Original Contributions |
From the Molecular Cardiology and Tupper Research Institutes (S.K.K., R.H.K., M.E.M.) and the Surgical Research Division (S.K.K.), New England Medical Center, Tufts University School of Medicine, Boston, Mass; the Maine Medical Center (V.L.), Portland, Me; and the Department of Medical Nutrition and Center for Biotechnology (G.G.J.M.K., J.-Å.G.), Karolinska Institute, Uppsala, Sweden.
Correspondence to Michael E. Mendelsohn, MD, Molecular Cardiology Research Center, 750 Washington St, #80, Boston, MA 02111. E-mail michael.mendelsohn{at}es.nemc.org
| Abstract |
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(ER
) is expressed in vascular cells from males and females and may
mediate some of the effects of estrogen on vascular tissue. However, we
recently found that estrogen is able to protect against vascular injury
in ovariectomized female ER
knockout mice. These mice express the
newly described estrogen receptor-ß (ERß) in their aortas,
suggesting that ERß may also mediate some of the direct effects of
estrogen on the vasculature. In this study, the level of expression of
ER
and ERß mRNA in male rat aortas was examined before and after
vascular injury using en face (Häutchen) preparations and in situ
hybridization. Little or no change in ER
expression was observed
after vascular injury in either vascular endothelial or
smooth muscle cells at any time point. In contrast, ERß mRNA was
found to be expressed markedly after balloon injury. In
endothelial cells, ERß was increased by 2 days after
injury, and high levels of expression were maintained at 8 and 14 days.
Furthermore, ERß expression was high in luminal smooth muscle cells
at 8 and 14 days after injury and had decreased to low levels by 28
days after injury. These data demonstrate the presence of ERß in male
vascular tissues and the induction of ERß mRNA expression after
vascular injury, supporting a role for ERß in the direct vascular
effects of estrogen.
Key Words: estrogen receptor vasculature knockout mouse vascular injury endothelium
| Introduction |
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Many of the effects of estrogen on its nonvascular target cells are
mediated through the first ER identified, a ligand-activated
transcription factor now called ER
(reviewed in References 1111 to
13). Very recently, a second ER capable of regulating gene expression,
ERß, was cloned from rat prostate tissue14 as
well as from mice15 and
humans.16 Although some domains of ER
and
ERß are homologous and share functional similarities, new data
suggest differences in their tissue localization and in the mechanisms
regulating their transcriptional
activities.14 15 16 17 ER
is known to be
expressed18 19 20 and
functional19 in vascular smooth muscle cells from
male and female animals and humans, and functional ER
has been
demonstrated recently in vascular endothelial cells as
well.21 22 23 However, ERß expression in vascular
tissues after injury has not yet been studied.
At least 2 direct effects of estrogen on vascular function have been
described: rapid effects on vasomotor tone and longer-term effects on
vascular cell proliferation and atherosclerosis
(reviewed in References 9 and 109 10 ). Physiological
estrogen replacement markedly suppresses the carotid
arterial response to injury to the same degree in female
wild-type and ER
KO mice.24 25 These female
ER
KO mice express mRNA for ERß in their
aortas,25 suggesting that this receptor may
mediate the protective effects of estrogen in the ER
KO mice. In the
present study, we sought to test the hypothesis that male animals
also express ERß and to explore the expression of ER
and ERß
mRNA in male blood vessels after vascular injury.
| Materials and Methods |
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In Situ Hybridization
For en face preparations, vessel segments were cut open
longitudinally, and the tissue was pinned out flat on
polytetrafluoroethylene (Teflon) cards
(luminal side facing up). Incubation with proteinase K (1 µg/mL,
37°C, Boehringer Mannheim Corp) took place for 15 minutes,
followed by prehybridization for 2 hours with 0.3 mol/L NaCl/20
mmol/L Tris (pH 7.5)/5 mmol/L EDTA/1x Denhardt's solution/10%
dextran sulfate/10 mmol/L dithiothreitol/50% formamide. T3 and T7
polymerase (Promega Corp) were used to generate both sense and
antisense strand 35S-UTPlabeled riboprobes. For
ER
, a 195-bp riboprobe corresponding to the 3' end (F domain) of the
published rat sequence31 was generated from
linearized pBluescript containing this B5TX1-EcoR1 fragment of the
receptor. For ERß, a 400-bp riboprobe
(EcoR1-AccI fragment) from the 5' untranslated
region of the rat ERß14 was generated. This
riboprobe has been validated by separate studies on nonvascular tissues
and gives results similar to 2 separate and distinct ERß riboprobes
in testis, prostate, and ovary tissue studies (see Reference 3232 ). For
ER
, 17 separate specimens were hybridized with the antisense probe,
and 5 were hybridized with the sense probe. For ERß, 18 separate
specimens were hybridized with the antisense probe, and 5 were
hybridized with the sense probe. Photographs were taken from 14
different animals, with
8 preparations per aorta. After hybridization
(at 55°C overnight), specimens were washed with 2x SSC/10
mmol/L ß-mercaptoethanol/1 mmol/L EDTA (twice for 10 minutes
each) (1x SSC contains 150 mmol/L NaCl and 15 mmol/L sodium
citrate, pH 7.0), treated with RNase A (20 µg/mL for 30 minutes at
37°C, Sigma Chemical Co), and washed in 2x SSC (as above), followed
by a high-stringency wash at 55°C for 2 hours (0.1x SSC/10
mmol/L ß-mercaptoethanol/1 mmol/L EDTA). Subsequent steps
followed the protocol as described.29 The
Häutchen procedure for en face preparations was carried out after
the probe hybridization. Slides were coated with
autoradiographic emulsion (Kodak, NTB2), exposed for 3
weeks, and then developed (Kodak, D-19). Vessel preparations were
observed under the light microscope using dark-field, bright-field, and
a combination of epiluminescence and bright-field illumination
(reflective light). Estimates of ER
and ERß mRNA were made by
comparison of sense or background signal with those at each of the
various time points and quantified according to an arbitrary scale (see
Table
legend) by an experienced observer (V.L.) blinded
to probe identity and animal treatment.
