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Endocrinology Vol. 143, No. 6 2052-2061
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Genomic and Nongenomic Mechanisms of Nitric Oxide Synthesis Induction in Human Endothelial Cells by a Fourth-Generation Selective Estrogen Receptor Modulator

Tommaso Simoncini, Gaetano Varone, Letizia Fornari, Paolo Mannella, Michele Luisi, Fernand Labrie and Andrea R. Genazzani

Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, 56100 Pisa, Italy; and Centre Hospitalier Universitaire de Québec, Laval University (F.L.), Québec G1V 4G2, Canada

Address all correspondence and requests for reprints to: Tommaso Simoncini, M.D., Ph.D., Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Via Roma 57, 56100 Pisa, Italy. E-mail: . t.simoncini{at}obgyn.med.unipi.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cardiovascular disease is the leading cause of morbidity and mortality in postmenopausal women. EM-652 (acolbifene) is a fourth-generation selective ER modulator (SERM) exerting complete antiestrogenic effects on the breast and uterus. EM-652 potently inhibits bone resorption and induces positive lipid modifications in estrogen-deficient animals. As most of the cardioprotective actions of estrogen are exerted directly at the vascular level, we studied the effects of EM-652 on endothelial production of nitric oxide (NO) in vitro and in vivo. EM-652 triggers NO release by human umbilical vein endothelial cells through nongenomic mechanisms, rapidly activating endothelial nitric oxide synthase (eNOS) via an ER-dependent sequential activation of MAPKs and PI3K/Akt pathways independently from gene transcription or protein synthesis. Moreover, EM-652 increases eNOS protein levels during prolonged treatments. Upon pharmacological comparison, EM-652 is markedly more potent than the SERMs raloxifene and tamoxifen in increasing NO synthesis from endothelial cells. In ovariectomized and fertile rats, EM-652 increases aortic eNOS expression and enzymatic activity at low, but not at higher, dosages. The present data show that EM-652 (acolbifene) has estrogen-like activity on the vascular wall, directly increasing NO production through genomic and nongenomic mechanisms in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASE IS a major burden for postmenopausal women in western countries, representing the leading cause of death and permanent infirmity (1). The endocrine modifications of the climacteric exert detrimental effects on the blood vessels, which add to the ageing process (1). Indeed, menopause has recently been advocated as a new independent cardiovascular risk factor in women (2, 3). Hormone replacement therapies (HRTs) have been shown to be associated with improved cardiovascular risk parameters (4), fewer cardiovascular events (5), and reduced cardiovascular mortality (6). However, this has been questioned by two large trials that provided evidence that HRT in elder women with established coronary disease has no beneficial effect on future cardiovascular events or survival (7, 8) as well as on atherosclerotic lesions progression (9), therefore raising an ongoing debate about the usefulness of these treatments for cardiovascular disease prevention (3, 10).

Although estrogen has traditionally been considered to exert cardioprotective actions through positive modifications of the lipid profile (4), there has been accumulating evidence supporting the concept that the major part of the beneficial effects of female sex hormones are exerted directly at the vascular level through lipid-independent mechanisms (11, 12). Indeed, several vascular cell types express ER{alpha} and ERß and are functionally regulated by estrogen (11, 12). Furthermore, estrogen is able to recruit and activate both transcriptional (genomic) signaling mechanisms as well as nontranscriptional (or nongenomic) pathways in vascular cells, thus regulating physiologically important functions such as nitric oxide (NO) synthesis by endothelial cells (13, 14, 15).

Selective ER modulators (SERMs) are molecules exerting estrogen agonistic or antagonistic activities in different tissues (16). EM-652 (acolbifene) is a fourth-generation SERM characterized by complete antiestrogenic effects on the mammary gland and endometrium while being a potent inhibitor of bone resorption in animal models (17). This unique endocrine profile of EM-652 heralds the possibility that this compound could be an effective tool for postmenopausal women (17). However, an ideal compound for HRT should mimic the effects of natural estrogen on the cardiovascular system. In this regard, it is known that the various SERMs induce different (and not always beneficial) modifications of the lipid profile in postmenopausal women (16), and we have shown that parent SERMs are not equally effective in exerting antiatherogenic effects on human endothelial cells (18). Although preliminary data indicate that EM-652 effectively corrects the lipid modifications associated with ovariectomy in rats (19, 20), no data on possible direct actions on the vessel wall have been described to date.

