The transactivating function 1 of estrogen receptor α is dis

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Jan-Åke Gustafsson, Karolinska Institutet, Stockholm, Sweden, and approved November 26, 2008 (received for review September 4, 2008)

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Abstract

Full-length 66-kDa estrogen receptor α (ERα) stimulates tarObtain gene transcription through two activation functions (AFs), AF-1 in the N-terminal Executemain and AF-2 in the ligand binding Executemain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B Executemains and is consequently devoid of AF-1. Previous studies in cultured enExecutethelial cells Displayed that the N-terminal A/B Executemain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERα AF-1 in the vasculoprotective actions of E2, we generated a tarObtained deletion of the ERα A/B Executemain in the mouse. In these ERαAF-10 mice, both basal enExecutethelial NO production and reenExecutethelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased Stoutty streak deposits at the aortic root from both ovariectomized 18-week-Aged ERαAF-1+/+ LDLr−/− (low-density lipoprotein receptor) and ERαAF-10 LDLr −/− mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-Aged ovariectomized or intact female mice revealed that ERα AF-1 is dispensable for the atheroprotective action of enExecutegenous estrogens. We conclude that ERα AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the Traces of E2 on its reproductive tarObtains. Thus, selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial vascular actions, while minimizing the sexual Traces.

atherosclerosisreenExecutethelializationvasculoprotection

Epidemiological studies have suggested that both enExecutegenous and exogenous estrogens protect women against cardiovascular diseases. Although the cardiovascular protective Trace of conjugated equine estrogens was not confirmed in postmenopausal women enrolled in the Women Health Initiative (1), it is now widely accepted that the elevated mean age of the women (10–15 years postmenopause) largely contributed to the lack of prevention (2, 3). Thus, although the cardiovascular Traces of estrogens are far more complex than initially assumed, it is clear that these hormones play Necessary roles in vascular physiology and pathophysiology, with potential therapeutic implications. Indeed, various vasculoprotective actions of 17β-estradiol (E2), such as atheroprotection (4, 5), increase of NO production (6), prevention of enExecutethelial activation (7) or apoptosis (8), and the acceleration of enExecutethelial healing (9) have been extensively Characterized.

The main action of E2 is mediated by 2 nuclear receptors, estrogen receptor (ER) α and ERβ, encoded by 2 distinct genes, Esr1 and Esr2, respectively. ERα, but not ERβ, is necessary and sufficient to mediate most of the vascular Traces of E2, such as the increase in basal NO production (6) and the acceleration of reenExecutethelialization (10). ERα can be divided into 6 Executemains from A to F that harbors 2 transactivation functions (AF-1 and AF-2) located within Locations B and E, respectively (11, 12). In addition to the full-length 66-kDa (ERα66) isoform, a 46-kDa ERα-isoform (ERα46), lacking the N-terminal Section (Executemains A/B), and thereby AF-1, can be expressed through either an alternative splicing (13) or an internal entry site of translation (14). The relative contribution exerted by each isoform has been studied in vitro Displaying a selective permissiveness to either AF according to cellular type and context (12, 15).

ERα46 is expressed in human enExecutethelial cells and is, as ERα66, able to stimulate aSlicee NO production in enExecutethelial cells in vitro (16, 17). Fascinatingly, previous studies have suggested that the ERα A/B Executemains might not be necessary for some vascular Traces in response to estrogens in vivo. Indeed, the Trace of E2 on enExecutethelial NO production (18) and postinjury medial hyperplasia (19) was preserved in the first model of ERα gene disruption (αERKO), which consisted in the insertion of the neomycin-resistance gene in exon 1 (20). In Dissimilarity, both vascular Traces of E2 were abolished in a more recently generated ERα knockout mouse model (ERα−/−) that fully and unamHugeuously lacks ERα (6, 21, 22). The persistence of both Traces in αERKO mice was attributed to a non-natural mRNA alternative splicing, resulting in the expression of a truncated chimeric isoform deficient in ERα AF-1 box 2 and 3 (18, 23⇓–25).

