Adult Tissue Angiogenesis: Evidence for Negative Regulation by Estrogen in the Uterus
W. Ma1,
J. Tan1,
H. Matsumoto,
B. Robert,
D. R. Abrahamson,
S. K. Das and
S. K. Dey
Departments of Molecular and Integrative Physiology (W.M., H.M.,
S.K.Dey), Anatomy and Cell Biology (B.R., D.R.A.), and Obstetrics and
Gynecology (J.T., S.K.Das), Ralph L. Smith Research Center, University
of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: S. K. Dey, Ph.D., Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu
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ABSTRACT
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Increased uterine vascular permeability and angiogenesis are
two major events of embryo implantation and placentation during
pregnancy. These latter processes require coordinated, uterine-specific
interactions between progesterone (P4) and estrogen (E)
signaling. Although roles of these steroids have long been suspected,
definitive functions of E and/or P4 in uterine angiogenesis
still remain elusive. We have therefore exploited the availability of
reporter and mutant mice to explore the regulation of
angiogenesis in response to steroid hormonal changes in
vivo. We present here molecular, genetic, physiological, and
pharmacological evidence that E and P4 have different
effects in vivo: E promotes uterine vascular
permeability but profoundly inhibits angiogenesis, whereas
P4 stimulates angiogenesis with little effect on vascular
permeability. These effects of E and P4 are mediated by
differential spatiotemporal expression of proangiogenic factors
in the uterus.
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INTRODUCTION
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UNDER PHYSIOLOGICAL CONDITIONS,
angiogenesis, the process by which new blood vessels originate
from preexisting vessels, primarily occurs in the uterus and ovary in
the adult during the reproductive cycle and pregnancy (1).
Indeed, increased vascular permeability and angiogenesis are essential
to successful implantation and placentation. Several studies have
provided indirect and descriptive evidence for the potential roles of
estrogen (E) and progesterone (P4) in these
processes in various species (reviewed in Refs. 2, 3, 4). As
described below, these studies primarily examined the changes in the
whole uterus and the expression of a number of gene products known to
control vascular permeability and angiogenesis, including vascular
endothelial growth factor (VEGF) and its receptors, without
investigating its angiogenic status. Thus, in vivo roles for
E and P4 in uterine angiogenesis are still
unclear. In this study, we have used a combination of molecular,
genetic, physiological, and pharmacological approaches to address this
question.
VEGF, originally discovered as a vascular permeability factor (reviewed
in Ref. 5), is also a potent mitogen for endothelial cells
and a key regulatory growth factor for vasculogenesis and angiogenesis
(6). Targeted disruption of even one allele of the
Vegf gene results in embryonic death in utero on
d 10.5 with aberrant blood vessel formation (7, 8).
Differential splicing of the Vegf gene generates several
VEGF isoforms in both humans and mice;
VEGF121 and VEGF165 are the
predominant isoforms in humans, whereas isoforms
VEGF120 and VEGF164, which
are shorter by one amino acid, are most abundant in mice (4, 9).
VEGF effects are primarily mediated by two tyrosine kinase receptors:
FLT1 (VEGF receptor 1) and FLK1/KDR (VEGF receptor 2)
(10, 11, 12, 13). Although FLT1 activation does not stimulate
endothelial cell mitosis, targeted disruption of the Flt1
gene produces impaired endothelial cell assembly into blood vessels and
embryonic lethality (14). FLK1 is the major transducer of
VEGF signals that induce chemotaxis, actin reorganization, and
proliferation of endothelial cells (6, 15, 16). Targeted
deletion of the Flk1 gene in mice produces defects in
hematopoietic and endothelial cell development leading to embryonic
death by d 9.5 (17).
Recently, another multifunctional VEGF receptor has been
identified as neuropilin-1 (NRP1). NRP1 was originally described as a
neuronal transmembrane receptor that participates in axonal guidance in
the developing nervous system (18, 19) and is a receptor
for the collapsin/semaphorin family of proteins (20, 21).
It is now known that NRP1 functions as a receptor for at least five
different ligands, collapsin-1/semaphorin-IIII/D, semaphorin-E,
semaphorin-IV, VEGF165, and placental growth
factor, which are involved in different biological processes such as
nervous system development, vasculogenesis, and angiogenesis (21, 22). NRP1 is expressed in human endothelial cells as a
VEGF165-specific receptor. When coexpressed in
endothelial cells with FLK1, NRP1 enhances the binding of
VEGF165 to FLK1 and
VEGF165-mediated chemotaxis severalfolds higher
than that of FLK1 alone (23). Conversely, inhibition of
VEGF165 binding to NRP1 inhibits its binding to
FLK1 and its mitogenic activity for endothelial cells.
