1 Section on Clinical
Pharmacology and 4 Department of
Histochemistry, To determine a potential role for the
renin-angiotensin system in the growth of human pulmonary artery (PA)
smooth muscle, we studied the localization of angiotensin (ANG)
II-receptor subtypes by autoradiography in sections of human PA and in
cultured PA smooth muscle cells (PASMCs) and examined the growth
responses to ANG II in vitro. Specific
125I-labeled
[Sar1,Ile8]ANG
II binding was demonstrated within the pulmonary arterial media, but
binding to cultured cells varied between isolates. Binding in tissues
and cells was inhibited by the ANG II type 1 (AT1) receptor antagonist
losartan but not by the type 2 (AT2) receptor antagonist
PD-123319. Microautoradiographic studies indicated that cultured PASMCs
exhibit heterogeneity with regard to ANG II binding sites. Addition of
ANG II to serum-deprived PASMCs, exhibiting a relatively high level of
125I-[Sar1,Ile8]ANG
II binding, led to a dose-dependent stimulation of DNA synthesis at 24 h and protein synthesis at 48 h. ANG II led to an increase in cell size
without an increase in cell number. These effects were inhibited by
losartan but not by PD-123319. In addition, ANG II led to rapid
activation of mitogen-activated protein kinase (MAPK), and ANG
II-stimulated DNA synthesis was inhibited by the specific inhibitor of
MAPK PD-98059. We conclude that the
AT1 receptor is expressed by human
PASMCs in vivo and in vitro and is coupled to activation of MAPK and
increased DNA and protein synthesis in vitro. These results are
consistent with the hypothesis that ANG II may be involved in human
pulmonary vascular remodeling.
mitogen-activated protein kinase; vascular remodeling; pulmonary
hypertension; angiotensin II type 1 receptors
A PREVIOUS STUDY IN ANIMALS (36) has
suggested that the renin-angiotensin system may contribute to the
development of pulmonary hypertension. Indeed, long-term administration
of the angiotensin-converting enzyme (ACE) inhibitor captopril to
patients with pulmonary hypertension has been shown to reduce pulmonary
vascular resistance in some studies (1, 30) but not in another (17).
More recently, Morrell et al. (24) and others (37) have
demonstrated that administration of captopril or the specific
angiotensin (ANG) II type 1 (AT1) receptor antagonist
losartan markedly attenuated the hemodynamic and structural changes of
hypoxia-induced pulmonary hypertension in the rat. Furthermore, it was
found that chronic hypoxia increased the expression of ACE, the enzyme
mainly responsible for the conversion of ANG I to ANG II, in the walls
of small pulmonary arteries undergoing remodeling (22).
These observations have led to the hypothesis that local production of
ANG II by ACE in the pulmonary arterial wall could contribute to smooth
muscle hypertrophy and/or hyperplasia seen in remodeled hypertensive
pulmonary arteries (22). However, although it is recognized that ANG II
can stimulate smooth muscle cell (SMC) growth (3, 10, 25) in addition
to its traditional role as a vasoconstrictor, the growth response to
ANG II is known to be highly variable depending on the species, the
vascular bed, and culture conditions (12). Moreover, the growth
response can vary widely between isolates (27). This variation may
reflect differences in receptor distribution and receptor coupling to signal transduction cascades (38).
The present study sought to determine whether ANG II could be a factor
that contributes to hypertensive remodeling of the pulmonary arterial
media in humans. The approach involved isolation of segments of the
proximal human pulmonary artery (PA) and determination of the tissue
distribution of ANG II-receptor subtypes by in vitro autoradiography.
Similar segments were used for isolation of medial PASMCs in culture.
Ligand binding studies were used to establish the ANG II-receptor
binding characteristics in cultured cells, and the growth responses to
ANG II were examined. The results demonstrate that isolates of human
PASMCs vary considerably in the relative density of ANG II binding
sites. In isolates that possess high-level binding, receptors are
predominantly of the AT1 subtype
and are coupled to activation of mitogen-activated protein kinase
(MAPK) and DNA and protein synthesis. Our findings are consistent with
the hypothesis that the renin-angiotensin system may contribute to
PASMC hypertrophy in human pulmonary hypertension and suggest that
inhibition of ANG II-generating pathways may prove useful in the
treatment of this condition.