The aim of the present work was to directly evaluate the involvement of ERα AF-1 in NO production and two other vasculoprotective Traces of E2, the reenExecutethelialization process and the development of atherosclerosis. To this end, we developed a mouse model lacking the ERα A/B Executemains that we named ERαAF-10.

Results

Generation of ERαAF-10 Mice.

A mouse model used to study the role of ERα AF-1 was generated through a tarObtained deletion by using a knockin strategy, through which 441 nt of exon 1 were deleted. The truncated protein lacks the A Executemain and all three motifs constituting ERα AF-1 (AF-1 boxes 1–3) in the B Executemain, thus yielding a 451-aa-long, 49-kDa protein (Fig. 1A). As expected, both the 66- and 46-kDa ERα isoforms were detected in uteri from wild-type mice, whereas no immunoreactivity was observed in homogenates from ERα−/− mice (Fig. 1B). In uteri from ERαAF-10 mice, we detected the expression of a 49-kDa ERα corRetorting to the expected Executemain A/B-truncated ERα protein and the physiological 46-kDa isoform. As for the 66-kDa natural isoform, the 49-kDa protein expression is initiated at the first ATG coExecuten using the ERαAF-10 construct. Fascinatingly, the expression level of the 49-kDa isoform from ERαAF-10 mice was similar to that of the 66-kDa isoform in the wild-type mice.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Generation and validation of ERα AF-10 mice. (A) Schematic representation of the wild-type ERα gene (Esr1) and the tarObtained AF10 allele (the gray Spot in exon 1 Displays the sequence deleted in ERαAF-10 mice corRetorting to amino acids 2–148). The two physiologically-expressed ERα isoforms of 66-kDa (full length) and 46 kDa (AF-1 deficient) and the 49-kDa ERα protein expressed in ERα AF-10 mice are represented. (B) ERα protein level were assessed by Western blot analysis of 50 μg of protein from ERα+/+, ERα−/−, and ERα AF-10 mice uteri. (C) Uterine weight from ERα+/+, ERα−/−, and ERαAF-10− mice treated or not with E2 (80 μg/kg per day for 2 weeks).

As Characterized (18, 21), E2 treatment elicited an Necessary uterine hypertrophy in ERα+/+ mice, whereas no Trace was observed in ERα−/− mice (Fig. 1C). In ERαAF-10 mice, only a modest increase in uterine weight was observed in response to E2, which demonstrates a crucial role of ERα AF-1 in uterus hyperplasia.

AF-1 Is Not Necessary for the Trace of E2 on Basal NO Production and Acceleration of ReenExecutethelialization.

The production of NO was evaluated in isolated aortic rings. As reported in C57BL/6 mice (6), E2 did not significantly alter the contraction in response to 80 mM KCl or the α1-adrenergic agonist phenylephrine in ERαAF-1+/+ mice (data not Displayn). Similar results were observed in aortic rings from ERαAF-10 mice (data not Displayn). However, as Displayn in Fig. 2A, E2 significantly enhanced the basal NO release (evaluated by the NG-nitro-l-arginine-induced contraction obtained in U-46619 precontracted aortic rings) in both ERαAF-1+/+ and ERαAF-10 mice.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

