© 1999 American Heart Association, Inc.
Vascular Biology |
Estrogen Mediates the Protective Effects of Pregnancy and Chorionic Gonadotropin in a Mouse Model of Vascular Injury
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Abstract |
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Abstract—To determine why pregnancy protects against intimal proliferation in a mouse model of vessel injury, we administered chorionic gonadotropin to intact and ovariectomized female mice. Chorionic gonadotropin markedly suppressed intimal proliferation in intact but not in ovariectomized female mice, indicating that the protective effects of chorionic gonadotropin require ovarian function. To test whether estrogen or progesterone might mediate the protective effects of pregnancy and chorionic gonadotropin, we administered estrogen and progesterone to ovariectomized mice. Estrogen administration to ovariectomized mice to achieve the elevated levels seen in pregnancy was sufficient to reproduce the marked suppression of intimal proliferation in response to vessel injury. Progesterone administration reduced intimal proliferation to a lesser degree and was correlated with increases in estrogen to levels seen in nonpregnant female mice. Staining for proliferating cell nuclear antigen suggested that estrogen reduced medial and intimal cell proliferation. Both the classic estrogen receptor-
and the recently discovered estrogen receptor-ß are
present in vascular tissue as assessed by immunohistochemistry,
providing a possible mechanism for the effects of estrogen. These
results suggest that the protective effects of estrogen do not plateau
at levels seen in normal females but increase further with estrogen
levels up through levels seen during pregnancy.
Key Words: vascular endothelium • atherosclerosis • estrogen • sex • pregnancy
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Introduction |
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There is a well-established sex difference in the incidence of atherosclerotic disease between men and premenopausal women.1 Much of the protective effect of female sex is mediated by estrogen.2 3 In experimental models of atherogenesis and vessel injury, estrogen reduces neointimal proliferation and lesion formation.4 5 6 7 Pregnancy represents another unique hormonal state, distinct from that in males and nonpregnant females. During pregnancy, the placenta produces chorionic gonadotropin and lactogen, and maternal production of estrogens and progesterone increases dramatically. To date, the effects of these hormonal changes on vascular intimal proliferation have not been well studied.
In prior studies, we applied a cuff model of vessel injury to wild-type and endothelial nitric oxide synthase (eNOS) –mutant mice to study the role of eNOS in the neointimal proliferation response.8 This model shows the expected sex difference between male and female mice, with more intimal proliferation observed in males than in females. We found unexpectedly that pregnant mice develop almost no intima in response to vessel injury. To study further the effect of sex and pregnancy on the vascular response to injury, we ovariectomized several mice and implanted subcutaneous pumps for infusion of hormones. Ovariectomized mice develop more neointima in response to cuff placement than do intact (nonovariectomized) female mice.
To test the possible role of circulating chorionic gonadotropin in suppressing the neointimal response during pregnancy, we administered human chorionic gonadotropin (hCG) to intact female and ovariectomized mice. hCG reduces intimal proliferation in intact female mice but not in ovariectomized mice. These results indicate that hCG requires intact ovarian function for its protective effects and does not directly suppress vascular smooth muscle proliferation. Estrogen replacement to levels seen in pregnancy and with hCG administration suppresses neointimal formation. Thus, elevated estrogen levels are sufficient to mediate the protection observed during pregnancy and with hCG treatment.
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Methods |
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Animals
Eight-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me) were used for cuff placement. Some female mice underwent bilateral ovariectomy at 6 weeks of age, 2 weeks before cuff placement. All procedures were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.
Vascular Cuff Injury Model
The cuff injury model was performed as described
previously.8 Mice were anesthetized with sodium
pentobarbital (50 mg/kg), and the femoral arteries were exposed and
isolated through a groin incision. A cuff 2.0 mm long, made from
longitudinally split polyethylene-50 tubing (Clayton), was placed
loosely around the left femoral artery and tied into place. The right
femoral artery was dissected and isolated, but no cuff was placed (sham
operation). The incisions were then closed with 4-0 sutures.