As endothelial cell-derived NO plays a critical role in cardiovascular physiology and disease, we studied whether EM-652 (acolbifene) has direct vascular effects in vitro on human endothelial cells as well as in vivo in fertile or ovariectomized female rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and experimental treatments
Human umbilical vein endothelial cells (HUVEC) were harvested enzymatically with type I A collagenase (1 mg/ml) as previously described (21) and maintained in phenol red-free DMEM (Life Technologies, Inc., Gaithersburg, MD), containing HEPES (25 mmol/liter), heparin (50 U/ml), endothelial cell growth factor (50 µg/ml), L-glutamine (2 mmol/liter), antibiotics, and 10% FBS. Before each experiment, HUVEC were kept for at least 48 h in DMEM containing 10% estrogen-deprived (by activated charcoal-stripping) FBS. All experiments were performed on confluent monolayers of endothelial cells. Before every experiment investigating rapid, nontranscriptional effects (up to 30-min treatments), HUVEC were serum-starved in DMEM containing no FBS for 8 h before treatment to avoid the confounding effects of serum. Whenever an inhibitor was to be used (ICI 182,780, actinomycin D, cycloheximide, PD 98059, or wortmannin), we added the compound 30 min before treatment with EM-652 or 17ß-E2.

Animal treatments
Fertile female Wistar rats, weighing 150–199 g, were purchased from Harlan Nossan (Italy). Animals were kept under 14 h of illumination/d (0600–2000 h) and free access to standard rat chow and tap water. After 14 d from arrival, some rats were ovariectomized during the same estrous cycle stage (as indicated by daily vaginal smears). Ovariectomy was performed with a minilaparotomic incision under pentobarbital anesthesia (20 mg/kg). After complete healing of the surgical wounds, ovariectomized rats as well as fertile animals were started on treatment with EM-652.HCl or E2 valerate (or both) for 14 d. EM-652.HCl was administered orally to all animals as a 0.4% (vol/vol) suspension in methylcellulose, E2 valerate was administered orally after suspension in pure ethanol and subsequent appropriate dilution in 0.9% NaCl solution. All fertile rats were in the same stage of the estrous cycle at the beginning of treatment. At the end of treatment, animals were killed by decapitation under pentobarbital anesthesia (30 mg/kg), and the abdominal aorta was obtained after careful dissection and isolation of the vessel. Aortas were snap-frozen in dry ice and stored at -80 C up to the time of assay. Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals (http://www. nap.edu/readingroom/books/labrats).

Endothelial NO synthase (eNOS) activity assay
Endothelial cells were harvested in ice-cold buffer containing 320 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonylfluoride. Rat aortas were homogenized in the same buffer, and after centrifugation to discard the cell debris, the supernatants were used for eNOS assay. eNOS activity was determined as conversion of [3H]arginine to [3H]citrulline by incubating cell extracts for 10 min at 30 C in 50 mM potassium phosphate, 1.2 mM L-citrulline, 1.2 mM CaCl2, 120 µM nicotinamide adenine dinucleotide phosphate (reduced), and 24 µM L-[3H]arginine. Converted citrulline was separated by unconverted arginine using the acidic ion exchange resin Dowex 50 W (200–400 mesh, 8% cross-linked, Sigma, St. Louis, MO) as previously described (22). Extracts incubated with the eNOS inhibitor, NG-nitro-L-arginine methyl ester (1 mM), served as the blank. Converted eNOS activity was obtained by subtracting the blank from the samples. The counts per min value of untreated samples was taken as 1, and all other values were normalized to untreated samples and expressed as the fold increase vs. the control value.

Nitrite assay
NO production by endothelial cells was determined by a modified nitrite assay using 2,3-diaminonaphtalene as described previously (23). The fluorescence of 1-(H)-naphtotriazole was measured with excitation and emission wavelengths of 365 and 450 nm, respectively. Standard curves were constructed with known amounts of sodium nitrite. Nonspecific fluorescence was determined in the presence of N{omega}-monomethyl-L-arginine (3 mM).