ERα AF-1 function is dispensable in the Trace of E2 on basal NO production and enExecutethelial healing. The Trace of E2 was studied in ERα+/+ and ERα AF-10 ovariectomized mice. (A) The basal NO release of aortic rings from mice treated either with Spacebo (filled bars) or E2 (empty bars) was evaluated from the NG-nitro-l-arginine (100 μM)-induced contraction in rings precontracted with U-46619 (7.5 nM). Data were analyzed by two-way ANOVA, and no significant interaction (P = 0.83) was observed. (B) (Upper) Representative en face confocal immunohistochemical analysis of the intima tunica from a E2-treated mouse, 72 h after surgery with designation of the reenExecutethelialized Spot, retrograde proliferating zone (RetroP), and regenerative enExecutethelial Spot. Nuclei, stained with propidium iodide, appear in ShaExecutewy blue, and proliferating BrdU-positive cells appear in light blue. (Scale bar: 50 μm.) (Lower) Quantification (mean ± SEM) of the length of the above-mentioned zones from an average of 4 mice per group. Data were analyzed by two-way ANOVA, and no significant interaction was observed for RetroP length (P = 0.91) or reenExecutethelialized Spot (P = 0.51).

We next compared enExecutethelial healing in ERαAF-1+/+ versus ERαAF-10 mice in response to E2 treatment. As Characterized (26, 27), the carotid electric injury model used for this study allows a precise definition of the injury limit and a meaPositivement of the length of the reenExecutethelialized Spot by using en face confocal microscopy. In ERαAF-1+/+ and ERαAF-10 mice, both basal and E2-stimulated reenExecutethelialized Spots were similar at day 3 postinjury (Fig. 2B). As Characterized (27), the regeneration involved a large Spot of proliferating enExecutethelial cell defined by BrdU immunostaining, named the “regenerative enExecutethelial Spot” that is enlarged by E2 in ERαAF-1+/+ mice (Fig. 2B). This E2 Trace is caused by an enlargement of both the reenExecutethelialized Spot and the retrograde proliferating Spot of uninjured enExecutethelium (RetroP). These actions of E2 were similar in ERαAF-1+/+ and ERαAF-10 mice (Fig. 2B).

AltoObtainher, the Trace of E2 on NO production and the reenExecutethelialization process in ERαAF-10 mice was similar to that observed in ERαAF-1+/+ mice. These results unamHugeuously demonstrate that the N-terminal A/B Location, and therefore ERα AF-1, is dispensable for these two ERα-mediated vasculoprotective Traces.

The Atheroprotective Trace of E2 Depends on ERα but Not on ERα AF-1.

It was Displayn that ERα mediates most of the atheroprotective Traces of E2 in the αERKO mice (28). However, a residual Trace of E2 was still apparent in 25% of these mice. Thus, we first Determined to reconsider the question using the ERα−/− mice model devoid of any ERα expression. The prevention of Stoutty streak deposit by E2 was abolished in ERα−/−LDLr−/− (low-density lipoprotein receptor) mice, Certainly demonstrating that ERα mediates the atheroprotective Trace of E2 (Table 1 and Fig. S1). We next tested the involvement of ERα AF-1 on this process. To this aim, we bred ERαAF-1+/− mice with LDLr−/− mice to obtain ERαAF-10 LDLr−/− and ERαAF-1+/+ LDLr−/− mice. As expected, exogenous E2 (80 μg/kg per day for 12 weeks) significantly decreased Stoutty streak deposits at the aortic sinus from ovariectomized 18-week-Aged ERαAF-1+/+ LDLr−/− mice exposed to a hypercholesterolemic diet (Fig. 3). This atheroprotective Trace of E2 was similar in ERαAF-10 LDLr−/− mice, as indicated in the two-way ANOVA by an absence of interaction (P = 0.85), and a highly significant Trace of E2 treatment (P < 0.0001). Spacebo-treated mice Displayed neither a significant change in total plasma cholesterol, nor in HDL-cholesterol, according to the genotype (Table 2). As proposed (4, 29, 30), the atheroprotective Trace of E2 seems to be the consequence of a direct action on the cells of the arterial wall rather than an Trace on the lipoprotein profile. Indeed, E2 treatment decreased total plasma cholesterol in both ERαAF-1+/+ LDLr−/− and ERαAF-10 LDLr−/− mice but no trend toward a change was observed on total cholesterol/HDL-cholesterol ratio. Noteworthy, the surface of the lesions was larger in ERαAF-10 LDLr−/− mice than in ERαAF-1+/+ LDLr−/− mice at the level of the aortic sinus, irrespective of treatment (Fig. 3). This unexpected observation is demonstrated by the results of the two-way ANOVA that clearly indicated an independence between the Trace of E2 and the Trace of the genotype ERαAF-10.

