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Estetrol (E4) acts as a typical estrogen (E) agonist in stimulating the endometrium, and when administered to postmenopausal women in various doses, reverses vaginal atrophy and decreases vasomotor symptoms. However, in contrast to other Es administered orally, E4 demonstrates a minimal hepatic effect and does not stimulate breast proliferation. Data in pre and postmenopausal women show minimal stimulatory effects on triglycerides, sex hormone binding globulin, corticosteroid-binding globulin, and angiotensinogen. Similarly, E4 exerts minimal effects on coagulation markers, and when combined with drospirenone as a combined oral contraceptive does not increase the generation of thrombin as measured by the activated protein C resistance ratio, unlike the use of ethinylestradiol. Because of the tissue selectivity of E4, this study sought to better delineate the molecular mode of action of E4. Selective E Receptor Modulators (SERMs) exhibit specific cell and tissue estrogenic, neutral or anti-estrogenic activities. SERMs interact with the ligand binding domain of the E Receptor alpha (ERα) in a way that is distinct from that of E. By preventing the kinking of helix 12 in ERα, SERMs recruit other co-receptor activators and inhibitors that are responsible for SERM action. We compared in replicates the binding pattern and affinities of 154 motifs of ERα co-regulator activators and repressors to complexes of ERα with Estradiol (E2), Estriol (E3) or E4 in a specific Pam Gene assay (1).The pattern of co-receptor binding in the presence of E4 was identical to that elicited by E2 and E3, but with a 50-fold lower potency. This pattern is strikingly distinct from that observed with 4-hydroxytamoxifen or raloxifen. These data together with previously published studies of the crystalline structures of ERα with E2, E3 or E4 complexes confirm that E4 interacts with nuclear ERα in a manner identical to that of the other Es (2). Previous studies have shown that E4 inhibits the activity of the membrane ERα, and blocks the stimulatory action of E2 in this membrane ERα subpopulation. In conclusion, E4 is a unique E with specific actions in tissues, demonstrating absence of specific membrane receptor effects, and no interaction with ERα in the way characteristic of SERMs. The unique profile of E4 activity with lower hepatic effects (minimal or no effects on triglycerides and coagulation) may ultimately translate into safer clinical use. 1) Broekema et al. Endocrinology. 2018; 159:2397. 2) Abot et al. EMBO Mol Med. 2014; 6:1328 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Estrogens exert pleiotropic effects on multiple physiological and behavioral responses. Male and female sexual behavior in rodents constitutes some of the best-characterized responses activated by estrogens in adulthood and largely depend on ERα. Evidence exists that nucleus- and membrane-initiated estrogen signaling cooperate to orchestrate the activation of these behaviors both in short- and long-term. However, questions remain regarding the mechanism(s) and receptor(s) involved in the early brain programming during development to organize the circuits underlying sexually differentiated responses. Taking advantage of a mouse model harboring a mutation of the ERα palmitoylation site, which prevents membrane ERα signaling (mERα; ERα-C451A), this study investigated the role of mERα on the expression of male and female sexual behavior and neuronal populations that differ between sexes. The results revealed no genotype effect on the expression of female sexual behavior, while male sexual behavior was significantly reduced, but not abolished, in males homozygous for the mutation. Similarly, the number of kisspeptin- (Kp-ir) and calbindin-immunoreactive (Cb-ir) neurons in the anteroventral periventricular nucleus (AVPv) and the sexually dimorphic nucleus of the preoptic area (SDN-POA), respectively, were not different between genotypes in females. In contrast, homozygous males showed increased numbers of Kp-ir and decreased numbers of Cb-ir neurons compared to wild-types, thus leading to an intermediate phenotype between females and wild-type males. Importantly, females neonatally treated with estrogens exhibited the same neurochemical phenotype as their corresponding genotype among males. Together, these data provide evidence that mERα is involved in the perinatal programming of the male brain.

