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Department of Cardiovascular Physiology, University of Goettingen, Goettingen, Germany
1Correspondence: Department of Cardiovascular Physiology, University of Goettingen, Humboldtallee 23, 37073 Goettingen, Germany. E-mail: hecker{at}veg-physiol.med.uni-goettingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-estradiol, caused a time- and concentration-dependent
decrease in expression of the NADPH oxidase subunit gp91phox (up to
60% inhibition at both the mRNA and protein level). This effect was
prevented by coincubation with the estrogen receptor antagonists
tamoxifen and ICI 182,780 (1 µmol/l each). Within the same
concentration range, E2 also up-regulated endothelial
nitric oxide synthase expression (
twofold). Moreover, preincubation
of the cells with E2 or a gp91phox antisense
oligonucleotide significantly decreased their capacity to generate
O2- on phorbol ester stimulation (i.e.,
assembly of the active NADPH oxidase complex). Blockade of NO synthase
activity, on the other hand, had no effect on phorbol ester-stimulated
O2- formation. In addition, E2
(100 nmol/l) inhibited the increase in adhesion molecule and chemokine
expression in cells exposed to cyclic strain. Cyclic strain enhanced
endothelial O2- formation, thereby offsetting
the inhibitory effect of NO on the expression of these gene products.
E2 thus seems to act as an antioxidant at the genomic level
which by improving the NO/O2- balance
normalizes expression of proatherosclerotic gene products in
endothelial cells.—Wagner, A. H., Schroeter, M. R., Hecker,
M. 17ß-Estradiol inhibition of NADPH oxidase expression in human
endothelial cells.
Key Words: endothelial nitric oxide synthase • estrogen • monocyte chemoattractant protein-1 • CD54 • superoxide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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, 2)
. It has long been suspected that
the level of circulating 17ß-estradiol (E2)
plays an important role in this gender-specific cardioprotective
effect, but the underlying mechanism of action of
E2 is still a matter of debate. Relevant
biological effects of estrogens are numerous and include, for example,
an improvement in lipid and lipoprotein profiles as well as
endothelium-dependent vasodilatation triggered by
E2 at physiological concentrations
(3)
. Moreover, there are recent reports of a significant
estrogen-stimulated increase in the bioavailability of nitric oxide
(NO) (4)
. Thus, E2 is capable of
augmenting endothelial NO release within minutes; this effect is
estrogen receptor (ER) dependent, but independent of changes in gene
expression, and the result of an activation of endothelial nitric oxide
synthase (eNOS) (5
, 6)
. On the other hand,
E2 has been shown to up-regulate eNOS mRNA
expression in fetal pulmonary artery endothelium and in rat and porcine
aortic endothelial cells (7
, 8)
. Because of the
well-documented antiatherogenic properties of NO (9)
, it
has been proposed that the cardioprotective effect of
E2 is based on an increased production of NO by
the endothelium (10)
.
Enhanced superoxide anion
(O2-) formation reduces the
bioavailability of NO in cardiovascular diseases, including
atherosclerosis, hence promoting the NO-sensitive expression of
proatherosclerotic gene products such as adhesion molecules or
chemokines in endothelial cells (11)
. NADPH oxidase is an
important source of O2- in
human endothelial cells (12
, 13)
, and an increasing body
of evidence demonstrates that the activity of this enzyme plays a
critical role in the early phase of atherosclerosis (14)
.
In human endothelial cells, the active enzyme is supposed to be of the
phagocyte type, i.e., a complex consisting of a membrane-bound
cytochrome b558 with two subunits (p22phox and
gp91phox), two cytosolic-activating factors (p47phox and p67phox), and
the small G-protein Rac (14)
.
Using primary cultures of human umbilical vein endothelial cells (HUVEC), we investigated the hypothesis that besides its acute effects on endothelial NO synthesis, the antiatherosclerotic action of E2 is brought about by an altered expression of enzymes responsible for the generation (e.g., NADPH oxidase) or degradation of O2- (e.g., superoxide dismutase), thereby increasing the bioavailability of NO further.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. They were cultured
in medium M199 without phenol red (c.c. pro, Neustadt/W., Germany)
containing 20% fetal bovine serum (Life Technologies, Karlsruhe,
Germany), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 U/ml
nystatin, 5 mmol/l HEPES, 5 mmol/l TES, 1 µg/ml heparin
(Sigma-Aldrich, Deisenhofen, Germany), and 40 µg/ml endothelial cell
growth factor (c.c. pro) on plastic dishes coated with gelatin (2 mg/ml
gelatin in 0.1 M HCl for 30 min at ambient temperature). Moreover,
cells were seeded onto BioFlexTM Collagen type I 6-well plates
(Flexcell, Hillsborough, NC) that had also been coated with gelatin.