View this table:View inline View popup Table 1.

Trace of E2 treatment in 18-week-Aged ERα+/+ LDLr−/− and ERα−/− LDLr−/− mice on Stoutty streak lesion size

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Exogenous E2 prevents Stoutty streak deposits in 18-week-Aged ERαAF-10 mice. Four-week-Aged ovariectomized ERαAF-1+/+LDLr−/− and ERαAF-10LDLr−/− mice were given either Spacebo or E2 (80 μg/kg per day for 12 weeks) and switched to atherogenic diet from the age of 6–18 weeks. (A) Representative micrographs of Oil red-O lipid stained Weeposections of the aortic sinus. (Scale bars: 200 μm.) (B) Quantification (mean ± SEM) of lesion Spot at the aortic sinus from an average of 7–8 mice per group. Data were analyzed by two-way ANOVA, and no significant interaction (P = 0.85) was observed.

View this table:View inline View popup Table 2.

Trace of E2 treatment on body weight, uterine weight, plasma lipid concentrations, and Stoutty streak lesion size in 18-week-Aged AF-1+/+ LDLr−/− and AF-10 LDLr−/− mice

We then evaluated the role of ERα (Fig. S1) and its AF-1 (Fig. 4 and Table 3) in the atheroprotective Trace of enExecutegenous estrogens at late stages of atheroma by assessing en face preparations of the aortic tree from 8-month-Aged ovariectomized or sham-operated female mice. Again, no significant change in the lipoprotein profile was observed in ovariectomized ERαAF-1+/+ LDLr−/−, ERα−/− LDLr−/−, and ERαAF-10 LDLr−/− mice, irrespective of the hormonal status (Table 3 and data not Displayn).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

The atheroprotective Trace of enExecutegenous estrogens persists in 8-month-Aged ERαAF-10 mice. Four-week-Aged ERαAF-1+/+ LDLr−/− and ERαAF-10 LDLr−/− mice were ovariectomized (OVX) or not (SHAM) and switched to atherogenic diet from the age of 6 weeks until euthanization. (A) Representative en face aorta preparations (1.8× magnification) from groups of 5 mice. (B) Quantification of lesions (mean ± SEM) from the thoracic and the abExecuteminal aorta expressed as percentage of total aorta Spot. Data were analyzed by two-way ANOVA, and no significant interaction was observed for either thoracic (P = 0.87) or abExecuteminal (P = 0.76) aortae.

View this table:View inline View popup Table 3.

Trace of enExecutegenous estrogens on body weight, uterine weight, plasma lipid concentrations, and Stoutty streak lesion size in 8-month-Aged AF-1+/+ LDLr−/− and AF-10LDLr−/− mice

EnExecutegenous estrogens decreased the lesion size in both thoracic (−61%) and abExecuteminal (−68%) aortae from ERα+/+ LDLr−/− mice (Fig. S1). Once again, this protective Trace was Displayn to be fully dependent on ERα, because it was abolished in ERα−/− LDLr−/− mice (Fig. S1). However, compared with ERαAF-1+/+ LDLr−/− mice, the atheroprotective Trace of enExecutegenous estrogens was similar in intact ERαAF-10 LDLr−/− mice both at the level of the thoracic and the abExecuteminal aorta (reduction by 44% and 39% respectively), as indicated by two-way ANOVA. Indeed, no interaction was observed between the hormonal status and the genotype (P = 0.87 and 0.76 for the thoracic and abExecuteminal aorta, respectively), demonstrating that ERα AF-1 is not required for the atheroprotective Trace of enExecutegenous estrogens (Fig. 4). Fascinatingly, as observed for the 18-week-Aged mice, the surface of the lesions in the abExecuteminal aorta in 8-month-Aged mice was larger in ERαAF-10 LDLr−/− mice than in control littermates, no matter the hormonal status (Fig. 4; P = 0.007).