Experimental Protocols
Mice were divided into 7 groups: (1) male mice, (2) intact
(nonovariectomized) female mice, (3) ovariectomized mice, (4)
ovariectomized mice receiving 17ß-estradiol (0.1 mg/d), (5)
ovariectomized mice receiving progesterone (0.25 mg/d), (6)
ovariectomized mice receiving ßhCG (8 ng/d), and (7) intact,
nonovariectomized mice receiving ßhCG (8 ng/d). Each group consisted
of 5 or 6 animals. 17ß-Estradiol, progesterone, and ßhCG were
obtained from Sigma Chemical Co and were delivered by a model 2002
mini-osmotic pump (Alzet) implanted subcutaneously between the scapulas
on the same day as cuff placement. The hormones were infused at a rate
of 0.5 µL/h during the 14-day period between cuff placement and
tissue harvesting.
Tissue Harvesting and Histology
Fourteen days after cuff placement, the mice were
anesthetized and euthanized (100 mg/kg pentobarbital IP). A
22-gauge butterfly angiocatheter was placed in the left cardiac
ventricle to allow in situ constant-pressure fixation at 100
mm Hg with 10% buffered formalin. The cuffed region and the
contralateral control region of the femoral arteries were harvested,
embedded in paraffin, and cut into continuous cross sections (10
µm). Parallel sections were subjected to hematoxylin and eosin
staining, elastin staining, trichrome staining, and
immunohistochemistry studies.
Serum Estradiol, Progesterone, and Cholesterol Assay
At the time of tissue harvesting, blood samples were obtained
from the carotid artery. Serum estradiol and progesterone levels were
measured with ELISA kits (estradiol and progesterone ELISA, Cayman
Chemical Co, Inc). Serum samples were purified with a Sep-Pak C18
cartridge (Waters Co), and the assays were conducted according to the
manufacturer's instructions. Standard curves were generated using
samples of known concentrations for estradiol and progesterone. The
assay sensitivity was 9.0 pg/mL, and the intra-assay and interassay CVs
were both 10%. Total cholesterol was measured
enzymatically (Sigma), and the intra-assay and interassay CVs were
1.4% and 1.9%, respectively.
Morphometry
Morphometric analyses were performed on
hematoxylin/eosin and elastin stained sections. Ten evenly spaced
sections of each cuffed and sham-operated control artery were
photographed and digitized using National Institutes of Health Image
software. For each section, 4 measurements were made: luminal area,
area inside the inner elastic lamina, area inside the outer elastic
lamina, and vessel circumference. The intima was defined as the area
between the lumen and the internal elastic lamina. The media was
defined as the area between the internal and external elastic laminas.
The mean vascular diameter was calculated as the vessel
circumference/
. Measurements were made for all 10 sections for each
vessel to obtain the mean for that vessel. Thickness and intima/media
volume (I/M) ratios reported for each group reflect the average of the
mean values obtained for each vessel. The measurements were done in a
blinded fashion with respect to the experimental groups.
Immunohistochemistry and Quantification of Proliferating Cell
Nuclear Antigen (PCNA) Activity
Parallel sections were used for immunohistochemistry studies.
Antibodies to -actin and CD31 were obtained from Dako Corp,
antibodies to factor VIII and PCNA were from Zymed, antibodies to the
progesterone receptor and estrogen receptor- came from Santa Cruz,
antibodies to estrogen receptor-ß were from Affinity Bioreagents,
Inc, and antibodies to eNOS were from Transduction Laboratories.
Immunoperoxidase staining was performed using the Vector Elite ABC kit
or the Zymed histoMouse kit. Before being stained, some sections were
pretreated with the antigen retrieval method by incubation with 0.01
mol/L citrate buffer (pH 6.0) at 100°C for 20 minutes, followed by
cooling to room temperature.9 10 The remainder of the
staining procedure was conducted at 4°C. Staining was visualized with
diaminobenzidine, diaminobenzidine with nickel, or aminoethylcarbazole,
and some of the sections were counterstained with hematoxylin.
