Hypertension. 2008;51:1210-1217
Published online before print February 7, 2008,
doi: 10.1161/HYPERTENSIONAHA.107.106807
(Hypertension. 2008;51:1210.)
© 2008 American Heart Association, Inc.
Prolonged Ovarian Hormone Deprivation Impairs the Protective Vascular Actions of Estrogen Receptor 
Agonists
Christian Pinna;
Andrea Cignarella;
Paola Sanvito;
Valeria Pelosi;
Chiara Bolego
From the Department of Pharmacological Sciences (C.P., A.C., P.S., V.P., C.B.), University of Milan, Milan, Italy; and the Department of Pharmacology and Anaesthesiology (A.C., P.S., V.P., C.B.), University of Padova, Padova, Italy.
Correspondence to Andrea Cignarella, Department of Pharmacology and Anaesthesiology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy. E-mail andrea.cignarella{at}unipd.it
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Abstract
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The vascular consequences of estrogen treatment may be driven
by its initiation timing. We tested the hypothesis that the
duration of ovarian hormone deprivation before estrogen reintroduction
affects the role of estrogen as mediator of endothelial function
and vascular relaxation in nondiseased vessels. Rats were ovariectomized
and implanted with 17β-estradiol (E
2) or oil capsules 1,
4, and 8 months after surgery. After the longest hypoestrogenicity
period, acetylcholine-mediated aortic relaxation was attenuated
and insensitive to E
2 administration despite endothelial integrity.
Whereas no rapid vasorelaxant responses were elicited by an
estrogen receptor (ER) β–selective agonist, responses
to E
2 and an ER
selective agonist waned postovariectomy at any
given time and were restored by E
2 treatment after 1 and 4 months
but not 8 months postovariectomy. Accordingly, endothelial ER
mRNA and protein expression declined
6-fold after prolonged
hypoestrogenicity and was restored by estrogen replacement starting
1 month but not 8 months postovariectomy. Furthermore, the amount
of active phosphorylated endothelial NO synthase rose significantly
after E
2 replacement after 1 and 4 months but not 8 months postovariectomy.
The present findings document that the functional impairment
of the ER
/endothelial NO synthase signaling network after an
extended period of hypoestrogenicity was not restored by E
2 administration, providing experimental support to early initiation
of estrogen replacement with preferential ER
targeting to improve
cardiovascular outcomes.
Key Words: endothelium • hormones • pharmacology • NO synthase • receptors
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Introduction
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In spite of a large body of preclinical studies attesting beneficial
actions of estrogenic treatment on the cardiovascular system,
large clinical trials of hormone therapy so far have failed
to improve clinical outcomes (reviewed in Reference
1). In attempting
to explain the apparent discrepancy between experimental and
clinical results, the timing of treatment initiation has been
deemed a critical factor. The timing hypothesis proposes that
the earlier an estrogenic treatment starts, the more likely
it is of being successful, because the time since menopause
is a major risk factor for the development and progression of
atherosclerosis.
2,3 This is consistent with observations that
cyclic or permanent changes in circulating concentrations of
estrogen in premenopausal and postmenopausal women, respectively,
affect vascular responses.
4,5 Of note, estrogen deprivation
in rats time-dependently impairs endothelial function, as assessed
by the loss of acetylcholine-mediated dilation, but this response
is restored by early 17β-estradiol (E
2) replacement.
6 In
addition, estrogen affects endothelial vasomotor responses per
se. We demonstrated previously that ovariectomy abolishes acute
estrogen dilation, which is restored by timely E
2 replacement.
7 Thus, vascular relaxation is a primary target of estrogen action
in the vessel wall. This is known to occur through rapid stimulation
of NO production and modulation of NO synthase genes.