HUVEC were identified by positive immunofluorescence for von Willebrand
factor, FACS analysis for platelet endothelial cell adhesion molecule
1, and negative immunofluorescence for smooth muscle -actin.
The human monocytic cell line THP-1 (ATCC TIB 202) was cultured in RPMI
1640 medium (Life Technologies) containing 10% fetal bovine serum and
antibiotics, as described before (15)
. Protein extracts
from cytokine-stimulated THP-1 cells were used as a positive control
for Western blot detection of gp91phox protein.
RT-PCR analysis
Total RNA was isolated from cells grown in 6-well plates
(1.5x106 cells/well) by solid-phase extraction
with the RNeasy kit from Qiagen (Hilden, Germany). Reverse
transcription and polymerase chain reaction for monocyte
chemoattractant protein 1 (MCP-1) and peptide elongation factor (EF-1)
were performed essentially as described previously (15)
.
Amplification of EF-1 cDNA served as an internal standard (housekeeping
gene). The sequences of the other primers used together with the
respective GenBank library accession number of the gene, the position
of the PCR product in the coding sequence, and predicted size are shown
in Table 1
. The identity of the amplification products for gp91phox, p22phox,
p67phox, p47phox, HSP-90, and eNOS was verified by direct sequencing
with a model 373 stretch DNA sequencer (Applied Biosystems,
Weiterstadt, Germany).
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Western blot analysis
Preparation and immunoblot analysis of protein extracts from
endothelial cells were performed as described (16)
. For
analysis of eNOS protein expression, cells were grown to near
confluence in 60 mm diameter Petri dishes (5x106
cells/dish). To detect gp91phox expression, HUVEC and THP-1 cells were
grown in 100 mm diameter Petri dishes (1.5x107
cells/dish), followed by preparation of a subcellular fraction
(microsomes) enriched in membrane proteins (17)
. Protein
extracts (10–30 µg protein per lane) were separated by denaturing
10% polyacrylamide gel electrophoresis in the presence of SDS
according to standard protocols and transferred to a BioTraceTM
polyvinylidene fluoride transfer membrane (Pall Corporation, Rossdorf,
Germany). Transferred proteins were probed by a monoclonal mouse
anti-human eNOS antibody (1:2000 dilution, BD Transduction
Laboratories, Heidelberg, Germany) or a monoclonal anti-gp91phox
antibody (1:500–1:5000 dilution, kindly provided by Dr. Mark
Quinn, Department of Veterinary Molecular Biology, Montana State
University, Bozeman; ref 18
). Visualization of the protein
bands was achieved by using a secondary anti-mouse IgG (whole molecule)
antibody conjugated to horseradish peroxidase (1:3000 dilution;
Sigma-Aldrich) and the SuperSignalTM chemiluminescent substrate (Pierce
Chemical, Rockford, IL), followed by exposure to an autoradiography
film (HyperfilmTM MP, Amersham Pharmacia Biotech, Freiburg, Germany).
Loading and transfer of equal amounts of protein in each lane were
verified by reprobing the membrane with a monoclonal anti-ß-actin
antibody from mouse ascites fluid (1:3000 dilution, Sigma-Aldrich),
followed by densitometry.
Detection of reactive oxygen species (ROS)
Measurement of intracellular ROS formation in human endothelial
cells seeded onto 20 x 20 mm gelatin-coated glass
coverslips was recorded by monitoring changes in
diclorofluorescein (DCF) fluorescence. HUVEC were loaded for 30
min with carboxy-2',7'dichlorodihydrofluorescein-diacetate
(H2DCFDA, 5 µmol/l, Molecular Probes, Leiden,
The Netherlands) in HEPES-Tyrode solution (composition in mmol/l: NaCl
137, KCl 2.7, CaCl2 1.4,
MgCl2 0.25,
NaH2PO4 0.4, Na-HEPES 10,
D-glucose 5) and time-dependent changes in fluorescence intensity were
monitored with a MicroMax CCD camera (Princeton Instruments, Trenton,
NJ) coupled to an Axiovert S100 TV microscope (Zeiss, Goettingen,
Germany) before and after the addition of phorbol dibutyrate (PDB, 1
µmol/l).
