Estradiol Alters Nitric Oxide Production in the Mouse Aorta Through the α-, but not β-, Estrogen Receptor
Although estradiol (E2) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E2 (80 μg/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E2 increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E2, whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E2. To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ERα or ERβ has been disrupted were ovariectomized and treated, or not, with E2. Basal NO production was increased and the sensitivity to acetylcholine decreased in ERβ knockout mice in response to E2, whereas this effect was abolished in ERα knockout mice. Finally, these effects of E2 on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E2 in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ERα, but not ERβ, mediates the beneficial effect of E2 on basal NO production.
The incidence of cardiovascular disease is higher in men than in premenopausal women but increases in postmenopausal women. Until recently, an abundance of epidemiological data suggested a role for estrogens in this atheroprotective effect.1,2⇓ However, the protective effects of estrogen on cardiovascular diseases has become controversial these last years. The Heart and Estrogen/progestin Replacement Study (HERS) recently demonstrated the failure of hormonal replacement therapy (HRT) to increase survival in secondary prevention.3 Because long-term trials evaluating the effect of HRT in primary prevention will conclude in several years, it is important to get insight into the vascular effects and mechanisms in the meantime.
The mechanism whereby this protection is attributable to favorable changes in blood lipids and lipoproteins accounts for approximatively one third of its effect,1 but a number of studies in humans4 as well as animals5–7⇓⇓ strongly suggest a direct action on the arterial wall.
Endothelium is now recognized to play a crucial role in the physiology of circulation. In particular, endothelium generates nitric oxide (NO). This free radical messenger inhibits platelet aggregation, interferes with mononuclear cell adhesion, and relaxes the underlying smooth muscle cells.8–10⇓⇓ NO is generated by a family of 3 isoenzymes: neuronal NO-synthase (type I), inducible NO-synthase (type II), and endothelial NO-synthase (type III). NO synthase (NOS) III activity can be stimulated by agonists (such as acetylcholine) and by a mechanical stimulus (shear stress). The importance of NO in vascular biology has prompted intensive research into the effects of estrogens in the vascular EDRF activity in several species. In rabbit, rat, and guinea pig, endothelium-derived relaxing factor (EDRF) was reported to be enhanced in ovariectomized 17β-estradiol (E2)–treated females compared with ovariectomized controls.11–16⇓⇓⇓⇓⇓ In these models, the increase in EDRF activity in response to E2 can be due to (1) an increase in NOS III expression,17,18⇓ (2) an increase in NOS III activity,15,19⇓ and (3) an increase in NO bioavailability,20 because the production of superoxide anion, one of the major mechanisms responsible for NO degradation, was found to be decreased in response to E2. Altogether, these studies suggest that the estrogen induced augmentation of NO production by vascular endothelium may contribute to its vasculoprotective effects.
It was reported that basal release of endothelium-derived NO was significantly higher in aortas of intact male mice compared with intact female mice.21 Surprisingly, and to the best of our knowledge, the effect of E2 on EDRF activity has never been reported in female mice. Ovariectomized female C57Bl/6 mice were then given either placebo or E2 and the thoracic aorta studied for EDRF activity, both under basal and stimulated conditions. Protein abundance of NOS III, II, and I was also investigated. The effect of E2 has long been thought to be mediated by activation of estrogen receptor (ER)-α. ERα is a ligand-dependent transcriptional activator that modulates gene expression in target cells not only in reproductive tissues, but also in bone and vessels. Moreover, ERβ recently discovered and cloned from rat prostate, was found to be expressed in many other tissues, including injured arteries.22 It has been suggested that ERα plays a role because the basal release of endothelium-derived NO is decreased in male ERα-knockout mice compared with wild-type mice.21 However, its role in the mediation of the E2 effect in females has not been reported. To define whether ERα or ERβ mediates the effect of E2, relaxation experiments were performed in ovariectomized transgenic mice in which either the ERα or ERβ has been disrupted.
