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1 Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697-4625; and 2 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Estrogen alters reactivity of cerebral arteries by modifying
production of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype is
unknown. ER-
knockout (ERKO) mice were used to test whether estrogen acts via ER-. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 mo
later. Estrogen increased levels of endothelial nitric oxide synthase
and cyclooxygenase-1 in vessels from wild-type mice but was ineffective
in ERKO mice. Endothelium-denuded middle cerebral artery segments
from all animals constricted when pressurized. In denuded arteries from
ERKO but not wild-type mice, estrogen treatment enhanced
constriction. In endothelium-intact, pressurized arteries from
wild-type estrogen-treated mice, diameters were larger compared with
arteries from untreated wild-type mice. In addition, contractile
responses to indomethacin were greater in arteries from wild-type
estrogen-treated mice compared with arteries from untreated wild-type
mice. In contrast, estrogen treatment of ERKO mice had no effect on
diameter or indomethacin responses of endothelium-intact arteries. Thus
ER- regulation of endothelial nitric oxide synthase and
cyclooxygenase-1 pathways appears to contribute to effects of estrogen
on cerebral artery reactivity.
cerebral circulation; estrogen receptor- knockout mice; nitric
oxide synthase; gonadal steroids; ovariectomy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS CLEARLY ESTABLISHED that in vivo exposure to estrogen affects blood vessel reactivity (5, 6, 9, 27, 29). In the cerebral circulation of rats and mice, a major target for estrogen action appears to be the vascular endothelium, with increased activity of endothelial-derived vasodilator factors in animals treated with estrogen. Foremost among the endothelial substances affected by estrogen treatment is nitric oxide (NO). Long-term estrogen treatment enhances NO-dependent dilation and increases levels of endothelial NO synthase (eNOS) in cerebral arteries (5, 6, 21, 27). Recently, we showed that in vivo treatment with estrogen also increases the function of a cyclooxygenase-1 (COX-1)-dependent vasodilatory substance in isolated mouse cerebral arteries (6). This COX-dependent factor, which is endothelium derived, appears to provide compensatory vasodilation following inhibition of NO synthesis, and this effect is enhanced with estrogen treatment. Thus estrogen modulates the production of endothelium-derived prostaglandins as well as NO.
Effects of long-term estrogen treatment are mediated through receptor signaling pathways that result in modulation of gene expression (2, 13, 15, 24). This process is thought to involve entry of estrogen into the cell, translocation to the nucleus, binding to a nuclear estrogen receptor (ER), dimerization, binding of the dimer to target genes, and transcriptional regulation (2, 24). Estrogen binding to an ER appears to be an essential step for many of the vascular effects of this hormone (4). For example, the ER antagonist ICI-182780 blocks estrogen-dependent vasoprotection, resulting in increased neointimal formation of balloon-injured rat carotid arteries (1). In cultured fetal pulmonary artery endothelial cells, ER antagonism with ICI-182780 completely inhibits the effect of estrogen on eNOS activity and transcription (18). ICI-182780 treatment also blocks estrogen-sensitive effects on eNOS activity and protein expression in cultured human umbilical vein endothelial cells (7).
Two ER subtypes are present in vascular smooth muscle and endothelial
cells: the classic ER- and the more recently discovered ER-
(8, 10, 12, 14, 17, 25). Available ER antagonists such as
ICI-182780, however, do not discriminate between the two ER subtypes.
Generation of mice that are homozygous for either ER- or ER- gene
mutation (2) provides another approach for defining the
function of ER subtypes in vascular regulation. Interestingly, recent
findings from studies using ER knockout mice ( or )
indicate that ER- and ER- may play redundant roles in mediating
vascular protective effects of estrogen (1, 10, 14). In
wild-type mice after carotid arterial injury, vascular medial area
increases, and there is proliferation of smooth muscle cells. These
effects are inhibited by estrogen. This effect of estrogen on the
response to carotid arterial injury persists in either ER- or ER-
knockout mice. Thus expression of either ER subtype appears adequate to mediate these effects of estrogen on vascular injury.