Immunoblotting
Cell lysates or rat aorta protein extracts were separated by SDS-PAGE. Specific antibodies vs. human eNOS (Transduction Laboratories, Lexington, KY; catalog no. N 30020), Akt (catalog no. 06-558), [Ser473]phospho-Akt (catalog no. 06-801), [Thr308]phospho-Akt (catalog no. 06-558, Upstate Biotechnology, Inc., Lake Placid, NY), von Willebrand factor (vWF; catalog no. SC 8068), PR (catalog no. SC 539), and AR (catalog no. SC 816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were incubated with the membranes overnight at 4 C. The blots were hybridized with secondary antibodies coupled to horseradish peroxidase as previously described (13). Immunodetection was accomplished using enhanced chemiluminescence.

Statistical analysis
All values are expressed as the mean ± SD. Statistical differences between mean values were determined by ANOVA, followed by Fisher’s protected least significance difference test for comparison of mean values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EM-652 rapidly activates NO synthesis in human endothelial cells
Estrogen-deprived HUVEC treated with increasing concentrations of EM-652 for 30 min released NO (measured as the stable metabolites nitrites) in the culture medium in a concentration-dependent fashion (Fig. 1AGo).



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  		Figure 1. EM-652 increases NO release through nongenomic mechanisms. Estrogen-deprived, serum-starved HUVEC were treated for 30 min with increasing concentrations of EM-652 (0.1–10 nM). Cell culture media were assayed for nitrite concentrations (A), and cell lysates were harvested for measurement of eNOS activity (B) or Western analysis of eNOS or vWF protein (C). D, Estrogen-deprived, serum-starved HUVEC were exposed to EM-652 (1 nM, 30 min) in the presence or absence of the RNA or protein synthesis inhibitors actinomycin D (ACT-D; 5 µM) and cycloheximide (CHX; 10 µM), and nitrite release was assayed. Both inhibitors were added 30 min before initiating treatment with EM-652. Asterisks indicate a significant difference (P < 0.05) vs. control. Exp A, B, and D were repeated 3 times in triplicate, and the mean ± SD of the 9 replicates are shown. The blots in C are representative of 3 different experiments, with comparable results. The bars over the blots express the mean ± SD densitometric values of the eNOS bands from the three different experiments, evaluated as the ratio over vWF (the control value was arbitrarily designated 1, and other values were correspondingly normalized).

 
EM-652 rapidly stimulates NO synthesis through a nongenomic activation of eNOS
As 30 min was possibly too short a time for a genomic effect, we checked whether EM-652 triggered an enzymatic activation of eNOS, independently from eNOS gene regulation. As shown in Fig. 1BGo, EM-652 treatment was associated with increased eNOS enzymatic activity in endothelial cell lysates. There was a perfect correlation between eNOS activation and NO release (Fig. 1Go, A and B), thus suggesting that within 30 min the increased NO synthesis was completely due to stimulated eNOS activity. As further proof, we checked for eNOS protein amount in endothelial cells treated with EM-652 for 30 min, and no differences were found (Fig. 1CGo), thus indicating the absence of a transcriptional action. As a loading control, the membrane was reblotted for the endothelial marker vWF, which is not induced or down-regulated under the investigated conditions (Fig. 1CGo).

Furthermore, we treated endothelial cells with the SERM in the presence or absence of the transcriptional inhibitor actinomycin D or the translational inhibitor cycloheximide. Under such conditions no inhibition of EM-652-induced NO release by the RNA or protein synthesis blockers was observed, thus confirming that rapid activation of eNOS by EM-652 is exerted through nontranscriptional and nontranslational mechanisms (Fig. 1DGo).

Characterization of eNOS nongenomic activation by EM-652
When HUVEC were treated with a low concentration (1 nM) of EM-652, there was a rapid increase in NO synthesis and release, which followed a two-step kinetic. An initial 2- to 2.5-fold significant increase in NO concentration was detectable in culture medium after 5 min (Fig. 2AGo). Subsequently, a significantly more substantial NO release (~5- to 6-fold vs. untreated cells) ensued after 20 min of challenge with EM-652 (Fig. 2AGo). The same kinetic profile was seen for eNOS activation in cell lysates (Fig. 2BGo), suggesting that different signal transduction pathways may be recruited by EM-652 during eNOS activation.