Finally, several studies recently investigated the short-term effect of E2 on NO production in cultured endothelial cells.23–29⇓⇓⇓⇓⇓⇓⇓ As the respective importance of the short- versus long-term effects of E2 has been reported only in cultured cells, we studied the effect of short-term E2 exposure in isolated aortic segments.
Materials and Methods
Ketamine (Ketalar) was from Parke Davis. l-phenylephrine (Phe, ref. P-6126), acetylcholine (ACh, ref. A-2661), sodium nitroprusside (SNP, ref. S-0501), A23187 (ref. C-7522), U46619 (9,11 dideoxy-11α, 9α-epoxy methano-prostaglandin, ref. D-8174), and NG-nitro-l-arginine (L-NA, ref. N-5501) and other materials were from Sigma. Adenosine-5′-triphosphate (ATP, ref. 127 523) was from Boehringer Mannheim. U46619 stocks were prepared in ethanol with final ethanol concentration of 0.01%.
All experimental protocols were performed in accordance with the recommendations of the European Accreditation of Laboratory Animal Care. The animals were housed in stainless steel cages in groups of 5, kept in temperature-controlled facilities on a 12-hour light-dark cycle and fed normal laboratory mouse chow. Only female mice were used this study. Wild-type mice were C57Bl/6 (weight 18 to 22 g; Iffa-Credo, Les Oncins, France). Targeted disruption (knockout) of mouse ERα and ERβ genes was performed by homologous recombination, resulting in ERα- and ERβ-homozygous deficient mice (ERα−/− and ERβ−/−).30 Six backcrosses with C57Bl/6 mice were performed. At 4-weeks old, the mice were anesthetized by injection of ketamine (150 mg/kg, IP) for bilateral ovariectomy. Two weeks later, the castrated animals received subcutaneously either placebo or a 0.1 mg 17β-estradiol (E2) pellet (Innovative Research of America) designed to release E2 at 80 μg/kg/day for 60 days. Fifteen days later, after 2 hours fasting, the mice were euthanized with an overdose of ketamine. The uterus was removed and weighed. The thoracic aorta was removed and cleaned of excess adventitial tissue. Serum hormone concentrations were measured as previously described.31
Isolated Vascular Ring Experiments
Four ring segments of 3 mm were obtained from the descending thoracic aorta (Figure 1). They were suspended in individual organ chambers filled with Krebs buffer (5 mL) with the following millimolar composition: 118.3 NaCl, 4.69 KCl, 1.25 CaCl2, 1.17 MgSO4, 1.18 K2HPO4, 25.0 NaHCO3, and 11.1 glucose, pH 7.40. The solution was aerated continuously with 95% O2/5% CO2 and maintained at 37°C. Tension was recorded with a linear force transducer. The resting tension was gradually increased to 1 g over a period of 45 minutes, and the ring segments exposed to 80 mmol/L KCl until the optimal tension for generating force during isometric contraction was reached. The vessels were left at this resting tension throughout the remainder of the study. They were contracted with Phe (3 μmol/L) to determine maximal contraction and then precontracted to 80% of that value (Phe, 0.25 μmol/L). When a stable contraction plateau had been reached, the rings were exposed cumulatively to either ACh (1 nmol/L to 30 μmol/L), ATP (0.1 to 30 μmol/L), A23187 (1 nmol/L to 1 μmol/L), or SNP (0.1 nmol/L to 0.3 μmol/L). Experiments were performed in the presence of diclofenac (2 μmol/L), except when indicated. For experiments without endothelium, the thoracic aorta was cannulated and dry air was insufflated for 5 minutes to desiccate, and thus destroy, the endothelium.
Basal NO production by aortic ring was evaluated from the contraction elicited after 30 minutes with L-NA (final concentration: 100 μmol/L) added to rings preconstricted for 30 minutes with the thromboxane A2 mimetic U46619 (7.5 nmol/L).21 Data were collected by Acknowledge software (Biopac System, Inc) and relaxation expressed as the percentage tension decrease below the tension elicited by precontracting the aortic rings with Phe.