At this point, it is not known which ER subtype mediates the effects of
estrogen on cerebrovascular reactivity. As noted above, estrogen
modulates reactivity of pressurized resistance-sized mouse cerebral
arteries through NOS- and COX-dependent mechanisms. Therefore, in the
present study, we used mice deficient in ER- (ERKO, ER-
knockout) to examine the hypothesis that estrogen modulates cerebral
artery diameter through ER--dependent mechanisms. Female mice
(ERKO and wild-type littermates) were either ovariectomized (OVX) or
ovariectomized with estrogen replacement (OVX+E). One month after
surgery, cerebral blood vessels from wild-type and ERKO mice were
isolated, and levels of eNOS and COX-1 were measured. Contractile tone
of isolated, pressurized middle cerebral arteries with and without
intact endothelium was also assessed. Comparison of OVX animals with
and without estrogen treatment enabled us to test whether actions of
estrogen depend on the presence of functional ER-.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
Animal procedures were approved by the Animal Care and Use Committee of
the University of California, Irvine. Female wild-type littermate
control and ERKO mice were supplied by the National Institutes of
Environmental Health Sciences (Research Triangle Park, NC)
(16). Mice were housed under a 12:12-h light-dark cycle
with food and water available ad libitum. Four groups of OVX mice were
used in the present study: wild-type OVX (n = 9), wild-type OVX+E (n = 9), ERKO OVX (n = 9), and ERKO OVX+E (n = 9). Ovariectomy and
ovariectomy with estrogen replacement were performed under anesthesia
(90 mg/kg ketamine and 10 mg/kg xylazine). Estrogen was replaced at the
time of ovariectomy by subcutaneous insertion of a 1-mm silicone
elastomer capsule made from Dow Corning Silastic medical grade tubing
(1.57 mm ID × 3.18 mm OD), sealed with silicone elastomer
adhesive type A (Dow Corning) and packed with 17-estradiol. Both OVX
and OVX+E animals were euthanized 1 mo after surgery.
Tissue preparation.
Mice were euthanized in the middle of the day by exposure to
CO2. The uterus was removed from each animal and weighed.
Brains were rapidly removed from the cranial cavity and placed in a
dissecting dish with cold oxygenated physiological salt solution (PSS)
containing (in mM) 118 NaCl, 4.8 KCl, 1.6 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, and 11.5 glucose. A 1- to 2-mm segment of the middle
cerebral artery, taken ~1 mm from the circle of Willis, was carefully
dissected for measurement of functional responses and immediately
placed in PSS (see Functional responses below). The
brain was then frozen at 
20°C until used for whole brain blood
vessel isolation and Western blot analysis.
Western blot analysis.
Levels of eNOS protein in isolated brain blood vessels were measured as
previously described (21). Briefly, two mouse brains were
pooled and homogenized gently with a Dounce tissue grinder. Blood
vessels were isolated by centrifugation through a dextran gradient
(15%, molecular weight of 38,400) and collected on a 50-µm nylon
mesh screen. The vessel preparation, as confirmed by light microscopy,
was a heterogeneous mixture of large arteries and veins, arterioles,
venules, and capillaries. The isolated vessels were glass homogenized
in a lysis buffer consisting of 50 mM -glycerophosphate, 100 µM
sodium orthovanadate, 2 mM magnesium chloride, 1 mM EGTA, 0.5% Triton
X-100, 1 mM DL-dithiothreitol, 20 µM pepstatin, 20 µM
leupeptin, 0.1 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride,
followed by incubation on ice for 20 min. Samples were centrifuged at
180 g (25 min at 4°C), and the supernatant was taken for
protein determination and analysis by SDS-PAGE/Western blot.
Functional responses. Middle cerebral artery segments were mounted in an arteriograph (Living Systems, Burlington, VT). Micropipettes were inserted into each end of the artery and secured in place with nylon ties. The proximal cannula was connected through a pressure transducer and windkessel to a reservoir of PSS equilibrated with 95% O2-5% CO2. The distal cannula was connected to a luer lock that was open during the initial equilibration to gently flush the luminal contents. After the equilibration period, the luer lock remained closed so that all experiments were conducted under no-flow conditions. A constant-flow peristaltic pump continuously superfused (30 ml/min) the artery with PSS. During a 60-min equilibration period, a pressure servo system maintained transmural pressure at 40 mmHg. The artery was viewed with an inverted microscope equipped with a video camera and monitor. A videoelectronic dimension analyzer was used to measure luminal diameter and wall thickness, defined as the distance from the inside arterial edge to the outside edge. Changes in transmural pressure and lumen diameter were digitized by a MacLab analog-to-digital converter and recorded on a Macintosh computer. All drugs, individually or in combination, were added to the superfusate in their final concentration.