Reagents and antibodies. Medium 199 (M199), type II collagenase, fetal bovine serum (FBS),
antibiotic-antimycotic solution, trypsin-EDTA, and four- and eight-well
slide chambers were purchased from GIBCO BRL (Paisley, UK). Optimal
cutting temperature mounting medium was obtained from H&E Histological
Equipment (Nottingham, UK). Losartan was provided by Merck Research
Laboratories (West Point, PA). PD-123319 was provided by Parke-Davis
(Wilmington, DE). PD-98059 was purchased from Calbiochem-Novabiochem
(Nottingham, UK). 125I-labeled
[Sar1,Ile8]ANG
II (2,200 Ci/mmol) was purchased from DuPont-NEN (Stevenage, UK);
[methyl-3H]thymidine
(6.7Ci/mmol) was from ICN Biomedicals (Thame, UK); L-[4-3H]phenylalanine,
Hyperfilm 3H autoradiography film,
and Hypercoat LM-1 emulsion were from Amersham; ANG II and
[Sar1,Ile8]ANG
II, monoclonal antibodies to Tissue preparation. Proximal segments
of the human PA (main, right and left, or lobar arteries) were obtained
from resected lung specimens from patients undergoing lung or
heart-lung transplantation for primary pulmonary hypertension
(n = 3 men and 1 woman; mean age 29 yr), congenital heart disease (n = 3 men and 2 women; mean age 32 yr), or underlying lung disease
(n = 7 men and 2 women; mean age 47 yr) comprising emphysema (n = 4),
sarcoid (n = 2), histiocytosis
(n = 1), fibrosing alveolitis
(n = 1), and cystic fibrosis
(n = 1). Additional samples of the
proximal PA were obtained from unused donors for transplantation
(n = 7 men and 4 women; mean age 31 yr). Further specimens were obtained from patients undergoing lobectomy
or pneumonectomy for bronchial carcinoma (n = 4 men; mean age 67 yr). Samples
of the proximal PA (pulmonary trunk or right or left PA) were placed in
M199 at 4°C for transport from the operating room to
the laboratory. An intact section of the PA (~0.5 cm long) was
removed from the original specimen in some cases for frozen sections
and in vitro autoradiographic studies. The segments were blotted dry
and placed in optimal cutting temperature mounting medium on cork
boards, then immediately frozen in isopentane cooled by liquid
nitrogen. Once frozen, the specimens were stored at The remaining samples of the PA were used for isolation of PASMCs (see
Isolation of PASMCs).
In vitro autoradiography of ANG II binding
sites. ANG-receptor expression in proximal PA segments
(n = 17) was characterized by receptor
autoradiography. The diagnoses in this group were as follows: primary
pulmonary hypertension (n = 4),
congenital heart disease (n = 4),
donors for heart-lung transplantation
(n = 5), and underlying lung disease
(n = 4). Ten-micrometer-thick cryostat
sections were incubated in 10 mM sodium phosphate buffer (pH 7.4)
containing the labeled antagonist
125I-[Sar1,Ile8]ANG
II as previously described (15). As in previous studies on human
tissues (15, 16), binding reached an apparent equilibrium within 90 min
at 20-22°C (data not shown). Nonspecific binding was defined
as that remaining in adjacent sections coincubated with either
10 Further anatomic resolution of the binding sites was achieved by tissue
microautoradiography as previously described (16). Sections were
incubated with ligand as described above, then apposed to
emulsion-coated coverslips for 2-3 wk at 4°C.
Isolation of PASMCs. The arteries were
opened to expose the endothelial surface, which was removed by gentle
scraping with a scalpel. The surrounding adventitia was then carefully
dissected from the tunica media. Cells were derived either from
explants or after tissue digestion. For the explant technique, the
pulmonary arterial media was cut into ~2-mm cubes and plated onto
25-cm2 tissue culture flasks.
Explants were left to adhere overnight and then were maintained in
M199, 20% FBS, and antibiotic-antimycotic solution (100 U/ml of
penicillin, 100 µg/ml of streptomycin, and 250 ng/ml of amphotericin
B). The cells were passed after ~2 wk into a single
75-cm2 flask and grown to
confluence in M199-10% FBS.