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  		Figure 2. Characterization of the rapid NO synthesis induction by EM-652. Estrogen-deprived, serum-starved HUVEC were treated with EM-652 (1 nM) for different times in the presence or absence of the pure ER antagonist, ICI 182,780 (10 µM), and nitrite release in cell culture medium (A) or eNOS activity in cell lysates (B) was assayed. Asterisks indicate significant differences (P < 0.05) vs. control. Double asterisks indicate significant differences (P < 0.05) between the indicated bars. Exp A and B were repeated 4 times in triplicate, and the mean ± SD of the 12 replicates are shown. Estrogen-deprived, serum-starved HUVEC were treated with EM-652 (1 nM) for different times in the presence or absence of the MAPK kinase-1/2 inhibitor PD 98059 (5 µM) or the PI3K inhibitor wortmannin (30 nM), and nitrite release in cell culture medium (C) or eNOS activity in cell lysates (D) were assayed. Asterisks indicate significant differences (P < 0.05) vs. the time-corresponding EM-652 alone bar. Exp C and D were repeated 3 times in triplicate, and the mean ± SD of the 9 replicates are shown. Estrogen-deprived, serum-starved HUVEC were treated with EM-652 (1 nM) for different times in the presence or absence of the pure ER antagonist, ICI 182,780 (10 µM), and wild-type or Ser473- or Thr308-phosphorylated Akt protein amounts in cell lysates were assayed (E). The blots are representative of 3 different experiments with comparable results. The bars over the blots express the mean ± SD densitometric values of the Ser473-Akt and Thr308-Akt bands from the 3 different experiments, evaluated as the ratio over wild-type Akt (the value at 0 min was arbitrarily designated 1, and the other values were correspondingly normalized). P, Phosphorylated.

 
EM-652 induced NO synthesis and eNOS activation via pathways requiring functional ER, as EM-652 signaling to eNOS was blocked by preincubating endothelial cells with a molar excess of the pure ER antagonist ICI 182,780 (Fig. 2Go, A and B).

Role of MAPKs and PI3K/Akt pathways in eNOS nongenomic activation by EM-652
As rapid activation of eNOS by estrogen can be mediated via activation of MAPK (24) and PI3K/Akt (13, 14, 15) pathways, we checked the roles of these cascades for EM-652-dependent eNOS activation. We therefore treated estrogen-deprived, serum-starved HUVEC with EM-652 for different times in the presence or absence of the MAPK kinase-1/-2 inhibitor PD 98059 (5 µM) or the selective PI3K inhibitor wortmannin (30 nM). The ultra-rapid (5–10 min) increase in NO synthesis (Fig. 2CGo) and eNOS activation (Fig. 2CGo) was prevented by the MAPK inhibitor, but not by wortmannin, whereas the reverse was observed at later time points. As upon activation of PI3K by liganded ER the downstream effector Akt was activated in endothelial cells (13), we tested whether the same ensued upon treatment with EM-652. Indeed, we found a significant and time-consistent phosphorylation of Akt on residues Ser473 and Thr308 (which trigger Akt enzymatic activation) in HUVEC after 20 min of EM-652 exposure (Fig. 2EGo). Akt phosphorylation on both residues was prevented by pretreatment with ICI 182,780 (Fig. 2EGo), thus showing that PI3K/Akt recruitment by EM-652 depends on ER. These results provide a mechanistic explanation for acute EM-652-dependent NO production by endothelial cells, suggesting that nongenomic activation of eNOS by EM-652 relies on a sequential activation of MAPK and of PI3K/Akt signaling cascades.