In all protocols, artery diameters at various transmural pressures (no flow) were measured. After the 60-min equilibration period, one of the following two protocols was performed. In each protocol, four separate series of pressure steps (each from 40 to 80 mmHg in 10-mmHg steps) were performed. In protocol 1, the first series of pressure steps was in PSS, the second in the presence of NG-nitro-L-arginine-methyl ester (L-NAME; 100 µM), the third in the presence of indomethacin (10 µM) plus L-NAME, and the fourth in zero Ca2+-EDTA (3 mM). In protocol 2, the first series of pressure steps was with endothelium-intact arteries in PSS, the second in the presence of L-NAME plus indomethacin, the third series followed endothelium removal in PSS with L-NAME and indomethacin, and the fourth was with endothelium-denuded arteries in zero Ca2+-EDTA (3 mM). Endothelium removal was accomplished by perfusing 1 ml of air through the artery lumen. All drugs were perfused for 20 min before the first pressure step, and each pressure step was maintained for 5-10 min to allow the vessel to reach a stable condition before the diameter was measured. Control arteries showed consistent responses to four sequential series of pressure steps. All drugs were purchased from Sigma Chemical (St. Louis, MO). Data are expressed as means ± SE. Statistical significance was determined using ANOVA with Scheffé's test. Acceptable level of significance was defined as P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Body and uterine weights.
Body weights and uterine weights for the various groups of animals
studied are shown in Table 1. One month
of estrogen treatment significantly increased the uterine weights of
wild-type OVX mice, but there was no effect of estrogen on uteri of
ERKO OVX mice. Body weights of ERKO OVX+E mice were significantly
greater than ERKO OVX, wild-type OVX, and wild-type OVX+E mice.
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Effect of estrogen on levels of eNOS and COX-1.
Our laboratory has previously demonstrated that estrogen treatment
increases levels of eNOS and COX-1 in rat cerebral arteries (21,
23). Therefore, we determined whether estrogen modulates levels
of either eNOS or COX-1 in cerebral vessels from the mouse by using
Western blot analysis. Estrogen treatment of wild-type OVX mice
resulted in a 3.8-fold increase in levels of eNOS (Fig. 1A) and 2.6-fold increase in
levels of COX-1 (Fig. 1B) compared with levels in cerebral
vessels from wild-type OVX mice. In contrast, estrogen had no
significant effect on either eNOS or COX-1 levels in arteries from
ERKO OVX mice (Fig. 1).
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Smooth muscle tone. Isolated, pressurized cerebral arteries of the mouse show significant smooth muscle tone, which is modulated by production of endothelial factors (6). Therefore, we first investigated whether ERs might modulate vascular smooth muscle cell tone in arteries denuded of endothelium. To confirm damage to the endothelium, arteries were preconstricted with Ba2+ (10 µM) and tetraethylammonium chloride (1 mM). Under these conditions, in intact arteries, addition of the endothelium-dependent dilator ADP (100 µM) increased the vessel diameter in arteries from all four groups of animals. After endothelial damage occurred, dilation to ADP was virtually eliminated in arteries from all animal groups (n = 6).
Endothelium-denuded arteries from OVX and OVX+E wild-type andERKO
mice were exposed to multiple pressure steps (Fig.
2) in PSS and in the presence of EDTA and
zero Ca2+ (passive response). Maximum passive diameters,
measured in the absence of Ca2+ at 80 mmHg, were not
significantly different among arteries from any of the four groups of
animals: wild-type OVX (160 ± 1.4 µm), wild-type OVX+E
(155 ± 1.2 µm), ERKO OVX (157 ± 1.7 µm), and ERKO
OVX+E (160 ± 1.9 µm) (P > 0.05, ANOVA) (Fig.
2). In PSS with Ca2+, arteries without endothelium from all
groups of wild-type and ERKO mice were substantially constricted
(Fig. 2). In wild-type OVX and OVX+E mice, diameters were similar
following endothelium removal, suggesting that estrogen treatment does
not affect vascular smooth muscle tone (Fig. 2, A and
B). In contrast to the situation in wild-type mice, there
was significantly enhanced smooth muscle tone in arteries from ERKO
OVX+E mice compared with ERKO OVX mice (Fig. 2, C and
D). For example, at 80 mmHg in endothelium-denuded arteries,
diameters were 82 ± 7 and 63 ± 3 µm for ERKO OVX and ERKO OVX+E mice, respectively. This suggests that, when ER- is
not functional, an additional effect of estrogen on smooth muscle tone
is manifest.
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Vascular tone in endothelium-intact arteries.
In arteries from all mouse groups, the presence of endothelium markedly
attenuated contractile tone in PSS (Fig.