O2- formation was determined by
monitoring lucigenin-enhanced chemiluminescence (final concentration of
250 µmol/l) in a Microlumat LB 99P microplate luminometer (Berthold,
Bad Wildbad, Germany) with HUVEC grown in a sterile 96-well multiwell
plate (IsoplateTM, Wallac, Turku, Finnland).
O2- production was stimulated
by the addition of PDB (1 µmol/l) after taking readings for the
background and basal O2-
formation, as described (19)
. The assay was calibrated by
monitoring the chemiluminescence signal of known amounts of
O2- generated by xanthine
oxidase (0.05 U) and xanthine (10–50 µmol/l). It was specific for
O2-; no light emission was
recorded in the presence of authentic NO or hydrogen peroxide.
Antisense oligonucleotide (ODN) treatment
HUVEC were treated with the single-stranded antisense ODN at
40% confluence. Briefly, the antisense ODN was premixed with 200
µg/ml Lipofectin reagent (Qiagen) at the desired concentration (4
µg/well) in medium M199 without heparin and endothelial cell growth
factor at room temperature. Medium supplements were added to the premix
and incubated with the cultured cells for 6 h at 37°C.
Thereafter, the ODN-containing medium was replaced by fresh medium and
the cells were allowed to recover for 14 h before measurement of
O2- formation. The gp91phox
antisense (AS) ODN had the sequence
5'-AACTGGGCTGTGAATGAGG-3', targeting base
pairs 7 to 25 downstream of the translation initiation start
in the coding sequence of gp91phox mRNA (GenBank library
accession No. NM_000397). The scrambled (SCR) control ODN had the
sequence 5'-CATTGTGGAGTGACAGGAG-3' (italic
letters denote phosphorothioate-bonded bases).
Data analysis
Unless indicated otherwise, results are expressed as means ± SE of n observations. One sample t
test, unpaired t test, or one-way analysis of variance,
followed by Bonferroni or Dunnett multiple comparisons test, was used,
where appropriate, to calculate differences between the means or the
means and control, with a P value < 0.05 considered
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-estradiol
or the estrogen receptor antagonists were used (cf. Figs. 4
and 5
c), however, all compounds including
E2 had to be dissolved in DMSO (0.05% (v/v)
final concentration). Moreover, expression of both the - and
ß-estrogen receptor in the cultured HUVEC was verified by RT-PCR
analysis (Fig. 1
).
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Effects of 17ß-estradiol on gp91phox, eNOS, and HSP-90 expression
E2 at physiological concentrations (1–100
nmol/l) caused a time- and concentration-dependent decrease in
expression of the NADPH oxidase subunit gp91phox [up to 60%
inhibition at both the mRNA (maximal after 8 h exposure,
Figs. 2a
, b
) and protein level (after 14 h exposure, Fig. 3a
)]. A significant albeit weaker inhibition at the mRNA
level could also be demonstrated after 8 h exposure to 100 nmol/l
E2 for the expression of p22phox (72±6% of
control, P<0.05, n=6), which together with
gp91phox forms the membrane-bound cytochrome b558 and
that of the cytosolic-activating factor p47phox (71±8% of control,
P<0.05, n=7). Expression of p67phox also seemed
to be attenuated (70±13% of control, n=5), but this effect
did not gain statistical significance. E2 also
did not significantly affect expression of the ROS-metabolizing enzymes
Cu2+/Zn2+-SOD,
Mn2+-SOD, GSH-peroxidase, or catalase (Fig. 2b
). On the other hand, E2 in a
concentration-dependent manner up-regulated expression of heat shock
protein 90 (HSP-90) at the mRNA level (up to twofold after 8 h
exposure, Figs. 2a
, b
) and that of eNOS at both the mRNA
(two- to threefold after 8 h exposure, Fig. 2a
, b
) and
protein level (after 14 h exposure, Fig. 3b
).
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Unlike E2, 17-estradiol (100 nmol/l) had no
effect on either gp91phox or eNOS mRNA expression (Fig. 4a
), whereas preincubation with the estrogen receptor
antagonists ICI 182,780 or tamoxifen (1 h, 1 µmol/l each) prevented
the E2-mediated down-regulation of gp91phox
expression as well as the increase in eNOS expression (Fig. 4b
).