Nrp1-deficient mice show peripheral nervous system
abnormalities and die in midgestation due to yolk sac vascular
insufficiency and developmental anomalies of the cardiovascular system
(24). Mice overexpressing NRP1 also show cardiovascular
abnormalities including increased number of blood vessels and abnormal
hearts (25).
We have recently shown that the genes encoding murine VEGF isoforms and
their receptors, FLT1, FLK1, and NRP1, are differentially expressed in
the mouse uterus in a spatiotemporal manner during implantation, and
that the predominant VEGF164 isoform interacts
with FLK1 and NRP1 (2, 4). These results provide evidence
that the VEGF system is important for uterine vascular permeability and
angiogenesis during implantation. Others have also shown the expression
of VEGF and its receptors in the uterus as a whole during pregnancy and
in response to steroid hormones (reviewed in Ref. 3). For
example, E rapidly induces uterine vascular permeability and
Vegf expression transcriptionally via nuclear ER (reviewed
in Refs. 3 and 26). In addition, the
Vegf gene contains E response elements (26).
P4 also up-regulates uterine Vegf
expression, but at a slower rate, via activation of its nuclear
receptor, PR (26). Because E rapidly stimulates uterine
vascular permeability and Vegf expression, and because
vascular permeability is considered a prerequisite for
angiogenesis, it is widely believed that E is a potent stimulator of
uterine angiogenesis during normal reproductive processes in
vivo. However, there is no experimental evidence to support this
conclusion.
In this paper, we have used a combination of approaches to address in
detail the roles of ovarian steroids on uterine angiogenesis in mice.
Unexpectedly, we found that although E promotes uterine vascular
permeability, it profoundly attenuates angiogenesis. In contrast,
P4 stimulates uterine angiogenesis with little
effect on vascular permeability.
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RESULTS
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E and P4 Differentially Regulate the Spatiotemporal
Expression of Vegf and Flk1 in the Uterus
In the mouse uterus, E elicits an early response (phase I) and a
late response (phase II) (27, 28). The phase I response is
characterized by increased uterine vascular permeability and edema that
reach a maximum after 6 h of E administration. The phase II
uterine response occurs between 12 and 24 h of E treatment and
represents the "true growth" phase, i.e. cellular
proliferation and hypertrophy (28, 29). Furthermore,
P4 attenuates the phase I estrogenic responses
(reviewed in Ref. 30). Because VEGF signaling is a strong
stimulator of vascular permeability, we therefore examined the temporal
and cell-specific expression of Vegf and Flk1 in
the mouse uterus in response to estradiol-17ß (E2) and/or
P4 by Northern and in situ
hybridization. Ovariectomized mice were given an injection of oil
(control), E2, P4, or E2 plus
P4, and uterine RNA was extracted at various
times. As previously reported (2), we detected multiple
transcripts of Vegf in uterine
poly(A)+ RNA samples with 4.2 kb as a major
transcript (Fig. 1
). When normalized to a
gene encoding ribosomal protein L7 (rpL7), the levels
of Vegf mRNA showed an early, but transient, increase after
E2 injection, with peak levels between 2 and 4 h (260% increase
over the oil-treated controls) followed by a decline reaching a nadir
by 24 h (Fig. 1
). In contrast, Vegf mRNA levels after a
P4 injection were modest and remained more or
less steady, except an increase (125% increase compared with controls)
at 12 h. The pattern of uterine levels of Vegf mRNA
after a combined injection of E2 and P4 was
similar to those of E2 treatment alone, except the levels were a little
lower (170% increase at 4 h). These results corroborate those of
a recently published report (26).

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Figure 1. Northern Blot Detection of Vegf and
Flk1 mRNAs in the Mouse Uterus
Poly(A)+ RNA samples (2 µg) of uteri after steroid
hormone treatments were separated by formaldehyde-agarose gel
electrophoresis, transferred to nylon membranes, UV cross-linked, and
hybridized to specific 32P-cRNA probes. The same blots were
stripped and rehybridized to a rpL7 (a house-keeping
gene) probe to confirm integrity of RNA samples.