|
| Results |
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and ERß mRNA Expression in Male Animals After Vascular
Injury
-disrupted (ER
KO) mice express mRNA
for ERß at qualitatively similar levels.25 To
examine the expression of mRNA for ER
and ERß after vascular
injury, the surface cells of uninjured or balloon-injured rat aortas
were studied using the en face (Häutchen)
technique.29 This procedure allows study of the
surface endothelial and intimal smooth muscle cells
migrating into the region as a single monolayer and provides a
sensitive method for studying gene expression in these 2 cell
populations. The en face technique was used to study expression of both
ER
and ERß in uninjured endothelial cells (the
intact and normal vascular lining) as well as in
endothelial cells localized to the advancing
endothelial cell edge at various time points after
injury. Since smooth muscle cells are observed in such preparations
only at 4 to 8 days after injury,29 ER
and
ERß expression in smooth muscle cells in the en face section were
examined and compared at 8, 14, and 28 days after balloon injury. The
Table
and ERß in
endothelial cells and smooth muscle cells in these
experiments. ER
expression by vascular endothelial
cells was very modest in all cases and was barely above background at
all time points examined (Table
mRNA after injury (Table
|
In contrast, ERß was clearly expressed in both vascular
endothelial cells and smooth muscle cells in the aortas
of male rats after injury. For endothelial cells, ERß
mRNA was moderately increased at the leading edge of
endothelial cells by 2 days after balloon injury, and
by 8 days leading edge endothelial cells showed high
levels of expression of ERß mRNA (Table
and Figure 2
). This high level of expression of
ERß in endothelial cells at the leading edge
persisted at day 14 after injury. Expression levels of ERß behind the
leading edge were similar to those seen in normal (uninjured)
endothelium in sections examined (data not shown).
|
Expression of ER
and ERß in luminal smooth muscle cells was also
examined. The en face technique does not allow determination of
baseline gene expression in smooth muscle cells of normal vessels and
allows study only of smooth muscle cells that migrate into the region
of the luminal surface
4 days after vascular injury. This population
of smooth muscle cells thus differs from other intimal or medial smooth
muscle cell populations in the vessel wall. However, once smooth muscle
cells have appeared on the luminal surface (ie, 8 days and 2 and 4
weeks), comparison of ER
and ERß mRNA expression between time
points is possible. After injury, ERß mRNA was moderately abundant in
luminal smooth muscle cells at both 8 and 14 days, and in all cases,
ERß message was significantly greater than that for ER
in these
cells (Table
and Figure 3
). By 28 days,
the level of expression of ERß mRNA in the smooth muscle cell
population had decreased to very low levels compared with those seen at
the 8-day and 2-week time points.
|
| Discussion |
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and ERß after balloon injury of
male rat aortas was strikingly different in these studies. ER
mRNA
was expressed only at very low levels in vascular
endothelial and smooth muscle cells after injury. ERß
mRNA expression, however, increased to high or very high levels in
vascular endothelial cells after injury. In the smooth
muscle cells that appeared at the luminal surface after injury, ERß
mRNA was abundant at both 8 days and 2 weeks but declined to near
baseline levels by 28 days after injury.
Estrogen inhibits the response to vascular injury in a variety of
animal models.24 25 33 34 35 36 37 38 39 40 41 In particular, rat
vascular tissues express functional estrogen,42
and estradiol inhibits myointimal proliferation after carotid balloon
injury in gonadectomized male rats,39 40 the
animal studied in these experiments. The high levels of ERß
expression in vascular cells of injured male vessels in these studies,
without significant changes in ER
expression, are striking and raise
the possibility that ERß may mediate some of the protective effects
of estrogen in the setting of vascular injury. This hypothesis is
consistent with the presence of ERß in the aortas of ER
KO
mice and the ability of estradiol to inhibit the vascular response to
injury in these animals to the same degree as in their wild-type
littermates.25
Upstream regulatory regions of the ERß gene important to the
observed induction of ERß message after vascular injury remain to be
defined, and the potential growth factors or other stimuli induced by
balloon injury that lead to increased ERß expression also are
presently unclear. However, the observed increase in mRNA for ERß
after balloon injury suggests that study of ERß may be important in
the understanding of the direct vascular protective effects of
estrogen. It will be important in subsequent studies to define
carefully the relative abundance of ERß mRNA and protein in vascular
cells and tissues from intact and ovariectomized female animals after
injury. Furthermore, careful analysis of ER
and ERß mRNA
protein in vascular cells and tissues under various conditions will be
important, including human cells and tissues from men, premenopausal
women, and postmenopausal women, both untreated and on hormone
replacement therapy. It will be especially important to examine cell
culture expression of the ERs and to correlate in vitro with in vivo
expression. Preliminary RT-PCR studies indicate that ERß is expressed
in male and female vascular cells (data not shown), but it is already
apparent that expression levels of ERß mRNA, like those of ER
,
vary with culture conditions, passage, and vascular bed of origin (see
Reference 2323 ). It will also be important to understand regional
differences in the vascular expression of ERß in vivo and to examine
specifically the role of ERß in mediating the vascular protective
effects of estrogen. The recent development of ERß-specific
antagonists and the ERß knockout mouse will allow this
latter topic to be studied directly.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received December 29, 1997; accepted April 16, 1998.
| References |
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and ß. Endocrinology. 1997;138:863870.
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