates pulmonary hypertension in rats.4 In contrast, no effects of 5-HT infusion were observed in animals maintained under normoxia. Moreover, the aggravating effect of 5-HT infusion on hypoxic pulmonary hypertension is prevented when rats are treated with dexfenfluramine, an inhibitor of 5-HT transport.5 These results suggest that the effects of hypoxia on pulmonary vascular remodeling are facilitated by 5-HT through an intracellular mechanism that requires internalization of the indolamine by the dexfenfluramine-sensitive high-affinity 5-HT transporter (5-HTT).
Although pulmonary endothelial cells play an important role in the removal of 5-HT from the circulation, pulmonary smooth muscle cells have also been shown to participate in this process. Indeed, using cells derived from bovine pulmonary arteries, Lee et al6 even found that 5-HT uptake by smooth muscle cells was 7- to 8-fold higher than uptake by endothelial cells.6 Moreover, uptake of 5-HT by both types of cells has also been reported to be potentiated by hypoxia.6 To date, the physiological role of 5-HTT in vascular smooth muscle cells has not yet been completely elucidated. In addition to contributing to the uptake and subsequent inactivation of 5-HT passing through the lung, 5-HTT might also mediate the proliferation of smooth muscle cells through its capacity to internalize the indolamine.7 As 5-HTT is also a target for drugs that have recently been shown to induce pulmonary hypertension in humans,8 9 the potential clinical relevance of this transporter provides further interest for studying both its genetic regulation and its role in the proliferation of pulmonary vascular smooth muscle cells.
The aim of the present study was to investigate 5-HTT expression in cultured pulmonary vascular smooth muscle cells exposed to hypoxia to assess whether 5-HTT could play a role in the control of their proliferation. Moreover, we also examined whether in vivo exposure to hypoxia could affect the expression of 5-HTT in remodeled pulmonary vessels.
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Isolation and Culture of Rat Pulmonary Artery Smooth Muscle Cells (PA-SMCs)
All animal care and procedures were in accordance with institutional and international guidelines. The method used for PA-SMC isolation and culture has been previously described.10 In brief, male Wistar rats weighing 250 to 300 g were killed by an overdose of pentobarbital. Lungs were immediately removed, and proximal pulmonary arteries were isolated under aseptic conditions. After removal of fat, adventitia, and connective tissue surrounding the arteries, the pulmonary arteries were cut into small pieces that were then incubated in DMEM supplemented with elastase type III (0.125 mg/mL), collagenase type I (1 mg/mL), and antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin). After a 90-minute incubation at 37°C, the tissue suspension was centrifuged, and the pellet was resuspended in DMEM supplemented with 15% (vol/vol) FCS, 2 mmol/L L-glutamine and the same antibiotics as above. Cells in the pellet suspension were cultured in 100-mm Petri dishes at 37°C in a humidified atmosphere of 5% CO2 and 95% air, up to confluency. The medium was changed every day, and the cells were harvested with trypsin (0.2 g/L)–EDTA (0.5 g/L). Cells after 4 to 5 passages were used for the experiments. All cells exhibited specific immunostaining by anti-
-smooth muscle actin
antibodies, as expected of PA-SMCs.10
Microculture Tetrazolium (MTT) Assay
The MTT assay was performed for evaluation of the effect of
hypoxia on PA-SMC viability and density.11 Cells
seeded in 96-well plates were cultured up to confluency, and the medium
was replaced by fresh serum-free medium. The cells were then exposed to
hypoxia (5% CO2 and 95%
N2) or normoxia (5% CO2,
20% O2, and 75% N2) for
24 hours. At the end of this period, MTT (0.2 mg/mL) was added to each
well, and incubation proceeded for 4 hours at 37°C. Thereafter, the
culture medium was removed and the cells were solubilized in 100 µL
of DMSO. The extent of reduction of MTT to formazan within cells was
quantified spectrophotometrically at 520 nm and taken as an indicator
of cell viability.11
Effect of 5-HT on [3H]Thymidine Incorporation by SMCs
Subjected to Normoxia or Hypoxia
SMCs in medium supplemented with 15% FCS were seeded in 24-well
plates at a density of 5x104 cells/well and
allowed to adhere. Then, cells were subjected to 48 hours of growth
arrest in medium containing 0.2% FCS. At the end of this period, cells
were incubated with 5-HT (10–8 to
10–6 mol/L) alone or in the presence of
platelet-derived growth factor (PDGF)-BB (10 ng/mL, Sigma) in
medium containing 0.2% FCS, antibiotics (as above), 0.6 mmol/L
ascorbic acid, 0.1 mmol/L iproniazid (a monoamine oxidase
inhibitor, Sigma), and 0.6 µCi/mL of
[3H]thymidine (50 Ci/mmol, Amersham). The
effect of 5-HT was also examined in the presence of 1 of 2 specific
5-HTT inhibitors,12 fluoxetine
(10–6 mol/L) and paroxetine
(10–7 mol/L), or in the presence of ketanserin
(10–6 mol/L), a 5-HT2A
receptor antagonist13 ; these were added 20
minutes before 5-HT. After 24 hours of incubation under normoxic (5%
CO2, 20% O2, and 75%
N2) or hypoxic (5% CO2 and
95% N2) conditions, cells were washed twice with
PBS, followed by ice-cold 10% trichloroacetic acid. Cells were then
dissolved in 0.1N NaOH (0.5 mL/well), and the incorporated
radioactivity was counted.