Effects of 17ß-estradiol on ROS formation in phorbol
ester-stimulated HUVEC
Incubation of the cultured HUVEC with the protein kinase C
(PKC) activator PDB (1 µmol/l) resulted in a marked increase in DCF
fluorescence that reached a maximum after 2–5 min (Fig. 5a
). A PDB concentration of 1 µmol/l was found to be optimal
for this effect to occur (19)
and therefore was chosen for
all further experiments. DCF fluorescence (mainly representing the
formation of hydrogen peroxide [dismutation product of
O2-] and possibly also
peroxynitrite [derived from the reaction of NO with
O2-]) was
predominantly located to the perinuclear region (Fig. 5b
).
Neither the NO synthase inhibitor
NG-nitro-L-arginine (L-NNA, 100 µmol/l) nor
sulfaphenazole (10 µmol/l), an inhibitor of cytochrome P450 2C9
thought to generate O2- in
endothelial cells in response to receptor-dependent agonists
(20)
, affected the PDB-stimulated change in DCF
fluorescence (not shown). Preincubation of the cells for 15 h with
E2, but not 17-estradiol (100 nmol/l each),
resulted in an 50% decrease in PDB-stimulated fluorescence, as
shown in Fig. 5c
. Preincubation with the estrogen receptor
antagonists tamoxifen or ICI 182,780 (1 h, 1 µmol/l) prevented the
inhibitory effect of E2 (Fig. 5c
).
A phagocyte-type NADPH oxidase is the source of phorbol
ester-stimulated O2- formation in human
endothelial cells
Incubation of the cultured HUVEC with PDB also resulted in a
prominent increase in O2-
formation (as judged by lucigenin-enhanced chemiluminescence), which
reached a maximum after 4–8 min (Fig. 6a
, b
). In the presence of recombinant bovine
Cu2+/Zn2+-SOD
(PeroxinormTM, 100 U/ml), the PDB-stimulated increase in
O2- formation was inhibited
by >90%, whereas the specific PKC inhibitor Ro 31–8220 (1
µmol/l) inhibited PDB-stimulated
O2- formation by 70% (Fig. 6c
). AEBSF (5 mmol/l) and phenylarsine oxide (PAO, 1
µmol/l), two inhibitors of the assembly of NADPH oxidase in
phagocytes (21
, 22)
, both attenuated PDB-stimulated
O2- formation by >90%
(Fig. 6c
). Neither L-NNA (100 µmol/l) nor sulfaphenazole
(10 µmol/l) significantly affected PDB-stimulated
O2- formation (Fig. 6c
). Diphenyleneiodonium (DPI, 1–100 µmol/l), an
inhibitor of flavoenzymes including NADPH oxidase, on the other hand
inhibited PDB-stimulated O2-
formation in a concentration-dependent manner (Fig. 6d
).
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Effects of 17ß-estradiol and down-regulation of gp91phox protein
expression on PDB-stimulated O2-
formation
A direct scavenging by E2 of xanthine
oxidase-derived O2- (0.01 U
xanthine oxidase and 50 µmol/l xanthine) could not be demonstrated
(Fig. 7a
). Incubation of the cultured HUVEC with
cyclodextrin-encapsulated E2 (100 nmol/l) for
14 h, on the other hand, significantly attenuated PDB-stimulated
O2- formation (up to 70%, Fig. 7b
). To confirm that the aforementioned decrease in gp91phox
protein expression was responsible for this effect, an anti-gp91phox
antisense ODN approach was used. Preincubation of the cultured HUVEC
with the antisense but not a scrambled control ODN for 20 h indeed
resulted in an inhibition of PDB-stimulated
O2- formation (70%)
comparable to the effect of E2 (Fig. 7c
). Note that the smaller rate of
O2- formation determined in the
antisense experiments was due to the lower density of the cultured
HUVEC (40–60% confluence), which is essential for successful
transfection of the cells with the antisense ODN. Western blot analysis
confirmed that the antisense ODN suppressed gp91phox protein expression
by 75% (Fig. 7 c
, inset).