PCNA-positive cells were counted in the total intimal and medial areas
in 3 sections from each vessel. The mean values of these 3 sections
were calculated for each vessel. For each group of animals, the mean
values for each vessel were averaged.
Statistical Analysis
All values were expressed as mean±SEM. Measurements were tested
by ANOVA with Scheffé's test. Comparisons were made using the
mean values for each vessel. Student's t test was used to
compare the estrogen and progesterone levels of each group of mice with
the levels seen in intact C57BL/6 female mice. For all statistical
analyses, P<0.05 was considered significant.
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Results |
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Sex Differences and the Effect of hCG on Intimal Proliferation
We used a nonocclusive cuff model to study the response to vessel injury in mice. Similar models have been well characterized in rabbits.11 12 The model is reproducible, easily quantitated, and reflects sex differences similar to those seen in humans. Cuff placement around the femoral artery results in the reproducible development of neointima after 14 days. The degree of neointimal proliferation can be quantitated by morphometry and expressed as the I/M ratio. Mouse femoral arteries contained no intima at baseline, and sham-operated vessels did not develop any intima.
As shown in Figure 1
, male C57BL/6 mice
developed an intima with an average thickness of 16.1±2.4 µm,
whereas intact female C57BL/6 mice developed an intima with an average
thickness of 11.2±1.3 µm. These values correspond to I/M ratios
of 0.43 for male and 0.27 for female mice. The difference between the 2
groups was significant (P<0.01). Ovariectomized mice
developed more intima in response to cuff injury than did intact female
mice, with an average thickness of 24.0±2.6 µm, corresponding
to an I/M ratio of 0.61.
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To test the hypothesis that circulating chorionic gonadotropins, which
are markedly elevated during pregnancy, suppress the intimal
proliferation response, we administered hCG to intact and
ovariectomized mice. As shown in Figure 1
, hCG suppressed the
proliferation response in intact female mice, reducing the average
intimal thickness to 2.9±1.1 µm (P<0.0001),
corresponding to an I/M ratio of 0.04. In contrast, hCG treatment of
ovariectomized mice did not inhibit intimal proliferation. The average
intimal thickness was 35.5±2.1 µm, even higher than in
untreated ovariectomized mice. Because the thickness of the media
(49.44±3.04 µm) was also higher than that of the untreated,
ovariectomized group (39.05±2.52, P<0.05), the I-M ratio
of hCG-treated, ovariectomized mice was the same as that of untreated,
ovariectomized mice, 0.61. Overall, these results indicate that the
vascular protective effect of hCG depends on intact ovarian
function.
Effect of Estrogen and Progesterone on Intimal
Proliferation
To assess how estrogen levels are affected by pregnancy, we
measured serum estrogen levels on the 7th, 14th, and 20th day of
pregnancy in a separate group of pregnant C57BL/6 mice. As shown in
Figure 2A, the 17ß-estradiol level
increases during pregnancy by 3- to 4-fold over basal levels. There is
an initial marked rise at 7 days, a slight decrease from the peak level
at 14 days, and increases at 17 and 20 days. Thus, during pregnancy,
the levels of estrogen are markedly elevated over those of nonpregnant
mice.
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To determine whether these elevated estrogen levels are sufficient to
mediate the protection seen in pregnancy and with chorionic
gonadotropin administration, we studied the effect of hormone
administration on ovariectomized mice. Replacement of estrogen or
progesterone by subcutaneous osmotic pumps reduced the intimal
response. The intimal thickness in ovariectomized mice that received
17ß-estradiol was 2.7±0.4 µm, corresponding to an I-M ratio
of 0.05 (Figure 1). Mice that received progesterone developed an
intimal thickness of 16.1±2.3 µm, with an I-M ratio of 0.30. In
these groups, the thickness of the media and the vessel diameter did
not vary significantly, so changes in intimal thickness accounted for
the changes in I-M ratio.
Figure 2B shows the estrogen levels of intact, ovariectomized,
estrogen-replaced, progesterone-replaced, and pregnant (day 7) mice.