Western Blotting Analysis
Thoracic aorta were frozen in liquid nitrogen, ground, and then suspended in boiling 2% SDS. Western blotting was performed has previously described,20 and blots were incubated with anti-NOS III antibody (Transduction laboratories; ref. N30020), anti-NOS II antibody (Transduction laboratories; ref. N31020), anti-NOS I antibody (Transduction laboratories; ref. N32020), or anti-ERα with antibody (Santa Cruz; ref. MC20 SC540).
Data were expressed as mean±SEM. Comparisons of concentration-response relaxations between the various aortic segments studied were made by 1-factor ANOVA. Comparisons of data between different groups were made with repeated measures and a Sheffe’s post hoc test used when differences were indicated. To test the respective roles of E2 treatment and genotype on NO production, a 2-way ANOVA was performed. When an interaction was observed between the 2 factors, the effect of E2 treatment was studied in each genotype using a t test. A value of P<0.05 was considered as statistically significant.
Basal NO Production and Relaxation of Upper and Lower Segments of the Thoracic Aorta From Ovariectomized Wild-Type Mice
The basal NO release, estimated from the additional L-NA–induced contraction obtained in U-46619 precontracted rings, was similar in the 4 segments (Figure 2 and online Table 2 in the online data supplement available at http://www.circresaha.org). The contraction in response to α1-adrenergic agonist phenylephrine was similar in the 4 segments studied (online Table 1). When the vessels were precontracted to 80% of the maximal contraction by phenylephrine, the maximal relaxation in response to acetylcholine was significantly decreased in the upper segment (a) as compared with the 2 lower segments (c and d), segment b being intermediate. The EC50 of the upper segment (a) was higher than that of the 2 lower segments (c and d). The responses of segments c and d were similar. In response to the calcium ionophore A23187, to ATP, and to sodium nitroprusside, EC50 and maximal relaxation were similar in all 4 segments (online Table 1).
Effect of E2 Treatment on Body and Uterus Weight
Ovariectomized mice implanted with a placebo pellet showed nondetectable (<5 pg/mL IE, <20 pmol/L) circulating levels of E2, whereas those implanted with a pellet-releasing E2 80 μg/kg/day showed plasma E2 concentrations averaging 0.5 nmol/L, irrespective of the genotype (online Table 2). Ovariectomized placebo-treated mice showed atrophied uterus (<20 mg), whereas both wild-type and ERβ−/− mice treated with E2 showed a significant increase in uterine weight, indicating the expected effect of E2 on this target sexual organ (online Table 2). In contrast, ERα−/− mice conserved an atrophied uterus even when treated with E2 (online Table 2).
Effect of E2 Treatment on Aortic Contraction and Relaxation in Wild-Type Mice
E2 did not significantly alter the contraction in response to phenylephrine and to U46619 (online Table 1). Basal NO release was significantly enhanced by E2 in the 4 segments (P=0.03) (Figure 2). EDRF activity, estimated from the relaxation of precontracted vessels in response to acetylcholine, was decreased by E2 treatment. The EC50 was significantly increased (on average 3-fold) and the maximal relaxation was significantly decreased (on average 30%) by E2 in comparison to the placebo group (P=0.03) (Figure 3 and online Table 1). The sensitivity to carbachol, a nonhydrolyzable activator of muscarinic receptors, was also decreased by E2. In contrast, there was no significant difference in relaxation between the 2 groups in response to ATP or to the calcium ionophore A23187 (Figure 3 and online Table 1). The maximal relaxation in response to the NO-releasing agent sodium nitroprusside (SNP) was not influenced by E2, whereas the EC50 was increased (2- to 3-fold) in the E2-treated group (Figure 3 and online Table 1). We also performed experiments in de-endothelialized aortic rings. As shown in Figure 3E, destruction of endothelium induced a leftward shift of the dose response of both placebo- and E2-treated groups. Comparison of E2 versus placebo-treated de-endothelialized rings revealed a slight but significant decrease in sensitivity to SNP (Figure 3E and online Table 1).
