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Saadia Eddahibi, PhD
Serge Adnot, MD
INSERM U492 and
Département de Physiologie
Hôpital H. Mondor
Créteil, France |
Introduction: Serotonin Signaling Pathway in
Pulmonary Hypertension
The effects of serotonin (5-hydroxytryptamine, 5-HT) on the
pulmonary circulation have been investigated because of the
reported increased risk of idiopathic pulmonary arterial
hypertension (IPH) in patients who used appetite suppressants
that interact with 5-HT.1 An association between the
anorexigen aminorex and IPH was first described in the
1960s. In the 1980s, use of the appetite suppressant fenfluramine
was shown to be associated with an epidemic of
IPH in France and Belgium.2 The serotonin hypothesis
received support from the occurrence of pulmonary hypertension
in Fawn-hooded rats, which have an inherited
platelet-storage defect.2
Early studies focused on circulating 5-HT and its potential
effects on the pulmonary vascular bed. In 1990, Herve
and colleagues reported elevated plasma 5-HT levels in a
patient with a platelet storage disease who developed IPH.
Five years later, this group of investigators showed that
patients with IPH had increased circulating serotonin levels
even after heart-lung transplantation.3 In addition to its
vasoactive effects, 5-HT exerts mitogenic and co-mitogenic
effects on pulmonary-artery smooth muscle cells. In contrast
to the constricting action of 5-HT on smooth muscle cells,
which is mainly mediated by 5-HT receptors (5-HT 1B/D,
2A, and 2B),2 the mitogenic and co-mitogenic effects of 5-
HT require internalization of 5-HT by the 5-HT transporter
(5-HTT).4,5 Accordingly, drugs that competitively inhibit 5-
HTT also block the mitogenic effects of 5-HT on smooth
muscle cells.6 The appetite suppressants fenfluramine, dfenfluramine,
and aminorex differ from selective serotonin
transporter inhibitors in that they not only inhibit serotonin
reuptake but also trigger 5-HT release and interact with 5-
HTT and 5-HT receptors in a specific manner.6,7
Serotonin Transporter (5-HTT) in
Pulmonary Hypertension
We recently tested the hypothesis that the 5-HTT in the lung
may be a key determinant of pulmonary vessel remodeling
because of its effects on pulmonary artery smooth muscle
cell growth. 5-HTT is abundantly expressed in the lung,
where it is predominantly located on pulmonary-artery
smooth muscle cells.8,9 It is encoded by a single gene
expressed in several cell types such as neurons, platelets,
and pulmonary vascular endothelial and smooth muscle
cells. The level of 5-HTT expression appears to be considerably
higher in human lung than in human brain, suggesting
that altered 5-HTT expression may have direct consequences
on pulmonary-artery smooth muscle cell function.
The requirement for 5-HTT as a mediator for the mitogenic
activity of 5-HT appears specific for pulmonary-artery
smooth muscle cells, since no such effect has been reported
with other smooth muscle cell types. Direct evidence that
5-HTT plays a key role in pulmonary vascular remodeling
was recently provided by studies showing that mice with targeted
5-HTT gene disruption developed less severe hypoxic
pulmonary hypertension than did wild- type controls and
that selective 5-HTT inhibitors attenuated hypoxic pulmonary
hypertension.10 Conversely, increased 5-HTT expression
was associated with increased severity of hypoxic pulmonary
hypertension.11 Although a heterogeneous population
of 5-HT2A, 5-HT2B, and 5-HT1B receptors exists in
pulmonary arteries, 5-HT receptor antagonists are not as
efficient as 5-HTT inhibitors in protecting against the development
of hypoxic pulmonary hypertension.12 Moreover,
expression of these 5-HT receptors is not altered in IPH.13
Taken together, these observations suggest a close correlation between
5-HTT expression and/or activity and the extent of pulmonary vascular
remodeling during experimental pulmonary
hypertension.