Pharmacological comparison of different SERMs on nongenomic induction of NO synthesis
As EM-652 has higher affinity for ER{alpha} and ERß than other SERMs and is more effective at inhibiting bone resorption, we compared the pharmacological potencies of EM-652, raloxifene, and tamoxifen on NO synthesis induction in endothelial cells. As shown in Fig. 3AGo, treatment with EM-652 induced a significant increase in NO release, with an already near-maximal stimulation by EM-652 at a concentration of 0.1 nM and slightly lower effects at the highest concentration tested, whereas raloxifene and tamoxifen had a first statistically significant effect at 100 and 1000 nM, respectively.



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  		Figure 3. Pharmacological properties of EM-652-dependent eNOS activation. A, Increasing concentrations of EM-652, raloxifene or tamoxifen were used to treat estrogen-deprived, serum-starved endothelial cells for 30 min, and nitrite concentrations in the culture medium were measured. Asterisks indicate significant differences (P < 0.05) vs. control. The experiment was repeated 4 times in triplicate, and the mean ± SD of the 12 replicates are shown. B, Estrogen-deprived, serum-starved HUVEC were exposed for 30 min to 17ß-E2 (10 nM) or to 17ß-E2 (10 nM) plus increasing concentrations of EM-652, and nitrites were measured in the medium. Asterisks indicate significant differences (P < 0.05) vs. E2 alone. The experiment was repeated 3 times in triplicate, and the mean ± SD of the 9 replicates are shown.

 
To check for the pharmacological behavior of rapid EM-652 treatment of endothelial cells in the presence of E2, we exposed HUVEC to either 17ß-E2 alone (10 nM) or together with EM-652 (at increasing concentrations) for 30 min. Nitrite measurements showed a synergistic effect of EM-652 addition (Fig. 3BGo), suggesting that rapid activation of eNOS by E2 is not antagonized, but, on the contrary, is potentiated, by the presence of EM-652.

Regulation of eNOS expression in endothelial cells by EM-652
Beyond triggering nongenomic eNOS activation, EM-652 also modulates NO synthesis regulating eNOS expression. As illustrated in Fig. 4AGo, NO production by endothelial cells is further increased after a 48-h treatment with low concentrations of EM-652. Although part of this NO production may still be related to increased eNOS activity through nongenomic mechanisms, when the amount of eNOS in HUVEC lysates was measured, a clear increase in eNOS protein could be recognized in association with a 48-h EM-652 treatment (Fig. 4BGo). The increase in eNOS by EM-652 was concentration dependent, but a significant induction was found even at the lowest concentration used (0.1 nM; Fig. 4BGo). The induction of eNOS by EM-652 was ER dependent, as ICI 182,780 completely blocked eNOS up-regulation (Fig. 4BGo). The induction of eNOS expression in endothelial cells was not a nonspecific effect of EM-652, because under the same experimental conditions there was no increase in the endothelial marker vWF expression (Fig. 4BGo).



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  		Figure 4. Regulation of eNOS expression by EM-652. A, Estrogen-deprived endothelial cells were treated with EM-652 (1 nM) for 48 h, and nitrite release was assayed in cell culture medium. The asterisk indicates a significant difference (P < 0.05) vs. control. The experiment was repeated 4 times in triplicate, and the mean ± SD of the 12 replicates are shown. B, HUVEC were exposed for 48 h to increasing concentrations of EM-652 in the presence or absence of the pure ER antagonist ICI 182,780 (10 µM). Cell lysates were immunoblotted with antibodies vs. eNOS or vWF. The blots are representative of 3 different experiments with comparable results. The bars over the blots express the mean ± SD densitometric values of the eNOS bands from the 3 different experiments, evaluated as the ratio over vWF (the control value was arbitrarily designated 1, and the other values were correspondingly normalized). C, Increasing concentrations of EM-652, raloxifene, or tamoxifen were used to treat endothelial cells for 48 h, and nitrite concentrations in the culture medium were measured. Asterisks indicate a significant difference (P < 0.05) vs. control. Experiments were repeated 3 times in triplicate, and the mean ± SD of the 9 replicates are shown. Estrogen-deprived HUVEC were exposed for 48 h to 17ß-E2 (10 nM) or to 17ß-E2 (10 nM) plus different concentrations of EM-652, and nitrites were measured in the medium (D) or eNOS activity was assayed in cell lysates (E). Asterisks indicate significant differences (P < 0.05) vs. E2 alone. The experiments were repeated 3 times in triplicate, and the mean ± SD of the 9 replicates are shown.