Measurement of [3H]5-HT Uptake
SMCs in medium containing 15% FCS were seeded in 24-well plates
at a density of 5x104 cells/well and allowed to
proliferate for 72 hours. At the end of this period, the medium was
removed, and cells were subjected to growth arrest in medium containing
0.2% FCS. After 8 to 24 hours of incubation under normoxic (5%
CO2, 20% O2, and 75%
N2) or hypoxic (5% CO2 and
95% N2) conditions, cells were washed twice with
PBS and exposed to 10 nmol/L
5-hydroxy[G-3H]tryptamine
creatinine sulfate ([3H]5-HT, 15 to
16 Ci/mmol, Amersham) in a medium containing (in mmol/L) NaCl 120,
KCl 5, CaCl2 1.2, MgSO4
1.2, glucose 5.6, Tris-HCl 4, HEPES 6.25, and ascorbic acid 0.5, pH 7.4
(uptake buffer). Under these conditions,
[3H]5-HT uptake by PA-SMCs was linear for at
least 15 minutes. Therefore, assays were performed for 10 minutes at
37°C in the absence or the presence of fluoxetine
(10–9 to 10–5 mol/L). At
the end of the incubation period, the medium was removed and cells were
washed 3 times with the uptake buffer. Cells were lysed by adding 0.5
mL of 0.1N NaOH, and radioactivity of lysates was counted by liquid
scintillation spectrometry. Uptake is expressed as fmol
[3H]5-HT taken up per mg protein (measured by
the method of Lowry et al,14 with BSA as standard).
Extraction of RNA From Cultured SMCs
To examine the effect of hypoxia on 5-HTT mRNA
expression, the medium of cells grown to confluency was removed and
replaced by serum-free medium. The cells were then exposed to
hypoxia (5% CO2 and 95%
N2) or normoxia (5% CO2,
20% O2, and 75% N2).
After a 2- to 24-hour exposure, cells were washed with PBS and lysed
with guanidinium isothiocyanate (Interchim). Total RNA was extracted
according to the method of Chomczynski and Sacchi15 and
electrophoresed in 1% agarose gel stained with ethidium bromide.
Quantification was performed with reference to a scale of total RNAs
prepared on a cesium chloride gradient16 and estimated by
optical density measurement at 260 nm.
Quantitative Determination of 5-HTT mRNA
The method was based on competitive polymerase chain reaction
(PCR),17 in which RNAs were reverse transcribed and the
synthesized cDNAs were amplified in the presence of an internal
standard consisting of the same target mRNA, synthesized with a
deletion of 
100 bases as described in detail
elsewhere.18
Total RNAs (0.8 µg per sample) and internal standard RNA (0.01 to 1 pg) were reverse transcribed (45 minutes at 48°C) and amplified using an Access reverse transcriptase (RT)-PCR kit (Promega) with the primers 5'-TTACACAGCATTCATGCG (nucleotides 2008 to 1991) and 5'-GGATCCCTGCTCACACTG (nucleotides 1541 to 1558)19 at 2.5 mmol/L MgCl2. Cycle amplifications were performed at 94°C, 56°C, and 72°C (1 minute each, 28 cycles). PCR products from the 5-HTT mRNA and the corresponding synthetic deleted RNA were of 484 and 400 bp, respectively. They were electrophoresed in 2% agarose gel stained with ethidium bromide and quantified with a gel analyzer (GDS 5000, UVP). RNA levels in smooth muscle cells exposed to hypoxia are expressed as a percentage of those found under normoxic control conditions.
