© 1997 American Heart Association, Inc.
Articles |
Estrogen Upregulates Endothelial Nitric Oxide Synthase Gene Expression in Fetal Pulmonary Artery Endothelium
From the Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas.
Correspondence to Philip W. Shaul, MD, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9063. E-mail PSHAUL{at}MEDNET.SWMED.EDU
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Abstract NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17ß caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10-10 to 10-6 mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.
Key Words: endothelium • estrogen • estrogen receptor • nitric oxide synthase • pulmonary circulation
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Introduction |
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Endothelium-derived NO, produced by eNOS, is a key modulator of vasodilation in the developing pulmonary circulation. It is critically involved in the 8- to 10-fold increase in pulmonary blood flow that occurs during cardiopulmonary transition at birth.1 Animal studies indicate that the capacity for pulmonary endothelial NO production is maximal at term because of a marked increase in pulmonary eNOS gene expression during late gestation and that this is followed by a fall in pulmonary eNOS expression in the postnatal period.2 3 4
The biphasic modulation of pulmonary eNOS expression in the perinatal period suggests that it is due to the effects of a factor that increases in activity in the lung during late gestation and then decreases in activity during early postnatal life. Such would be the case for a placentally derived hormone or growth factor acting in the lung in a paracrine manner. One possible factor is estrogen, which increases in abundance in the fetal blood during the latter phase of gestation in many species.5 6 7 For example, fetal plasma levels of unconjugated estradiol in sheep increase 5-fold, from 80 to 140 days of gestation (term, 145 days), achieving concentrations in the range of 10-9 mol/L.5 In addition, there is evidence that prolonged systemic elevations in estrogen, either related to gender, pregnancy, or chronic administration of the hormone, cause enhancement of basal endothelial NO production in a variety of nonpulmonary organs.8 Furthermore, the latter effects are related to increases in eNOS enzymatic activity.9 10 However, the basis for estrogen-mediated eNOS upregulation is unclear, and it is not known if estrogen modulates eNOS expression in the fetal pulmonary endothelium.
To better understand the molecular mechanisms underlying the upregulation in pulmonary eNOS expression during late fetal life, the present investigation was designed to determine the direct effects of estrogen on fetal PAEC eNOS expression. Since estrogen has cardiac and systemic effects that may indirectly alter endothelial cell eNOS expression through changes in shear stress,11 12 13 studies were performed in early-passage cultured ovine fetal PAECs. We have used this cell culture model previously in investigations of oxygen modulation of eNOS expression.14 Based on the observation in both rats and sheep that pulmonary eNOS expression increases during late gestation as fetal plasma estrogen levels are rising2 3 4 5 6 7 and the previously documented upregulation in nonpulmonary eNOS enzymatic activity with systemic estrogen administration,9 10 the hypothesis was raised that estrogen causes direct upregulation of eNOS expression in ovine fetal PAECs. In addition to testing this hypothesis, experiments were performed to address the following questions: (1) Is eNOS expression modified by estrogen at physiological concentrations? (2) What is the time course of eNOS modulation by estrogen? (3) What are the molecular mechanisms underlying estrogen modulation of eNOS expression?
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Materials and Methods |
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Cell Culture and Treatment
PAECs were obtained from mixed-breed fetal lambs at 125 to 135 days of gestation (term, 144±4 days; n=12), using methods that we have previously described.15 The procedures followed in the care and euthanasia of the study animals were approved by the Institutional Review Board for Animal Research. The PAECs were propagated in RPMI medium containing 10% iron-supplemented calf serum, 10% lamb serum, 1% L-glutamine, 1% antibiotic-antimycotic mixture, 0.15% nystatin, 0.15% gentamicin, and 0.10% tylosin, in a humidified incubator with 5% CO2 in air at 37°C. The identity of the cells was confirmed by phenotype (cobblestone appearance and contact inhibition), by immunofluorescence studies with antibody to factor VIII–related antigen, and by examinations of acetylated low-density lipoprotein uptake. Under quiescent conditions, the PAECs express eNOS and not iNOS.14
Near-confluent cells at passages 4 to 6 were placed in phenol red–free
serum-free medium for 18 hours to remove the effects of the
estrogen-like activity of phenol red and serum-derived estrogen. The
cells were then placed in phenol red–free medium containing 20%
charcoal-stripped serum. The charcoal stripping removes estrogen
metabolites and other steroid hormones.16 The cells were
treated for up to 96 hours with either control medium or medium
containing varying concentrations of E2ß or
E2
, ranging from 10-10 to
10-6 mol/L. Preliminary experiments
revealed that the vehicle for the estrogen compounds, ethanol, had no
effect on PAEC eNOS activity or expression. Culture medium was replaced
every 48 hours, and estrogen treatment was repeated every 24 hours.