Ovariectomy resulted in a drop in circulating estrogen levels, from
390.6±22.8 to 193.1±53.1 pg/mL (P<0.05). Estrogen
replacement in ovariectomized mice at the doses used in our study
resulted in levels of estrogen comparable to those seen during
pregnancy. Progesterone replacement in ovariectomized mice resulted in
an increase of serum estrogen to physiological
levels. Thus, estrogen replacement in ovariectomized mice to levels
seen in pregnancy decreases cuff-induced intimal proliferation to the
same degree as in pregnancy. hCG treatment of intact female mice also
increased estrogen levels to degrees comparable with estrogen
replacement and in pregnancy. hCG treatment had no effect on the
estrogen levels of ovariectomized mice.
The serum progesterone level was not altered by ovariectomy or by
ovariectomy plus estrogen treatment (Figure 2C). hCG treatment
of intact female mice elevated the serum progesterone to levels similar
to those due to progesterone treatment itself, but hCG treatment had no
effect on progesterone levels in ovariectomized mice.
Histology and Cellular Proliferation
To characterize the histology of the intimal response, we
performed a histological analysis and
immunostaining of cuff-injured vessels, as shown in
Figure 3. Elastin stains, which stain the
elastin fibers black, and Accustain trichrome, which stains the
collagen fibers green, did not reveal differences in the amount or
arrangement of collagen or elastin fibers in the medial and adventitial
layers among the groups. The medial and intimal cells were stained by
using a monoclonal antibody directed against smooth muscle -actin.
The cells closest to the lumen were stained by using an antibody
directed against factor VIII–related antigen and CD31,
consistent with their being endothelial cells.
There were no differences detected in the staining of
endothelial cells with the use of an anti-eNOS antibody
among the groups.
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We examined the expression of PCNA as a marker of cell proliferation in
the vessel wall. PCNA staining for each of the groups is shown in
Figure 4. The mean number of
PCNA-positive cells in the intima and media is shown in Figure 5A and 5B. There were significantly more
PCNA-positive cells in the intimas of male mice (102±3.1) after cuff
injury than in female mice (59±5.2, P<0.01). Ovariectomy
increased the number of PCNA-positive cells in the intima (116.1±11.9,
P<0.001). Estrogen and progesterone treatments of
ovariectomized mice suppressed the number of PCNA-positive cells to
14.3±1.7 (P<0.0001) and 58.5±7.8 (P<0.0003),
respectively, paralleling the effects of this treatment on intimal
thickness and I-M ratio. hCG treatment reduced the number of
PCNA-positive intimal cells in intact females (13.8±2.8,
P<0.01), but not in ovariectomized females (113.8±7.3). In
most of the groups, the number of intimal PCNA-positive cells was
correlated with the increase in intimal thickness. However, in
ovariectomized mice and in ovariectomized mice treated with hCG, the
increase in intimal thickness was more pronounced than the increase in
PCNA-positive cells, suggesting that there may be an increase in the
amount of tissue matrix as well.
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Expression of Estrogen and Progesterone Receptors
Most tissues that respond to estrogen contain estrogen receptors.
To determine whether cells within the vasculature express estrogen
receptors that might mediate the protective effects of estrogen, we
used immunohistochemistry to stain for the estrogen receptors- and
-ß in the femoral artery. Staining of sections from ovary and oviduct
were used as positive controls and for comparison. Estrogen
receptors- and -ß were both found in femoral arteries and veins of
male and female mice. The cells that stain for these receptors include
endothelial cells, smooth muscle cells, and fibroblasts
in the adventitia, as well as cells lining small vessels within the
adventitia (Figure 6). The staining
intensity for both receptors was stronger in the
endothelial cells than in smooth muscle cells. Both
nuclear staining and cytoplasmic staining were observed. Progesterone
receptor staining was present in the nuclei of intimal cells,
medial smooth muscle cells, and adventitial cells. Negative control
sections did not show nonspecific staining.