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Because signaling of VEGF via FLK1 is an essential component of
angiogenesis, we also examined the uterine expression of the gene
encoding FLK1 in response to steroid hormones (Fig. 1
). An E2 injection
resulted in an early increase in uterine Flk1 mRNA (
7.0
kb) levels that peaked at 6 h (205% over the controls). This was
followed by a decline reaching a level similar to that of the control
(oil) at 24 h. After an injection of P4,
however, an increase in the level of Flk1 mRNA was observed
at 6 h, reaching a peak at 12 h (165% increase over the
controls), and sustained through 24 h. The pattern of levels in E2
plus P4-treated uteri was similar to those of E2
treatment alone. These results suggest that whereas E2 regulates the
uterine expression of Vegf and its receptor Flk1
in an early and transient manner, P4 has more
sustained effects on the expression of these genes.
The early but transient rise in uterine levels of Vegf and
Flk1 mRNAs seen within 6 h of an E2 injection (Fig. 1
)
is consistent with a role for VEGF in uterine vascular permeability.
However, Northern analysis gives no indication of the uterine cell
types that are responding by increased gene expression, and levels of
whole uterine mRNAs by Northern hybridization may have limited meaning
because of the dilution effects resulting from heterogeneous uterine
cell types in which myometrial cells constitute the major cell
population. We therefore examined the cell-specific expression of
Vegf and Flk1 mRNAs by in situ
hybridization in the ovariectomized uterus at different hours after an
E2 injection. Indeed, differential cell-specific expression of
Vegf and Flk1 mRNAs were observed with changing
time. As shown in Fig. 2A
, Vegf
mRNA accumulation was markedly up-regulated, primarily in stromal
cells, within 2 h of an E2 injection and persisted through 6
h. In contrast, whereas the levels of Flk1 mRNA were very
low at 2 h, a marked increase was noted at 6 h of E2
treatment. The prompt increase in Vegf expression at 2 and
6 h was accompanied by an up-regulation of Flk1
expression in the stromal bed at 6 h after an injection of E2
(Fig. 2A
). This result suggests that VEGF is important for uterine
vascular permeability changes that are induced by E during the phase I
response.

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Figure 2. In Situ Hybridization of
Vegf and Flk1 mRNA in Mouse Uteri After
Steroid Hormone Treatments
A, Two and 6 h after oil (control) or E2 treatment. B, Twenty-four
hours after oil, E2, P4, or E2 plus P4
treatment. Representative dark-field photomicrographs of longitudinal
sections of uteri are shown (bar, 60 µm). le, luminal
epithelium; s, stroma; myo, myometrium.
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Because vascular permeability is normally followed by angiogenesis, we
next examined the expression of Vegf at 24 h after an
injection of oil (control), E2, P4, or E2 plus
P4 (Fig. 2B
). Low to modest levels of
Vegf mRNA accumulation were noted in both the epithelial and
stromal cells of ovariectomized mice receiving an injection of oil
(control). Surprisingly, Vegf expression was primarily
limited to the luminal epithelium at 24 h of E2 treatment; the
stromal expression was very low. In contrast, Vegf mRNA
accumulation was prominent and primarily stromal in response to
P4 alone. The expression pattern in response to
P4 plus E2 was similar to that of
P4 alone, but at lower levels (Fig. 2B
). The
differential cell-specific expression of Vegf in response to
E2 and/or P4 prompted us to examine the
cell-specific expression of Flk1 in steroid-exposed
ovariectomized uteri. As shown in Fig. 2B
, the accumulation of
Flk1 mRNA in stromal endothelial cells was very low in
oil-treated control uteri and was not altered 24 h after an E2
injection. In contrast, the accumulation of Flk1 mRNA was
remarkably up-regulated in endothelial cells of the stromal bed 24
h after an injection of P4. The combined
injection of P4 with E2 resulted in an
accumulation pattern similar to that of P4 alone,
albeit at lower levels. The late induction of stromal Vegf
and Flk1 by P4 alone suggests that
P4 positively regulates uterine angiogenesis. E,
on the other hand, appears to be a negative regulator of uterine
angiogenesis, because Vegf and Flk1
expression in the stromal bed is very low in response to this steroid
during the late phase, and P4-induced changes are
attenuated by E.