NOS Activity
After treatment, PAECs were washed with ice-cold PBS, pelleted,
and resuspended in ice-cold 50 mmol/L Tris buffer (pH 7.4)
containing 1.0 mmol/L EDTA, 5 mmol/L
mercaptoethanol, 10 µg/mL pepstatin A, 10 µg/mL
leupeptin, 90 µg/mL phenylmethylsulfonyl fluoride, and
1.0 µmol/L tetrahydrobiopterin. The cells were disrupted
by repeated freeze-thawing in liquid nitrogen. NOS activity in the cell
lysate was determined by measuring the conversion of
[3H]arginine to
[3H]citrulline.14 Briefly, 50 µL of cell
lysate was added to 50 µL of buffer, yielding final concentrations of
reagents as follows: 2 mmol/L ß-NADPH, 2
µmol/L tetrahydrobiopterin, 10 µmol/L flavin
adenine dinucleotide, 10 µmol/L flavin
mononucleotide, 0.5 mmol/L CaCl2 in
excess of EDTA, 15 nmol/L calmodulin, 2
µmol/L cold L-arginine, and 2.0 µCi/mL
[3H]L-arginine. After incubation at 37°C
for 30 minutes, the assay was terminated by the addition of 400 µL of
40 mmol/L HEPES buffer (pH 5.5) with 2 mmol/L
EDTA and 2 mmol/L EGTA. The terminated reactions were
applied to 1-mL columns of Dowex AG50WX-8 (Tris form) and eluted with 1
mL of the 40 mmol/L HEPES buffer.
[3H]Citrulline was collected in scintillation vials and
quantified by liquid scintillation spectroscopy. NOS activity was
linear with time for up to 1 hour, and it was fully inhibited by
2.0 mmol/L nitro-L-arginine methyl ester. The
calcium dependence of NOS activity was evaluated by the addition of
2.5 mmol/L EGTA. The protein content of the samples was
determined by the method of Bradford, using bovine serum
albumin as the standard.17
Immunoblot Analysis
The methods used for immunoblot analysis
generally followed those we have previously reported.14
After washing and pelleting, PAECs were resuspended in the 50
mmol/L Tris buffer described above and ultrasonically disrupted
(Branson Ultrasonics). The protein content of the preparation was
determined, SDS/polyacrylamide gel electrophoresis was
performed on 25 µg protein with 7% acrylamide, and the
proteins were electrophoretically transferred to nylon filters. The
filters were blocked for 1.5 hours in buffer containing 150
mmol/L NaCl and 10 mmol/L Tris (pH 7.5) with 0.5%
Tween 20 and 5% dried milk and incubated overnight at 4°C with a
1:2000 dilution of primary antiserum to eNOS. The eNOS antiserum was
the kind gift of Dr Thomas Michel (Department of Medicine, Brigham
and Women's Hospital, Harvard Medical School, Boston, Mass). After
incubation with primary antiserum, the nylon filters were washed with
the 150 mmol/L NaCl buffer with Tween 20 and incubated for
1.5 hours with a 1:5000 dilution of a donkey anti-rabbit immunoglobulin
antibody–horseradish peroxidase conjugate (Amersham). The filters were
washed in the 150 mmol/L NaCl buffer with Tween 20, and the
bands for eNOS were visualized by chemiluminescence (ECL Western
Blotting Analysis System, Amersham) and quantified
densitometrically. Under the conditions used, there was a linear
relationship between the protein load and the densitometric values for
eNOS (r=.96 to .98).