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Discussion |
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Pregnancy is associated with vascular changes, including systemic vasodilation, increased cardiac output, and increased blood volume. These effects are thought to be mediated by hormonal changes. Chorionic gonadotropin, placental lactogen, and prolactin are newly synthesized, and maternal estrogen levels and progesterone levels rise. We had previously found an unexpected marked reduction in the neointimal response to cuff injury in pregnant mice, a protective effect that overrode even the effect of eNOS gene deletion.8 This led us to investigate whether changes in chorionic gonadotropin or ovarian hormones might mediate this protection.
Chorionic gonadotropin, made by the syncytiotrophoblast, "rescues"
the corpus luteum and stimulates progesterone production.
Levels of chorionic gonadotropin rise early and remain detectable
throughout pregnancy. Chorionic gonadotropin is a dimer of - and
ß-subunits. The -subunit is shared with luteinizing hormone,
follicle-stimulating hormone, and thyroid-stimulating hormone, whereas
specificity is conferred by the ß-subunit. Thus, we used the
ß-subunit of hCG (ie, ßhCG) in our studies. We found that ßhCG
suppressed the intimal response of intact female mice but not of
ovariectomized mice. Thus, chorionic gonadotropin does not act directly
on vascular smooth muscle but rather causes its effects by modulating
ovarian hormone production.
Estrogens may mediate this effect, since ovarian production of estrogen increases markedly during pregnancy. We found that estradiol levels in pregnant mice increased to 1801 pg/mL, compared with levels of 390 pg/mL in nonpregnant mice. The estrogen level of hCG-treated, intact female mice, but not of hCG-treated ovariectomized mice, rose to levels comparable to those in pregnant mice. Estrogen replacement in ovariectomized mice to achieve estradiol levels in the same range as seen in pregnancy also led to a marked reduction in neointimal proliferation. These results indicate that increases in estrogen levels are sufficient to account for the protection seen in pregnancy.
The mouse cuff model demonstrates a protective effect of female sex.8 In this study, we have shown that ovariectomy obliterates the protective effect of female sex. The I/M ratio of ovariectomized mice was twice as high as that in intact female mice and was even higher than that in normal male mice. Estrogen replacement reduced neointimal proliferation in this model, as it does in other models of vessel injury, including balloon injury models of the rabbit common iliac artery4 and rat carotid artery,5 the filament injury model of the mouse carotid artery,6 and the cuff injury model of the rat femoral artery.7 The estradiol levels in estrogen-replaced, ovariectomized mice in this study exceeded those in nonpregnant female mice and reached levels seen during pregnancy. Suppression of the neointimal response also exceeded the degree observed in nonpregnant female mice and reached the degree seen during pregnancy. Estradiol levels were correlated with the suppression of neointimal proliferation.
The protective effects of estrogens on the cardiovascular system have been reviewed recently.2 3 13 Estrogen may reduce atherogenesis by several mechanisms, including direct effects on vascular smooth muscle cell growth, effects on lipid profile, antioxidant effects, and increased bioavailability of endothelial NO. In humans, estrogen is favorable to the lipid profile and decreases plasma LDL and increases plasma HDL and VLDL.2 3 14 Estrogen also enhances cellular uptake and degradation of LDL and reduces LDL oxidation in vivo.15 We did not find any differences in cholesterol levels between the different groups of animals (data not shown), suggesting that hormonal effects on the cuff model of vessel injury are not caused by changes in cholesterol levels. Lipid profile changes may be less relevant in this relatively acute model of vessel injury.
Estrogen also increases eNOS production16 17 18 and restores endothelium-dependent vasodilation.19 Increased NO levels would protect against atherogenesis by inhibiting smooth muscle proliferation, thrombosis, and leukocyte activation. However, estrogens may have additional effects that do not depend on NO. The prevention of fatty streak formation in apoE–deficient mice by 17ß-estradiol is not affected by NOS inhibitors.20 A sex difference in vascular response to cuff injury persists in eNOS-mutant mice, showing that some estrogen effects do not depend on eNOS.8 In the cuff model of vessel injury, the most relevant protective effects of estrogen appear to be direct effects on vascular smooth muscle growth and proliferation. Estrogens reduce thymidine uptake in porcine coronary artery explants.21 Effects on balloon-injured vessels in mice are reduced by estrogens.1 5 In the current study, we used immunohistochemistry for PCNA as a measure of cell proliferation.22 PCNA staining of both the media and intima were dramatically reduced after estrogen treatment.