Effects of E and P4 on Uterine Expression of Angiogenic
Factors are Mediated by Their Nuclear Receptors
E and P4 effects in the uterus are primarily
mediated via activation of nuclear ER
and PR, respectively. Because
these steroids differentially regulate uterine expression of
Vegf and Flk1 in a spatiotemporal manner, we
sought to examine whether the effects of E or P4
on the expression of these genes are direct and mediated via their
nuclear receptors. Thus, we examined the expression of Vegf
and Flk1 in uteri of mice with a null mutation for the
ER
or PR gene by in situ
hybridization. Our results show distinct expression of stromal
Vegf and endothelial Flk1 in the endometrial bed
in ER
(-/-) mice (Fig. 3A
). In
contrast, uterine expression of Vegf and Flk1 in
PR(-/-) mice was very scanty (Fig. 3B
), and the response
was not altered in ovariectomized PR(-/-) mice by
treatment with E2 or P4 (data not shown). The
level and pattern of expression of Flk1 mRNA in
ER
(-/-) and PR(-/-) uteri correlate well
with lacZ-stained endometrial blood vessels under a
Flk1 promoter in ER
(-/-) x
Flk1(+/-)lacZ and
PR(-/-) x Flk1(+/-)lacZ
double mutant female mice, respectively (compare Fig. 3
vs.
Fig. 6A
). Uteri of ER
(-/-) mice contain PR and respond
to P4 with respect to gene expression and
decidualization (31, 32, 33). The results provide genetic
evidence that higher Flk1 expression in
ER
(-/-) uteri is primarily due to
P4 effects, whereas the attenuated expression in
PR(-/-) uteri is the result of predominant E action. We
next sought to determine the effects of E and/or
P4 on uterine angiogenesis.

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Figure 3. In Situ Hybridization of
Vegf and Flk1 mRNAs in Uteri of Intact
ER (-/-) (A) and PR(-/-) (B) Mice
Representative bright- and dark-field photomicrographs of longitudinal
uterine sections are shown (bar, 60 µm). le, luminal
epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.
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Figure 6. lacZ-Stained Endometrial Blood
Vessels in ER (-/-) x
Flk1(+/-)lacZ or
PR(-/-) x
Flk1(+/-)lacZ Double Mutant Mice
A, Photomicrographs of lacZ-stained endometrial
blood vessels in representative longitudinal uterine sections and
cross-sections of the ovary, brain, and skeletal muscle from intact
ER (-/-) x
Flk1(+/-)lacZ or
PR(-/-) x
Flk1(+/-)lacZ double mutant mice are
shown (bar, 60 µm). B, lacZ-stained
endometrial blood vessels in representative cross-sections of
ovariectomized PR(-/-) x
Flk1(+/-)lacZ double mutant mice in
response to steroid hormones are shown (bar, 60 µm).
Ovariectomized mice were injected with oil or steroids once daily for
2 d and killed 24 h after the last injection.
Arrowheads indicate the location of
lacZ-stained blood vessels. le, luminal epithelium; ge,
glandular epithelium; s, stroma; myo, myometrium.
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E and P4 Differentially Regulate Uterine
Angiogenesis
FLK1 (VEGF receptor 2) is a well established marker of endothelial
cell development and angiogenesis (34, 35). Mice in which
the Flk1 gene has been disrupted with an insertion of the
Escherichia coli ß-galactosidase (lacZ)
gene (17) were used to study uterine angiogenesis.
Although the Flk1(-/-)lacZ embryos
die in utero,
Flk1(+/-)lacZ embryos are viable, and
Flk1(+/-)lacZ females have normal
fertility. Thus, Flk1(+/-)lacZ mice serve
as a powerful genetic model, with ß-galactosidase expression as a
read-out for Flk1 promoter activity and as an endothelial
cell marker to examine uterine angiogenesis under different
physiological and experimental conditions.
To examine the status of uterine angiogenesis in response to E2
and/or P4, ovariectomized
Flk1(+/-)lacZ mice received
injections of oil, P4, and/or E2 once daily for
2 d and were killed 24 h after the last injection. When
compared with oil-treated (control) mice, endometria of mice treated
with P4 showed an increased density of
lacZ-stained blood vessels (Fig. 4A
). In contrast, mice treated with E2
exhibited a remarkably reduced number of such endometrial blood
vessels, even lower than those of the oil-treated mice. Mice that
received both P4 and E2 injections displayed a
number of lacZ-stained endometrial blood vessels
intermediate between P4-
and E2-treated mice (Fig. 4A
). To quantify the
extent of endometrial angiogenesis, we measured the area of uterine
sections occupied by lacZ-stained blood vessels. The mean
percentage of endometrial area occupied by lacZ-stained
blood vessels is shown in Fig. 4B
. Furthermore, the inhibitory response
of E2 or stimulatory effects of P4 on uterine
angiogenesis were reversed by IC1182,780, an ER antagonist, or
RU-486, a PR antagonist (Fig. 4C
). These results suggest that whereas
E2 is profoundly inhibitory, P4 is stimulatory to
uterine angiogenesis. Furthermore, E2 is capable of counteracting the
stimulatory effects of P4 on uterine
angiogenesis. This observation prompted us to examine the long-term
effects of sustained stimulation of the uterus with
P4 or E2 using SILASTIC brand (Dow Corning Corp, Midland, MI) implants (36). In
ovariectomized Flk1(+/-)lacZ mice with
SILASTIC brand implants containing P4 or E2 for
4 d, a similar pattern of lacZ staining was noted as
observed for steroid treatments of shorter duration (data not shown).
