Research ArticleEndocrinologyVascular biology Open Access | 10.1172/jci.insight.161284

Morgane Davezac,1 Rana Zahreddine,1 Melissa Buscato,1 Natalia F. Smirnova,1 Chanaelle Febrissy,1 Henrik Laurell,1 Silveric Gilardi-Bresson,1 Marine Adlanmerini,1 Philippe Liere,2 Gilles Flouriot,3 Rachida Guennoun,2 Muriel Laffargue,1 Jean-Michel Foidart,4 Françoise Lenfant,1 Jean-François Arnal,1 Raphaël Métivier,5 and Coralie Fontaine1

1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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1I2MC, Institut National de la Santé et de la Recherche Médicale (INSERM) U1297, University of Toulouse 3, Toulouse, France.

2INSERM U1195, University Paris-Saclay, Le Kremlin-Bicêtre, France.

3Institut de Recherche en Santé, Environnement et Travail (Irset), INSERM UMR_S 1085, EHESP, University of Rennes, Rennes, France.

4Department of Obstetrics and Gynecology, University of Liège, Liège, Belgium.

5Institut de Génétique de Rennes (IGDR), UMR 6290, CNRS, University of Rennes, Rennes, France.

Address correspondence to: Jean-François Arnal, INSERM/UPS U1297 - I2MC, Institut des Maladies Métaboliques et Cardiovasculaires, 1 avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. Phone: 33.5.31.22.40.98; Email: jean-francois.arnal@inserm.fr.

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Published February 2, 2023 - More info

Published in Volume 8, Issue 5 on March 8, 2023
JCI Insight. 2023;8(5):e161284. https://doi.org/10.1172/jci.insight.161284.
© 2023 Métivier, et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published February 2, 2023 - Version history
Received: April 22, 2022; Accepted: February 1, 2023 View PDF Abstract

The main estrogen, 17β-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4’s action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.

Graphical Abstract Introduction

A large body of evidence indicates that the main endogenous estrogen, 17β-estradiol (E2), exerts beneficial effects on the endothelium (1). In particular, E2 accelerates carotid artery endothelial healing after either endovascular or perivascular injuries (2). The capacity of the endothelium to regenerate following injury is essential to ensure its role as a semipermeable barrier, and thus to prevent various vascular diseases, including atherosclerosis, restenosis, and thrombus formation (3). This beneficial effect of E2 relies on the activation of estrogen receptor α (ERα) in both endothelial and hematopoietic cells (4). As a member of the nuclear receptor superfamily, ERα is primarily considered a ligand-regulated transcription factor but besides its genomic action, a pool of ERα also localizes at the plasma membrane and mediates rapid signaling through interaction with other proteins such as endothelial nitric oxide synthase (eNOS), SRC, or several other kinases (5). Combinations of several transgenic mouse models with pharmacological tools demonstrated the pivotal role of nongenomic effects of ERα in E2-induced endothelial healing (6–9). In particular, we previously highlighted the loss of E2-mediated protection against endothelial injury in the C451A-ERα mouse model in which ERα is unable to localize to the plasma membrane due to a point mutation of its palmitoylation site (6). More recently, we also demonstrated the crucial role of arginine 264 of ERα, involved in PI3K and G protein interaction, in mediating this E2 effect (7).

In addition to E2, which is mainly produced by the ovaries in premenopausal women, 2 other natural estrogens, estriol (E3) and estetrol (E4) are produced during pregnancy by the placenta and the fetal liver, respectively. E3 is used to reduce genitourinary symptoms in postmenopausal women (10), and E4 was recently approved by the Food and Drug Administration and the European Medicines Agency for oral contraception and is in a phase III clinical trial for the hormone treatment of menopause (11–14). Indeed, E4 induces fewer effects on liver-derived coagulation factors than classic estrogens, and thereby could not increase the risk of venous thromboembolism (15, 16). All 3 estrogens display distinct ERα activation profiles due to differential receptor affinities, metabolism (half-life), and subfunction activation (17). Even though both E3 and E4 are commercialized for therapeutic purposes, their effects on arteries remain largely undescribed compared with E2. In 2014, we reported that E2, but not E4, was able to accelerate reendothelialization after perivascular injury of the carotid artery and that E4 was unexpectedly able to inhibit this effect (17). Along with other experiments, we concluded that despite similarities in nuclear ERα’s actions, E4 not only fails to elicit, but is even able to antagonize the membrane-initiated effects of ERα mediated by E2 (5, 17). As endothelial injury and increased endothelial turnover are key events in arterial areas prone to atheroma (18–20), lack of endothelial healing capacity could represent a disadvantage of E4 compared with E2, or even a deleterious effect in an organism with endogenous E2. Altogether, these data prompt clarification of the role of E4 in endothelial injury in a different model and exploration for the first time of the still-unknown impact of E3 in this process. To this aim, we used 2 different models of carotid artery injury to compare the involvement of each cell type (i.e., endothelial cells versus smooth muscle cells, SMCs) in response to each ligand: (a) the perivascular model induced by an electrical injury, in which both the endothelium and media are destroyed, to confirm the previous results of E2 and E4 and to evaluate the effect of E3; and (b) the endovascular model, consisting of an intraluminal injury where the endothelium is removed while SMCs are preserved. This latter endovascular model better reflects an endothelial injury induced by smoking, hyperglycemia, or hypertension, all of which lead to vascular diseases.