Women now live more than a third of their lives after the onset of menopause. The decline in endogenous estrogen production during this period is accompanied by functional disorders that affect quality of life. These symptoms may be relieved by menopausal hormone therapy (MHT) initially based on the administration of equine conjugated estrogens (mainly in the United States, oral route) or the natural estrogen, 17β-estradiol (in Europe, transdermal route). Estrogen receptor α (ERα), but not ERβ, mediates most of the physiological effects of estrogens. ERα belongs to the superfamily of nuclear receptors and regulates the transcription of genes via its activation functions AF1 and AF2. In addition to these classical genomic actions, estrogens can activate a subpopulation of ERα present at the cell membrane and thereby induce rapid signals. In this review, we will summarize the evolution of MHTs in last decades, as well as treatments that use various selective estrogen receptor modulators (SERMs). Next, we will describe recent advances in the understanding of the mechanisms of estrogen action, in particular the respective roles of nuclear and membrane ERα as well as the potential implications for future therapies.

Estrogens (E), in oral contraceptives (COCs) and hormone replacement therapy (HRT) drugs used for the relief of climacteric symptoms of menopause, increase the synthesis of clotting factors, decrease the levels of coagulation inhibitors, and increase the risk of venous thromboembolic events (VTE). Ischemic stroke incidence in postmenopausal women during HRT use is also increased and is probably due to a thrombotic event. This suggests that a safer estrogen may reduce stroke and VTE incidence, with lower impact on hemostasis.

Les femmes vivent désormais plus d’un tiers de leur vie après la survenue de la ménopause. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne fréquemment de troubles fonctionnels qui affectent la qualité de vie. Ces symptômes peuvent être soulagés par un traitement hormonal (THM) initialement fondé sur l’administration d’œstrogènes conjugués équins (principalement aux États-Unis, par voie orale) ou d’un œstrogène naturel, le 17β-estradiol (en Europe, notamment par voie transdermique). Le récepteur des œstrogènes α (REα) relaye la majorité des effets physiologiques des œstrogènes. REα appartient à la superfamille des récepteurs nucléaires. Il régule la transcription de gènesviases fonctions activatrices (AF1 et AF2). Outre ces actions génomiques classiques, les œstrogènes peuvent aussi activer une sous-population de récepteurs REα présents à la membrane des cellules et ainsi induire des signaux rapides. Dans cette revue, nous résumerons l’évolution des THM depuis les débuts de la substitution hormonale jusqu’aux nouvelles molécules émergentes fondées sur une modulation sélective du REα. Nous décrirons également les progrès récents sur la compréhension des mécanismes d’action des œstrogènes, en détaillant les rôles respectifs des REα nucléaire et membranaire et les développements thérapeutiques possibles qui pourraient en découler.

International audience; AimsDefects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects.Methods and resultsUsing in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine.ConclusionAltogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing.

Background Although estrogen receptor α ( ER α) acts primarily as a transcription factor, it can also elicit membrane‐initiated steroid signaling. Pharmacological tools and transgenic mouse models previously highlighted the key role of ER α membrane‐initiated steroid signaling in 2 actions of estrogens in the endothelium: increase in NO production and acceleration of reendothelialization. Methods and Results Using mice with ER α mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ER α plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear versus membrane‐initiated steroid signaling in the arterial protection conferred by ER α. ERaC451A mice were fully responsive to estrogens to prevent atheroma and angiotensin II –induced hypertension as well as to allow flow‐mediated arteriolar remodeling. By contrast, ER α AF 20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ER α with estetrol was able to prevent hypertension and to restore flow‐mediated arteriolar remodeling. Conclusions Altogether, these results reveal an unexpected prominent role of nuclear ER α in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ER α agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women.

Women now spend more than one-third of their lives in the postmenopausal years, and the decline of endogenous estrogen production during menopause is accompanied by a series of functional disorders that affect the quality of life. These symptoms could be alleviated or even totally suppressed by menopausal hormone therapy (MHT), initially based on natural estrogens extracted from the urine of pregnant mares (mainly in the USA, using the oral route) and later from the synthesis of the natural estrogen, 17β-estradiol (mainly in Europe, in particular using the transdermal route). Estrogen receptor (ER) α is the main receptor mediating the physiological effects of estrogens. ERα belongs to the nuclear receptor superfamily and activates gene transcription in a time and tissue-specific manner through two distinct activation functions (AF), AF1 and AF2. In addition to these classical genomic actions, ERα also mediates membrane initiated signaling enabling rapid actions of estrogen, potentially along or in interaction with other receptors. Here, we provide a brief historical overview of MHT, and we then highlight recent advances in the characterization of new treatments based on the association of estrogens with selective estrogen receptor modulators (SERMs) or on the modulation of nuclear or membrane ERα.