We also examined by in situ hybridization the expression of
Vegf and Nrp1 in uteri of these mice (Fig. 5A
). In ovariectomized uteri without the
steroid treatment, the expression of Vegf and
Nrp1 was modest throughout the endometrium. E2 treatment
again suppressed stromal expression of Vegf; the expression
was primarily restricted to epithelial cells, as observed at 24 h
after an E2 injection (compare Fig. 2B
vs. Fig. 5A
). In
contrast, the expression was distinct and mostly stromal in
P4-treated mice. The expression of
Nrp1 in E2-treated uteri was observed in both epithelial and
stromal cells at very low levels. In contrast, Nrp1 was
abundantly expressed in stromal cells of
P4-treated mice (Fig. 5A
). Our observation of
up-regulated expression of Nrp1 in the stroma by
P4 is consistent with a recent report of
up-regulated Nrp1 expression in the rat uterus by
P4, but not by E2 (37). Although the
observation of heightened expression of Vegf,
Flk1, and Nrp1 in the stromal bed in response to
P4 suggests its stimulatory role, their
attenuated expression in the stromal bed by E2 suggests its inhibitory
role in uterine angiogenesis. Immunolocalization of the platelet
endothelial cell adhesion molecule (PECAM), another endothelial cell
marker (35), showed similar distribution patterns as
lacZ staining in steroid-treated uteri (Fig. 5B
).

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Figure 4. lacZ-Stained Endometrial Blood
Vessels in Flk1(+/-)lacZ
Mice After Steroid Hormone Treatments
Ovariectomized mice were treated with oil, E2,
P4, or E2 plus P4 once daily for 2 d and
killed 24 h after the last injection. A, Photomicrographs of
lacZ staining of representative uterine cross-sections
are shown. B, Quantification of lacZ staining is shown
as mean percentage of stained endometrial area. Four to six sections
from two to three mice in each group were evaluated for quantification.
Results are presented as mean ± SEM. Values were
statistically significant from each other (P <
0.05, ANOVA). C, Ovariectomized mice were treated for 2 d with
either P4, E2, P4 plus RU-486, or E2 plus
ICI-182,780 (ICI). RU-486 and ICI were injected 30 min prior to steroid
injections. Mice were killed 24 h after the last injection.
Photomicrographs of lacZ staining of representative
uterine longitudinal sections are shown (bar, 60 µm).
Arrowheads indicate the location of
lacZ-stained blood vessels. le, luminal epithelium; ge,
glandular epithelium; s, stroma; myo, myometrium.
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Figure 5. In Situ Hybridization of
Vegf and Nrp1 mRNAs and
Immunolocalization of PECAM in Mouse Uteri After Long-Term E2 or
P4 Treatment
Ovariectomized mice carrying SILASTIC brand implants containing oil,
E2, or P4 were killed after 4 d, and uteri were
processed for in situ hybridization and
immunohistochemistry. A, Representative dark-field autoradiographic
signals of in situ hybridization in longitudinal uterine
sections are shown (bar, 75 µm). B, Frozen
longitudinal uterine sections (10 µm) were subjected to
immunostaining using a rat-antimouse monoclonal antibody to PECAM
(PECAM-1). Red color indicates the sites of
immunoreactive PECAM (bar, 60 µm). le, luminal
epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.
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Heightened Angiogenesis with the Loss of ER
Function Is
Consistent with Reduced Angiogenesis with the Loss of PR Function
in the Uterus
Although uterine estrogenic effects are virtually absent in
ER
-deficient mice, uteri in PR-deficient mice are highly
estrogenized due to the absence of P4 effects.