We found that E3, similar to E2, accelerated endothelial healing in both models. In agreement with our previous study (17), E4 did not accelerate reendothelialization in the perivascular injury model. However, surprisingly, E4 accelerated endothelial healing in the endovascular injury model. We then used a combination of transgenic mouse models harboring ERα proteins mutated for specific subfunctions or with tissue-specific deletion of ERα to assess the mechanisms underlying the particular action of E4.

Results

E4 accelerates endothelial healing after endovascular but not perivascular injury of the carotid artery. First, to compare the effects of E2, E3, and E4 (Figure 1A) on endothelial healing, we adjusted E3 and E4 concentrations in homemade pellets to achieve estrogenic impregnation similar to that of E2, taking into account the difference in ERα affinity between estrogenic compounds (17) and using the uterotrophic effect of estrogens as an endogenous bioassay of estrogen activity. As expected, control ovariectomized mice (vehicle treated) displayed an atrophied uterus, while E2, E3, and E4 induced similar increases in uterine weight (Figure 1B). Moreover, similar vaginal impregnation (Figure 1C) and thymic atrophy (Figure 1D) were observed across treatments, supporting altogether a comparable estrogenic action of the 3 ligands under these experimental conditions. Estrogen plasma concentrations were measured by gas chromatography–tandem mass spectrometry. Importantly, no interconversion between these 3 estrogens was detected across the samples (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.161284DS1).

Figure 1

17β-Estradiol (E2), estriol (E3), and estetrol (E4) accelerate endothelial healing following carotid artery endovascular injury. Four-week-old female mice were ovariectomized and 2 weeks later implanted subcutaneously with vehicle (Veh), E2, E3, or E4 pellets for 2 weeks. Mice were then subjected to endovascular injury of the carotid artery. Carotid reendothelialization was analyzed 5 days after injury (n = 7–11 per group). (A) Chemical structures of E2, E3, and E4. (B) Uterine weight. (C) Vaginal weight. (D) Thymic weight. (E) Representative Evans blue staining of carotids with outlined deendothelialized areas (scale bar: 1 mm) and (F) quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared with day 0. ECs, endothelial cells. Results are expressed as mean ± SEM. To test the effect of the different treatments, Kruskal-Wallis test (B and D) or 1-way ANOVA (C and F) was performed. *P < 0.05, **P < 0.01, ****P < 0.0001 versus Veh-treated group.

We evaluated endothelial healing by Evans blue staining after endovascular injury of the carotid artery in ovariectomized mice treated with E2, E3, or E4, a model in which SMCs are totally preserved (Figure 1E) as previously described (2). As expected, estrogenic impregnation with E2 promoted endothelial healing since quantification of reendothelialized areas showed 30% endothelial regeneration in vehicle-treated mice compared with day 0 and approximately 80% in E2-treated mice (Figure 1F). Both E3 and E4 treatments also increased endothelial healing, but no statistically significant differences in reendothelialization rates were observed between the E2-, E3-, and E4-treated groups (Figure 1, E and F). E4’s effect on endothelial healing was confirmed using VE-cadherin staining (Supplemental Figure 1). This beneficial effect of E4 contrasts at first glance with our previous work reporting the failure of E4 to promote reendothelialization after perivascular injury (17). However, in contrast to the endovascular model, the perivascular injury induces a complete decellularization of the arterial wall, including both endothelial cells and the underlying SMCs. We confirmed here that E4 is not able to accelerate endothelial healing in this perivascular model (Figure 2, A and B). In addition, we show that E3, like E2, promoted endothelial healing in this perivascular model, in striking contrast to E4. Importantly, coadministration of E4 with either E2 or E3 abrogated the accelerative effect of these 2 estrogens on endothelial regeneration (Figure 2, A and B). Altogether, these results demonstrate that the 3 endogenous estrogens, E2, E3, and E4, are able to accelerate endothelial healing in the mouse carotid artery, but the presence of underlying SMCs is specifically required for E4 to mediate this vascular action.