For tissue digests, the medial layer was cut into small pieces with a
scalpel and incubated in type II collagenase (1,000 U/ml) in serum-free
medium at 37°C for 4 h. The action of collagenase was stopped by
the addition of M199-20% FBS. The cells were then passed through a
filter (pore size 100 µm) and centrifuged at 200 g for 5 min, then resuspended in
M199-20% FBS before being plated in
25-cm2 flasks. Subsequent passages
were carried out at confluence, dividing one flask into four. Cells
were used for experiments between passages 3 and 10.
Additional experiments were conducted with a commercially available
human PASMC line (Clonetics, TCS Biologicals, Bucks, UK) previously
reported (11) to undergo hypertrophy in response to ANG II stimulation.
Cells were grown and passaged according to instructions from and with
reagents provided by the supplier.
The phenotype of isolated cells was investigated with antibodies to
smooth muscle-specific antigens: monoclonal anti- Ligand binding studies on cultured
cells. For ligand binding studies in cultured cells,
PASMCs were seeded at a density of 20,000 cells/well in 24-well plates.
The cells were allowed to grow to 70-80% confluence in
M199-10% FBS. For the binding assays, the cells were intially
washed twice for 5 min in serum-free M199. The cells were then
incubated in M199-0.1% (wt/vol) BSA containing 10 Competition curves were derived by incubating the cells with
10 Microautoradiography in cell
monolayers. In some isolates, binding of
125I-[Sar1,Ile8]ANG
II to single cells was examined by microautoradiography in SMC
monolayers. Cells were plated at a density of 2,000/well in four-well
slide chambers and grown for 48 h in M199-10% FBS. Monolayers were
washed 2 × 5 min in PBS before the addition of M199-0.1% BSA
containing 10 Growth responses to ANG II. Growth of
human PASMCs was determined by
[methyl-3H]thymidine
and
L-[4-3H]phenylalanine
incorporation, representing DNA and protein synthesis, respectively.
Cells from passages
3-10 were used in
all experiments. For
[methyl-3H]thymidine
studies, the cells were washed with PBS, trypsinized with 0.25%
trypsin-EDTA for 5 min, and suspended in M199-10% FBS. The cells
were then seeded in 24-well plates at a density of 20,000/well and
grown to 70-80% confluence. At this stage, the cells were washed
three times with warm PBS and quiesced by incubation with M199-0.1% FBS for 48 h. The medium was then exchanged for fresh M199-0.1% FBS containing
10 Protein synthesis was determined at 24, 48, and 72 h with a similar
protocol as for
[methyl-3H]thymidine
incorporation except that the cells were incubated with 0.4 mM
L-phenylalanine for 30 min
before the addition of 2 µCi/well of
L-[4-3H]phenylalanine
for the final 24 h of exposure to the test conditions.
For studies of cell proliferation, the cells were incubated with
10 To determine whether ANG II increased cell size, we measured the
relative size distribution by flow cytometry. Cells were grown to
70-80% subconfluence in
75-cm2 flasks and quiesced in
0.1% serum for 3 days before incubation with
10 MAPK activity. Activation of MAPK was
assayed by immunoblotting. The cells were grown to 70-80%
confluence in 35-mm petri dishes, washed, and then quiesced in
M199-0.1% FBS for 48 h. The medium was replaced with fresh
M199-0.1% FBS for 90 min and then treated with M199-0.1% FBS
either with or without the addition of
10 In additional experiments, the effect of PD-98059 on ANG II-stimulated
MAPK activation was examined. The cells were treated as above except
that 10 For immunoblotting, the samples were thawed and sonicated for 10 min at
4°C to shear DNA and reduce sample viscosity. The samples were then
microcentrifuged for 10 min at 13,000 rpm, and the supernatant was
collected. Protein content was determined as above. The samples (20 µg of total protein) were subjected to SDS-polyacrylamide gel
electrophoresis (12%). The proteins were then electrotransferred to
nitrocellulose membrane and incubated in 10% Marvel in Tris-buffered
saline-Tween (TBS-T), pH 7.4, overnight at 4°C. The membrane was
washed 4 × 10 min in TBS-T, followed by a 1-h incubation with
polyclonal anti-phosphorylated MAPK primary antibody (1:1,000; New
England Biolabs). These antibodies detect p42mapk and
p44mapk only when catalytically
activated by phosphorylation at
Tyr204. The membrane was again
washed before a 1-h incubation with a horseradish peroxidase-conjugated
secondary antibody (1:2,000; New England Biolabs). After four further
washes in TBS-T, the membrane was incubated in electrochemiluminence
reagent (Amersham) for 1 min before being exposed to film for ~2 min.