Northern Analysis
Total RNA obtained from PAECs by a single extraction method with
an acid guanidinium thiocyanate–phenol–chloroform mixture was
subjected to oligo-dT affinity chromatography to obtain
poly-A(+) RNA, which was size-fractionated on 1.0% agarose
formaldehyde gels and transferred to nylon membranes.14
The quantity of poly-A(+) RNA used ranged from 5 to 15 µg, with
identical amounts loaded for all samples in a given experiment. The RNA
was cross-linked to the membranes by UV irradiation. After
prehybridization, the membranes were hybridized overnight at 42°C in
the presence of random-primed 32P-labeled human
endothelial NOS cDNA (1.2 kb). After hybridization, the
blots were washed, and autoradiography was performed
and quantified densitometrically. Northern analysis for the
housekeeping gene MDH was performed using a 0.5-kb random-primed
32P-labeled cDNA probe to verify equivalent
RNA stability and loading between samples. Densitometric values for
eNOS mRNA were normalized to the abundance of MDH mRNA before
comparisons were made between treatment groups.
ER Inhibition
The effects of estrogen on gene expression are mediated by the
activation of ERs, which include the classical ER, ER, and the
recently discovered ER, ERß.8 18 19 To determine the
role of ERs in the modulation of eNOS by the hormone, additional
experiments were performed in the absence or presence of ICI 182,780
(10-5 mol/L), a pure ER
antagonist.20 Since recently described
ERß-mediated transcriptional transactivation is inhibited by the
related antagonist ICI 164,384,18 it is likely
that the ICI 182,780 compound inhibits both ER- and ERß-related
gene expression. This agent was chosen instead of tamoxifen because the
latter compound has agonist-like effects in some
systems.20 ICI 182,780 was the kind gift of Dr B.M. Vose
(Zeneca Pharmaceuticals, Cheshire, UK).
ER Immunocytochemistry
To determine if fetal PAECs express ER protein,
immunocytochemistry was performed using an antiserum directed against
the estrogen-binding domain of ER. Since the ligand-binding domains
of ER and ERß are highly homologous,18 19 the
antiserum is likely to recognize either receptor subtype. PAECs grown
in six-well plates (Beckton Dickinson & Co) were washed with PBS and
fixed with freshly prepared PBS-buffered 4%
paraformaldehyde (pH 7.4) for 30 minutes at RT. After
applying a protein-blocking agent (Immunon) for 10 minutes at RT, the
cells were incubated 2 hours at RT with either 1:100 human ER
monoclonal antibody (Chemicon International, Inc) or preimmune serum
(DAKO Corp) in dil-uent (Cell Marque). After quenching of
endogenous peroxidase activity with 3%
H2O2 for 30 minutes at RT, sequential 10-minute
incubations with a biotinylated secondary link antibody and a
streptavidin-labeled conjugate of horseradish peroxidase were performed
(DAKO Corp). The detection of the ER was visualized using the chromogen
3',3'-diaminobenzidine (Research Genetics, Inc) after 15 minutes of
development and subsequent rinses with H2O.
Immunostaining was visualized by light microscopy.
Transfection Assays
To determine if endothelial ERs are capable of
estrogen-induced transcriptional transactivation, transient
transfection assays were performed with a specific estrogen-responsive
reporter system. One reporter plasmid contains three copies of the
Xenopus vitellogenin ERE proximal to the thymidine kinase
promoter driving the expression of firefly luciferase cDNA (ERE-Luc),
and the other reporter plasmid, TK-Luc, is identical to ERE-Luc but
lacks the ERE.21 PAECs grown to 50% to 60% confluence in
six-well plates were preincubated in OptiMem medium (Life Technologies)
for 30 minutes at 37°C. ERE-Luc or TK-Luc (1 µg) and a plasmid
containing simian virus 40–driven ß-galactosidase (pSV-ß-Gal,
Promega Corp) to normalize for transfection efficiency were mixed with
Lipofectamine (10 µL/well, Life Technologies) and incubated in a
total volume of 200 µL for 30 minutes at room temperature. The
lipid-coated DNA and 800 µL OptiMem were then added to each well of
PAECs. After 5 hours, 1 mL of growth medium containing 20%
iron-supplemented calf serum and 20% lamb serum was added to each
well, and the following day the medium was replaced with growth medium
containing phenol red–free RPMI and 2% iron-supplemented calf serum.