5,8,9
The cellular responses to estrogens are mediated by interactions with either nuclear- or membrane-located estrogen receptor (ER) or ERβ,10 of which the expression in the vasculature is highly regulated both by endocrine status and pathological conditions. For instance, ER gene expression decreases after ovariectomy in rat cerebral arteries and increases after E2 replacement therapy11 without changes in ERβ gene expression.12 The 2 ER isoforms not only are differentially modulated by circulating hormones but also mediate distinct actions in the vascular wall. In fact, ER conveys both short-term effects including endothelial dilation7 and long-term anti-inflammatory actions of E2.13 In this regard, we demonstrated previously that ER-selective agonists, unlike ERβ-selective agonists, induce acute vascular relaxation in the aorta from intact female or E2-replaced ovariectomized (OVX) rats.7 Such an activity, however, is undetectable in preparations from untreated OVX rats, suggesting that physiological levels of circulating E2 are essential for rapid vascular responses to be induced.7,14 This likely occurs through regulation of the levels of ER and cellular effectors thereof, such as endothelial NO synthase (eNOS) after E2 administration.15
Thus, the present study was designed to investigate whether incremental periods of estrogen deprivation affect rapid functional vasomotor responses to estrogenic agents and/or long-term effects of E2 treatment. In particular, we examined the impact of timing of E2 treatment initiation after ovariectomy on endothelial function by measuring the following: (1) rapid ex vivo aortic vasorelaxation induced by nonselective (E2) and novel selective ER agonists; (2) accumulation of the eNOS protein and its active phosphorylated form (peNOS); and (3) expression of ER.
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Methods
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Drugs and Reagents
Noradrenaline (NA), acetylcholine chloride, and E
2 were purchased
from Sigma-Aldrich. Propylpyrazole triol (PPT) and diarylpropionitrile
(DPN) was purchased from Tocris Cookson Inc. NA and acetylcholine
were dissolved in distilled water, whereas E
2 and PPT were freshly
dissolved in ethanol at a concentration of 1 mmol/L; further
dilutions were obtained in Krebs’ solution. The final
ethanol concentration in organ baths did not exceed 0.01%.
Animal Protocol
Experiments were performed on isolated aortic rings excised from female Sprague-Dawley rats initially weighing 200 to 225 g (Charles River, Calco, Italy). The animals were kept in temperature-controlled facilities on a 12-hour light/dark cycle and fed normal chow. Bilateral ovariectomy was performed under ketamine (40 mg/kg IP) and xylazine (20 mg/kg IP) anesthesia. OVX animals were divided into 3 groups and euthanized after 1, 4, and 8 months since ovarian ablation. Each group received subcutaneous implant of 2 silastic capsules containing 25 µL of vehicle (peanut oil) or E2 (235 µg/mL; 0.86 mmol/L) for 5 days before sacrifice.16,17 Plasma E2 concentrations after treatment approached the normal rat proestrus level.16 All of the procedures were performed in accordance with the guidelines for laboratory animal care of Milan University.
Isolated Organ Bath Experiments
The aorta was carefully removed, cleaned of fat and connective tissue, and cut into 5- to 6-mm rings. Vessels were suspended in 5-mL organ baths containing Krebs’ solution at 37°C, continuously bubbled with 95% O2 and 5% CO2. The Krebs’ solution had the following composition: 118 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.2 mmol/L of KH2PO4, 1.1 mmol/L of MgSO4, 2.5 mmol/L of CaCl2, 25 mmol/L of NaHCO3, and 5.5 mmol/L of glucose (pH 7.4). The rings were connected to isometric tension transducers (Ft 10, WPI) coupled with a digital recording system (PowerLab 8SP, ADInstruments). Vascular tissues were equilibrated for 30 minutes and contracted with 0.1 µmol/L of NA to develop a maximal response. Preparations were then washed with fresh Krebs’ solution, and the equilibration period was allowed to continue for an additional 30 minutes. Experiments were carried out on tissues precontracted with NA to 60% of maximal contraction. Cumulative concentration-response curves for E2 and PPT (ER selective agonist) were obtained over the concentration range 0.01 pmol/L to 100 nmol/L. To test vehicle effects, cumulative additions of equivalent ethanol dilutions were also performed. Cumulative concentration-response curves were also obtained for acetylcholine (10–9 to 10–5 mol/L). Relaxant responses were expressed as the percentage of relaxation of NA-precontracted tissues.