Statistics. Differences between
control and experimental conditions were analyzed by one-way analysis
of variance and a post hoc Tukey's honestly significant difference
test. Conventional significance was considered to have been achieved
when P was <0.05. All results are
expressed as means ± SE. Binding curves were analyzed with GraphPad
Prism version 2.01 (GraphPad Software, San Diego, CA).
Distribution of ANG II-receptor subtypes by
autoradiography. To determine the ANG II-receptor
subtype(s) in the intact pulmonary arterial media, autoradiographic
studies were employed. These studies demonstrated specific binding of
125I-[Sar1,Ile8]ANG
II to the pulmonary arterial media in all arteries studied (n = 17; Fig.
1,
A-C).
This binding was inhibited by incubation with an excess of unlabeled
ANG II or with the specific
AT1-receptor antagonist losartan.
However, binding was unaffected by incubation with
10
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-smooth muscle actin (clone 1A4) and
smooth muscle myosin (clone hSM-V), FITC-conjugated anti-mouse IgG,
L-phenylalanine, and all other
reagents were purchased from Sigma (Poole, UK).
40°C
before being sectioned.
6 M unlabeled
[Sar1,Ile8]ANG
II or ANG II. Binding sites were characterized further by incubating
consecutive sections with 2.5 × 10
10 M
125I-[Sar1,Ile8]ANG
II in the presence of the
AT1-receptor antagonist losartan or the AT2-receptor antagonist
PD-123319. Macroautoradiographic images were obtained by apposing
labeled sections to Hyperfilm 3H
for 3-4 days at 4°C.
-smooth muscle
actin (clone 1A4) and anti-smooth muscle myosin (clone hSM-V). For
immunostaining, the cells were grown to subconfluence in eight-well
slide chambers. The cells were fixed in acetone at
20°C for
10 min, then washed in phosphate-buffered saline (PBS) for 3 × 5 min. The cells were incubated with primary antibody for 1 h at room
temperature, then with anti-mouse FITC-conjugated secondary antibody
for 1 h, again at room temperature. Between steps, the slides were
thoroughly rinsed in PBS for 3 × 5 min at room temperature. The
cells were then mounted in a solution of PBS and glycerol (1:1) and
visualized by fluorescence microscopy.
10 M
125I-[Sar1,Ile8]ANG
II. Preliminary studies showed that binding reached an apparent equilibrium within 90 min at 37°C (data not shown). Nonspecific binding was defined as that remaining bound to cells incubated with
10
6 M unlabeled
[Sar1,Ile8]ANG
II, and ANG-receptor subtypes were characterized by incubation with
either 10
6 M losartan or
10
6 M PD-123319. The cells
were washed twice for 1 min with serum-free M199 at 4°C and lysed
with 0.2 M NaOH-0.1% SDS. The lysates were transferred to 5-ml
polystyrene vials and counted in a scintillation counter. Protein
concentration was assayed with the Bio-Rad DC protein assay (Bio-Rad,
North Yorkshire, UK) according to the manufacturer's instructions.
10 M
125I-[Sar1,Ile8]ANG
II in M199-0.1% BSA in the presence of increasing concentrations (10
11 to
10
7 M) of unlabeled
[Sar1,Ile8]ANG
II. To obtain estimates of maximal binding capacity and dissociation constant, saturation curves were generated by incubating the cells with
increasing concentrations of
125I-[Sar1,Ile8]ANG
II (10
11 to
10
9 M) in the presence and
absence of 10
6 M
[Sar1,Ile8]ANG II.
10 M
125I-[Sar1,Ile8]ANG
II with and without the addition of
10
6 M losartan,
10
6 M PD-123319, or
10
6 M ANG II. After 90 min,
the cells were washed 2 × 1 min in M199 and 1 × 10 s in
distilled H2O. The slides were
then air-dried, and emulsion was applied to the slide with a wire loop.
The slides were developed after 7 and 14 days, mounted, and examined by
dark- and light-field microscopy.