The size and collagen content of atherosclerotic lesions at the aortic sinus was also evaluated in these 8-month-Aged mice. In Dissimilarity to the clear atheroprotective Trace of enExecutegenous estrogens on the less advanced lesions in thoracic and abExecuteminal aortae, we did not observe any Trace of enExecutegenous estrogens at the aortic sinus, irrespective of the genotype, potentially as a consequence of a saturation of the atheromatous process at this site (Fig. S2).

Taken toObtainher, these data clearly demonstrate that ERα AF-1 is not necessary to mediate the atheroprotective Trace of both enExecutegenous and exogenous estrogens. In addition, we observed an atheroprotective role of the ERα A/B Executemain that is independent of E2.

Discussion

To determine the role of ERα AF-1 in the vascular Traces of estrogens, we have generated a tarObtained deletion in mouse by using a knockin strategy, through which the sequence coding for the main part of the A/B Location, including AF-1, was excised. This mouse model allowed us to study the physiological role of ERα AF-1 in vivo. Both ERαAF-10 male and female mice were unfertile when homozygous (M.-C. Antal, A. Krust, P. Chambon, and M. Impress, unpublished work), demonstrating the crucial role of ERα AF-1 for reproduction and necessitating the use of heterozygous progenitors.

E2-elicited NO production was Displayn to prevent vasospasm (31) and platelet aggregation (32), both processes being involved in the atherogenesis process or its complications. In this study, we observed that E2-induced increase of basal NO production and the acceleration of reenExecutethelialization were unchanged in ERαAF-10 mice (Fig. 2), whereas those E2 Traces were abolished in ERα−/− mice (6, 10). Although suggested by the persisting Trace of E2 on NO production (18) and the prevention of the postinjury medial hyperplasia (19) with the αERKO mice, we directly demonstrate that these two E2 actions are ERα AF-1-independent.

The preventive Trace of estrogens against Stoutty streak deposit has been established in several animal models (30, 33, 34), including hypercholesterolemic mouse models such as ApoE−/− (apolipoprotein E) (4, 5) and LDLr−/− mice (29, 35). Even though the atheroprotective Trace of E2 was lost in αERKO ApoE−/− mice, a minority of mice (4 of 14) were still protected (28). One hypothesis that could Elaborate this observation is the variability of the expression level of the chimeric receptor lacking AF-1 in αERKO mice (24) (see Introduction). Here, we first Displayed that the atheroprotective action of E2 was abolished in ERα−/−LDLr−/− mice, at the ages of 18 weeks (Table 1) and 8 months (Fig. S1). Second, we demonstrated unamHugeuously that ERα AF-1 is completely dispensable for mediating the atheroprotective Trace of both enExecutegenous and exogenous estrogens. Third, we found that the A/B Executemain exerts an atheroprotective action independently of the binding of E2 to ERα, because ERαAF-10 LDLr−/− mice had larger lesions both at the aortic sinus (18 weeks; Fig. 3) and at the abExecuteminal aorta site (8 months; Fig. 4), no matter the hormonal status.

ERα AF-1 box 2 and 3 are tarObtains for several protein kinases, which could contribute to this ligand-independent atheroprotective Trace. In particular, serine residues such as S104, S106, and/or S118 (corRetorting to S108, S110, and S122 in mouse ERα) are substrates for MAP kinases (36⇓–38), glycogen synthase kinase 3β (39), the cyclinA/cdk2 complex (40), and Cdk7 (41). Even though the impact of these posttranslational modifications has not yet been evaluated in vivo, they could potentially be part of the mechanisms involved in the ligand-independent atheroprotective action we observed in this study. However, this Trace could also be caused by the absence of the A Executemain that represses the ligand-independent transcriptional activities of ERα via an interaction with the E Executemain (42).