3) compared with responses of denuded
arteries (Fig. 2). There were no significant differences in passive
diameters of the intact arteries among the four animal groups.
Endothelium-intact arteries from wild-type OVX mice, however, had
significantly smaller diameters in PSS at all levels of pressure
compared with their passive responses (Fig. 3A). After
estrogen treatment (OVX+E), intact arteries from wild-type mice
exhibited even larger diameters in PSS; in this case, the diameters
were not significantly different from passive diameters at any level of
intraluminal pressure (Fig. 3B). Thus, as reported
previously by our laboratory (6), in wild-type OVX mice,
constrictor tone of pressurized arteries is reduced by prior estrogen
treatment when the endothelium is intact. Middle cerebral arteries from
ovary-intact female wild-type mice also show no significant
constriction to pressure when the endothelium is intact
(6); therefore, the effect of OVX to increase constriction is reversed by estrogen treatment.
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ERKO mice. Arteries isolated from ERKO OVX and
ERKO OVX+E mice had similar diameters in PSS, and these diameters
were not significantly different from their respective passive
diameters (Fig. 3). Preliminary studies with pressurized cerebral
arteries from ovary-intact ERKO mice indicate that they have more
tone than either of the ERKO OVX groups. For example, passive
diameters and diameters in PSS of arteries (60 mmHg) from ovary-intact
ERKO mice were 150 ± 1.9 and 145 ± 1.8 µm,
respectively (P < 0.05; n = 5). In
comparison, passive diameters and diameters in PSS of arteries (60 mmHg) from ERKO OVX mice were 153 ± 1.4 and 152 ± 1.8 µm, respectively. Thus, in contrast to wild-type mice, ovariectomy in
ERKO mice appears to attenuate cerebral artery constriction;
however, the effect of ovariectomy is not reversed by estrogen replacement.
Response to inhibition of COX.
Our previous studies of mouse cerebral arteries suggest that COX is a
major target of estrogen action (6). This effect is
apparent following inhibition of NOS with L-NAME. Indeed,
in arteries from wild-type mice, treatment with estrogen had a major effect on diameter responses to indomethacin in the presence of L-NAME (Fig. 4). In arteries
from wild-type OVX mice, addition of indomethacin to
L-NAME-treated arteries did not significantly affect
diameter at any pressure compared with L-NAME alone (Fig. 4A). However, in arteries from wild-type OVX+E animals,
indomethacin treatment significantly decreased diameter compared with
diameters in L-NAME alone (Fig. 4B;
P < 0.05, ANOVA).
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ERKO OVX and OVX+E mice were then compared to
determine whether the effect of estrogen treatment on COX pathways is
dependent on ER-. Addition of indomethacin to ERKO OVX and OVX+E
arteries pretreated with L-NAME caused diameters to
significantly decrease at all pressures compared with
L-NAME alone. Thus, in arteries from ERKO mice, there
was no effect of estrogen treatment on the degree of constriction
caused by addition of indomethacin in the presence of
L-NAME.
Effect of estrogen on artery wall thickness.
We also explored the possibility that estrogen treatment affects blood
vessel wall structure. At 80 mmHg in zero Ca2+ plus EDTA,
artery wall thickness was not significantly different between wild-type
OVX and OVX+E or between ERKO OVX and ERKO OVX+E mice (Fig.
5). Thus treatment with estrogen did not
affect artery wall thickness in either animal group, that is, in either the wild-type or ERKO mice.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our laboratory (6) has demonstrated previously that
estrogen treatment in vivo increases endothelium-dependent vasodilator mechanisms in mouse cerebral arteries. We now report that treatment with estrogen for 1 mo also elevates levels of enzymes responsible for
synthesis of vasodilators, eNOS and COX-1, in cerebral arteries. Furthermore, we confirm that estrogen treatment of wild-type OVX mice
upregulates vasodilator activity, dependent on an intact endothelium.
In this study, we used ERKO mice to assess whether the ER-
subtype mediates these cerebrovascular effects of estrogen. In contrast
to results in wild-type mice, estrogen treatment of ERKO mice had no
significant effect on levels of eNOS and COX-1 or vascular reactivity
in cerebral arteries. These data suggest that the effects of estrogen
on cerebral arteries from OVX mice depend on functional ER-.