These findings led us to examine the status of uterine angiogenesis in
mice homozygous for ER
or PR null mutations
but heterozygous for the Flk1lacZ
mutation. ER
(+/-) or PR(-/-) males
were mated with Flk1(+/-)lacZ
females to generate ER
(-/-) x
Flk1(+/-)lacZ or
PR(-/-) x Flk1(+/-)lacZ
double mutant female mice. Our results of lacZ staining show
that the density of endometrial blood vessels in
ER
(-/-) x Flk1(+/-)lacZ
mice was remarkably higher than that observed in PR(-/-)
x Flk1(+/-)lacZ mice (Fig. 6A
). In fact, the lower density of
endometrial vessels in the latter group of mice was similar to that
observed in ovariectomized Flk1(+/-)lacZ
mice treated with E2 (Fig. 4A
vs. Fig. 6A
). The density of
these endometrial blood vessels was even higher in ovariectomized
PR(-/-) x Flk1(+/-)lacZ
mice, and P4 did not alter this response.
However, treatment of such ovariectomized mice with E2 reduced the
number of lacZ-stained endometrial blood vessels, and as
expected, treatment with P4 did not alter the E2
response (Fig. 6B
). However, the angiogenic status in the ovarian theca
was similar in both ER
(-/-) x
Flk1(+/-)lacZ and PR(-/-) x
Flk1(+/-)lacZ mice. Likewise, there was
no significant alteration in lacZ staining in adult brains
or skeletal muscles of these double mutant mice (Fig. 6A
).
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DISCUSSION
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The present investigation provides molecular, genetic,
physiological, and pharmacological evidence that E is a potent
inhibitor of uterine angiogenesis in mice, and surprisingly, that
P4 is a stimulator of uterine angiogenesis.
Furthermore, these effects of E and P4 are
mediated via activation of their cognate nuclear receptors and are
specific to the uterus, because angiogenesis in the ovary, brain, and
skeletal muscle were similar in mice with null mutations for the
ER
or PR gene. These findings challenge the
prevailing notion of E-induced stimulation of uterine angiogenesis
(reviewed in Ref. 3). This notion is built on the
correlative findings that vascular permeability is followed by
increased angiogenesis and that E rapidly induces uterine vascular
permeability coincident with increased VEGF expression. Indeed, our
observation of rapid, but transient, induction of Vegf and
Flk1 in the uterine stromal bed by E supports the idea that
E increases uterine vascular permeability during the phase I response.
However, our findings of reduced uterine vascular density and decreased
stromal endothelial Flk1 expression during the late
estrogenic response suggest that this steroid has an inhibitory role in
uterine angiogenesis. This is further confirmed by genetic evidence in
ER
(-/-) mice that display increased density of
lacZ-stained endometrial blood vessels and increased stromal
expression of Vegf and Flk1 in the absence of
ER
functions. The role of ERß is questionable, because uterine
expression of ERß is very low in ER
(-/-)
mice. Moreover, an ER antagonist (ICI 182,780), which negates both
ER
and ERß functions (38), reversed the inhibitory
effects of E2 on uterine angiogenesis. Thus, our findings demonstrate
that uterine vascular permeability is not always followed by
angiogenesis. This is consistent with the recent observation that
although mice deficient in individual Src family kinases show normal
angiogenesis, mice lacking in pp60c-src or pp62c-yes, but not fyn,
failed to exhibit VEGF-mediated vascular permeability
(39).
The restricted expression of Vegf in the uterine epithelium
with a very low or undetectable level of expression in the stroma
during the late phase of estrogenic stimulation suggests that VEGF is
not readily available in the stroma for increased angiogenesis. In
contrast, our observation of increased vessel density in
P4-treated
Flk1(+/-)lacZ mice, as well as increased
accumulation of Vegf mRNA in stromal cells and
Flk1 mRNA in stromal endothelia of wild-type mice after
P4 treatment, suggests that this steroid
stimulates uterine angiogenesis. This steroid participates in this
process via its nuclear receptor, PR, because the vessel density is
severely compromised in uteri of PR(-/-) x
Flk1(+/-)lacZ double mutant mice with
manifestation of predominantly E actions, and reversal of
P4 effects by a PR antagonist RU-486 can be
observed in wild-type mice.
P4 has recently been shown to attenuate in
vitro proliferation of human endothelial cells derived from
various tissues (40). This P4 effect
appears to be mediated via PR, because these cells express PR, and a PR
antagonist compromises this P4 effect.
Furthermore, using PR(-/-) mice, these investigators
provided evidence that P4 also interferes with
re-endothelialization of injured aortae, again suggesting the
inhibitory role of P4 in endothelial cell
proliferation (40). The discrepancy between this study and
our present investigation is most likely due to the differences in
experimental designs and parameters used and cell types studied.