 
Pharmacological analysis of SERMs on delayed activation of NO synthesis
Upon comparison with raloxifene and tamoxifen, EM-652 showed a high potency, similar to the rapid, nongenomic effects described above. Indeed, EM-652 was markedly more potent than the other SERMs and showed a bell-shaped curve of efficacy, with a decreasing stimulatory effect at the highest concentration tested, whereas raloxifene and tamoxifen exerted stimulatory effects at much higher concentrations (Fig. 4CGo).

When HUVEC were treated for 48 h with 17ß-E2 alone or together with different concentrations of EM-652, a peculiar behavior was found for NO release and eNOS activation, with synergic action at lower concentrations that is lost at higher ones (Fig. 4Go, D and E), suggesting that EM-652 may have an in vitro antiestrogenic effect at high concentrations.

In vivo effects of EM-652 on eNOS expression and activity
To test whether the findings of the effects of EM-652 on endothelial cells are also observed in vivo, we treated ovariectomized or fertile female Wistar rats for 14 d, and eNOS protein content and enzymatic activity in abdominal aortas were assayed.

Ovariectomized rats have markedly lower amounts of eNOS in aortic tissues compared with fertile animals (Fig. 5Go). The loss of eNOS after castration is dependent on estrogen deficiency, as it can be reversed by oral administration of E2 valerate in a dose-dependent fashion (Fig. 5Go). No changes were seen under the same conditions for vWF expression (Fig. 5Go).



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  		Figure 5. In vivo aortic eNOS expression is estrogen dependent. Ovariectomized Wistar rats were treated orally for 14 d with vehicle or different dosages of E2 valerate, and abdominal aorta tissue extracts were assayed for eNOS and vWF protein contents. Each band represents the blotted protein extract from a different animal’s aorta. A total of 12 animals for each experimental condition were assayed with consistent results, and representative blots are shown. The bars show the mean ± SD densitometric values of the eNOS bands from the different experiments, evaluated as the ratio over vWF (the ovariectomized value was arbitrarily designated 1, and other values were correspondingly normalized).

 
In fertile rats administration of EM-652 was associated with slightly increased levels of eNOS protein (Fig. 6AGo) as well as of eNOS activity at the lowest dose used (Fig. 6BGo), whereas higher doses were associated with eNOS protein levels slightly below the baseline (Fig. 6AGo) and with aortic eNOS activity similar to that in untreated rats (Fig. 6BGo). No effects of EM-652 were found on other relevant proteins, such as AR, PR, or vWF (Fig. 6AGo).



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  		Figure 6. In vivo effects of EM-652 on aortic eNOS in fertile rats. Fertile Wistar rats were treated orally for 14 d with different dosages of EM-652, and abdominal aorta tissue extracts were assayed for eNOS, AR, PR, or vWF protein contents (A) and eNOS activity (B). Ovariectomized Wistar rats were treated orally with EM-652 for 14 d, and abdominal aorta tissue extracts were assayed for eNOS, AR, PR, or vWF protein contents (C) and eNOS activity (D). Each band in A and C represents the aortic protein extract from a different animal. A total of 12 animals for each experimental condition were assayed with consistent results, and representative blots are shown. The bars show the mean ± SD densitometric values of the eNOS bands from the different experiments, evaluated as the ratio over vWF (the control value was arbitrarily designated 1, and other values were correspondingly normalized). Asterisks in B and D indicate a significant difference (P < 0.05) vs. control. Exp B and D were performed on 12 aortic extracts per condition in duplicate, and the mean ± SD of the 24 samples are shown.

 
EM-652 administration restored aortic expression of eNOS in ovariectomized animals, with maximal induction observed with the 0.1 µg/kg·d dose and decreasing efficacy at higher dosages (Fig. 6CGo). Enzymatic eNOS activity in the aortic homogenates followed a similar pattern, with a significant increase at the low dose and lower efficacy at higher doses, although the decrease at high doses was not statistically significant (Fig. 6DGo). Again, no effects on AR, PR, or vWF was detected (Fig. 6CGo).