Seventy-two hours after transfection, the cells were lysed, and the
extracts were centrifuged at 10 000g to remove
unbroken cells and debris. Luciferase activity was measured with a
luminometer (Monolight 2010, Analytical Luminescence
Laboratory),22 and ß-galactosidase activity was measured
spectrophotometrically (at 420 nm) by the generation of
o-nitrophenol from the substrate,
o-nitrophenyl-ß-D-galactopyranoside.23
The results are normalized as relative luciferase light
units/ß-galactosidase activity. In selected wells, the cells were
transfected with pGL2–control vector (Promega Corp) containing a
simian virus 40 promoter and enhancer to serve as a positive control
for luciferase expression. Reporter activity was assessed in control
cells and cells treated with either 10-8
mol/L E2ß or E2ß plus
10-5 mol/L ICI 182,780 for 48 hours.
The ERE-Luc and TK-Luc plasmids were the kind gift of Drs Richard Karas
and Michael Mendelsohn in the Department of Medicine, Tufts University
School of Medicine, Boston, Mass.
Statistical Analysis
Data for the NOS activity assays and the immunoblot
and Northern analyses are presented as the percentage
of the value observed in control PAECs. Treatment groups were compared
using the Mann-Whitney U test for nonparametric
data or nonparametric ANOVA.24 The experiments
evaluating transcription activation were analyzed by
parametric ANOVA and Newman-Keuls post hoc testing. Results are
expressed as mean±SEM. Significance was accepted at
P<.05.
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Results |
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NOS Activity
The effect of E2ß on NOS activity in PAECs is shown in Fig 1A
. Cells were treated with
control medium or medium with 10-8
mol/L E2ß for 48 hours, and NOS activity was then
determined in cell lysates. NOS activity in control cells ranged from
15 to 50 pmol · mg protein-1 ·
min-1 in four independent experiments.
E2ß treatment caused a 2.5-fold increase in NOS enzymatic
activity. Both control and E2ß-stimulated NOS activity
were fully inhibited by calcium chelation (data not shown).
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The dose response to E2ß is depicted in Fig 1B
. NOS
activity rose in a dose-dependent manner, with a threshold
concentration of 10-10 mol/L, a maximal
effect (2.5-fold increase) at 10-8
mol/L, and a submaximal effect at 10-6
mol/L. E2, the less active stereoisomer of
estrogen, had no effect on NOS activity over the same concentration
range (data not shown).
The time course of the effect of E2ß on NOS activity is
shown in Fig 1C. After 24 hours of treatment, there was no discernible
change in NOS activity. Maximal upregulation of NOS activity was
evident at 48 hours. The effect of E2ß persisted for at
least 96 hours.
eNOS Protein Expression
The effect of E2ß on eNOS protein expression in
PAECs is depicted in Fig 2. In the
representative immunoblot shown (Fig 2A),
the protein was detectable at the expected size of 135 kD, and there
was enhanced protein expression in cells exposed to
10-8 mol/L E2ß for 48
hours. Quantitative densitometry for four independent experiments
revealed that eNOS protein expression in E2ß-treated
cells was increased to 190% of control values (Fig 2B).
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eNOS mRNA Expression
To determine the basis for E2ß-induced upregulation
of eNOS protein expression and enzymatic activity, eNOS mRNA abundance
was determined by Northern analysis of poly-A(+) RNA from PAECs
exposed to control medium or medium with 10-8
mol/L E2ß for 48 hours (Fig 3). Similar to eNOS protein expression
and NOS activity, eNOS mRNA abundance was increased in
E2ß-treated cells (Fig 3A). These results were confirmed
in four separate studies (Fig 3B), which showed a 3-fold increase in
eNOS mRNA abundance with E2ß treatment when corrected for
the abundance of mRNA for the housekeeping gene MDH.
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Role of ERs
To determine whether the upregulation of eNOS expression involves
ER activation, additional experiments were performed evaluating the
effects on NOS activity of E2ß
(10-8 mol/L for 48 hours) in the
absence or presence of the ER antagonist ICI 182,780 (Fig 4). In this series of experiments,
E2ß alone caused a 4.4-fold increase in NOS activity. In
contrast, the addition of ICI 182,780 fully inhibited the upregulation
of NOS activity by E2ß.
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To determine if fetal PAECs express ER protein, immunocytochemistry was
performed using an antiserum directed against the estrogen-binding
domain of ER. Positive immunostaining was evident in
most cells (Fig 5A). The
immunostaining was primarily nuclear, but cytoplasmic
staining was also evident. Immunostaining was absent
when preimmune serum was substituted for the primary antiserum (Fig 5B).