10 to
10
6 M ANG II and 0.5 µCi/well of
[methyl-3H]thymidine
for 24 h with and without the addition of
10
6 M losartan or
10
6 M PD-123319. Additional
experiments were performed with ANG II with and without the addition of
the selective inhibitor of MAPK activation PD-98059 (3-30 µM)
(5). The cells were rapidly washed three times with PBS at 4°C
followed by the addition of 1 ml/well of 10% TCA at 4°C for 30 min. The TCA was then discarded, and 0.5 ml of 0.2 M NaOH was added to
each well. The plate was stored at 4°C overnight, the resulting
lysates were transferred to scintillation vials, and thymidine
incorporation determined by scintillation counting.
6 M ANG II for 1-4
days. Because ANG II may be degraded by peptidases in culture, fresh
ANG II was added daily to achieve a final concentration of
10
6 M. The cells were
counted with a hemocytometer, and viability was assessed by trypan blue exclusion.
6 M ANG II for 48 h. The
cells were then harvested by trypsinization and centrifugation.
Single-cell suspensions of equal density were prepared in serum-free
M199 and then immediately measured for relative size distribution based
on forward scatter (FACS Vantage, Becton Dickinson). Forward scatter
has been shown to correlate with cell size (14).
7 M ANG II. At specified
time points (1, 5, 10, 20, 30, 60, and 120 min), the medium was removed
and the cells were snap-frozen in liquid nitrogen, then harvested in
0.5 ml of lysis buffer (containing 20 µM E64, 1 µM pepstatin A, 2 µg/ml of aprotinin, 10 µg/ml of leupeptin, 62.5 mM
-glycerophosphate, 50 mM NaF, 2.5 mM
Na3VO4, 0.1% Triton X-100 in 20 mM Tris, and 2 mM EDTA, pH 7.4) with a cell
scraper. The lysate was divided into aliquots and stored frozen at
20°C before blotting.
7 M ANG II was added
for 10 min with and without preincubation (30 min) with 30 µM
PD-98059.
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
6 M PD-123319, the
specific AT2-receptor antagonist.
These findings indicate that the majority of ANG II binding sites in
the normal or diseased human pulmonary arterial media are of the
AT1 subtype. Microautoradiographic
studies on sections of PA demonstrated an apparently homogeneous
pattern of radioligand binding within the media (data not shown).
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Fig. 1.
Autoradiographs of a section of main pulmonary artery from a patient
with primary pulmonary hypertension
(A-C)
and a pulmonary artery smooth muscle cell (PASMC) monolayer
(D-F)
showing distribution of
125I-[Sar1,Ile8]ANG
II binding either alone (total binding;
A and
D) or in presence of ANG II type 1 (AT1) receptor antagonist
losartan (B and
E) or
AT2-receptor antagonist PD-123319
(C and
F). Virtually all activity bound to
arterial media
(A-C,
open arrows), and cell monolayers could be displaced by losartan,
whereas PD-123319 had no effect, indicating predominance of
AT1-receptor subtype. Relatively
little binding was detected in neointima (*) of hypertensive artery.
Microautoradiography in cell monolayers demonstrated a mixed population
of cells with different binding densities within the same isolate
(D-F);
intense labeling with
125I-[Sar1,Ile8]ANG
II is observed in some cells, whereas neighboring groups of cells had
only low-level or no binding (solid arrows). Bars, 3 mm in
A-C
and 75 µm in
D-F.
To assess the possibility that the different PASMC isolates were heterogeneous with respect to ANG II-receptor expression (see ANG II binding sites on cultured human PASMCs), we conducted microautoradiographic studies in cell monolayers in isolates with high and low levels of binding. These studies showed a mixed population of PASMCs within the same isolate, with individual cells exhibiting different binding densities. Relatively more cells demonstrated a higher binding density in those isolates with a high degree of specific binding (Fig. 1, D-F).
ANG II binding sites on cultured human
PASMCs. Cells derived by the explant or digest
technique yielded cells with the typical morphology of vascular SMCs.
The smooth muscle phenotype of these cells was confirmed by positive
immunofluorescence with anti-smooth muscle myosin and anti--smooth
muscle actin.