Transfection of SMCs
SMCs were transfected with a plasmid pGL3 in which the promoter
of the human-5-HTT was fused to the reporter luciferase
gene.20 21 The human 5-HTT promoter genomic fragment (bp
–1428 to +217 with respect to the transcription initiation site) is
defined by a TATA-like motif and several potential binding sites for
transcription factors including activator protein (AP)-1,
AP-2, SP1, and a cAMP response element (CRE)–like
motif.20 To correct for variations in the transfection
yields, cells were cotransfected with a plasmid pCMV-LacZ reporter gene
(Invitrogen). SMCs (5x106 in DMEM without FCS
were electroporated in the presence of 5 µg of pCMV-LacZ gene and 10
µg of human 5-HTT promoter-luciferase gene or 10 µg of pGL3 control
vector (Promega). A combination of a voltage of 260 V and a capacitance
of 1800 µF for a relaxation time of 30 ms was used in these
experiments (Equibio; Eurogentec). After electroporation, cells were
collected and diluted in DMEM containing 15% FCS and antibiotics.
Cells were then seeded in two 25-cm2 plates and
incubated at 37°C for 48 hours to allow expression of transfected
luciferase- and LacZ-encoding genes. At the end of this period, the
medium was changed to serum-free DMEM, and cells were incubated under
either normoxic or hypoxic conditions for 2 to 24 hours. At the end of
exposure, cells were lysed, and the activities of luciferase (Promega)
and ß-galactosidase (Boehringer Mannheim) were measured
according to the manufacturer's protocols. Briefly, luciferase
activity was measured by addition of 10 µL of lysate samples to 100
µL luciferin reagent. Chemiluminescence was counted 15 s after
reagent mixing in a liquid scintillation spectrometer.
ß-Galactosidase activity was measured after incubation of 50 µL of
lysate samples with chlorophenol
red-ß-D-galactopyranoside at a final concentration of
5 mmol/L. Absorbance at 574 nm was measured after a 60-minute
incubation of the assay mixture at room temperature.
Exposure of Rats to Chronic Hypoxia
Male Wistar rats, weighing 250 to 300 g at the beginning of
the experiments, were exposed to chronic hypoxia (10%
O2; 2 weeks) in a ventilated chamber (volume=500
L; Flufrance) as previously described.22 To establish the
hypoxic environment, the chamber was flushed with a mixture of room air
and nitrogen, which was recirculated. The chamber environment was
monitored with an oxygen analyzer (Servomex OA150). Carbon
dioxide was removed by soda lime granules, and excess humidity was
prevented by cooling of the recirculation circuit. The temperature of
the chamber was maintained at 22°C to 24°C. The chamber was opened
every day for 1 hour so that the cages could be cleaned and food and
water provided. Normoxic rats were kept in the same room, with the same
light-dark cycle. Rat chow and tap water were provided ad libitum.
After 15 days of hypoxia or normoxia, rats were deeply anesthetized with sodium pentobarbital (40 mg/kg IP). Proximal pulmonary arteries were removed and frozen immediately at -80°C. Total RNA was extracted from pulmonary arteries of 4 rats in each group. Lungs isolated from normoxic or chronically hypoxic rats were used for total RNA extraction or in situ hybridization histochemistry.
Preparation of 35S-Labeled Antisense 5-HTT
Riboprobe
For the hybridization of 5-HTT mRNA in lung sections, a partial
rat 5-HTT cDNA (nucleotides 1540 to 2007) was cloned into
the plasmid vector Bluescript SKII–
(Stratagene). The plasmid was linearized and used as template for the
synthesis of the 35S-labeled antisense RNA probe
with the Ampliscribe T7 transcription kit (Tebu) in the presence of 125
µCi of 35S-labeled -UTP (1500 Ci/mmol, New
England Nuclear). Anti-sense 35S-labeled cRNA
riboprobe was purified and suspended in RNase-free water.