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To determine if endothelial ERs are capable of
estrogen-induced transcriptional transactivation, the
estrogen-responsive reporter plasmid ERE-Luc or the control plasmid
TK-Luc was introduced into PAECs. In the absence of estrogen (control),
reporter activity was similar in transfections with TK-Luc and ERE-Luc
(Fig 6A). In contrast, ERE-Luc reporter
activity was 7-fold greater than TK-Luc reporter activity in cells
treated with 10-8 mol/L
E2ß (Fig 6B). The activation of transcription by
E2ß was completely inhibited by the addition of ICI
182,780 (Fig 6C). Similar findings were obtained in three independent
experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have demonstrated that E2ß causes marked upregulation of NOS activity in fetal PAECs. We have shown that this occurs at a threshold concentration of 10-10 mol/L and that maximal upregulation is obtained with 10-8 mol/L E2ß, indicating that the effect occurs at levels of the hormone that are achieved in the fetal plasma during late gestation.5 We have also demonstrated that E2
, the less active stereoisomer of estrogen, has no
effect on NOS activity over the same concentration range. Furthermore,
we have shown that E2ß-stimulated increases in NOS
activity are evident by 48 hours and that they persist for at least 96
hours. These observations reveal that prolonged estrogen exposure has
direct stereospecific effects on the capacity of the fetal
pulmonary endothelium to produce the critical
vasodilator NO. We have also performed studies to determine the isoform of NOS that is involved. The ovine fetal PAECs express eNOS exclusively under quiescent conditions.14 However, iNOS is expressed in endothelial cells under certain conditions, particularly after cytokine activation.1 Since NOS activity in both control and estrogen-treated fetal PAECs was fully inhibited by calcium chelation, the observed increase in NOS activity is related to upregulation of calcium-dependent eNOS and not calcium-independent iNOS.1 In addition, since NOS enzymatic activity was determined in cell lysates in the presence of excess quantities of required substrates and cofactors, the increase in activity represents an increase in eNOS enzyme abundance. This is confirmed by the increase in eNOS protein evident by immunoblot analysis.
To determine the basis for estrogen-mediated upregulation of eNOS enzyme activity and protein expression in the fetal PAECs, we assessed eNOS mRNA abundance in control and estrogen-treated cells. In parallel with the enhancement in NOS enzymatic activity and eNOS protein expression, estrogen caused a 3-fold rise in PAEC eNOS mRNA abundance. This finding indicates that the mechanism by which estrogen modulates eNOS gene expression in the fetal PAECs is at the level of gene transcription or mRNA stability.
We also performed studies to determine whether ERs play a role in the
effect of estrogen on PAEC eNOS. The highly specific ER
antagonist ICI 182,780 completely inhibited the
upregulation of eNOS by E2ß, indicating that this effect
requires stimulation of ERs, which are ligand-activated
transcription factors.8 18 19 In addition, ER protein
expression was evident by immunocytochemistry, and it was found
primarily in nuclei. The two ER subtypes, ER and ERß, are highly
homologous, particularly in the DNA-binding domain (95% to 96%) and
in the C-terminal ligand-binding domain (55% to
58%).18 19 Since both ER and ERß activation are
inhibited by the ICI compounds8 18 and the
immunostaining may recognize either ER or ERß, the
observed effects of estrogen on PAEC eNOS may be mediated by either or
both receptor subtypes. Studies of ER subtype expression in adult rat
lung reveal the presence of both ER and ERß mRNA, with the latter
subtype predominating.25 The pulmonary cell
specificity of subtype expression is yet to be determined. Further
studies are now indicated to distinguish the role of ER and ERß in
eNOS upregulation in PAECs.
To determine if endothelial ERs are capable of estrogen-induced transcriptional transactivation, studies were performed using an ERE–luciferase reporter gene construct. These experiments revealed that estrogen stimulates gene transcription 7-fold in fetal PAECs and that this is fully inhibited by ICI 182,780. These findings collectively indicate that fetal PAECs express ERs that are active in the regulation of gene transcription in general and in the regulation of eNOS expression specifically. To our knowledge, these are the first studies to directly demonstrate estrogen-mediated activation of gene transcription in endothelial cells.