Figure 2

In contrast to E2 and E3, E4 does not accelerate endothelial healing after carotid artery perivascular injury. Four-week-old female mice were ovariectomized and 2 weeks later were implanted subcutaneously with vehicle (Veh), E2, E3, or E4 pellets or a combination of 2 of these estrogens for 2 weeks. Mice were subjected to perivascular injury of the carotid artery. Carotid reendothelialization was analyzed 3 days after injury (n = 5–9 per group). (A) Representative Evans blue staining of carotids with outlined deendothelialized areas (scale bar: 1 mm) and (B) quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared with day 0. ECs, endothelial cells. Results are expressed as mean ± SEM. To test the effect of the different treatments, 1-way ANOVA was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh-treated group; ††P < 0.01 for difference between E2 and E2+E4; §§§P < 0.001 for difference between E3 and E3+E4.

ERα in SMCs is required to accelerate endothelial healing in response to E4, independently of membrane-initiated signaling. In order to assess the mechanism underlying the particular action of E4 and to directly evaluate the role of ERα in SMCs, we used a mouse model selectively invalidated for ERα in SMCs using the inducible Cre-ERT2 fusion gene system under the control of the α-smooth muscle actin (α-SMA) promoter (αSMACreERT2+ERαlox/lox mice) (21). We confirmed the efficiency and specificity of ERα deletion in SMCs from the aorta and the uterus of αSMACreERT2+ERαlox/lox compared with control littermate αSMACreERT2–ERαlox/lox mice (Supplemental Figure 2). Estrogen receptor 1 (Esr1) gene expression was reduced by 93% in the isolated media from the aorta of αSMACreERT2+ERαlox/lox mice, whereas no change was observed in the adventitia (Supplemental Figure 2, A and B). Similarly, we confirmed the specific deletion of ERα in SMCs in another tissue, as ERα staining revealed specific deletion of ERα in the myometrium of the uterus from αSMACreERT2+ERαlox/lox mice (Supplemental Figure 2C). As we used an inducible model, we additionally confirmed that tamoxifen injections did not alter reendothelialization rates after endovascular injuries in vehicle- and E4-treated wild-type (WT) mice (Supplemental Figure 3).

We next evaluated endothelial healing in αSMACreERT2–ERαlox/lox and αSMACreERT2+ERαlox/lox ovariectomized female mice supplemented or not with E4 (Figure 3A). In these mice, E4 treatment led to a similar uterine impregnation in both genotypes (Supplemental Table 2). As expected, E4 promoted reendothelialization after endovascular injury of the carotid artery in littermate control mice (40% of reendothelialization in vehicle-treated mice as compared with 80% in E4-treated mice) (Figure 3B). This accelerative effect was completely abrogated in αSMACreERT2+ERαlox/lox mice (Figure 3B), demonstrating that ERα in SMCs is required to promote E4’s effect on endothelial healing. Altogether, unlike what we have shown for E2 (22) and E3 (Figure 3C), SMCs appear to be the main target cells for the accelerative effect of E4 on endothelial healing.

Figure 3

ERα in smooth muscle cells is necessary for E4’s effect on endothelial healing but dispensable for E3’s effect. (A) Four-week-old ovariectomized αSMACreERT2+ERαlox/lox female mice and their respective control littermates were implanted with vehicle (Veh), E4, or E3 pellets for 2 weeks and subjected to endovascular injury of the carotid artery. Quantitative analysis of reendothelialization 5 days after injury, relative to day 0, are depicted in response to (B) E4 (n = 5–6 per group) or (C) E3 (n = 5–7 per group). Results are expressed as mean ± SEM. To test the effect of E4 and E3 treatments in each genotype, 2-way ANOVA was performed. **P < 0.01 versus Veh-treated group.