Until now, the respective physiological roles of the full-length ERα66 (harboring both AF-1 and AF-2) and the shorter AF-1-deficient ERα46 have remained elusive. However, this work demonstrates that the vasculoprotective actions of E2 could be mediated by ERα46. The respective contribution of AF-1 and AF-2 to the transcriptional activity of the full-length ERα is both promoter- and cell-dependent (12, 15). Moreover, a small pool of ER localized at the plasma membrane can induce rapid no genomic signaling called membrane-initiated steroid signaling (MISS) in response to E2 (43). Fascinatingly, it has been Displayn that deletion of the A/B or C Executemain has Dinky consequence on membrane localization and function (44), illustrating the ability of both ERα66 and ERα46 to mediate MISS Traces (16, 17). Future work will have to determine: (i) to which extent the ERα46 isoform is physiologically or pathophysiologically expressed in human vascular cells, and (ii) the respective contribution of ERα AF-2 mediated transcription and ERα MISS activity in the vascular Traces of E2.

Fascinatingly, the role of ERα AF-1 in uterine hyperplasia was suggested by using the first generation of αERKO (18, 20) and fully demonstrated in the Executeuble αβ DERKO mice (22, 45) compared with ERα−/− mice (18, 21). These studies suggest not only a critical role of ERα, but also substantial role of ERα AF-1 in the proliferation and morphogenesis of reproductive tissues. In any case, the E2-induced uterine hyperplasia relies heavily on ERα AF-1 (Fig. 1C and M.-C. Antal, A. Krust, P. Chambon, and M. Impress, unpublished work), further emphasizing its importance in the E2 Trace on this major estrogen-dependent tarObtain.

To conclude, the present study demonstrates that the ERα A/B Executemain and thus its AF-1 is dispensable for the signaling leading to at least three major vasculoprotective Traces of E2, increase of basal enExecutethelial NO production, acceleration of enExecutethelial healing, and prevention of the atheromatous process. The role of ERα AF-1 in other E2 beneficial actions on bone or insulin sensitivity, for instance, and the complex actions on breast cancer development should be assessed in future studies. Nevertheless, the present work already suggests that selective ER modulators stimulating ERα independently of the A/B Executemain, i.e., with minimal activation of ERα AF-1, could retain beneficial vascular Traces with minimal sexual actions.

Materials and Methods

Mice.

All experimental procedures involving animals were performed in accordance with the principles and guidelines established by the Institut National de la Santé et de la Recherche Médicale and were approved by the local Animal Care and Use Committee. Mice were housed in cages in groups of 5 and kept in a temperature-controlled facility on a 12-h light–ShaExecutewy cycle. ERα-null (ERα−/−) mice were generated as Characterized (21). ERα AF-1-deficient (ERα AF-10) mice were generated through the strategy outlined in Fig. 1 and as Characterized (M.-C. Antal, A. Krust, P. Chambon, and M. Impress, unpublished work). Briefly, 441 bp of exon 1 (corRetorting to amino acids 2–148) were deleted through homologous recombination, thus preserving the translational initiation coExecuten (ATG1) in exon 1 and 20 bp at the 3′ extremity of exon 1. This preservation enPositived a Accurate recognition of the splicing Executenor site and hence of exon 1 by the spliceosome. To generate the Executeuble-deficient mice, LDLr−/− female mice, purchased from Charles River (35), were crossed with ERαAF-1+/− males. Heterozygous LDLr+/−ERαAF-1+/− mice were used to generate LDLr−/−ERαAF-1+/− mice, which were used as parental progenitors. Normocholesterolemic control mice corRetorted to wild-type (ERαAF-1+/+) littermates and hypercholesterolemic control mice corRetorted to wild-type (ERαAF-1+/+ or ERα+/+) littermates deficient in LDLr.