Vázquez et al. (40) studied the influence
of P4 on proliferation of human dermal and
coronary endothelial cells and of mouse brain endothelial cells in
culture, whereas our study examined the effects of
P4 and/or E2 on endometrial angiogenesis in mice
in vivo. They did not examine the effects of
P4 on proliferation of PR-expressing human
endometrial endothelial cells. We speculate that ovarian steroids
influence endothelial cell functions with respect to angiogenesis in a
tissue-specific manner. Because the uterus is a major target for E and
P4, and because heterogeneous cell types of the
uterus respond differently to these hormones in a dynamic manner, we
suspect that uterine endothelial cells respond to these steroids
differently from cells in extrauterine sites. It is also highly
possible that angiogenic factors generated by the action of steroid
hormones on uterine cell types act on endothelial cells for
angiogenesis in a paracrine manner. However, such paracrine effects are
absent in endothelial cells in culture.
P4-induced angiogenesis in the uterus is
physiologically meaningful, because this steroid is an essential
hormone for the initiation and maintenance of pregnancy in all mammals
examined, and uterine angiogenesis is an essential component during
pregnancy. Although E and P4 are critical to
implantation and pregnancy maintenance in mice and rats,
P4 alone is sufficient for these events in
several species including hamsters, guinea pigs, rabbits, and pigs
(reviewed in Ref. 41). This suggests that
P4 is a prime regulator of uterine angiogenesis
during early pregnancy in these species. Furthermore, uterine
expression of proangiogenic factors is very low on d 1 of pregnancy in
mice when the uterus is under the dominance of preovulatory ovarian E
secretion. However, dramatic increases in the expression of these
factors are observed from d 4 onward, with rising
P4 levels by the newly formed corpora lutea
(2, 4). Thus, perhaps a balance between negative and
positive influences resulting from coordinated interactions between E
and P4 during early pregnancy determines the
normal angiogenic status of the uterus during early pregnancy in
mice.
Our results suggest that E-induced early endometrial vascular
permeability and stromal edema are mediated by an early induction of
VEGF and FLK1 in the stromal bed, whereas the attenuated uterine
angiogenesis during the late estrogenic growth phase is the result of
suppressed Vegf, Nrp1, and Flk1
expression in the stromal bed. In contrast,
P4-induced uterine angiogenesis is executed by
late and sustained induction of Vegf and Flk1 in
conjunction with Nrp1 (Fig. 7
).
Our results also suggest that an increased vascular permeability is not
always a prerequisite condition for increased uterine angiogenesis. In
conclusion, we provide here the first evidence for the regulation of
angiogenesis in a physiologically relevant adult organ system in
vivo. This information will be important for comparing normal
physiological angiogenesis with the process during pathological
conditions such as uterine adenocarcinoma, endometriosis, and
dysfunctional uterine bleeding. In this respect, it is interesting to
note that xenografts resulting from a human endometrial cancer cell
line overexpressing ER
showed reduced tumor growth and angiogenesis
(42).

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Figure 7. A Scheme Depicting Uterine Vascular Permeability
and Angiogenesis in Response to Steroids
This scheme suggests that in the stromal bed, E2 rapidly, but
transiently, induces Vegf and Flk1,
whereas P4 induces these genes in the stroma in a more
delayed and sustained manner.
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MATERIALS AND METHODS
|
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Animals and Treatments
Adult CD-1 mice were purchased from the Charles River Laboratories, Inc. (Raleigh, NC). Flk1-deficient mice were
generated by disruption of the Flk1 gene using homologous
recombination in embryonic stem cells (17). A targeting
vector was constructed in which the translated portion of the first
coding exon and the proximal part of the first intron of the
Flk1 gene were replaced by a promoterless
ß-galactosidase gene from E. coli, leaving the
Flk1 promoter intact. Therefore, ß-galactosidase
expression is used as a read-out for Flk1 promoter activity.
ER
-deficient mice (129/J/C57BL/6J) and PR-deficient mice
(129SvEv/C57BL/6) were generated as previously described (31, 43) and were kindly provided by Dennis Lubahn (University of
Missouri, Columbia, MO) and Bert OMalley (Baylor College of Medicine,
Houston, TX), respectively, for establishing our colonies.
ER
(-/-) x Flk1(+/-)lacZ
double mutant mice were generated by crossing
Flk1(+/-)lacZ females with
ER
(+/-) males, whereas
PR(-/-)/Flk1(+/-)lacZ double mutants
were generated by crossing Flk1(+/-)lacZ
females with PR(-/-) males. PCR analysis of the genomic
DNA determined the genotypes. All mice were housed in the Animal Care
Facility at the University of Kansas Medical Center (Kansas City, KS)
according to NIH and institutional guidelines for laboratory
animals.