Although eNOS was originally considered to be a constitutive gene, it
has become evident more recently that its expression is regulated by a
variety of stimuli. With the exception of tumor necrosis factor-,
which alters eNOS mRNA stability, eNOS modulation by physical factors
such as cyclic strain and by growth factors such as transforming growth
factor-ß occurs at the level of transcription.26 27 The
present observations that estrogen causes upregulation of eNOS
mRNA, that the eNOS upregulation is inhibited by ER antagonism, and
that ER activation stimulates PAEC gene transcription suggest that
estrogen increases the transcription rate of the eNOS gene. Inspection
of the 5' flanking sequence of the human eNOS gene reveals that there
are 11 copies of the estrogen half-palindromic motif (TGACC or its
reverse complement) within 
2 kb of the initiation
ATG.28 Widely spaced half-palindromic motifs of this type
function synergistically in certain genes to form an
ERE.29 Thus, estrogen may upregulate eNOS gene
transcription in PAECs through the binding of occupied ERs to the
estrogen half-palindromic motifs in the gene promoter. Functional
analysis of the eNOS promoter in fetal PAECs is now warranted
to assess this possible mechanism.
There is considerable evidence from investigations in intact animal models that estrogen enhances the capacity for NO production in nonpulmonary endothelium. For example, Gisclard et al30 demonstrated in rabbits that E2ß treatment for 4 days causes an increase in endothelium-dependent relaxation in femoral artery rings. Using NOS antagonists, Kauser and Rubanyi31 showed that bioassayable endothelium-derived NO is higher in thoracic aortas isolated from female versus male rats. In addition, in studies of sheep uterine arteries isolated after 3 days of E2ß treatment, Veille et al32 demonstrated that the enhanced NO-dependent relaxation is related to greater NOS enzymatic activity. Furthermore, studies by Weiner and colleagues9 10 of guinea pig skeletal muscle during pregnancy and after E2ß treatment suggest that this is related to the upregulation of endothelial cell eNOS mRNA expression. However, the cell specificity of eNOS upregulation in the skeletal muscle was not examined, and this may be an important issue because it has been demonstrated that the eNOS isoform is expressed in skeletal myocytes.33 In addition, since estrogen has cardiac and systemic effects that may alter endothelial cell eNOS expression through changes in shear stress,11 12 13 it may be difficult to assess the direct effects of the hormone on endothelial cell eNOS in whole-animal models.
To avoid these confounding variables, investigators have performed studies in cultured nonpulmonary endothelial cells. Hayashi et al34 demonstrated E2ß-induced increases in NO release and eNOS protein in cultured human umbilical vein and bovine aortic endothelium, and Hishikawa et al35 obtained similar findings in human aortic endothelium. However, Arnal et al36 have more recently been unable to replicate the observed eNOS upregulation in bovine aortic endothelium. The basis for these discrepancies is not evident. The present study is the first to demonstrate that estrogen upregulates pulmonary endothelial eNOS expression. It is also the first to show in either pulmonary or nonpulmonary endothelium that this is due to an upregulation in eNOS mRNA expression that is most likely secondary to enhanced eNOS gene transcription.
Although a variety of phenotypic characteristics relevant to the regulation of pulmonary endothelial NO production are conserved in the early-passage PAECs,14 15 a degree of caution may be warranted in the direct extrapolation of the present findings in the cultured cells to processes in the intact fetal lung. For example, the level of oxygenation present in the cell culture system may differ from that in the lung, and we have previously demonstrated that eNOS gene expression in this cell type is modulated by oxygen.14 In addition, the level of ER expression in the cultured PAECs may not accurately reflect the abundance of the receptor in the intact endothelium. However, since studies of ER abundance in human umbilical vein and bovine aortic endothelium indicate that receptor density falls with cell passage,34 the response of endothelium in situ may actually be more robust than that presently observed in passage-4 to -6 cells. In addition, the use of the cultured cells enables us to evaluate the direct effects of single factors such as estrogen on PAEC phenotype.
With these potential limitations in mind, there are important
physiological implications of estrogen modulation
of PAEC eNOS expression in both the adult and the fetus. In studies of
adult dogs, pregnancy or prolonged estrogen treatment blunts the
pulmonary vasoconstrictor responses to acute hypoxia
and prostaglandin F2
infusion.37 In addition, estrogen treatment in adult rats
protects against the development of pulmonary hypertension with
monocrotaline administration or chronic
hypoxia.38 39 The present observations suggest
that these effects may be due to enhanced eNOS gene expression in the
adult pulmonary endothelium. In the fetus,
estrogen-mediated effects on eNOS expression may underlie the
developmental upregulation in pulmonary eNOS gene expression
during late gestation.2 3 4 Fetal plasma estrogen
concentrations increase markedly during late gestation to levels in the
range of 10-9 mol/L because of enhanced
placental production of the hormone,5 and we have
now demonstrated that such levels of the hormone upregulate eNOS in
fetal PAECs. This may indeed occur in vivo, because T. Parker and S.