To check for the possible antiestrogenic effects suggested by in vitro experiments, we administered to ovariectomized rats either E2 valerate alone or together with different doses of EM-652 and were able to demonstrate the same phenomenon observed in vitro, with a synergic action of EM-652 at the lower dose, where it further potentiates E2-dependent eNOS induction, but the loss of such effect at the higher doses (Fig. 7Go). This supports the possibility of an optimal dose for EM-652, beyond which the pharmacological efficacy on endothelial eNOS expression decreases.



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  		Figure 7. Effects of EM-652 on estrogen-dependent rat aortic eNOS expression. Ovariectomized Wistar rats were treated orally for 14 d with vehicle or with E2 valerate alone or together with different doses of EM-652, and aortic extracts were assayed for eNOS and vWF protein contents. Each band represents the protein extract from a different aorta. Twelve animals for each experimental condition were assayed with consistent results, and representative blots are shown. The bars show the mean ± SD densitometric values of the eNOS bands from the different experiments, evaluated as the ratio over vWF (the ovariectomized value was arbitrarily designated 1, and the other values were correspondingly normalized).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cardiovascular disease is a major concern for postmenopausal women, and estrogen deficiency represents an important concurrent factor in the pathophysiology of vascular degeneration. HRT is beneficial to postmenopausal women, but the side-effects may be significant, and the fear of developing uterine and breast cancer is an important reason for noncompliance. The development of compounds that act as estrogens selectively on the desired tissues, whereas behaving as estrogen antagonists on the mammary gland and endometrium thus has major clinical significance, but one of the required goals for an ideal molecule is that the positive effects of estrogens on the cardiovascular system not be lost.

Estrogen actions on the cardiovascular system are multifactorial, but the main part of the beneficial effects is exerted through actions on vascular cells (11, 12). Thus, ideally, a proposed cardiovascular-active SERM should be able to exert positive effects on the vascular wall (12). Our current knowledge of the vascular effects of SERMs is still very limited (12); however, there are indications that not all SERMs have the same effects at this level (18).

NO synthesis by endothelial cells is of paramount importance for the regulation of vascular tone and blood flow and for control of the hemostatic process (25). Furthermore, endothelium-derived NO is a potent antiinflammatory and antiatherogenic factor, being able to prevent endothelial cell dysfunction and vascular degenerative processes (25).

NO is a primary vascular target of estrogens. In fact, estrogens stimulate the synthesis and release of NO through the stimulation of eNOS gene expression (26) as well as through nontranscriptional activation of eNOS enzymatic activity (13, 14, 15). We have recently shown that the SERM raloxifene induces NO synthesis in endothelial cells (23). This finding is of potential pathophysiological importance, as this SERM has been shown to relax rabbit coronary arteries through a NO-dependent mechanism (27).

Our present data show that the fourth-generation SERM, EM-652 (acolbifene), which acts as a complete estrogen antagonist in the mammary gland and endometrium, exerts a typical estrogen-like effect on endothelial cells, triggering NO synthesis. We also demonstrate that EM-652 has a double mechanism of action, inducing a rapid nongenomic stimulation of eNOS activity as well as an up-regulation of eNOS protein upon longer exposure.

Nongenomic signaling through ERs is of primary importance for estrogen action. This is true also in endothelial cells, where we and others have recently shown that rapid NO synthesis upon exposure to estrogen relies on nongenomic activation of MAPK and PI3K pathways (13, 14, 15). EM-652-induced NO release by endothelial cells follows the same kinetic as natural estrogen. Indeed, upon challenge with EM-652, a biphasic increase in NO release from endothelial cells can be found; the first part (between 0 and 10 min) can be blocked by MAPK inhibitors, whereas the second (between 20 and 30 min) and more substantial part is sensitive to the presence of PI3K inhibitors. PI3K activation leads to the recruitment of Akt, which is responsible for the Ser/Thr phosphorylation and rapid activation of eNOS (28, 29). Our data show that EM-652 triggers Akt activation in endothelial cells that is time-consistent with eNOS activation and that can be prevented by blocking ER signaling. It is thus possible that ER activation by the fourth-generation SERM EM-652 triggers the sequential recruitment of MAPK and PI3K/Akt pathways in human endothelial cells, leading to rapid nongenomic eNOS activation.