Lack of Involvement of COX Pathway or Endothelium-Derived Hyperpolarizing Factor (EDHF) in Mouse Aortic Relaxation
In the placebo group, incubation of aortic segments with L-NA (100 μmol/L) completely blocked ACh-elicited relaxation, demonstrating that NO was responsible for the relaxation. As all the previous experiments were performed on thoracic aorta contraction in the presence of the cyclooxygenase inhibitor diclofenac (2 μmol/L), experiments were repeated in its absence. Similar results were obtained for all the parameters reported above in online Table 1, either in placebo- and E2-treated animals (not shown). Experiments were also performed in the presence of 40 mmol/L KCl. The effect of E2 on ACh-elicited relaxation persisted in the presence of 40 mmol/L KCl, indicating that EDHF did not account for the observed effect of E2.
E2 Does Not Alter NOS I, II, and III Protein Abundance in Thoracic Aorta
Western blotting quantification did not reveal any changes in NOS III immunoreactivity in control or E2 groups (Figure 4). The NOS II protein was slightly detected in thoracic aorta homogenates from both control and E2 groups without significant differences between the 2 groups (Figure 4). Whereas the antibody against NOS I recognized mouse NOS I in cerebellum homogenates, we did not detect any signal in the thoracic aorta homogenates, suggesting that the abundance of the immunoreactive protein was below the threshold of detection.
Effect of E2 Treatment on Aortic Contraction and Relaxation in Mice Deficient in Either ERα or in ERβ
Contraction was similar in all categories of mice implanted with a placebo pellet, irrespective of the genotype (online Table 2). Two-factor ANOVA revealed an interaction (P=0.02) between E2 treatment and the genotype for basal NO release. E2 treatment significantly increased basal NO release in wild-type and ERβ−/− mice, but had no effect in ERα−/− animals (Figure 5A and online Table 2).
Likewise, in lower segments cd, 2-factor ANOVA revealed an interaction between E2 treatment and the genotype for the EC50 and Emax of ACh-elicited relaxation (P=0.05 and P=0.036, respectively). E2 treatment significantly decreased the sensitivity to ACh in wild-type mice (EC50, P=0.014; Emax, P<0.001), but had no effect in ERα−/− (EC50, P=0.56; Emax, P=0.51) (Figure 5B and online Table 2). Although the alteration of ACh-elicited relaxation tended to be greater in E2-treated ERβ−/− mice than in E2-treated wild-type mice, the difference was not statistically significant (EC50, P=0.35). Analogous results were obtained with segments a and b.
Abundant ERα immunoreactivity was detected in the uterine homogenates from wild-type and ERβ−/− at the expected size (65 kDa), whereas it was undetectable in ERα−/− mice (Figure 5C). ERα immunoreactivity was also detected in the thoracic aorta homogenates from the wild-type, and E2 treatment had no effect on ERα abundance. ERα was undetectable in ERα−/− mice. ERα immunoreactivity was also detected in the aorta homogenates from ERβ−/− mice (not shown).
Effect of Short-Term E2 Exposure on Aortic Rings
To investigate the extent to which short-term E2 exposure elicits an alteration of NO production similar to that observed in long-term impregnation, 2 kinds of experiments in aortic segments from castrated mice were undertaken. First, 10 nmol/L E2 was added to U46619-precontracted aortic rings from both placebo- and E2-treated mice. No change in vessel tone was detected in the 30 minutes following the addition of E2 in the organ bath (n=3, not shown). Second, the relaxation in response to ACh was studied before and after a 15-minute period of incubation with 10 nmol/L E2. Again, no change was detected after short-term E2 exposure (n=3, not shown).