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17ß-Estradiol attenuation of MCP-1 and ICAM-1 expression induced
by cyclic strain
Exposure to E2 (100 nmol/l) for 14 h
also inhibited the increase in MCP-1 and intercellular adhesion
molecule 1 (ICAM-1) expression in HUVEC cultured on BioFlexTM
elastomers and exposed to cyclic strain (3 h, 20% elongation,
Fig. 8a
). Mechanical deformation transiently triggers NADPH
oxidase-dependent O2- formation
in these cells, which appears to offset the inhibitory effect of NO on
expression of these gene products (Fig. 8b
). Moreover, HUVEC
exposed for 6 h to endogenous (100 µmol/l L-NNA) or exogenous
oxidative stress (0.01 U/ml xanthine oxidase and 50 µmol/l xanthine)
also respond with a significant increase in MCP-1 mRNA expression (Fig. 8c
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. Several
reports suggest that this protective effect of estrogen is mediated by
an increase in the local concentration of NO (4
, 23)
through up-regulation of eNOS expression (24)
and/or a
nongenomic but nonetheless estrogen receptor-mediated activation of
eNOS, e.g., via the phosphatidiylinositol-3-kinase/Akt kinase pathway
(6)
. On the other hand, there is mounting evidence that
NADPH oxidase, a major source of ROS, namely
O2-, in endothelial cells
(13
, 25)
, plays a critical role in the development of
atherosclerosis (14)
. By way of peroxynitrite formation,
NO and O2- readily neutralize
each other, thereby reducing the bioavailability of NO
(26)
. The aim of our study was therefore, to examine
whether E2 affects the
NO/O2- balance in endothelial
cells by influencing NADPH oxidase expression (and/or activity) or that
of the main ROS-metabolizing enzymes
Cu2+/Zn2+-SOD,
Mn2+-SOD, catalase or GSH peroxidase.
The main findings of our study were that in human cultured endothelial
cells E2 at physiological concentrations (range:
up to 2.8 nmol/l in nonpregnant premenopausal woman and 70 nmol/l
during pregnancy; ref 27
) significantly inhibited
expression of the NADPH subunit gp91phox whereas that of eNOS, as
predicted, was increased. Because of the decrease in gp91phox
expression (but not the increase in eNOS expression), phorbol
ester-stimulated (i.e., NADPH oxidase-mediated)
O2- formation was significantly
attenuated in estrogen-treated cells, and this effect was mimicked by
blocking gp91phox protein synthesis with an appropriate antisense ODN.
Moreover, results obtained with 17-estradiol, the estrogen receptor
antagonists tamoxifen and ICI 182,780, and a cell free
O2--generating system clearly
show that the antioxidative effect of E2 is
specific, estrogen receptor mediated, and due to neither an
O2--scavenging effect nor a
genomic effect on the major ROS-metabolizing enzymes.
NADPH oxidase as the main source of O2-
Although lucigenin-enhanced chemiluminescence has been
widely used to assess O2-
formation in living cells, it may not be a useful tool under certain
conditions (28)
. This is why we also used DCF fluorescence
analysis to corroborate these results. DCF primarily reacts with
peroxides such as hydrogen peroxide, the product of the spontaneous or
enzymatic dismutation of O2-,
or peroxynitrite, the reaction product of endogenous NO and
O2- (29)
. That
endothelial NADPH oxidase is indeed the source of
O2- under the chosen
experimental conditions was evidenced by the following: 1)
O2- formation was stimulated by
activation of PKC, which facilitates assembly of the active NADPH
complex by phosphorylation of p47phox (30)
; 2)
PKC-dependent O2- formation was
sensitive to AEBSF and PAO, two inhibitors of the assembly of NADPH
oxidase in phagocytes (21
, 22)
, to DPI, a general
flavoenzyme inhibitor that also affects NADPH oxidase activity
(31)
, and to the broad but specific PKC inhibitor RO
31–8220; 3) other potential sources of
O2-, such as the recently
described cytochrome P450 2C9 epoxygenase (20)
or eNOS
itself, could be ruled out by the lack of effect of sulfaphenazole and
L-NNA, respectively.
Critics could say that phorbol ester-stimulated
O2- formation may well be an
index for the capacity of the endothelial cells to generate
O2- through NADPH oxidase, but
that this manner of O2-
formation does not occur in vivo. One must consider the hemodynamic
forces that endothelial cells are exposed to in vivo, especially at
sites in the vascular system, which are prone to develop
atherosclerosis such as the bifurcations of the main conduit arteries.
Here, the endothelial cells are subjected to an enhanced cyclic strain,
which by way of PKC-dependent NADPH oxidase activation (32
this study) may trigger a substantial increase in
O2- formation.