Endothelium Isolation
The thoracic aorta was longitudinally opened on a Petri dish. The endothelial layer was removed by gently scraping the lumen and transferred into a 1.5-mL Eppendorf tube containing PBS. The tissue pooled from 2 aortas was centrifuged at 8000 rpm for 5 minutes and the supernatant discarded. The pellet was dissolved in RNA lysis solution and frozen at –80°C until RNA analysis or resuspended in 30 µL of lysis buffer18 and frozen until protein analysis. Immunodetection of the endothelial marker eNOS in 15 µg of lysate protein gave similar results to that performed in 15 µg of protein from cultured rat aorta and human umbilical vein endothelial cells.7
Histology and Immunohistochemistry
The thoracic aorta was gently removed, cleaned, and washed in PBS-7% saccharose overnight. Aortic rings were then included in OCT embedding compound and frozen in liquid nitrogen. Ten-micrometer sections were fixed with cold acetone and stored at –80° until use. Endothelium integrity was assessed using hematoxylin-eosin staining (Mayer’s Hematoxylin, Sigma, Eosin Y, BHD). For immunohistochemical staining, sections were incubated in blocking buffer for 40 minutes. To assess the presence of eNOS, peNOS, and ER in the aortic endothelium, sections were incubated overnight with an appropriate primary antibody (anti-eNOS and anti-ER, 1:10, Santa Cruz Biotechnology Inc; anti-peNOS, 1:10, Zymed). After washing with PBS, tissues were incubated with biotinylated secondary antibody (1:1000, anti-rabbit) for 30 minutes and then washed. Endogenous peroxidase activity was blocked with H2O2 before incubation with streptavidin-conjugated horseradish peroxidase for 30 minutes. Sections were then stained with diaminobenzidine (Vector) to detect immunoreactivity. The presence of a brown precipitate indicated positive findings for primary antibodies. Negative controls were treated with rabbit IgG instead of primary antibodies.
Western Blot Analysis
After 4 freeze-thaw cycles, endothelial protein lysates were boiled for 5 minutes and centrifuged at 13 000 rpm for 15 minutes. Total protein was determined using Lowry’s method. Cell lysate protein was size fractionated on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto Hybond ECL membranes (GE HealthCare). After blocking with 5% nonfat dry milk (Bio-Rad), membranes were hybridized overnight with anti-eNOS (1:800, Santa Cruz Biotechnology), anti-peNOS (pS1177, phospho-specific, BD Transduction Laboratories), or anti-ER antibody (1:500, Santa Cruz Biotechnology); washed; and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. After extensive washing, membranes were developed using enhanced chemiluminescence reagents (GE HealthCare). The uniformity of protein load and transfer efficiency across the test samples was verified with Ponceau staining.
Quantitative Real-Time PCR
RNA was extracted using a commercially available kit (Ambion). RNA (1 µg) was reverse transcribed using random hexamer primers and SuperScript (Invitrogen). Amplification was performed on the Taqman 7000 (Applied Biosystems) using FastStart TaqMan Probe Master (Roche Applied Sciences), gene-specific TaqMan probes (Applied Biosystems) and primers, and a standard 2-steps thermal cycler protocol (95°C for 15 seconds and 60°C for 1 minute, repeated 40 times). Relative quantification of gene expression was calculated by the comparative CT method and normalized to the eukaryote housekeeping gene 18S.
Statistical Analysis
All of the data are expressed as means±SEMs of 4 to 5 independent experiments, each value representing means±SEMs of duplicate or triplicate determinations. Concentration-response curves were obtained with software Prism (GraphPad Software Inc). Potency (pD2) values of and maximal responses (Emax) to pharmacological agents were compared by 1-way ANOVA followed by Bonferroni’s posthoc test as appropriate, using Minitab software. Western blot and real-time PCR data are the means±SEMs of 3 independent experiments. The comparison between groups (OVX versus estrogen-replaced) was performed by 1-way ANOVA. Values of P<0.05 were considered significant.
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Results
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The hormone regimen used in this study produces circulating
E
2 concentrations of 52 to 58 pg/mL.
16 E
2 treatment in OVX animals
restored uterine weight at all of the time points, whereas it
had no effect on body weight (
Table 1).