There are conflicting reports on whether progesterones, used clinically to counter the potential neoplastic effects on the endometrium of unopposed estrogens, attenuate the vasculoprotective effects of estrogen. Two studies that have shown such an effect include a balloon injury model of the rat carotid artery23 and an ovariectomized, cholesterol-fed model in rabbits.24 In contrast, another study in rabbits did not show such an effect of progesterone.25 In our study, progesterone did not appear to interfere with the vasculoprotective effect of estrogen. Progesterone levels were nearly doubled by hCG treatment of intact female mice, whereas they were not elevated at all in ovariectomized mice treated with estrogen. Despite these differences, the degree of protection against neointimal formation was equivalent in these 2 groups with supraphysiological estrogen levels. Progesterone also did not interfere with the protective effect of estrogen at physiological levels. Progesterone administration to ovariectomized mice also doubled progesterone levels, yet the degree of intimal proliferation was equivalent in this group and in intact females.
Receptors for estrogen mediate some of its biological
effects.26 27 28 Both the classic estrogen receptor- and
the recently identified estrogen receptor-ß29 have a
similar high affinity for estradiol. However, some ligands show
different relative affinities for the 2 receptors, and their tissue
distribution and relative levels also vary. We found specific staining
for both and ß in the ovaries, oviduct, brain, and blood vessels.
Estrogen receptor- and -ß staining was seen in intimal cells,
medial smooth muscle cells, and adventitial cells. Estrogen
receptor-ß staining was predominantly more nuclear than cytoplasmic
in the vessels. Staining in vessels was weaker than in the ovary and
oviduct for both receptors (Figure 6), and progesterone receptor
staining was stronger than estrogen receptor staining in vessels.
Progesterone receptor staining was predominantly nuclear and appeared
in the endothelium, medial smooth muscle, and
adventitia. Progesterone receptors are present in human
vessels,30 although their role in modulating the events
involved in atherogenesis are not clear.23 24 25
Our results demonstrate that the protective effect of pregnancy in the cuff injury model can be replicated by administration of chorionic gonadotropin or by elevated levels of estradiol. The effect of chorionic gonadotropin depends on intact ovarian function, because hCG had no vasculoprotective effect in ovariectomized mice. hCG itself has growth-stimulatory effects, which may explain the increased intimal and medial thicknesses of the hCG-treated ovariectomized mice compared with untreated ovariectomized mice. However, the I/M volume ratios were comparable. Our study does not address the potential effects of other hormones present during pregnancy, such as placental lactogen, which has growth hormone and prolactin-like effects. The estradiol levels of pregnant female mice, hCG-replaced intact females, and estrogen-replaced ovariectomized mice were all similar and were higher than physiological levels seen in normal, intact, cycling female mice. Estrogen levels were correlated with the degree of vascular protection, suggesting that ovarian estrogen synthesis may mediate the protective effects of pregnancy and hCG treatment. Furthermore, the protective effects of estrogen did not plateau at levels seen in female mice but continued to increase with higher estrogen levels, as seen in pregnant mice, hCG-treated, intact female mice, and estrogen-treated, ovariectomized mice. With further advances in our understanding of the molecular mechanisms by which estrogen acts, it may be possible to safely and specifically suppress the vascular responses to injury beyond the degree naturally observed in nonpregnant females.
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Acknowledgments |
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We gratefully acknowledge the support of National Heart, Lung, and Blood Institute (Bethesda, Md) grant HL-57818 and National Institute of Neurologic Diseases and Stroke NS-33335 (to P.L.H.). P.L.H. is an Established Investigator of the American Heart Association. We thank Attila Fabian for excellent technical assistance.
Received November 12, 1998; accepted January 29, 1999.
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