In Situ Hybridization Histochemistry
Lungs isolated from normoxic or hypoxic rats were distended by
infusion of Tissue-Tek (Miles) diluted in PBS (1:1) into the trachea,
rapidly frozen in isopentane at -30°C, and stored at -80°C. Lung
sections (15 µm) were cut at -20°C in a cryostat and thaw
mounted onto ready-to-use Super Frost plus slides (Consortium de
Matériel pour Laboratoire, Nemours, France). Sections were
then fixed for 30 minutes in ice-cold 4%
paraformaldehyde in PBS, dehydrated in a graded series
of ethanol solutions (60%, 80%, 95%, and 100%), and stored at
-80°C until use.
For the hybridization step, each lung section was covered with hybridization buffer containing 0.6 mol/L NaCl, 10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 µg/mL denatured salmon sperm DNA, 50 µg/mL yeast total RNA, 2.5x Denhardt's solution, 10% dextran sulfate, 10 mmol/L DTT, 50% formamide, and 4x106 cpm of 35S-labeled antisense riboprobe. Incubation then proceeded overnight in a humid chamber at 57°C. Nonhybridized probe was subsequently eliminated by RNase A (50 µg/mL in 100 mmol/L Tris-HCl and 0.5 mol/L NaCl, pH 8.0) for 60 minutes at 37°C, followed by extensive washes.23 Then, sections were dipped in Kodak autoradiographic emulsion (Amersham) and stored in the dark at 4°C before development 1 to 2 weeks later. Sections were finally counterstained with hematoxylin phloxin saffron and examined using light- and dark-field microscopy.
Statistical Analysis
[3H]5-HT uptake after various times of
exposure to normoxia were compared using 1-way ANOVA. Since there were
no significant differences between these values, they were pooled.
Values obtained after various times of exposure to hypoxia were
compared, with the pooled normoxic values using 1-way ANOVA followed by
Dunnett's t test. [3H]Thymidine
incorporation was compared at various concentrations of 5-HT under
normoxic or hypoxic conditions using a 2-way ANOVA, followed, in case
of significant interaction, by the Mann-Whitney
nonparametric test for comparison between normoxic and
hypoxic conditions at each concentration of 5-HT.
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Effect of 5-HT on [3H]Thymidine Incorporation by PA-SMCs During Normoxia or Hypoxia
In quiescent cells maintained in a normoxic environment and incubated with serum-free medium (0.2% FCS), 5-HT produced a concentration-dependent increase in [3H]thymidine incorporation (Table 1
). In the presence of PDGF-BB (10
ng/mL), the stimulatory effect of 5-HT was still observed, despite a
13-fold increase in [3H]thymidine incorporation
induced by PDGF-BB alone. Addition of 10–6 mol/L
5-HT to the culture medium increased DNA synthesis by 5-fold and
3.5-fold in the presence of 0.2% FCS and PDGF-BB, respectively.
Pretreatment of the cells with a 5-HT transport inhibitor,
either fluoxetine (10–6 mol/L) or paroxetine
(10–7 mol/L), completely inhibited the increase
in [3H]thymidine incorporation induced by 5-HT
in the presence of either 0.2% FCS or PDGF-BB (Figure 1A and 1B). In contrast, the response to
5-HT was unaltered in the presence of the 5-HT2A
receptor antagonist ketanserin at
10–6 mol/L. Neither fluoxetine/paroxetine nor
ketanserin affected the response to PDGF-BB alone (data not shown).
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A decrease (–30% and –37%, P<0.05) in
[3H]thymidine incorporation was observed in
PA-SMCs exposed to hypoxia in the absence and in the presence
of PDGF-BB. However, the stimulatory effect of 5-HT on
[3H]thymidine incorporation persisted under
hypoxic conditions. Indeed, in cells incubated with 0.2% FCS,
[3H]thymidine incorporation in response to low
(10–8 mol/L) and intermediate
(10–7 mol/L) concentrations of 5-HT was greater
under hypoxic than normoxic conditions (Table 1
and Figure 1). Hypoxia also potentiated the stimulatory effect of
10–8 mol/L 5-HT in cells incubated with PDGF-BB.