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Table 1. Body and Uterine Weight in OVX Rats Implanted 1, 4, or 8 Months After Surgery With Oil or E2 Capsules for 5 Days
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The functional capacity of the endothelium ex vivo was tested using arterial rings isolated from thoracic aorta by obtaining concentration-response curves to acetylcholine, an endogenous endothelium-dependent vasodilator triggering eNOS-mediated NO production. Acetylcholine induced full vascular relaxation with comparable efficacy and potency in precontracted aortic tissues from oil- and E2-treated rats after 1 and 4 months of hypoestrogenicity (Figure 1A and 1B). By contrast, acetylcholine-induced relaxation was impaired in aortic tissues from 8-month OVX oil-treated rats and was not improved by E2 replacement (Figure 1C). The pharmacological parameters of acetylcholine efficacy (Emax) and pD2 in treatment groups are shown in Table 2.
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Table 2. Pharmacologic Assessment of Emax and pD2 (log EC50) From Concentration-Response Curves to Acetylcholine, E2, and PPT in Aortic Rings From OVX Rats Implanted 1, 4, or 8 Months After Surgery With Oil (n=4) or E2 (n=5) Capsules for 5 Days
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After endothelial function assessment, rapid responses to increasing concentrations of exogenous E2 (0.01 pmol/L to 100 nmol/L) were recorded in aortic preparations. No vasodilating response was induced by E2 in tissues from oil-replaced rats irrespective of the duration of estrogen deprivation (Figure 2A through 2C). A significant 22% relaxation to the exogenous hormone was observed in tissues from E2-replaced rats that underwent surgery 1 and 4 months earlier (Figure 2A and 2B). By contrast, no significant relaxant responses (P>0.05 versus oil) were elicited by exogenous E2 in aortic tissues from 8-month OVX E2-replaced rats (Figure 2C). The pharmacological parameters of E2 efficacy (Emax) and pD2 in treatment groups are shown in Table 2. Because the rapid vascular relaxation to E2 is mediated solely by ER,7 we also obtained concentration-response curves to the ER selective agonist PPT. The relaxant responses to this agent were entirely overlapping to those elicited by the physiological nonselective ER ligand E2 (Figure 2A through 2C). The pharmacological parameters of PPT efficacy (Emax) and pD2 in treatment groups are shown in Table 2. To confirm that ERβ is not involved in rapid vasorelaxation, we obtained concentration-response curves to the ERβ selective agonist diarylpropionitrile, which failed to affect vascular tone irrespective of OVX duration and E2 replacement (Figure 3A through 3C). The pharmacological parameter of diarylpropionitrile efficacy (Emax) in treatment groups is shown in Table 3.
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Figure 2. Concentration-response curves to the nonselective ER agonist E2 (triangles) and the ER-selective agonist PPT (squares) in aortic rings isolated from rats that were ovariectomized and implanted 1 month (A), 4 months (B), or 8 months (C) later with E2 (black symbols) or oil (white symbols) capsules for 5 days. Aortic rings were precontracted with NA to 60% of maximal contraction before cumulative ER agonist addition. Data are expressed as the percentage of relaxation±SEM of 4 to 5 sets of independent experiments.
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Table 3. Pharmacologic Assessment of Emax and pD2 (log EC50) From Concentration-Response Curves to Diarylpropiolnitrile in Aortic Rings From OVX Rats Implanted 1, 4, or 8 Months After Surgery With Oil (n=4) or E2 (n=5) Capsules for 5 Days
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To test whether morphological changes in the vasculature after 8-month ovariectomy rendered the vessel nonresponsive to E2-induced relaxation, sections of the aorta were obtained at different time points. Hematoxylin-eosin staining (Figure 4) provided no evidence for visible injury or gross vascular changes irrespective of OVX duration and E2 replacement, suggesting the occurrence of functional rather than morphological impairment of the endothelium after long-term ovariectomy.
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Figure 4. Histological analysis of aortic tissues from rats that were ovariectomized and implanted 1, 4, or 8 months later with E2 or oil capsules for 5 days. Ten-micrometer sections were stained with hematoxylin (blue, nuclei) and eosin (pink, cytoplasm). No differences were observed among groups with regard to endothelial integrity. Bar=50 µm.