We first studied the relaxation of the thoracic aorta at the 4 different levels in castrated mice given placebo. The ACh-induced relaxation in the lower parts of the aorta was significantly higher than that in the upper segments. A similar observation had been previously made in rabbit aorta32 as well as in rat aorta.33 It has obvious methodological implications, in particular the necessity to analyze the 4 segments separately, although the 2 lower segments (c and d) can be pooled. The lack of alteration following cyclooxygenase or EDHF inhibition, combined with the total abolition of relaxation after incubation with the NO synthase inhibitor L-NA, confirmed that ACh-induced relaxation was mainly dependent on NO.34 Similarly, the contraction elicited after NO blockade by L-NA was not influenced by COX or EDHF inhibition.
We then studied long-term exposure to physiological levels of E2 (ie, 0.5 nmol/L on average) on basal aortic NO production and relaxation. The contraction in response to U46619 and phenylephrine were not altered by E2, allowing the comparison of basal NO production and relaxation. Basal NO production is increased in castrated mice given E2. A similar effect of E2 was previously reported in rabbit19 and in rat.15 The contraction elicited by NO blockade was independent of both cyclooxygenase derivatives and EDHF. It was also independent of extracellular superoxide, as superoxide dismutase (SOD) did not alter endothelium-dependent relaxation or basal NO production (not shown). Thus, decreased NO breakdown through an antioxidant effect of E2, previously reported in cultured bovine endothelial cells,20 cannot account for the enhanced basal NO release in mouse.
Surprisingly, we found that E2 decreased ACh-elicited relaxation in all segments. This is the first report of a decrease in NO release in response to ACh on E2 treatment. In other species, E2 induced either an enhancement of ACh-elicited relaxation15 or had no effect.19,35⇓ Because similar results were obtained in response to carbachol, a nonhydrolyzable activator of muscarinic receptor, an increase in ACh degradation by acetylcholine esterase did not account for the observed effect. The sensitivity of the relaxation in response to SNP was slightly but significantly decreased in the E2-treated group, suggesting some degree of hyposensitivity to NO as a consequence of long-term E2 exposure. This was observed both in intact and de-endothelialized rings. The hyposensitivity of vessel from E2-treated mice could have been the consequence of enhanced basal NO production, and this probably represents the counterpart to the classical observation of a supersensitivity to NO donors after suppression of endogenous NO.36 Moreover, ACh did not show any constrictive effect on smooth muscle cells in the presence of L-NA or when the endothelium was removed irrespective of whether they had been treated with E2, showing that the endothelium is the sole target of ACh. Although the decrease in sensitivity to NO of E2-treated rings could contribute to the impairment of the relaxation in response to ACh, only part of this alteration could be explained by this factor for at least 2 reasons: (1) both the receptor-dependent (ATP) and the receptor-independent (A23187) endothelium-mediated relaxation remained normal after E2 treatment; and (2) the magnitude of the alteration of the ACh-induced relaxation is larger than that to SNP.
Overall, these data suggest that the alteration elicited by E2 in the aorta could be restricted to the M3 muscarinic receptor or to its signal transduction pathway. The modulation of M3 receptor–dependent signaling has been shown in rat myometrium.37 Thus, the muscarinic receptor pathway can be a target for E2. However, in the aorta, this alteration appears to be not only restricted to 1 receptor type (ie, the muscarinic receptor) but also to 1 species (ie, the mouse). This again questions the physiological relevance of ACh for evaluation of endothelial function. ACh remains a pharmacological tool, and it has never been shown that a physiological stimulation of the muscarinic receptor of the endothelial aorta occurs in vivo. Moreover, in the present study, its effect does not correlate to the response to other more relevant pathophysiological stimuli such as ATP, which is released during platelet aggregation for instance. Finally, the effect of E2 on ACh-elicited NO release is strictly the opposite of that induced on basal NO release. In order to clarify the effect of E2 on endothelial NO production, we assessed the abundance of the 3 NO synthases. Western blot and subsequent quantification did not reveal any change in NOS III immunoreactivity in response to E2 treatment. In addition, NOS II was detected although at a low level, whereas NOS I was not detected. The absence of change in NO synthase expression in aorta homogenates fits with the lack of effect of E2 on A23187-induced relaxation.