Despite its antagonistic effect on membrane ERα signaling, E4 accelerates endothelial healing in the presence of exogenous and endogenous estrogens. Since membrane ERα mediates the acceleration of reendothelialization in response to E2 (6, 22), we then decided to evaluate the role of this pathway in response to E4. To this end, we used 2 different mouse models targeting ERα membrane-initiated signaling, in which acceleration of endothelial regeneration in response to E2 was shown to be abrogated (7, 22). In C451A-ERα mice, ERα does not localize to the plasma membrane due to the point mutation of its palmitoylation site, leading to the loss of global membrane-initiated ER signaling. In this model, endothelial regeneration was approximately 20% in the vehicle-treated group, and E4 increased reendothelialization to 60% independently of genotype (Figure 4A), demonstrating that E4 promotes endothelial healing independently of membrane ERα. We extended this result using a second mouse model, i.e., R264A-ERα mice, targeting the second major amino acid involved in membrane ERα signaling (7). Similarly, we found no difference in reendothelialization rates between control and R264A-ERα female mice following E4 treatment (Figure 4B). In these 2 mouse models, uterine impregnation in response to E4 was similar in all genotypes (Supplemental Table 2). In addition, we used an immortalized human aortic endothelial cell line (TeloHAEC) to directly evaluate membrane ERα signaling in response to E2, E3, and E4 in endothelial cells. Since TeloHAECs (like other immortalized endothelial cell types) have no detectable ERα expression, we generated stably transduced TeloHAECs expressing full-length ERα (ERα-TeloHAECs) (Supplemental Figure 4, A–C). To evaluate membrane ERα signaling in these cells, we measured ERα interaction with the tyrosine kinase SRC using the proximity ligation assay (PLA) technique. Interaction of ERα with SRC is indicated by the presence of red dots in the cytoplasm of ERα-TeloHAECs. Importantly, no dots were detected using either only one antibody or both antibodies in TeloHAECs that do not express ERα, validating the specificity of the technique (Supplemental Figure 4, D–F). E2 and E3 increased the ERα-SRC interaction, whereas E4 failed to elicit this membrane ERα effect (Figure 4, C and D, and Supplemental Figure 5). Importantly, when administered together, E4 completely abrogated the stimulatory effect of E2 and E3 on the ERα-SRC interaction, highlighting that E4 antagonizes membrane ERα signaling in endothelial cells, as suggested in the model of perivascular injury (Figure 2).

Figure 4

E4 does not require membrane-initiated ERα signaling to accelerate endothelial healing and antagonizes this pathway in endothelial cells. Four-week-old ovariectomized (A) C451A-ERα (n = 6–7 per group) and (B) R264A-ERα (n = 7–11 per group) female mice and their respective control WT littermates were implanted with vehicle (Veh) or E4 pellets for 2 weeks and subjected to endovascular injury of the carotid artery. Schematic representation of each mouse model and quantitative analysis of reendothelialization 5 days after injury relative to day 0 are depicted. Results are expressed as mean ± SEM. To test the effect of E4 treatments in each genotype, 2-way ANOVA was performed. (C) Estrogen-deprived ERα-TeloHAECs were incubated with DMSO, E2 (1 × 10–8 M), E4 (1 × 10–6 M), or a combination of E2 and E4 for 5 minutes. Proximity ligation assay for ERα-SRC interaction was performed. Interactions are represented by red dots. Nuclei were counterstained with DAPI (scale bars: 20 μm). (D) Quantification of the number of dots per ERα-positive cell from 1 representative experiment. The experiment was replicated 3 times. Results are expressed as mean ± SEM. To test the effect of the different treatments, 1-way ANOVA was performed. *P < 0.05, **P < 0.01, ****P < 0.0001 versus Veh-treated group. ††††P < 0.0001 for difference between E2 and E2+E4.

The results presented above demonstrate that, on one hand, E2 and E4 act on different cell types to accelerate endothelial healing, and on the other hand E4 antagonizes E2-induced membrane ERα activity necessary for the effect of E2 on endothelial healing. As E4 is commercialized for contraception, which implies its use in the presence of endogenous estrogens, it is therefore important to assess whether its agonistic or antagonistic effects will be observed in this clinical setting. We thus decided to evaluate the impact of E4 on reendothelialization after endovascular injury of the carotid artery in the presence of exogenous or endogenous E2 (gonad-intact mice) (Figure 5). In contrast to the results obtained after perivascular injury (Figure 2), the coadministration of E4 with E2 still led to accelerated reendothelialization after endovascular injury, with no difference compared to E2 and E4 alone (Figure 5, A–C). In order to better model the use of E4 for contraception, we also administered E4 to gonad-intact female mice in which endogenous estrogens are present (Figure 5D). We validated that E4 led to the arrest of ovarian function by analyzing estrous cycles in vehicle- and E4-treated gonad-intact mice (Figure 5E). While vehicle-treated mice presented with regular estrous cycles, E4-treated mice were blocked in the estrus stage, validating our experimental model of contraception. Importantly, in gonad-intact mice, E4 accelerated endothelial healing (80% of reendothelialization compared with 30% in control mice; Figure 5F). Altogether, these results suggest that even though E4 antagonizes E2’s effects on membrane ERα in endothelial cells, the specific agonistic effect of E4 on ERα in SMCs is sufficient to mediate a beneficial endothelial healing effect compared with control mice.

Figure 5