During hypoxia, the stimulatory effect of 5-HT on DNA synthesis
was completely abolished by fluoxetine or paroxetine but unaltered by
ketanserin. Thus, in cells incubated with 0.2% FCS or PDGF-BB,
[3H]thymidine incorporation remained unchanged
when 5-HT (10–6 mol/L) was associated with
pretreatment with fluoxetine or paroxetine under both normoxic and
hypoxic conditions (Figure 1).
Effect of Hypoxia on [3H]5-HT Uptake by
SMCs
[3H]5-HT uptake remained stable in SMCs
that had been exposed to normoxia for various times (8, 16, or 24
hours). In contrast, a progressive increase in
[3H]5-HT uptake was observed in cells exposed
to hypoxia (Figure 2). Thus, at
its maximal value that was reached after hypoxia for 16 hours,
[3H]5-HT uptake was 3-fold higher than that
found under normoxic conditions (P<0.001, Figure 2).
Fluoxetine caused a concentration-dependent inhibition of
[3H]5-HT uptake, with similar
IC50 values under normoxic and hypoxic conditions
(2.2x10–8 and 2.8x10–8
mol/L in normoxia and after 24 hours of exposure to hypoxia,
respectively). At the highest concentration of fluoxetine tested
(10–5 mol/L), residual
[3H]5-HT uptake was similar under hypoxic
versus normoxic conditions.
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Effect of Hypoxia on 5-HTT mRNA Levels in PA-SMCs
Cells exposed to hypoxia showed no evidence of cellular
damage. In particular, the number of cells incubated in serum-free DMEM
remained similar after 24 hours of exposure to normoxia or
hypoxia (MTT measurements). Levels of 5-HTT mRNA did not change
significantly with time in cells exposed to normoxic conditions. In
contrast, exposure to hypoxia for 2 hours resulted in a
2.4-fold increase of 5-HTT mRNA levels (Figure 3). A significant increase (+50%) was
still observed after 4 hours of hypoxia; then 5-HTT mRNA levels
returned down to the values measured in PA-SMCs exposed to normoxic
conditions (Figure 3).
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Induction by Hypoxia of the 5-HTT Promoter Gene Construct
Transfected to SMCs
In cells cotransfected with human 5-HTT promoter-luciferase and
pCMV-LacZ genes, exposure to hypoxia led to a marked increase
in luciferase activity compared with normoxic controls, whereas
ß-galactosidase activity remained unchanged (Figure 4). As illustrated in Figure 4, the increase in luciferase activity developed progressively during
hypoxia, up to a maximum that was reached at 16 to 24 hours. At
this time, luciferase activity in cells exposed to hypoxia was
5- to 6-fold higher than that measured in cells maintained under
normoxic conditions (Figure 4). However, exposure to
hypoxia of cells cotransfected with pCMV-LacZ and
pGL3-luciferase reporter vectors did not change the activities of
expressed enzymes compared with the values obtained under normoxic
environment (not shown).
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Effect of Chronic Hypoxia on 5-HTT mRNA Levels in
Pulmonary Arteries and Lungs
mRNA encoding 5-HTT was present in proximal pulmonary
arteries from chronically hypoxic rats but was not detected in
arterial tissue from normoxic animals (Table 2). In lungs from chronically hypoxic
rats, the levels of 5-HTT mRNA, measured by competitive RT-PCR, were
found to be significantly higher (+38%, P<0.05) than those
determined in the same tissues of normoxic rats (Table 2).
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As illustrated in Figure 5, in situ
hybridization histochemistry performed on rat lung sections showed that
5-HTT mRNA was predominant in pulmonary vascular walls. In rats
that had been maintained under hypoxic conditions, the hybridization
signal was observed in both endothelial and SMCs and
was more marked than in arteries from normoxic rats.