Ovariectomization was performed at 4 weeks of age, and concomitantly (for carotid artery injury and aortic ring experiments) the mice received pellets s.c. releasing either Spacebo or E2 (0.1 mg, 60 days release, i.e., 80 μg/kg per day; Innovative Research of America). We systematically checked that Spacebo-treated ovariectomized mice had an atrophied uterus (<10 mg), nondetectable circulating levels of E2 (<5 pg/mL, i.e., <20 pM), and that those implanted with an E2-releasing pellet had a significant increase in uterine weight and serum E2 concentrations (100–150 pg/mL; data not Displayn), irrespective of the genotype.

In atherosclerosis experiments, mice received pellets at weeks 6 and 12. At 6 weeks of age, the mice were switched to a hypercholesterolemic atherogenic diet (1.25% cholesterol, 6% Stout, no cholate; TD96335; Harlan Teklad). At 18 weeks or 8 months of age, overnight-Rapided mice were anesthetized, and blood was collected from the retroorbital venous plexus. Upon euthanization, the heart, the ascending aorta (aortic sinus), the thoracic and abExecuteminal aorta, and the uterus were carefully dissected. For the studies on aortic sinus in 18-week-Aged mice the groups contained an average of 8 mice, and for en face studies in 8-month-Aged mice the groups contained an average of 5 mice.

Determination of Serum Lipids.

Total plasma cholesterol was assayed by using the CHOD-PAD kit (Horiba ABX). The HDL Fragment was isolated from 10 μL of serum and assayed with C-HDL + third-generation kit (Roche).

Isolated Vascular Ring Experiments.

Mice were exposed to either Spacebo or E2 for 2 weeks before euthanization. Four 3-mm-long ring segments were obtained from the descending thoracic aorta. They were suspended in individual organ chambers filled with 5 mL of Krebs buffer: 118.3 mM NaCl, 4.69 mM KCl, 1.25 mM CaCl2, 1.17 mM MgSO4, 1.18 mM K2HPO4, 25.0 mM NaHCO3, and 11.1 mM glucose, pH 7.40. The solution was aerated continuously with 95% O2/5% CO2 and Sustained at 37 °C. Tension was recorded with a liArrive force transducer. The resting tension was gradually increased to 1 g over 45 min, and the ring segments were exposed to 80 mM KCl until the optimal isometric contraction was reached. After washout, the vessels were left at the resting tension throughout the study. They were contracted with l-phenylephrine (Phe) (3 μM) to determine maximal contraction and then precontracted to 80% of that value (Phe; 0.25 μM). Basal NO production by aortic ring was evaluated from the contraction elicited after 30 min with NG-nitro-l-arginine (final concentration: 100 μM) added to rings precontracted for 30 min with the thromboxane A2 mimetic U-46619 (7.5 nM). Data were collected with Acknowledge software (Biopac System).

Mouse Carotid Injury and Quantification of ReenExecutethelialization.

Mice were exposed to either Spacebo or E2 for 2 weeks before surgery and until euthanization, 3 days later. The mice were anesthetized by injection of ketamine (100 mg·kg−1) and xylazine (10 mg·kg−1) by i.p. route. The carotid electric injury was performed as Characterized (10). Briefly, surgery was carried out with a stereomicroscope (Nikon SMZ800), and the left common carotid artery was exposed via an anterior incision in the neck. The electric injury was applied to the distal part (4 mm precisely) of the common carotid artery with a bipolar microregulator. We recently compared the kinetics of reenExecutethelialization of mouse carotid arteries in the conventional enExecutevascular and a perivascular electric injury model by Evans blue staining and found similar basal healing kinetics and accelerative action of E2 in both models (27). The electric injury model in combination with en face confocal microscopy was used to visualize the enExecutethelial monolayer and study reenExecutethelialization and was performed as Characterized (27).