The current study extends our previous findings (6) by demonstrating that estrogen treatment increases levels of eNOS and COX-1 in cerebral vessels from wild-type OVX mice, as well as increasing endothelial-dependent vasodilation. As previously demonstrated in our laboratory, contractile tone of pressurized cerebral arteries in vitro is clearly modulated by simultaneous release of endothelium-derived factors. When endothelium is absent, the degree of vasoconstriction in pressurized arteries is substantially greater than that found in arteries with intact endothelium. Thus the true smooth muscle tone can only be observed when endothelial factors are eliminated. When the endothelium is intact, vascular tone of pressurized arteries reflects the net effect of smooth muscle-derived contractile tone and the activity of released endothelial factors.
Our laboratory (6) has previously shown that ovariectomy in wild-type mice results in more constricted diameters of isolated, endothelium-intact cerebral arteries, an effect that is reversed by in vivo estrogen treatment. The present study confirms the ability of estrogen treatment to enhance endothelium-dependent vasodilation in cerebral arteries from wild-type OVX mice. We also demonstrate in wild-type mice that changes in diameter after inhibition of COX (in the presence of a NOS inhibitor) are increased by estrogen treatment. Thus, in wild-type mice, estrogen treatment enhances the vasodilator function of the endothelium and increases levels of eNOS and COX-1.
Mechanism of estrogen effect. Estrogen-mediated increases in protein levels and endothelium-dependent vasodilation imply that eNOS and COX-1 protein stability and/or gene transcriptional mechanisms are targeted by estrogen treatment (15). Although our current study is unable to distinguish among these possible mechanisms to explain the eNOS and COX-1 protein data, published reports have suggested that estrogen does affect the expression of both eNOS and COX-1 in cultured endothelial cells. A study of human endothelial cells found that the effect of estrogen on eNOS is not a consequence of changes in eNOS mRNA stability but is due to increased eNOS transcription (15). In the COX-1 pathway, estrogen has been demonstrated to enhance PGI2 production though tamoxifen-sensitive mechanisms in primary cell cultures from human umbilical vein (22). Similar effects of estrogen on PGI2 release, COX-1 protein, and COX-1 mRNA expression were found in cultured fetal pulmonary artery endothelial cells (13). Thus there is convincing evidence that estrogen can affect genomic regulation of both eNOS and COX-1.
Estrogen and reactivity of cerebral arteries in ERKO mice.
In marked contrast to our findings in wild-type mice, estrogen
treatment had absolutely no effect on levels of eNOS or COX-1 or
endothelium-dependent vasodilation in arteries from ERKO OVX mice.
Contractile responses to an inhibitor of COX were also identical in
arteries from ERKO OVX and ERKO OVX+E mice. This lack of effect
of estrogen on reactivity of cerebral arteries in ERKO mice strongly
suggests that estrogen modulates endothelial-dependent vasodilatory
pathways through ER--mediated mechanisms.
subtype
plays an important role in endothelium- and NOS-dependent modulation of
vascular reactivity. Deletion of ER- was shown to affect
NOS-sensitive modulation of vascular tone in isolated mouse aorta
(25). In ERKO mice, endothelium-dependent constriction to inhibition of NOS was significantly decreased compared with wild-type mice. In a study on mouse hearts subjected to global ischemia and reperfusion (31), nitrite production
and coronary flow rate were also decreased in hearts from ERKO mice
compared with wild-type animals. A direct association of ER- with
upregulation of eNOS gene expression was demonstrated in a study of
bovine aortic endothelial cells transfected with the ER- gene
(28). ER- gene transfection increased eNOS mRNA and
protein levels as well as NOS activity, and this effect was sensitive
to inhibition by ICI-182780. Interestingly, in this system, addition of
estrogen had no further effect on eNOS, suggesting that the ER-
transfected into the cultured cells was constitutively active in the
absence of ligand.
In contrast to the above studies and our current findings, several
studies have concluded that ER- deletion has no effect on
cardiovascular function, particularly in models of vascular injury. For
example, the effect of experimental ischemic stroke on infarct
volume and cerebral blood flow was compared in brains from female
control and ERKO mice (26). In that study, infarct volume was not affected by ER- deletion. Furthermore, ER-
deletion did not reduce regional blood flow in either ischemic
or nonischemic areas. In another study, control and ERKO
mice were used to explore the effect of estrogen on carotid artery
responses to injury (10). 17-Estradiol significantly
inhibited all measures of smooth muscle growth after mechanical injury
in both control and ER-deficient OVX mice. Clearly, these studies
suggest that estrogen can prevent vascular injury by a mechanism
independent of the ER-. Thus, at this point, it is not entirely
clear which ER may be responsible for each of the diverse vascular
effects of estrogen.