To examine the effects of E and/or P4 on
uterine expression of proangiogenic genes or uterine angiogenesis
(lacZ staining), ovariectomized mice were injected with
sesame oil (0.1 ml/mouse), estradiol-17ß (E2) (100 ng/mouse),
P4 (2 mg/mouse), E2 and P4,
E2 and ICI 182,780 (an ER antagonist), or P4 and
RU486 (a PR antagonist). ICI 182,780 and RU486 were injected at a dose
of 500 µg and 1 mg per mouse per day, respectively. In another set of
experiments, ovariectomized mice were implanted sc with SILASTIC brand
implants (0.1 x 1 cm) filled with E2, or implants (0.1 x 2
cm) filled with P4, for 4 d. At termination
of the treatments, uteri were processed for subsequent analysis. For
systemic injections, steroids and antagonists were dissolved in sesame
oil and injected sc.
Probes
The cDNA clones for Vegf, Flk1,
Nrp1, and rpL7 have been previously described
(2, 4). For Northern hybridization, antisense
32P-cRNA probes were generated, whereas for
in situ hybridization, sense or antisense
35S-cRNA probes were generated using appropriate
polymerases. Probes had specific activities of about 2 x
109 dpm/µg.
Northern Hybridization
For Northern hybridization, total RNA (6.0 µg) or
poly(A)+ RNA (2.0 µg) was denatured and
separated by formaldehyde/agarose gel electrophoresis, transferred to
nylon membranes, and UV cross-linked. Northern blots were
prehybridized, hybridized, and washed as previously described (2, 4). Quantification of hybridized bands was analyzed by
densitometric scanning.
In Situ Hybridization
In situ hybridization was performed as
previously described (2, 4). In brief, frozen sections (10
µm) were mounted onto (poly)L-lysinecoated
slides and fixed in cold 4% paraformaldehyde in PBS. The sections were
prehybridized and hybridized at 45 C for 4 h in 50% formamide
hybridization buffer containing the 35S-labeled
antisense cRNA probes. RNase A-resistant hybrids were detected by
autoradiography. Sections were post-stained with eosin and hematoxylin.
Sections hybridized with the sense probes did not result in any
positive hybridization.
lacZ Staining and Quantification
The expression of ß-galactosidase was assessed by
lacZ staining as previously described (44). In
brief, small pieces of tissues were fixed in 0.2% paraformaldehyde
solution followed by infusion in 30% sucrose at 4 C overnight. Tissues
were embedded in OCT and snap-frozen. Frozen sections were mounted onto
glass slides and stained overnight at 37 C using
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
as a substrate. Sections were counterstained with eosin. Uterine area
occupied by lacZ-stained blood vessels was quantitated.
Random sections of uteri were used for lacZ staining;
digital images were obtained, and measurements were made using the
Scion Image program (Scion Corp., Frederick, MD). For
consistency of measurements, the total uterine stromal area was defined
by subtracting the section area occupied by the uterine luminal and
myometrial layers, and the percentage of uterine stromal area occupied
by lacZ-positive vascular structures was measured for each
section.
Immunohistochemical Localization of PECAM
Frozen longitudinal uterine sections (10 µm) were subjected to
immunostaining using a rat-anti-mouse monoclonal antibody to PECAM
(PECAM-1, BD PharMingen, San Diego, CA) at a dilution of
1:50 using a Histostain-SP kit (Zymed Laboratories, Inc.,
San Francisco, CA) as previously described (45). Red color
indicates the sites of immunoreactive PECAM.
 |
ACKNOWLEDGMENTS
|
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We thank Brigid Hogan for her valuable advice.
 |
FOOTNOTES
|
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This work was supported by NIH Grants HD-12304, HD-29968, HD-33994-06,
Mellon Foundation (to S.K.Dey), ES-07814 (to S.K.Das), and DK-52483 (to
D.R.A.), and an American Heart Association grant (to B.R.). A center
grant in Mental Retardation provided core facilities. S.K.Dey is the
recipient of an NIH MERIT award.
1 These authors contributed equally to this work. 
Abbreviations: E, Estrogen; E2, estradiol-17ß; NRP1,
neurophilin 1; P4, progesterone; PECAM, platelet
endothelial cell adhesion molecule; rpL7, ribosomal
protein L7; VEGF, vascular endothelial growth factor.
Received for publication June 28, 2001.
Accepted for publication August 8, 2001.
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