Estrogen receptors (ER) are used as therapeutic targets, either for contraception or for the hormonal replacement therapy in menopausal women, but also in physiopathology for breast cancer treatment. It is therefore important to understand the tissue-specificity of the actions of ERα to optimize the benefits/risks ratio in each tissue. Besides the conventional nuclear ERα acting as a transcription factor, many studies have demonstrated that ERα is also able to mediate extra nuclear signaling, enabling rapid actions of estrogen. Recently, new transgenic mouse models were used to study these effects, and allowed to genetically segregate membrane versus nuclear actions of a steroid hormone receptor, demonstrating their in vivo tissue-specific roles.

Problem Platelet reactivity has not been evaluated in integrated functional testing during normal pregnancy. Here, we analysed platelet functions under arterial shear rate in comparison with static conditions. Method of study Thirty pregnant women with uncomplicated pregnancies and 30 healthy non-pregnant women were enrolled in this study. Platelet adhesion to collagen and fibrinogen and subsequent thrombus formation were measured at arterial shear rate in whole blood using a microfluidic and imaging system. Standard light transmission aggregometry, flow cytometry of activation markers in washed platelets and impedance aggregometry in whole blood were also used to assess platelet responsiveness in static conditions. Results Compared to non-pregnant controls, thrombus formation on collagen fibres and firm platelet adhesion on fibrinogen under arterial shear rate were significantly reduced in pregnant women. Platelet aggregometry assays in suspension showed a slight increase in platelet reactivity in pregnant women. Conclusion While platelet aggregometry and platelet activation markers in static conditions show little changes in platelet reactivity, monitoring of platelet adhesion and thrombus growth on collagen or fibrinogen under flow condition in whole blood indicates a significant decrease in pregnant women compared to controls. This decrease might contribute to counteract a hypercoagulable state and to reduce the risk of arterial thrombosis.

Resume Objectif Evaluer l’apport de l’œstrogenotherapie locale (OL) vaginale sur les troubles urogenitaux lies a la menopause ainsi que sur ses effets indesirables a travers une revue de la litterature. Materiel Une revue de la litterature a ete realisee dans la base de donnees Pubmed en utilisant les mots cles suivants : vaginal estrogen , urinary incontinence , urgency , urinary infection, vulvovaginal atrophy, dyspareunia, breast cancer, endometrial cancer, thrombosis . Resultats L’OL a demontre son efficacite dans la prevention des infections urinaires, le traitement de l’hyperactivite vesicale et des troubles genitaux de la femme menopausee a travers d’importantes etudes randomisees ainsi que des meta-analyses. Les effets indesirables locaux (pertes vaginales, erythemes, metrorragies, etc.) sont rares. La diffusion systemique de l’OL faible dose est limitee et autorise ainsi sa prescription chez la femme menopausee sans surveillance particuliere. Cependant, l’utilisation d’une OL doit etre evitee chez la femme avec un antecedent de carcinologique du sein du fait d’une absence d’etude controlee ayant evalue le risque de survenue de cancer du sein. En dehors du cas particulier de la femme a haut risque, il n’existe pas d’augmentation du risque de thrombose chez la femme menopausee sous OL. Conclusion La prescription de l’OL faible dose dans le traitement des troubles urogenitaux post-menopausiques de la femme est sure et efficace. L’utilisation de l’OL chez la patiente avec un antecedent de cancer mammaire ou a haut risque de thrombose devrait cependant etre evitee.

The life expectancy of women has risen in the past century from 48years to more than 80. The decline of endogenous estrogen production (in particular, the principal circulating physiological hormone, 17β-estradiol) at menopause (which occurs at an average of 51years) is often accompanied by a series of functional disorders that affect quality of life (QoL). This estrogen deficiency affects different tissues and results in an increase in the prevalence of various disorders, including but not limited to osteoporosis and cardiovascular disease. Hormone therapy for menopause is a relatively recent biomedical challenge, which underwent a downturn after the Women Health Initiative study of older postmenopausal women. We will summarize the WHI findings in the first part of this article. At Inserm unit 1048, we are working on understanding the protective effects of estrogen against the development of atherosclerosis and type 2 diabetes in murine models. We have also focused in recent years on modeling the impact of estrogen in thrombosis models, to attempt to clarify the complex relation between estrogen and thrombotic risk. In part II of this article, we will describe a new strategy of hormone therapy for menopause, combining estrogens and selective estrogen receptor modulators (SERM). We review the scientific underpinnings of this strategy, which may enable the renewal of hormone therapy for menopause.