One aspect of our findings that merits comment is that vascular
reactivity and eNOS and COX-1 levels from ERKO OVX mice resembled those found in wild-type OVX+E mice. Of course, there is no particular reason to believe that cerebral blood vessels in mice lacking ER-
throughout development should necessarily resemble vessels from
wild-type mice. It should not be surprising that there would be
substantial differences in baseline function of vessels from these two
groups of animals. Regardless of the mechanism, however, it is remotely
possible that upregulation of eNOS and COX-1 in arteries of ERKO
mice does not allow us to observe a further effect of estrogen. If
there were such a ceiling effect, however, it would seem surprising to
find it in two separate systems, both COX-1 and eNOS.
As to the mechanism for this variation in baseline function in arteries
of ERKO mice, our unpublished data strongly suggest that ovarian
overproduction of testosterone in ERKO mice (2) may affect cerebral arterial responses. In the present study, all mice
were ovariectomized. However, recent preliminary data suggest that
endothelium-intact cerebral arteries from ovary-intact ERKO mice are
more constricted in vitro compared with arteries from ERKO OVX mice
(Geary, Krause, and Duckles, unpublished observations). Thus changes in
vascular reactivity after ovariectomy of ERKO mice may reflect a
loss of testosterone. Indeed, we have previously found that the loss of
testosterone in orchiectomized male rats significantly reduces vascular
reactivity through enhanced endothelium-dependent vasodilatory
mechanisms (5). Other studies have shown that genetic
females taking high-dose androgens have impaired flow-mediated dilation, suggesting abnormal endothelial function (20).
Future studies may help to determine whether unopposed testosterone
affects cerebral artery reactivity in ERKO mice.
Estrogen sensitivity of other responses.
Our studies clearly show that, in ERKO OVX mice, estrogen has no
effect on levels of eNOS and COX-1 and no effect on the response to
pressure of endothelium-intact cerebral arteries. However, we did
detect two effects of estrogen in ERKO mice. First, body weights of
ERKO OVX+E mice were significantly greater than those shown in the
other groups. This is similar to the findings of Couse and Korach
(2), which showed that body weights of ovary-intact
ERKO mice were significantly greater than wild-type mice.
ERKO OVX+E compared with untreated ERKO
OVX mice. This is in contrast to our current findings with wild-type
mice as well as with the previous study from our laboratory of
wild-type mice, in which all effects of estrogen on cerebral arteries
were abolished after endothelial damage (6). Although we
do not know how estrogen may have enhanced smooth muscle tone, the
density of L-type Ca2+ channels has been shown to be
greater in cardiac ventricular myocytes from male ERKO mice compared
with myocytes from wild-type controls (11). Because smooth
muscle tone of cerebral arteries may depend on influx of
Ca2+ through L-type channels (3), estrogen
treatment of ERKO OVX mice might have affected the density of
vascular smooth muscle Ca2+ channels through an
ER--independent pathway. Alternatively, estrogen treatment may have
influenced other basic components of vascular smooth muscle cell
function in ERKO mice.
Estrogen and cerebrovascular disease.
The physiological implications of the effects of estrogen on the
cerebral circulation remain to be determined. It is well known that
women experience fewer cerebrovascular incidents than men of similar
age (30). Estrogen may provide long-term protection against ischemic stroke through endothelium-dependent
mechanisms designed to maintain cerebrovascular reserve capacity
(19). Thus the current findings begin to elucidate the ER
transduction pathway, which may affect the incidence and severity of
cerebrovascular disease. Interestingly, expression of ER- was found
to be lower in atherosclerotic coronary arteries compared with normal
vessels from premenopausal women (17), suggesting that
ER- deficiency plays a role in vascular pathology. Our results point
to the importance of ER- in vasodilator mechanisms in the cerebral circulation.
ERKO mice. Developmental processes or ovariectomy of ERKO mice may have altered
endothelial-dependent reactivity through yet to be identified
mechanisms. Nevertheless, our findings support the hypothesis that the
action of estrogen to modulate endothelial vasodilator mechanisms in
cerebral arteries is mediated by ER-.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-50775.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. P. Duckles, Dept. of Pharmacology, College of Medicine, Univ. of California, Irvine, Irvine, CA 92697-4625 (E-mail: spduckle{at}uci.edu).
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. Section 1734 solely to indicate this fact.
Received 27 April 2001; accepted in final form 30 July 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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