To determine the presence and subtype of ANG II binding sites on
cultured cells, we performed radioligand binding studies with
125I-[Sar1,Ile8]ANG
II. In five isolates, we detected a high level (>90%) of specific
125I-[Sar1,Ile8]ANG
II binding: two from patients with congenital heart disease, one from a
patient transplanted for sarcoidosis, and two from control subjects.
The relative binding density in these isolates was 81 ± 23 fmol/mg
protein (range 41-158 fmol/mg protein). Ligand binding to these
isolates could be inhibited by prior incubation with losartan but not
with PD-123319 (Fig. 2), suggesting that in
vitro, the predominant ANG II receptor was of the
AT1 subtype, similar to that in
the intact media. Competition binding experiments demonstrated that the
binding was both saturable and specific. Analysis of the
saturation-binding curves yielded a mean maximal binding capacity of
1,131 ± 72 fmol/mg and a dissociation constant of 0.32 ± 0.05 nM for
125I-[Sar1,Ile8]ANG
II (Fig. 3). The relative binding density
in these isolates was found to be unaffected by the increasing passage
number of cells in culture (repeated measurements made up to
passage 13).
|
|
Of the remaining isolates, 21 showed relatively low-level (<50% specific) binding of 125I-[Sar1,Ile8]ANG II, with a mean binding density of 0.76 ± 0.16 fmol/mg protein (range 0.08-2.25 fmol/mg), and 7 demonstrated no specific binding.
Binding studies to the commercial human PA cell line revealed only low-level binding, with a relative binding density of 4.34 ± 0.12 fmol/mg protein.
Growth responses to ANG II and effects of specific
receptor antagonists. Incubation with ANG II for 24 h
resulted in a dose-dependent increase in
[methyl-3H]thymidine
in the isolates found to have high-level specific binding sites for
125I-[Sar1,Ile8]ANG
II (Fig.
4A). In
these cells, the effect of
107 M ANG II was
significantly inhibited by
10
6 M losartan but was
unaffected by the presence of
10
6 M PD-123319, suggesting
that the observed increase in DNA synthesis was mediated via the
AT1-receptor subtype (Fig.
4B). The addition of ANG II
(10
6 M) to these isolates
caused no increase in cell number over a 4-day period despite the daily
addition of fresh ANG II to the culture medium. Further experiments
were conducted for up to 10 days, again with no increase in cell number
(data not shown).
|
In the same isolates, protein synthesis as measured by incorporation of
L-[4-3H]phenylalanine
was stimulated by ANG II in a dose-dependent manner after 48 and 72 h
of incubation (Fig.
5A) but
not by 24 h of incubation. The response was again inhibited by losartan
but not by PD-123319 (Fig. 5B).
|
The above experiments suggested that ANG II induced hypertrophy rather
than proliferation of PASMCs. To support this, we measured the relative
cell size by flow cytometry in isolates with a high-level binding of
125I-[Sar1,Ile8]ANG
II. Incubation with ANG II led to an ~10% increase in mean relative
cell size compared with that in control cells (Fig.
6).
|
In the remaining isolates that demonstrated only low-level or absent binding to 125I-[Sar1,Ile8]ANG II, no measurable increase in [methyl-3H]thymidine or L-[4-3H]phenylalanine incorporation was detected in response to incubation with ANG II.
Role of MAPK. To determine whether
MAPK is involved in the ANG II-induced growth response in cells with
relatively high-level 125I-[Sar1,Ile8]ANG
II binding, we studied the time course of activation of MAPK by
immunoblotting and the effect of inhibition of MAPK activation on ANG
II-mediated DNA synthesis. Incubation of PASMCs with 3-30 µM
PD-98059 led to a dose-responsive inhibition of ANG II-induced [methyl-3H]thymidine
incorporation (Fig. 7). Immunoblotting for
phosphorylated p42mapk and
p44mapk demonstrated rapid
(1-min) activation of both isoforms, which peaked at 5 min and declined
over the subsequent 10-60 min (Fig. 8A).
Pretreatment of the cells with PD-98059 markedly attenuated the ANG
II-induced activation of MAPK (Fig.
8B), confirming the inhibitory
effect of this compound on MAPK activation.