Overexpression of 5-HTT Responsible
for Pulmonary-Artery Smooth Muscle Cell Hyperplasia in IPH
 Figure
1. Left panel: 5-HTT-like immunoreactivity in lung
sections from control subjects and patients with idiopathic
pulmonary arterial hypertension (IPH). Panel A shows a lung
section from a control subject, with weak to moderate 5-HTT-like
immunoreactivity in pulmonary arterial endothelial cells and
strong immunoreactivity in smooth muscle cells. Panels B, C,
and D show lung sections from patients with IPH: 5-HTT-like
immunoreactivity is much stronger, especially in the medial
layer of pulmonary arteries with marked muscular hypertrophy
(B); no 5-HTT immunostaining is detected in intimal fibrosis
(C); in lesions with onionskin arrangement, 5-HTT-like immunoreactivity
is prominent at sites of intense pulmonary-artery smooth muscle
cell proliferation (D). Panel E shows no immunoreactivity in
a section incubated with secondary antibody but no primary
antibody. Scale bar: 100 µm in A, B, C, and E; 200 µm
in D. Right panel: Individual platelet [3H]citalopram-binding
in normal controls and in patients with IPH.9
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Evidence that the 5-HTT plays an important role in
the pathogenesis of human IPH has recently been demonstrated.
5-HTT expression was shown to be increased in platelets
and lungs from patients with IPH, where it predominated in
the media of thickened pulmonary arteries and in onion-bulb
lesions (Figure 1).9 Interestingly, the higher
level of 5-HTT protein and activity persisted in cultured smooth
muscle
cells isolated from pulmonary arteries of patients with IPH,
as compared to cells from controls (Figure 2).9 Moreover,
pulmonary-artery smooth muscle cells from patients with
IPH grew faster than those from controls when stimulated by
serotonin or serum (which contains micromolar concentrations
of serotonin), as a consequence of increased expression of the
serotonin transporter (Figure 2).9 In the presence
of 5-HTT inhibitors, the growth-stimulating effects of serum
and serotonin were markedly reduced, and the difference
between growth of pulmonary-artery smooth muscle cells
from patients and controls was abolished. The proliferative
response of pulmonary-artery smooth muscle cells to various
growth factors such as PDGF, EGF, TGFß, FGFa, and IGF did
not differ between patients with primary pulmonary hypertension
and controls9. From these studies in can be concluded
that 5-HTT overexpression and/or activity in pulmonary-
artery smooth muscle cells from patients with IPH is
responsible for the increased mitogenic response to serotonin
and to serum.
 Figure
2. Left panel: 5-HTT activity in pulmonary-artery smooth
muscle cells (PA-SMCs) from patients with idiopathic pulmonary
arterial
hypertension (IPH) and from controls. Right panel: PA-SMC proliferation
as assessed by [3H]thymidine incorporation into cells from
patients with IPH (solid bars) and from controls (open bars). The
cells were incubated with increasing concentrations of 5HT (10-8
to
10-6 mol/L) in the presence of 0.2% serum. The response was also
measured in the presence of ketanserin (10-6 mol/L), a 5-HT2A
receptor antagonist, GR 127935 (10-6 mol/L), a 5-HT1B/1D receptor
antagonist, fluoxetine (10-5 mol/L), or citalopram (10-5 mol/L).9 |
5-HTT Gene Polymorphism and Increased
Expression in Pulmonary Arteries in IPH
That 5-HTT expression is genetically controlled has been
convincingly demonstrated: a polymorphism in the promoter
region of the human 5-HTT gene alters the level of transcription.