Estrogen receptor alpha (ERα) has been demonstrated to play a key role in reproduction but also to exert numerous functions in nonreproductive tissues. Accordingly, ERα is now recognized as a key regulator of energy homeostasis and glucose metabolism and mediates the protective effects of estrogens against obesity and type 2 diabetes. This chapter attempts to summarize our current understanding of the mechanisms of ERα activation and their involvement in the modulation of energy balance and glucose metabolism. We first focus on the experimental studies that constitute the basis of the understanding of ERα as a nuclear receptor and more specifically on the key roles played by its two activation functions (AFs). We depict the consequences of the selective inactivation of these AFs in mouse models, which further underline the prominent role of nuclear ERα in the prevention of obesity and diabetes, as on the reproductive tract and the vascular system. Besides these nuclear actions, a fraction of ERα is associated with the plasma membrane and activates nonnuclear signaling from this site. Such rapid effects, called membrane-initiated steroid signals (MISS), have been characterized in a variety of cell lines and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS as well as the generation of mice expressing an ERα protein impeded for membrane localization has just begun to unravel the physiological role of MISS in vivo and their contribution to ERα-mediated metabolic protection. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators.

Estrogens play an important role in bone growth and maturation as well as in the regulation of bone turnover in adults. Although the effects of 17β‐estradiol (E2) are well documented in long bones and vertebrae, little is known regarding its action in the mandible. E2 actions could be mediated by estrogen receptor (ER) α or β. ERs act primarily as transcriptional factors through two activation functions (AFs), AF1 and AF2, but they can also elicit membrane‐initiated steroid signaling (MISS). The aim of the present study was to define ER pathways involved in E2 effects on mandibular bone. Using mice models targeting ERβ or ERα, we first show that E2 effects on mandibular bone are mediated by ERα and do not require ERβ. Second, we show that nuclear ERαAF2 is absolutely required for all the actions of E2 on mandibular bone. Third, inactivation of ERαMISS partially reduced the E2 response on bone thickness and volume, whereas there was no significant impact on bone mineral density. Altogether, these results show that both nuclear and membrane ERα are requested to mediate full estrogen effects in the mandible of growing mice. Finally, selective activation of ERαMISS is able to exert an effect on alveolar bone but not on the cortical compartment, contrary to its protective action on femoral cortical bone. To conclude, these results highlight similarities but also specificities between effects of estrogen in long bones and in the mandible that could be of interest in therapeutic approaches to treat bone mass reduction. © 2018 American Society for Bone and Mineral Research.

Objective— Chronic nonhealing wounds are a substantial medical concern and are associated with morbidity and mortality; thus, new treatment strategies are required. The first step toward personalized/precision medicine in this field is probably in taking sex differences into account. Impaired wound healing is augmented by ischemia, and we previously demonstrated that 17β-estradiol exerts a major preventive effect against ischemia-induced skin flap necrosis in female mice. However, the equivalent effects of testosterone in male mice have not yet been reported. We then investigated the role of steroid hormones in male mice using a skin flap ischemia model. Approach and Results— Castrated male mice developed skin necrosis after ischemia, whereas intact or castrated males treated with testosterone were equally protected. Testosterone can (1) activate the estrogen receptor after its aromatization into 17β-estradiol or (2) be reduced into dihydrotestosterone, a nonaromatizable androgen that activates the androgen receptor. We found that dihydrotestosterone protected castrated wild-type mice by promoting skin revascularization, probably through a direct action on resistance arteries, as evidenced using a complementary model of flow-mediated outward remodeling. 17β-estradiol treatment of castrated male mice also strongly protected them from ischemic necrosis through the activation of estrogen receptor-α by increasing skin revascularization and skin survival. Remarkably, 17β-estradiol improved skin survival with a greater efficiency than dihydrotestosterone. Conclusions— Testosterone provides males with a strong protection against cutaneous necrosis and acts through both its estrogenic and androgenic derivatives, which have complementary effects on skin survival and revascularization.