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The present results show that, in rat PA-SMCs, the mitogenic action of 5-HT depends on the 5-HTT activity. Exposure of PA-SMCs to hypoxia increases the transcriptional rate of the 5-HTT gene and potentiates the growth-promoting effect of 5-HT. Increased 5-HTT gene expression by PA-SMCs is also observed in remodeled pulmonary arteries of rats subjected to chronic hypoxia and developing pulmonary hypertension. These in vitro and in vivo data support a model whereby low oxygen tension induces the expression of 5-HTT, which, in turn, may sensitize PA-SMCs to the mitogenic action of 5-HT.
The effects of 5-HT on pulmonary vessels is currently the matter of renewed interest, notably because anorectic drugs acting through inhibition of 5-HT transport have been reported to cause an increased risk of developing pulmonary hypertension in humans.8 9 Studies have long been concerned with the vasoactive properties of 5-HT, mediated mainly through its binding to specific receptors on pulmonary vascular endothelial and SMCs. More recently, 5-HT was reported to promote growth of pulmonary vascular SMCs as well as of other cell types.7 24 25 Serotonin is a known mitogen for SMCs isolated from bovine, porcine, and rat aorta as well as rat and bovine pulmonary arteries.7 25 26 The mechanism by which 5-HT causes smooth muscle cell proliferation may vary with cell types and species. Several studies have concluded that the mitogenic action of 5-HT is initiated through its binding to a cell surface receptor, notably the 5-HT2A type,25 whereas evidence has also been provided that it results from an energy-dependent transport of 5-HT into the cell.7 In the present study, we found that 5-HT was a potent inducer of rat PA-SMC proliferation and that this effect was dose-dependently inhibited by the highly selective inhibitors of 5-HT transport, paroxetine, and fluoxetine,12 27 but not by the 5-HT2A receptor antagonist ketanserin.13 Indeed, fluoxetine and paroxetine inhibited [3H]5-HT uptake and 5-HT–induced cell proliferation at similar concentrations, suggesting that both phenomena were tightly related. These results are consistent with those of Lee et al,7 showing that the proliferative response of bovine pulmonary vascular SMCs to 5-HT was inhibited by agents that block the transport of 5-HT but not by 5-HT receptor antagonists. In accordance with these previous studies, we found that the mitogenic response to 5-HT also occurred when rat SMCs were incubated in the presence of PDGF-BB. Both fluoxetine and paroxetine inhibited the proliferative action of serotonin in the presence of PDGF-BB, whereas none of these inhibitors affected PDGF-BB–induced cell proliferation. Taken together, these results clearly indicate that low concentrations of 5-HT stimulate proliferation of PA-SMCs and that this effect is dependent on the active transport of 5-HT within cells. At this time, the mechanisms by which 5-HT exerts its mitogenic effect after being transported inside SMCs remain speculative. Lee et al28 have observed that 5-HT–induced DNA synthesis is associated with tyrosine phosphorylation of GTPase-activating protein and that both effects are blocked by 5-HT transport or tyrosine kinase inhibitors. Therefore, although 5-HT–induced mitogenesis in SMCs requires cellular internalization through 5-HTT rather than binding to a membrane receptor, tyrosine phosphorylation of GTPase-activating protein appears as a downstream intermediate in the signaling pathway. Recently, involvement of superoxide anion formation in association with 5-HT transport has also been suggested to play a role in the mitogenic effects of 5-HT.29
Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling. One possible mechanism that may account for this effect is a direct action of hypoxia on the expression of specific genes involved in smooth muscle cell proliferation. We found that exposure of PA-SMCs to hypoxia resulted in a rapid and transient increase in the level of 5-HTT mRNA, followed by a prolonged 2.5- to 3.0-fold increase in 5-HT transport activity. While the increase in 5-HTT mRNA levels peaked at 2 hours, the maximal increase in 5-HT transport was measured at 16 to 24 hours of hypoxia, a finding consistent with the time required for protein synthesis and integration of the transporter into the plasma membrane. Interestingly, the IC50 of fluoxetine remained unchanged in cells that had been subjected to hypoxia, indicating that the pharmacological properties of the transporter were not affected by hypoxia. Previous studies already documented an increased rate of 5-HT uptake in cultured endothelial and SMCs derived from bovine pulmonary arteries during exposure to hypoxia.6 30 31 Whether this was due to an increased activity or expression of the transporter protein was not specified. In the present study, we provided evidence that the increased 5-HT uptake was directly related to an increased transcriptional rate of the 5-HTT gene. Thus, in cells transfected with a luciferase-reporter