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DISCUSSION |
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The main findings of this study are that the AT1-receptor subtype is the predominant ANG receptor in the human pulmonary arterial media and isolated human PASMCs and that ANG II is capable of activating MAPK and stimulating DNA and protein synthesis via the AT1 receptor in these cells. We used in vitro autoradiography to show that the human pulmonary arterial media and PASMC monolayers possess specific binding sites for the radiolabeled ANG II analog 125I-[Sar1,Ile8]ANG II. The displacement of this ligand by the specific AT1-receptor subtype antagonist losartan but not by the AT2-receptor antagonist PD-123319 suggests that the human pulmonary arterial media and isolated PASMCs express ANG II receptors predominantly of the AT1 subtype. Furthermore, we found that primary cultures of human PASMCs exhibited heterogeneity in the relative binding density for 125I-[Sar1,Ile8]ANG II between isolates. This heterogeneity may be due to the presence of a mixed population of cells within a given isolate containing varying proportions of cells with relatively high- and low-level binding as demonstrated by microautoradiograpy in cell monolayers.
The distribution of ANG II-receptor subtypes in the human main PA has not been previously studied. Although at least four ANG II-receptor subtypes have been identified, only two, the AT1 and AT2 subtypes, have been studied in detail in vascular tissue. The mRNA for the AT1 subtype has been localized to fetal rat PASMCs (23) and is expressed at lower levels in adult PAs. The AT2 subtype predominates over the AT1 subtype during development of the rat aorta but is expressed at very low levels in adult arteries (33). However, reexpression of the AT2 subtype has been reported in balloon-injured adult rat arteries (13). Furthermore, the AT2 receptor has been localized to rat endothelial cells where it may mediate growth (29). Our autoradiographic studies in tissue sections, receptor binding studies in cultured cells, and microautoradiographic studies in cell monolayers indicate that ANG II binding sites in the media of large human PAs and isolated PASMCs are of the AT1 subtype. This is consistent with a previous report from this laboratory (35) demonstrating predominantly AT1 binding sites in the media of human coronary arteries.
One explanation for the finding of PASMC isolates with high- and low-level 125I-[Sar1,Ile8]ANG II binding is cellular heterogeneity in the expression of ANG II receptors. The concept of cellular heterogeneity within the arterial media is well recognized in the bovine PA (7, 8). The use of microautoradiography in cell monolayers allowed us to demonstrate the presence of individual PASMCs with high- and low-level binding within the same isolate. Isolates with relatively high-level 125I-[Sar1,Ile8]ANG II binding were enriched by a relatively greater proportion of individual PASMCs with a high level of binding. In contrast, in vitro macro- and microautoradiography of radioligand binding in the intact PA did not suggest regional heterogeneity of receptor expression. However, autoradiography does not allow sufficient resolution to confirm differences in ANG II-receptor binding between individual cells in the arterial media. Therefore, possible explanations for our findings include 1) heterogeneity of PASMC AT1-receptor expression in vivo and in vitro and 2) homogeneous PASMC AT1 expression in vivo, with heterogeneity in the capacity of the cells to retain receptor expression in culture. Our data would suggest the latter but cannot exclude the former.
When PASMCs with a relatively high level of
125I-[Sar1,Ile8]ANG
II binding were incubated with ANG II, a dose-dependent increase in DNA
synthesis was observed at 24 h and in protein synthesis at 48 and 72 h.
Stimulation of DNA and protein synthesis by ANG II was inhibited by
coincubation with losartan but not with PD-123319, suggesting that ANG
II-induced DNA and protein synthesis is mediated via the
AT1-receptor subtype. However, ANG
II did not increase PASMC number despite prolonged incubation with
106 M ANG II and daily
addition of fresh ANG II to the culture medium. These findings suggest
that stimulation by ANG II leads to hypertrophy rather than hyperplasia
in human PASMCs, similar to previous findings in rat systemic vascular
SMCs (9, 10). A hypertrophic effect of ANG II in human PASMCs is
supported by the increase in relative cell size we observed by flow cytometry.
In further studies, we addressed the potential role of activation of MAPK, an essential part of the pathways that mediate cell growth, in ANG II-stimulated DNA synthesis. These studies demonstrated that a specific inhibitor of MAPK activation, PD-98059, dose dependently inhibited ANG II-induced DNA synthesis. Furthermore, with immunoblotting for phosphorylated p42mapk and p44mapk, we found that ANG II rapidly activated these isoforms, an effect that was inhibited by PD-98059. The time course of activation of MAPK by ANG II is similar to that previously reported in rat aortic SMCs (6). Taken together, these results provide strong evidence for a growth-promoting effect of ANG II on human PASMCs.