Abman (unpublished data, 1997) have recently found that the
intrapulmonary infusion of E2ß for 48 to 72 hours
causes a marked increase in pulmonary blood flow in the fetal
lamb that is NO-mediated. Furthermore, since estrogen concentrations in
the newborn fall to baseline levels by 24 hours after
birth,7 withdrawal from the effects of estrogen may
underlie the downregulation in pulmonary eNOS in the newborn
period.2 3
Similarly, there are potential pathophysiological implications of the present findings, particularly in situations in which fetal plasma estrogen levels are abnormally decreased. Experiments in fetal rhesus monkeys reveal that the normal rise in blood estrogen occurring before birth is completely absent in pregnancies in which intrauterine infection is induced with group B streptococcus.40 In addition, estrogen levels are markedly decreased in the cord blood of postmature human infants compared with term control infants.41 It is postulated that in pregnancies complicated by placental dysfunction, such as that associated with intrauterine infection or postmaturity, attenuated placental estrogen synthesis may lead to diminished fetal pulmonary eNOS expression, thereby contributing to the pathogenesis of persistent pulmonary hypertension of the newborn. Further studies of estrogen-mediated regulation of eNOS gene expression in fetal PAECs will continue to advance our knowledge of the role of this hormone in both the pulmonary circulation and other estrogen-responsive vascular beds.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HD-30276 and HL-53546. The project was done during an Established Investigatorship of the American Heart Association (Dr Shaul). We are indebted to Marilyn Dixon for preparing this manuscript.
Received February 21, 1997; accepted June 25, 1997.
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References |
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C. R. Rosenfeld, R. E. White, T. Roy, and B. E. Cox Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H319 - H328. [Abstract] [Full Text] [PDF]
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T. Hayashi, M. Jayachandran, D. Sumi, N. K. Thakur, T. Esaki, E. Muto, H. Kano, Y. Asai, and A. Iguchi Physiological Concentration of 17{beta}-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury : Role of NO Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1613 - 1621. [Abstract] [Full Text] [PDF]
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W. A. Salhab, P. W. Shaul, B. E. Cox, and C. R. Rosenfeld Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2134 - H2142. [Abstract] [Full Text] [PDF]
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 P. V. GUILLOT, L. LIU, J. A. KUIVENHOVEN, J. GUAN, R. D. ROSENBERG, and W. C. AIRD Targeting of human eNOS promoter to the Hprt locus of mice leads to tissue-restricted transgene expression Physiol Genomics, March 13, 2000; 2(2): 77 - 83. [Abstract] [Full Text] [PDF]
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 Z. German, K. L. Chambliss, M. C. Pace, U. A. Arnet, C. J. Lowenstein, and P. W. Shaul Molecular Basis of Cell-specific Endothelial Nitric-oxide Synthase Expression in Airway Epithelium J. Biol. Chem., March 10, 2000; 275(11): 8183 - 8189. [Abstract] [Full Text] [PDF]
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E. Wight, C. F. Kung, P. Moreau, H. Takase, N. A. Bersinger, and T. F. Luscher Aging, Serum Estradiol Levels, and Pregnancy Differentially Affect Vascular Reactivity of the Rat Uterine Artery Reproductive Sciences, March 1, 2000; 7(2): 106 - 113. [Abstract] [PDF]
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T. A. Parker, D. Dunbar Ivy, H. L. Galan, T. R. Grover, J. P. Kinsella, and S. H. Abman Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, February 1, 2000; 278(2): L374 - L381. [Abstract] [Full Text] [PDF]
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I. Dumont, P. Hardy, K. G. Peri, X. Hou, S. Molotchnikoff, D. R. Varma, and S. Chemtob Regulation of endothelial nitric oxide synthase by PGD2 in the developing choroid Am J Physiol Heart Circ Physiol, January 1, 2000; 278(1): H60 - H66. [Abstract] [Full Text] [PDF]
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J. A. Whitney, Z. German, T. S. Sherman, I. S. Yuhanna, and P. W. Shaul Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, January 1, 2000; 278(1): L131 - L138. [Abstract] [Full Text] [PDF]