gene construct containing the human 5-HTT promoter, exposure to hypoxia was associated with a marked increase in luciferase activity. This effect on the 5-HTT promoter was specific, because when the cells were transfected with the same luciferase reporter gene under the dependence of the SV40 promoter, hypoxia did not change luciferase activity. These results can be taken as evidence that hypoxia-mediated activation of 5-HTT transcription is not restricted to the rat but may also apply to humans. Several mechanisms could account for this effect. Previous studies have indicated that expression of the AP-1 proteins c-Jun and c-Fos is upregulated by hypoxia.32 Since there are 2 potential AP-1–binding sites in the promoter region of 5-HTT,33 it can be speculated that induction of 5-HTT expression under hypoxic conditions is mediated by AP-1. A more specific mechanism of hypoxia-induced gene expression might involve the transcription factor hypoxia-inducible factor or HIF-1, which binds to identified hypoxia-sensitive elements in the promoter of several hypoxia-inducible genes.34 35 The consensus sequence of these elements is 5'-TACGTGCT-3'. Interestingly, our computer-aided search in the 5-HTT promoter revealed in 2 locations, the presence of core sequence 5'-CGTG-3', which has previously been shown to bind HIF-1.36 Accordingly, it can be inferred that hypoxia increases the transcriptional rate of the 5-HTT gene probably through one of these potential mechanisms.
Previous studies have shown that hypoxia reduces the proliferating effect of growth factors on cultured SMCs.37 38 In line with these data, we found that PA-SMC proliferation in response to PDGF-BB was attenuated in hypoxic cells compared with their normoxic controls. In contrast, 5-HT added to the cultured cell medium stimulated PA-SMC proliferation to a greater extent under hypoxia than under normoxic conditions. This increased growth-stimulatory effect of 5-HT was directly related to the increased expression of the transporter, since it was completely suppressed in the presence of fluoxetine or paroxetine. That 5-HT may act as a mitogenic factor with increased efficiency during hypoxia should have physiological relevance regarding in vivo stimulation of smooth muscle cell proliferation and vascular remodeling. In particular, the cell proliferation due to 5-HT might contribute to neomuscularization of nonmuscular pulmonary vessels, which occurs as a consequence of prolonged hypoxic exposure.4 5
An important finding of the present study was that the 5-HTT gene was expressed in pulmonary vessels of rats exposed to chronic hypoxia. Our in situ hybridization studies confirmed that the 5-HTT transcript is predominantly located in the media of newly remodeled distal pulmonary arteries in pulmonary hypertensive rats. Interestingly, RT-PCR allowed no detection of basal 5-HTT expression in proximal pulmonary arteries of control normoxic rats, whereas 5-HTT transcripts could be easily detected in the same tissues of hypoxic rats. The absence of 5-HTT mRNA in SMCs from proximal pulmonary arteries of normoxic rats is at variance with results obtained in cultured cells from the same tissue showing expression of 5-HTT mRNA under basal conditions. These data suggest that in vivo PA-SMCs do not phenotypically express 5-HTT under normoxic conditions and that induction occurs in response to hypoxia. In that case, one would infer that 5-HT may behave as a mitogenic factor for PA-SMCs only during conditions of increased 5-HTT mRNA expression. Previous in vivo studies performed in our laboratories are consistent with this hypothesis.4 Indeed, we recently observed that 5-HT promoted the development of pulmonary hypertension when infused continuously in rats during a 2-week exposure to 10% O2.4 Thus, rats treated with 5-HT demonstrated a greater degree of pulmonary hypertension, right ventricular hypertrophy, and structural distal pulmonary artery remodeling than saline-treated rats exposed to a similar level of hypoxia. In contrast, no effect was seen in normoxic rats subjected to the same infusion of 5-HT. Moreover, Fawn-Hooded rats, which have a deficit in platelet 5-HT storage and increased plasma 5-HT levels, developed pulmonary hypertension when they were raised at the altitude of Denver, Colo (an environment with a mild reduction in oxygen tension), whereas Sprague-Dawley rats exhibited normal pulmonary artery pressure under the same conditions.2 Interestingly, exposure of Fawn-Hooded rats to a moderately O2-enriched environment prevented the development of pulmonary hypertension at the altitude of Denver. It is also noteworthy that treatment of hypoxic rats with dexfenfluramine, an inhibitor of 5-HTT transport, prevented the exacerbation of pulmonary hypertension normally expected from the drug-induced increase in plasma 5-HT levels. These findings strongly suggest that 5-HT may act as a potent mitogenic factor in vivo, leading to pulmonary vascular remodeling and pulmonary hypertension, provided that 5-HTT is active in PA-SMCs.