Estrogen activation of the PI3K/Akt/eNOS cascade is pathophysiologically important in vivo, preventing leukocyte adhesion to the endothelium in animals (13). ER{alpha}/PI3K interaction takes place once ER{alpha} has undergone a proper ligand-induced conformational shift (13). However, each ligand induces a different ER conformation upon entering the hormone-binding pocket (30, 31), indicating that not all SERMs may be able to generate the right shape in the ER{alpha}. Therefore, the evidence of an activation of this cascade by EM-652 is probably of clinical importance.

The present data show that EM-652 is able to increase eNOS amounts in vitro in human endothelial cells as well as in vivo in rat aortas. Control of eNOS expression by estrogen is exerted at the transcriptional level through partially unknown mechanisms. Endothelial NOS gene promoter region contains no bona fide estrogen response elements, and estrogen has been shown to increase eNOS transcription by increasing the promoter-binding activity of the transcription factor Sp1 (26). On the other hand, increased eNOS levels after EM-652 treatment may also depend on posttranscriptional or posttranslational modifications stabilizing eNOS RNA or protein.

We here show that ovariectomy is associated with a dramatic decrease in eNOS amount in the aorta, and that estrogen supplementation in ovariectomized animals is sufficient to prevent this loss, supporting the pathophysiological importance of estrogen depletion for postmenopausal cardiovascular disease as well as the potential positive role of estrogen replacement in maintaining blood vessel function after menopause.

As some SERMs regulate the expression of target genes through mechanisms that are at least partially distinct from the ones activated by natural estrogen (32), it is important to observe that EM-652 is able to increase eNOS expression, its potency being much higher than that of raloxifene.

EM-652-induced eNOS up-regulation is ER dependent, as it is blocked by an excess of the pure antiestrogen ICI 182,780. The concentration of EM-652 required to increase eNOS synthesis is extremely low, as even at 0.1 nM eNOS induction is nearly maximal, confirming the observation of the high biological potency of this compound (17). The effects in vivo on fertile or ovariectomized rats are of particular interest. Indeed, there is a differential effect of low or high dosages of the compound, with the lower doses causing increases in eNOS levels as well as eNOS activity in aortas from estrogen-deficient and fertile rats. Higher doses, however, are associated with lesser increases in aortic eNOS protein in ovariectomized animals and a decrease in intact rats (although in the presence of unchanged eNOS enzymatic activities in the tissue homogenates), thus suggesting that in vivo there may be an optimal concentration at which EM-652 may exert full estrogen-like activity, whereas at higher concentrations it may have partial antiestrogenic effects. This is further supported by the dose-dependent effects of EM-652 on ovariectomized, estrogen-replaced animals, in which low doses of SERM act synergically with estrogen replacement therapy, producing a further increase in aortic eNOS, whereas higher doses have opposite effects.

In conclusion, we show that a fourth-generation SERM, EM-652 (acolbifene), is able to exert direct actions on the vascular wall in vitro and in vivo, thus increasing through nongenomic as well as genomic mechanisms endothelial NO production. The present data suggest that EM-652 may have direct cardiovascular antiinflammatory and antiatherogenic effects. Furthermore, our present data contribute to the debate on the differences between SERMs. Although these compounds are considered similar due to their common denomination, there are profound differences in the effects of the these molecules, including the ability to recruit nongenomic ER signaling or to exert direct regulatory actions on important tissues, such as the vascular wall, as well as the presence of dramatic differences in pharmacological properties, all of which may be of great clinical importance.


   Acknowledgments
 
We are indebted to Drs. M. Stomati and E. Casarosa for their help with the in vivo studies.


   Footnotes
 
This work was supported by a Young Researcher Grant from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (to T.S.).

Abbreviations: eNOS, Endothelial nitric oxide synthase; HRT, hormone replacement therapy; HUVEC, human umbilical vein endothelial cells; NO, nitric oxide; SERM, selective ER modulator; vWF, von Willebrand factor.

Received July 23, 2001.

Accepted for publication December 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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