The genitourinary syndrome of menopause has a negative impact on quality of life of postmenopausal women. The treatment of vulvovaginal atrophy includes administration of estrogens. However, oral estrogen treatment is controversial because of its potential risks on venous thrombosis and breast cancer. Estetrol (E4) is a natural estrogen synthesized exclusively during pregnancy by the human fetal liver and initially considered as a weak estrogen. However, E4 was recently evaluated in phase 1 to 2 clinical studies and found to act as an oral contraceptive in combination with a progestin, without increasing the level of coagulation factors. We recently showed that E4 stimulates uterine epithelial proliferation through nuclear estrogen receptor (ER) α, but failed to elicit endothelial responses. Herein, we first evaluated the morphological and functional impacts of E4 on the vagina of ovariectomized mice, and we determined the molecular mechanism mediating these effects. Vaginal epithelial proliferation and lubrication after stimulation were found to increase after E4 chronic treatment. Using a combination of pharmacological and genetic approaches, we demonstrated that these E4 effects on the vagina are mediated by nuclear ERα activation. Altogether, we demonstrate that the selective activation of nuclear ERα is both necessary and sufficient to elicit functional and structural effects on the vagina, and therefore E4 appears promising as a therapeutic option to improve vulvovaginal atrophy.

Estrogen receptor alpha (ER alpha) regulates gene transcription through two activation functions (ER alpha-AF1 and ER alpha-AF2). We recently found that the protection conferred by 17 beta-estradiol against obesity and insulin resistance requires ER alpha-AF2 but not ER alpha-AF1. However, the interplay between the two ER alpha-AFs is poorly understood in vivo and the metabolic influence of a specific ER alpha-AF1 action remains to be explored. To this end, wild-type, ER alpha-deficient, or ER alpha-AF1 deficient ovariectomized female mice were fed a high fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ER alpha-AF1 agonist/ER alpha-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ER alpha-deficient and ER alpha-AF1 deficient mice, revealing the specific role of ER alpha-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ER alpha-AF1 deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ER alpha-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ER alpha-AF2 in the metabolic actions of 17 beta-estradiol. This redundancy in the ability of the two ER alpha-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ER alpha-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation.

International audience; A promising alternative to conventional hormone therapy for postmenopausal symptoms is treatment combining Bazedoxifene (BZA), a third-generation selective estrogen receptor modulator (SERM), and conjugated equine estrogen (CE). This combination is also known as a tissue-selective estrogen complex (TSEC). Understanding the tissue-specific actions of SERMs and the TSEC remains a major challenge to try to predict their clinical effects. The aim of this study was to compare acute versus chronic treatment with BZA, CE or CE + BZA in two major targets of estrogens, the uterus and the liver. In these two tissues, acute treatment with CE, but not with BZA, induced similar gene expression change than the most important endogenous estrogen, 17-β estradiol (E2). Acute induction of gene expression by E2 or by CE was antagonized by the addition of BZA. Concomitantly, BZA alone or in combination with E2 or CE induced a partial degradation of ERα protein after acute exposure. In uterus, chronic treatment of BZA alone had no impact on tissue weight gain or on epithelial cell proliferation, and also antagonized CE-effect in uterus, thereby mimicking the acute effect. By contrast, in the liver, chronic BZA and CE + BZA elicited agonistic transcriptional effects similar to those of CE alone. In addition, at variance to BZA acute effect, no change in ERα protein abundance was observed after chronic treatment in this tissue. These experimental in vivo data highlight a new aspect of the time-dependent tissue-specific action of BZA or TSEC, i.e. they can act acutely as antagonists but become agonists after chronic treatment. This shift was observed in liver tissue, but not in proliferative sex target such as the uterus.