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Acknowledgments |
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This research was supported by grants from INSERM. V.F. was a recipient of a fellowship from Direction des Recherches, Etudes et Techniques-Délégation Générale pour l'Armement during the performance of this work. We thank Dr Denis Ravel and Nicole Laudignon from Servier Laboratory for financial support. We are grateful to Prof K. Peter Lesch (Department of Psychiatry, University of Würzburg, Germany) for his generous gift of the recombinant plasmid with the reporter luciferase gene under the control of the human 5-HTT promoter. We also acknowledge the gifts of drugs (fluoxetine, paroxetine, and ketanserin) from pharmaceutical companies (Lilly, SmithKline Beecham, and Janssen, respectively).
Received August 10, 1998; accepted October 21, 1998.
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S. Eddahibi, A. Chaouat, N. Morrell, E. Fadel, C. Fuhrman, A.-S. Bugnet, P. Dartevelle, B. Housset, M. Hamon, E. Weitzenblum, et al. Polymorphism of the Serotonin Transporter Gene and Pulmonary Hypertension in Chronic Obstructive Pulmonary Disease Circulation, October 14, 2003; 108(15): 1839 - 1844. [Abstract] [Full Text] [PDF]
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M.-A. Pascaud, F. Griscelli, W. Raoul, E. Marcos, P. Opolon, B. Raffestin, M. Perricaudet, S. Adnot, and S. Eddahibi Lung Overexpression of Angiostatin Aggravates Pulmonary Hypertension in Chronically Hypoxic Mice Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 449 - 457. [Abstract] [Full Text] [PDF]
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E. Marcos, S. Adnot, M. H. Pham, A. Nosjean, B. Raffestin, M. Hamon, and S. Eddahibi Serotonin Transporter Inhibitors Protect against Hypoxic Pulmonary Hypertension Am. J. Respir. Crit. Care Med., August 15, 2003; 168(4): 487 - 493. [Abstract] [Full Text] [PDF]
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S. Eddahibi, N. Morrell, M-P. d'Ortho, R. Naeije, and S. Adnot Pathobiology of pulmonary arterial hypertension Eur. Respir. J., December 1, 2002; 20(6): 1559 - 1572. [Abstract] [Full Text] [PDF]
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 Y. Mitani, A. Mutlu, J. C. Russell, D. N. Brindley, J. DeAlmeida, and M. Rabinovitch Dexfenfluramine protects against pulmonary hypertension in rats J Appl Physiol, November 1, 2002; 93(5): 1770 - 1778. [Abstract] [Full Text] [PDF]
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S. L. Lee, A. R. Simon, W. W. Wang, and B. L. Fanburg H2O2 signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L646 - L652. [Abstract] [Full Text] [PDF]
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S. Eddahibi and S. Adnot Endothelins and pulmonary hypertension, what directions for the near future? Eur. Respir. J., July 1, 2001; 18(1): 1 - 4. [Full Text] [PDF]
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S. Eddahibi, S. Adnot, E. Frisdal, M. Levame, M. Hamon, and B. Raffestin Dexfenfluramine-Associated Changes in 5-Hydroxytryptamine Transporter Expression and Development of Hypoxic Pulmonary Hypertension in Rats J. Pharmacol. Exp. Ther., April 1, 2001; 297(1): 148 - 154. [Abstract] [Full Text]
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 A. Kereveur, J. Callebert, M. Humbert, P. Herve, G. Simonneau, J.-M. Launay, and L. Drouet High Plasma Serotonin Levels in Primary Pulmonary Hypertension : Effect of Long-Term Epoprostenol (Prostacyclin) Therapy Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2233 - 2239. [Abstract] [Full Text] [PDF]
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