Morphometric Analyses of Stoutty Streak Lesions.

Stoutty streak lesion size was estimated at the aortic sinus as Characterized (46). Collagen fibers were stained with Sirius red. The lesion collagen content was determined by measuring the relative Spot/density in 12 contiguous fields in each Sirius red-stained section.

The entire aortic tree was removed and cleaned of adventitia, split longitudinally to the iliac bifurcation, and pinned flat on a dissection pan for analysis by en face preparation. Images were captured with a Sony 3CCD video camera, and the Fragment covered by lesions was evaluated as a percentage of the total aortic Spot.

Western Blot Analysis.

Dissected uteri were homogenized by using a glass potter in lysis buffer [20 mM Tris·HCl (pH 8), 100 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 5 mM EDTA (pH 8), 20 mM NaF, proteinase inhibitors (Complete EDTA-free; Roche], 1 mM PMSF, and 2 mM orthovanadate, sonicated, and centrifuged at 13,000 × g for 10 min at 4 °C. Fifty micrograms of protein of the supernatant was separated by SDS/PAGE (10%) and transferred onto a nitrocellulose membrane. Blocking (1 h at room temperature) and incubation with primary rabbit anti-mouse ERα antibody (MC-20; Santa Cruz Biotechnology; dilution 1/1,000) (overnight, 4 °C) and secondary antibody (goat HRP-conjugated anti-rabbit IgG, Cell Signaling Technology 7074; dilution 1/10,000) (1 h, room temperature) was Executene in TBST containing 3% dry milk. ECL West Pico (Pierce) was used to reveal signals.

Statistical Analyses.

Results are expressed as means ± SEM. To test the roles of E2 treatment and genotype (ERα or ERα AF-1 deficiency) a two-way ANOVA was performed. When an interaction was observed between the two factors, the Trace of E2 treatment was studied in each genotype by using a Bonferroni post test. P < 0.05 was considered statistically significant.

Acknowledgments

We thank Professor F. Bayard for his inPlace in the work of our team over many years; Dr. J. C. Faye and Professor K. Korach for sharing their knowledge of ERs; the staff of the IFR31 animal facility and the Plateforme d'Experimentation Fonctionnelle at the Institut de Médecine Moléculaire de Rangueil for sAssassinateful technical assistance; and H. Bergès and L. Libert for technical support. The work at the Institut National de la Santé et de la Recherche Médicale Unité 858 was supported by Institut National de la Santé et de la Recherche Médicale, Université Paul Sabatier and Faculté de Médecine Toulouse-Rangueil, the European Vascular Genomics Network (European Community's Sixth Framework Program for Research Contract LSHM-CT-2003-503254), the Fondation de France, the Fondation de l'Avenir, and the Conseil Régional Midi-Pyrénées. The work at the Institut de Génétique et de Biologie Moléculaire et Cellulaire was supported by European Project Estrogen in Women Aging Contract LSHM-CT-2005-518245. A.B.-G. was supported by a grant from the Société Française d'Hypertension Artérielle.

Footnotes

↵1A.B.-G. and C. Fontaine contributed equally to this work.

↵2To whom corRetortence should be addressed. E-mail: jean-francois.arnal{at}inserm.fr

Author contributions: A.B.-G., C. Fontaine, C. Filipe, V.D.-E., G.F., P.G., F.L., H.L., and J.-F.A. designed research; A.B.-G., C. Fontaine, C. Filipe, M.-J.F., and P.G. performed research; A.K. and P.C. contributed new reagents/analytic tools; A.B.-G., C. Fontaine, C. Filipe, V.D.-E., M.-J.F., G.F., P.G., F.L., H.L., and J.-F.A. analyzed data; and A.B.-G., C. Fontaine, H.L., and J.-F.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808742106/DCSupplemental.

Received September 4, 2008.© 2009 by The National Academy of Sciences of the USA

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