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Because eNOS is a key determinant of endothelium-dependent relaxation, as well as an effector of estrogen action in the vessel wall, we assessed the amount of the active peNOS in endothelial lysates from rats at different time since ovarian surgery. The effectiveness of E2 treatment was witnessed by the 3- to 4-fold increase in peNOS versus oil in the endothelium from rats that were deprived of estrogen for 1 or 4 months (Figure 5A) as shown by Western blotting experiments. By contrast, the amount of endothelial peNOS did not increase after E2 treatment in the endothelium from rats that had been deprived of estrogen for 8 months (Figure 5A). Total eNOS protein levels in the aortic endothelium of oil-treated rats were also increased by E2 treatment after 1 or 4 months postovariectomy (Figure 5B), yet to a lower extent as compared with peNOS. As shown with peNOS above, E2 reintroduction after 8 months led to nonsignificant changes in eNOS protein levels (Figure 5B).
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Figure 5. Western analysis of Ser1177-peNOS (A) and total eNOS (B) of endothelial lysates isolated from rats that were ovariectomized and implanted 1, 4, or 8 months later with E2 or oil capsules for 5 days. Each band is from a 15-µg protein sample from whole-cell lysate, whereas loading control was performed through β-actin immunodetection. For reference, the same protein amount from human umbilical vein endothelial cells (HUVEC) and rat smooth muscle cells (RSMC) was applied in the right lanes. Data are from 3 sets of independent experiments. *P<0.05 vs timing-matched oil.
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In attempting to define a potentially unifying explanation for the functional and biochemical events in the arterial wall described so far, we found that E2 treatment doubled endothelial ER mRNA expression in tissues from 1-month OVX rats as assessed by real-time PCR (Figure 6). Although a similar relative increase in ER mRNA levels was observed in the endothelium of 8-month OVX E2-treated rats, ER expression dropped dramatically in 8 months as compared with 1-month OVX rats (Figure 6). ER protein as measured by Western blotting was upregulated by E2 treatment in keeping with real-time PCR data (Figure 7) and was identical to a standard recombinant ER protein (Figure 7). Finally, immunohistochemical analysis for ER, peNOS, and eNOS in aortic tissues from 1-month as compared with 8-month OVX E2-treated animals revealed marked negative regulation of the former proteins with less apparent changes in the levels of the more abundantly expressed eNOS (Figure 8). This suggests that prolonged hypoestrogenicity disrupted the ER/eNOS signaling network in nondiseased rat arteries.
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Figure 7. Western blot analysis of ER of endothelial lysates isolated from rats that were ovariectomized and implanted 1 month later with E2 or oil capsules for 5 days. Each band is from a 15-µg protein sample from whole-cell lysate, whereas loading control was performed through β-actin immunodetection. For reference, the same protein amount of recombinant origin was applied in the right lanes. Data are from 3 sets of independent experiments. *P<0.05 vs oil.
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Discussion
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To the best of our knowledge, this is the first demonstration
that E
2 treatment did not exert beneficial effects on the biology
of the arterial wall in rats after prolonged estrogen deprivation,
such as that ensuing after 8 months postovariectomy, while retaining
efficacy when administered within a window of 4 months after
ovariectomy. Although the vascular actions of E
2 are abrogated
in surgically postmenopausal atherosclerotic monkeys,
19 the
present study provides possible mechanisms whereby the vascular
actions of exogenous E
2 in nondiseased vessels were abrogated
simply on prolonged deprivation of the endogenous hormone. In
this context, 2 key players were identified in dictating vascular
estrogen efficacy, namely, ER
and eNOS. The 2 proteins are organized
into a functional signaling module in caveolae as demonstrated
in cultured endothelial cells.
15,20 Although it is well established
that eNOS is modulated by E
28,21,22 through both genomic and
nongenomic activation of ER
,
23,24 the present findings render
ER
and eNOS as primary targets for the loss of efficacy of delayed
E
2 reintroduction after ovarian hormone deprivation. In addition,
the present findings are relevant to rapid actions of ER agonists,
as well as long-term effects of E
2 replacement against the background
of estrogen deprivation. The fact that ER
agonists, at least
in part, induce their protective effects via NO, as first shown
by Rosselli et al,
25 suggest that NO or endothelial-dependent
relaxation may serve as an important marker to assess whether
estrogen therapy should be initiated or not.