14 This polymorphism consists of two common alleles,
a 44-bp insertion or deletion, designated the L and S
allele, respectively. The L allele drives a two- to threefold
more active
 Figure
3. 5-HTT activity as assessed by [3H]5-HT uptake (left
panel)
and [3H]thymidine incorporation (right panel) in response to
5-HT
(10-6 mol/L) into smooth muscle cells (SMCs) from controls
with the
SS, LS, or LL genotype. Each bar is the mean±SEM of
data obtained
in six individuals in each group. *P <.05, **P <.01,
and ***P <.001
compared with respective values for the SS genotype. [3H]5-HT
uptake and [3H]thymidine incorporation were also greater in
LL cells
than in LS cells (P <.01).9
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transcription of the 5-HTT gene than the S
allele. In studies of pulmonary-artery smooth muscle cells
from controls, we found that cells from LL subjects
expressed twofold more 5-HTT mRNA than did cells from SS
subjects, and that LS subjects had an intermediate level of
expression (Figure 3).9 Accordingly, the growth-stimulating
effects of 5-HT or serum were more marked in cells from
subjects with the LL genotype than those with LS or SS
genotypes, indicating that the capability of pulmonary-artery
smooth muscle cells to proliferate in response to serotonin
or serum was directly linked to the functional polymorphism
of the 5-HTT gene promoter (Figure 3). We have studied a
large population of patients with IPH (n = 89) and found
that 65% of patients, compared to only 27% of controls,9
were homozygous for the L allelic variant of the 5-HTT gene
promoter, which is associated with 5-HTT overexpression
and increased pulmonary-artery smooth muscle cell growth.
This finding suggests that the LL allele confers susceptibility
to IPH. Several questions, however, remain unanswered.
Although the long allele of the 5-HTT gene promoter is
strongly associated with IPH, this does not fully explain the
increased 5-HTT expression in patients with IPH. Thus,
additional factors are probably needed to produce 5-HTT
overexpression. Whether this overexpression results from an
alteration in the 5-HTT gene itself or from alterations in
other factors involved in regulating 5-HTT gene expression
remains to be determined. No preponderance
of the LL genotype has been found in familial IPH. Preliminary
results suggest that the bone morphogenetic
protein receptor II (BMPR2) agonists BMP4 and
BMP6 may inhibit 5-HTT expression. An attractive hypothesis
is that decreased BMPR2 function leads to increased 5-
HTT expression. Loss of BMPR2 and increased 5-HT transport
into pulmonary-artery smooth muscle cells may also
interact directly to synergistically produce pulmonary hypertension.
There is a pressing need for studies exploring the
molecular pathways that connect BMPR2 mutant genotypes,
5-HTT expression, and the IPH phenotype.
5-HTT Gene Polymorphism and
Mechanisms of Increased Expression in Other Forms
of Pulmonary Hypertension
In most forms of pulmonary hypertension in adults, as well
as in persistent pulmonary hypertension in neonates, a
genetic predisposition has been suggested. Since many factors,
such as inflammation or appetite suppressants,15 interact
with 5-HTT expression, we sought to determine whether
the associations linking 5-HTT overexpression to pulmonary
arterial hypertension, and 5-HTT gene polymorphism to susceptibility
to pulmonary arterial hypertension, existed in
other types of pulmonary hypertension in humans. In recent
studies, we investigated lung transplant recipients with various
forms of pulmonary hypertension, including pulmonary
venoocclusive disease, scleroderma, sarcoidosis, sickle cell
disease, bronchiectasis, and histiocytosis X.13 We found
increased 5-HTT expression in both the lungs and pulmonary-
artery smooth muscle cells from these patients, as
well as an increased growth response of pulmonary artery
smooth muscle cells to 5-HT or serum. The L-allelic variant
of the 5-HTT gene promoter polymorphism was present in
14/25 (56%) of lung transplant recipients with these other
causes of pulmonary hypertension but in only 27% of controls.
13 Thus, the frequency of the LL genotype was significantly
higher in this population of lung transplant recipients
with pulmonary hypertension than in a control population,
suggesting that the LL genotype may confer genetic susceptibility
to pulmonary arterial hypertension as well as to
severe pulmonary hypertension associated with other disorders.