A growth effect of ANG II on systemic vascular SMCs is well recognized (3, 9, 18). However, the reported effects of ANG II as a growth factor appear to be highly variable depending on the species (21) and vascular bed from which the cells are derived as well as on the conditions under which the cells are cultured (12). Relatively few studies (3, 20, 21) have been conducted in primary human vascular SMCs. Generally, ANG II has been found to exert relatively weak effects on DNA synthesis in human systemic SMCs (21), some reports (3, 20) suggesting that ANG II requires the presence of serum to produce a mitogenic effect, whereas another (2) reports no effect of the peptide on mitogenesis. None of these previous studies determined the relative density of SMC binding sites for ANG II. Because we observed considerable variation in ANG II binding between isolates, with a minority of isolates demonstrating high-level binding, it is possible that the reported differences in the growth-promoting properties of ANG II in human cells are related to differences in ANG II-receptor density.
In cultured rat SMCs derived from the systemic circulation, DNA
synthesis induced by ANG II frequently has a delayed onset (34),
suggesting that it exerts its effects mainly via induction of other
growth factors (10, 31, 34). Indeed, there is evidence that the
hypertrophic or proliferative response to ANG II is determined by the
autocrine production of transforming growth factor-1 (10). In the
present study, we observed that ANG II stimulated relatively rapid
(within 24 h) synthesis of DNA in primary cultures of human PASMCs
under serum-deprived conditions. This observation may indicate the
presence of diverse signaling mechanisms involved in the growth response to ANG II in rat and human cells, although whether such differences exist remains to be determined.
One previous study (11) reported a weak stimulatory effect of ANG II on protein synthesis as assessed by [3H]leucine incorporation from the same commercial source of human PASMCs used in the present study. In contrast to the previous report, we were unable to demonstrate ANG II-induced DNA or protein synthesis in these cells, although it is unlikely that we were using cells from exactly the same line. The absence of ANG II-stimulated growth in our cell line was probably due to the relatively low binding density.
Studies in animals (22, 24, 32, 36) have suggested a role for the renin-angiotensin system in the development of hypoxia-induced pulmonary hypertension. The ACE inhibitor captopril and the specific ANG II-receptor antagonist losartan prevented the hemodynamic and structural changes of chronic hypoxia-induced pulmonary hypertension in the rat (24, 37). Furthermore, it has been demonstrated that expression of ACE protein and mRNA is increased in small PAs of rats with hypoxia-induced pulmonary hypertension (22), suggesting that increased local production of ANG II may contribute to vascular remodeling in this animal model. In general, inhibition of endogenous ANG II by losartan does not appear to prevent pulmonary hypertension induced by ingestion of the pyrrolizidine alkaloid monocrotaline (4, 32). Interestingly, however, in a recently developed monocrotaline and high-flow model of pulmonary hypertension (26), which is characterized by the formation of a neointima, ACE inhibitors and losartan both delay the neointimal proliferation. Taken together, these studies perhaps indicate a specific role for endogenous ANG II in the complex cellular events leading to pulmonary vascular remodeling.
Evidence for involvement of the renin-angiotensin system in clinical pulmonary hypertension remains limited. Schuster et al. (28) found evidence for increased local expression of ACE in the endothelium and neointima of elastic PAs of patients with primary pulmonary hypertension. A handful of studies (1, 30) in patients with pulmonary hypertension have indicated that inhibition of ACE may reduce pulmonary vascular resistance during long-term administration. The present study has demonstrated that ANG II is capable of stimulating growth of SMCs in at least a subpopulation of cells derived from the human pulmonary circulation and therefore may contribute to pulmonary vascular remodeling in humans. These studies suggest a potential role for the use of AT1-receptor antagonists in the treatment of clinical pulmonary hypertension.
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ACKNOWLEDGEMENTS |
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This study was supported by British Heart Foundation Grant PG/96121.
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FOOTNOTES |
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N. Morrell is a Medical Research Council Clinician Scientist Fellow.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. W. Morrell, Section on Clinical Pharmacology, Division of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (E-mail: nmorrell{at}rpms.ac.uk).
Received 10 November 1998; accepted in final form 1 April 1999.
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