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G. New, S. J. Duffy, R. W. Harper, and I. T. Meredith Estrogen improves acetylcholine-induced but not metabolic vasodilation in biological males Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2341 - H2347. [Abstract] [Full Text] [PDF]
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M. Garcia-Duran, T. de Frutos, J. Diaz-Recasens, G. Garcia-Galvez, A. Jimenez, M. Monton, J. Farre, L. S. de Miguel, F. Gonzalez-Fernandez, M. d. M. Arriero, et al. Estrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human Neutrophils Circ. Res., November 26, 1999; 85(11): 1020 - 1026. [Abstract] [Full Text] [PDF]
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 I. S. Yuhanna, A. N. MacRitchie, R. L. Lantin-Hermoso, L. B. Wells, and P. W. Shaul Nitric Oxide (NO) Upregulates NO Synthase Expression in Fetal Intrapulmonary Artery Endothelial Cells Am. J. Respir. Cell Mol. Biol., November 1, 1999; 21(5): 629 - 636. [Abstract] [Full Text]
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 A. M. McNeill, N. Kim, S. P. Duckles, D. N. Krause, and H. A. Kontos Chronic Estrogen Treatment Increases Levels of Endothelial Nitric Oxide Synthase Protein in Rat Cerebral Microvessels • Editorial Comment Stroke, October 1, 1999; 30(10): 2186 - 2190. [Abstract] [Full Text] [PDF]
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S. Nuedling, S. Kahlert, K. Loebbert, P. A. Doevendans, R. Meyer, H. Vetter, and C. Grohe 17{beta}-Estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo Cardiovasc Res, August 15, 1999; 43(3): 666 - 674. [Abstract] [Full Text] [PDF]
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E. Tan, M. V Gurjar, R. V Sharma, and R. C Bhalla Estrogen receptor-{alpha} gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration Cardiovasc Res, August 15, 1999; 43(3): 788 - 797. [Abstract] [Full Text] [PDF]
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M. C. Pace, K. L. Chambliss, Z. German, I. S. Yuhanna, M. E. Mendelsohn, and P. W. Shaul Establishment of an immortalized fetal intrapulmonary artery endothelial cell line Am J Physiol Lung Cell Mol Physiol, July 1, 1999; 277(1): L106 - L112. [Abstract] [Full Text] [PDF]
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J. Zhang, G. A. Massmann, C. P. Mirabile, and J. P. Figueroa Nonpregnant Sheep Uterine Type I and Type III Nitric Oxide Synthase Expression Is Differentially Regulated by Estrogen Biol Reprod, May 1, 1999; 60(5): 1198 - 1203. [Abstract] [Full Text]
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E. A. Kirsch, I. S. Yuhanna, Z. Chen, Z. German, T. S. Sherman, and P. W. Shaul Estrogen Acutely Stimulates Endothelial Nitric Oxide Synthase in H441 Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 1999; 20(4): 658 - 666. [Abstract] [Full Text]
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H. J. Knot, K. M. Lounsbury, J. E. Brayden, and M. T. Nelson Gender differences in coronary artery diameter reflect changes in both endothelial Ca2+ and ecNOS activity Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H961 - H969. [Abstract] [Full Text] [PDF]
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K. E. Vagnoni, C. E. Shaw, T. M. Phernetton, B. M. Meglin, I. M. Bird, and R. R. Magness Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1845 - H1856. [Abstract] [Full Text] [PDF]
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R. R. Magness, T. M. Phernetton, and J. Zheng Systemic and uterine blood flow distribution during prolonged infusion of 17beta -estradiol Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H731 - H743. [Abstract] [Full Text] [PDF]
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G. G. Geary, D. N. Krause, and S. P. Duckles Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H292 - H300. [Abstract] [Full Text] [PDF]
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 B. O Okoye, P. D Losty, M. J Fisher, I. Wilmott, and D. A Lloyd Effect of dexamethasone on endothelial nitric oxide synthase in experimental congenital diaphragmatic hernia Arch. Dis. Child. Fetal Neonatal Ed., May 1, 1998; 78(3): 204F - 208. [Abstract] [Full Text]
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X. Wang and A. A. Abdel-Rahman Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2309 - H2315. [Abstract] [Full Text] [PDF]
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