Consistent with our finding in IPH, the L/S polymorphism
of the 5-HTT gene promoter was only partly responsible for
the increased 5-HTT expression in pulmonary hypertension
associated with other illness: pulmonary-artery smooth muscle
cells from these patients exhibited higher 5-HTT levels
than same-genotype cells from controls. No additional promoter
sequence alterations were found. This finding indicates
that differences in 5-HTT expression between patients
and controls cannot be ascribed entirely to 5-HTT gene promoter
polymorphism. Additional studies revealed that the
sequences of the main regulatory regions of the 5-HTT gene,
including the promoter region and intron 2, were not altered
in patients with pulmonary hypertension compared to controls.
Thus, 5-HTT gene overexpression in pulmonary-artery
smooth muscle cells from patients with pulmonary hypertension
may not result from alterations in the regulatory
sequences of 5-HTT gene. Complex mechanisms are probably
involved, such as alterations in related genes or signaling
pathways involved in regulating 5-HTT expression, and
additional studies will be required to understand this
process. 5-HTT Gene Polymorphism in Pulmonary
Hypertension Complicating COPD
 Figure
4. Individual values of pulmonary artery pressure
(Pap) measured
in patients with COPD classified according to their
LL, LS,
or SS genotype. Values are mean Pap recorded during
right heart
catheterization. The mean of each group of values is
indicated by a
horizontal bar. * P <.05 ** P <.01 compared with
respective values
for the LS genotype; §P <.01, compared with
respective values for
the SS genotype. The level of Pap did not differ between
patients
with the LS or SS genotype.
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The pathogenesis of pulmonary hypertension in patients
with advanced chronic obstructive pulmonary disease
(COPD) is still incompletely understood. Chronic hypoxemia
is considered the major contributing factor, in association
with inflammation and morphological changes in lung
parenchyma. However, pulmonary artery pressure varies
greatly among individuals with COPD, with some patients
developing severe pulmonary hypertension out of proportion
to the severity of their underlying disease.16 Because we
previously showed that hypoxia was a strong inducer of 5-
HTT gene expression, we investigated whether 5-HTT gene
promoter polymorphism, in combination with hypoxia, determined
the extent of pulmonary vascular remodeling and,
consequently, the severity of pulmonary hypertension in
patients with advanced hypoxemic COPD.17 Investigating a
large series of patients with advanced COPD (n = 103), we
found that pulmonary hypertension severity in these patients
was closely related to LL genotype of the 5-HTT gene promoter
polymorphism. Mean or systolic pulmonary artery
pressure was more than 10 mm Hg higher, and pulmonary
vascular resistance twice as high, in patients homozygous
for the L allelic variant compared to those with LS or SS
genotype.17 No significant differences in pulmonary artery
pressure or pulmonary vascular resistance were found
between LS and SS individuals, in keeping with previous
evidence that the S variant may exert a dominant influence. The association between the LL genotype
and pulmonary hypertension prompted us to compare the LL, LS,
and SS
groups regarding variables potentially related to its development.
We found no differences regarding blood gas variables
or smoking history. Surprisingly, airflow limitation, as
a percent
reduction in FEV-1, was more severe in SS or LS
patients than in LL patients.17 Conceivably, COPD patients
with severe pulmonary hypertension may become symptomatic
earlier in the course of their disease, at a stage when
they have less airflow limitation than do LS or SS patients,
although this requires additional investigation. Since COPD
severity is mainly determined by the FEV-1 value, this unexplained
difference also indicates that pulmonary hypertension
in COPD develops independently from the severity of
the underlying lung disease. Given the prognostic impact
of
pulmonary hypertension, this suggests that early identification
of patients with COPD at risk for developing pulmonary
hypertension might improve the management of the disease
by prompting measures targeting pulmonary hypertension
progression.
Conclusion
The present results support a crucial role for enhanced
5-HTT activity in the pathogenesis of pulmonary vascularremodeling
and suggest that functional polymorphism of the
5-HTT gene promoter may confer susceptibility to various
forms of pulmonary hypertension. Agents capable of selectively
inhibiting 5-HTT-mediated pulmonary artery smooth
muscle cell proliferation deserve to be investigated as potential
treatments for pulmonary hypertension.
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