Significance of gp91phox
The NADPH oxidase subunits gp91phox and p22phox in endothelial
cells seem to differ from their plasma membrane-bound counterparts in
polymorphonuclear neutrophils in that they are associated with the
endoplasmic reticulum instead (33)
. Our DCF fluorescence
data tend to corroborate this notion, as peak fluorescence intensities
after phorbol ester stimulation of the cultured HUVEC were
preferentially detected in the perinuclear endoplasmic reticulum-rich
region. Moreover, as the results with the antisense ODN allude to, the
abundance of gp91phox seems to be most critical for the NADPH
oxidase-dependent O2--forming
capacity of human endothelial cells. This notion is reinforced by the
fact that two-thirds of patients with chronic granulomatous disease, a
rare inherited immunodeficiency syndrome, have mutations in the CYBB
gene encoding the gp91phox subunit of the phagocyte NADPH oxidase. As a
result, these phagocytes are characterized by their inability to
produce (sufficient) amounts of ROS to combat pathogenic bacteria
(34)
.
Role of the different estrogen receptors
HSP-90 seems to play an important role in the rapid,
estrogen-induced increase in eNOS activity in endothelial cells
(35)
. It is not clear what the consequences of the
observed up-regulation by E2 of HSP-90 expression
in this context are, but it can be viewed as a typical estrogen
receptor-mediated genomic effect of the sex hormone. Moreover, the
effects of E2 on gp91phox and eNOS expression
could be reversed by the nonselective estrogen receptor (ER) antagonist
ICI 182,780 and the partial antagonist tamoxifen (36)
,
indicating that these potentially atheroprotective effects of the
estrogen (see below) are indeed ER mediated. To date, two estrogen
receptors have been identified, designated ER and ERß; both of
these receptors are expressed in vascular cells (for review, see ref
27
), including human umbilical vein endothelial cells
(37
, 38
; this study). Apparently, detection of estrogen
receptor expression in HUVEC is not a consistent finding (39
, 40)
. However, the clear effects of the ER antagonists in these
cells (6
; this study) further support their existence.
Because neither the cellular localization of the two receptors nor
their sensitivity to the two antagonists has been elucidated with
certainty, it does not seem possible at this time to attribute the
observed genomic effects of E2 on, e.g., gp91phox
and eNOS expression, to stimulation of ER, ERß, or both. Recent
studies using a membrane-impermeable bovine serum
albumin-E2 conjugate
(E2BSA) indicate that
E2-stimulated endothelial NO release might occur
via ER located in plasmalemmal caveolae (6
, 41)
. In a
separate series of experiments, we were indeed able to reproduce
E2-mediated inhibition of gp91phox expression
with this cell-impermeant E2BSA (not shown),
pointing to the involvement of a membrane-bound ER. However, these data
should be interpreted with caution, as it is not clear how much free
E2 is present in the E2BSA
preparation (42)
. There is also the possibility that a
third estrogen receptor, ER
, exists (43)
.
Clinical implications
The known risk factors for coronary heart disease have all been
associated with ‘endothelial dysfunction’ (44)
. Enhanced
oxidative stress may be another factor contributing to this
dysfunction, although controversy exists on this issue
(44)
. However, a recent study with normotensive woman
showed that endothelial dysfunction secondary to acute endogenous
estrogen deprivation is caused by a reduction in the bioavailability of
NO (45)
. Moreover, E2 is capable of
attenuating O2- formation in
phagocytes (46)
and endothelial cells (this study). In
endothelial cells, this effect can occur independent of the
simultaneous stimulatory effect of E2 on eNOS
gene expression (47
; this study).
One consequence of the E2-mediated improvement in
the endothelial NO/O2- balance
is that the expression of proatherosclerotic gene products is reduced
which is controlled by the local concentration of NO. As mentioned
before, a potent stimulus for enhancing endothelial
O2- formation via the PKC-NADPH
oxidase pathway (32
; this study) and, in turn, the
expression of proatherosclerotic adhesion molecules and chemokines
is the overt cyclic strain endothelial cells are exposed to at arterial
bifurcations. In fact, it has been known for some 150 years that
atherosclerosis preferentially develops at these sites
(48)
. The present findings in human cultured endothelial
cells of an E2-mediated decrease in
deformation-induced up-regulation of ICAM-1 and MCP-1 expression, two
gene products whose expression is attenuated by NO (49
, 50)
but enhanced by O2-
(51
, 52
; this study), support the contention that this
effect of the sex hormone is relevant for the pathogenesis of
atherosclerosis.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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and/or ERß mediated.
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ACKNOWLEDGMENTS |
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Received for publication March 30, 2001.
Revision received June 5, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J. Clin. Invest. 103,401-406[Medline]
localized in caveolae. Biochem. Biophys. Res. Commun. 263,257-262[Medline]
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P<0.05 vs. E2).