Acetylcholine is often used to determine the functional capacity of the endothelium. In postmenopausal women, intracoronary infusion of estrogen selectively enhances endothelium-dependent dilation by acetylcholine, supporting a role for estrogen in acetylcholine-mediated responses.26 In the present study, E2 reintroduction after 8 months postovariectomy failed to enhance attenuated acetylcholine-mediated dilation because of prolonged E2 deprivation, differently from what observed previously in 3- to 18-month OVX rats with estrogen replacement starting on the day of surgery.6 Furthermore, E2 reintroduction after 8 months but not 1 or 4 months postovariectomy failed to restore the rapid relaxant response to nonselective and ER-selective agonists that was abolished by estrogen deprivation (Figure 2 and Table 2). Because endothelial integrity was not affected by long-term ovariectomy (Figure 4), these results emphasize that uncontrolled long-term ovariectomy impaired endothelial function so that it became insensitive to estrogen replacement.
Our results also indicate that a major pathway of E2’s beneficial actions in the arterial wall was enhanced eNOS activation through phosphorylation of Ser1177, which is mediated by the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells.20,27 Accordingly, the increase in eNOS and even more so in peNOS levels in response to E2 replacement in 1- and 4-month OVX animals was blunted in 8-month OVX animals most likely because of the marked fall in ER expression observed in the same tissues, resulting in functional impairment of the ER/eNOS signaling network. Consistent with the functional assessment using nonselective and ER-selective agonists, ER mRNA and protein expression sharply decreased after long-term as compared with short-term ovariectomy and was positively regulated by E2 treatment regardless of timing. This is in contrast to the previous observation of nonsignificant changes in ER expression as evaluated (not quantitatively) in the thoracic aorta (not in the endothelium) after OVX and E2 treatment,28 although the schedule of ovariectomy and treatment with E2 in that study largely differed from ours. Our data also illustrate that the positive regulation of ER mRNA and protein levels by E2 treatment after 8 months postovariectomy restored only 20% of the ER mRNA levels seen in the endothelium of oil-treated 1-month OVX rats (Figures 6 and 8
), making it unlikely that such a reduced ER pool was capable of mediating biological actions of E2 therapy. Thus, loss of ERs in the artery wall occurs not only in atherosclerotic arteries29 but also in nondiseased vessels on prolonged estrogen deprivation. Although ERβ expression was not assessed in the present study, this ER isoform does not appear to be relevant to rapid vasorelaxant responses to ER agonists (Figure 3 and Reference 7). On the other hand, the efficacy of our E2 replacement regimen was documented by the uterotrophic effect independent of hypoestrogenicity duration. Overall, prolonged hypoestrogenicity induced genomic effects on ER expression that had profound impacts on its signaling network and vascular biology in terms of outcome of E2 administration and rapid endothelium-dependent relaxation to estrogenic agents. It should be pointed out, however, that additional ER-dependent and independent mechanisms, including inhibitory effects on smooth muscle cell growth, may account for protective effects of E2 on the vasculature.
Perspectives
This study provides experimental proof of concept that the timing of initiation of E2 treatment after loss of ovarian hormone production is critical to therapeutic cardiovascular outcomes.2 Accordingly, recent animal studies show that prolonged hypoestrogenicity suppresses the neuroprotective and anti-inflammatory actions of E2.30 Beyond basic science contributions, the timing issue was raised by observational studies31 and validated to some extent by secondary analysis of the large-scale Women’s Health Initiative Trial,32 with further trials ongoing. Thus, the time may have come to integrate findings from fundamental research into clinical practice to successfully exploit the beneficial actions of estrogenic agents on the cardiovascular system.
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Acknowledgments
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Sources of Funding
This study was supported by grants from the University of Milan (to C.P.) and the University of Padova (to A.C.).
Disclosures
None.
Received December 3, 2007;
first decision December 12, 2007;
accepted December 19, 2007.
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Abstract
Introduction
) or oil (
) capsules for 5 days. Aortic rings were precontracted with NA to 60% of maximal contraction before cumulative ACh addition. Data are expressed as the percentage of relaxation±SEM of 4 to 5 sets of independent experiments.
P<0.01 vs 1-month E2.