It is tempting to link the increase in basal NO production in E2-treated mice to that recently reported by several independent groups in cultured endothelial cells.23–27⇓⇓⇓⇓⇓ Caulin-Glasser and colleagues28 initially reported that E2 rapidly increased NO production and the same group subsequently reported that the association between HSP-90 and NOS III following E2 exposure could play a role in NOS III activation. Other groups29 reported that within 5 minutes, E2 induces a translocation of NOS III from the membrane to intracellular sites close to the nucleus. However, on more prolonged exposure to E2, most of the NOS III returns to the membrane, and thus probably cannot account for the long-term effects observed in the present work. Finally, Shaul’s group24 also reported that the nongenomic activation of NOS III by E2 involves MAP kinase–dependent mechanisms and demonstrated that ERα, localized in caveolae, can directly activate NOS III.27 HSP-90 binds to both NOS III and ERα, and these interactions could to promote the short-term E2-induced NOS III activity demonstrated in vitro.38,39⇓ In this series of experiments, a physiological concentration of E2 stimulated NO production within 5 to 30 minutes, according to the cultured endothelial cells models used. However, in the present study, short-term incubation with physiological levels of E2 did not induce any changes in mouse aorta NO production. Thus, native endothelial cells in mouse (present study) and rat aorta (unpublished data) do not appear to respond to short-term E2 as do cultured endothelial cells.
Transgenic mice allowed us to determine which ER gene was involved in the effect of E2 on arterial NO production. ERα has long been considered as the unique target of E2, as it has been characterized in reproductive tissues, bone, and vessels, particularly in the endothelial cells.40 However, it was initially suggested that ERβ could mediate some effects of E2 in the vessel wall.41 The present work demonstrates that ERα, but not ERβ, mediates the effects of E2 on endothelial NO production, ie, the enhancement of basal NO release by endothelium and the alteration of ACh sensitivity. In addition, we recently demonstrated that ERα mediates the acceleration of reendothelialization of heat-damaged vessels in response to E2.31 These studies indicate the prominence of the α isoform of ER in the effect of E2 on endothelium.
Does this increase in basal NO release contribute to the vasculoprotective effect of E2? Several years ago, we tested this hypothesis in apolipoprotein E–deficient mice by blocking the production of NO. We found that the inhibition of NO synthase by L-NAME did not alter the protective effect of E2 on fatty streak deposit.42 Although we did not assess the decrease of NO production in the hypercholesterolemic mice, the prevention of fatty streak formation by E2 appears independent of NO. In addition, in an external vascular cuff model, endogenous E2 prevented the neointimal proliferation independently of NOS III.43 However, this does not exclude that, in more advanced stages of atherosclerosis, the increase of NO production and/or the prevention of endothelial dysfunction could contribute to protecting the vessel against platelet aggregation and vasospasm, 2 major mechanisms leading to clinical cardiovascular events. Taking into account the crucial role of the endothelium in the maintenance of vascular integrity, endothelial ERα should be considered as a primordial target in pharmacological studies concerning cardiovascular diseases.
The work at INSERM U397 was supported in part by the INSERM, the Ministère de la Recherche et de la Technologie, the Fondation de France, the Fondation de l’Avenir, the Fondation pour la Recherche Médicale, and the Conseil Régional Midi-Pyrénées. The work at Institut de Génétique et de Biologie Moléculaire et Cellulaire was supported by a grant from the CNRS, the INSERM, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche Médicale, the Fondation pour la Recherche Médicale, and by Groupement d’Intérêt Public/Hoechst-Marion-Roussel.
Original received July 5, 2001; resubmission received October 23, 2001; revised resubmission received January 3, 2002; accepted January 3, 2002.
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