Estrogen receptor alpha (ERα) has been demonstrated to play a key role in reproduction but also to exert numerous functions in nonreproductive tissues. Accordingly, ERα is now recognized as a key regulator of energy homeostasis and glucose metabolism and mediates the protective effects of estrogens against obesity and type 2 diabetes. This chapter attempts to summarize our current understanding of the mechanisms of ERα activation and their involvement in the modulation of energy balance and glucose metabolism. We first focus on the experimental studies that constitute the basis of the understanding of ERα as a nuclear receptor and more specifically on the key roles played by its two activation functions (AFs). We depict the consequences of the selective inactivation of these AFs in mouse models, which further underline the prominent role of nuclear ERα in the prevention of obesity and diabetes, as on the reproductive tract and the vascular system. Besides these nuclear actions, a fraction of ERα is associated with the plasma membrane and activates nonnuclear signaling from this site. Such rapid effects, called membrane-initiated steroid signals (MISS), have been characterized in a variety of cell lines and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS as well as the generation of mice expressing an ERα protein impeded for membrane localization has just begun to unravel the physiological role of MISS in vivo and their contribution to ERα-mediated metabolic protection. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators.

Article en libre accès; Women and men, female and male animals and cells are biologically different, and acknowledgement of this fact is critical to advancing medicine. However, incorporating concepts of sex-specific analysis in basic research is largely neglected, introducing bias into translational findings, clinical concepts and drug development. Research funding agencies recently approached these issues but implementation of policy changes in the scientific community is still limited, probably due to deficits in concepts, knowledge and proper methodology. This expert review is based on the EUGenMed project (www.eugenmed.eu) developing a roadmap for implementing sex and gender in biomedical and health research. For sake of clarity and conciseness, examples are mainly taken from the cardiovascular field that may serve as a paradigm for others, since a significant amount of knowledge how sex and oestrogen determine the manifestation of many cardiovascular diseases (CVD) has been accumulated. As main concepts for implementation of sex in basic research, the study of primary cell and animals of both sexes, the study of the influence of genetic vs. hormonal factors and the analysis of sex chromosomes and sex specific statistics in genome wide association studies (GWAS) are discussed. The review also discusses methodological issues, and analyses strength, weaknesses, opportunities and threats in implementing sex-sensitive aspects into basic research.

Objective— Flow (shear stress)-mediated outward remodeling (FMR) of resistance arteries is a key adaptive process allowing collateral growth after arterial occlusion but declining with age. 17-β-estradiol (E2) has a key role in this process through activation of estrogen receptor α (ERα). Thus, we investigated the impact of age and timing for estrogen efficacy on FMR. Approach and Results— Female rats, 3 to 18 months old, were submitted to surgery to increase blood flow locally in 1 mesenteric artery in vivo. High-flow and normal-flow arteries were collected 2 weeks later for in vitro analysis. Diameter increased by 27% in high-flow arteries compared with normal-flow arteries in 3-month-old rats. The amplitude of remodeling declined with age (12% in 18-month-old rats) in parallel with E2 blood level and E2 substitution failed restoring remodeling in 18-month-old rats. Ovariectomy of 3-, 9-, and 12-month-old rats abolished FMR, which was restored by immediate E2 replacement. Nevertheless, this effect of E2 was absent 9 months after ovariectomy. In this latter group, ERα and endothelial nitric oxide synthase expression were reduced by half compared with age-matched rats recently ovariectomized. FMR did not occur in ERα −/− mice, whereas it was decreased by 50% in ERα +/− mice, emphasizing the importance of gene dosage in high-flow remodeling. Conclusions— E2 deprivation, rather than age, leads to decline in FMR, which can be prevented by early exogenous E2. However, delayed E2 replacement was ineffective on FMR, underlining the importance of timing of this estrogen action.

Introduction In total, 17β-Estradiol (E2) exerts numerous beneficial effects in vascular disease. It regulates gene transcription through nuclear estrogen receptor α (ERα) via 2 activation functions, AF1 and AF2, and can also activate membrane ERα. Objective and method Using mice with ERα mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ERα plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20), we sought to fully characterize the respective roles of nuclear versus membrane-initiated steroid signaling in the arterial protection conferred by ERα. Results Whereas the increase in NO production and the acceleration of endothelial healing in response to E2 were abrogated in ERaC451A mice, the protection against atheroma, neointimal hyperplasia, angiotensin II-induced hypertension, as well as the flow-mediated arteriolar remodelling, were preserved. In contrast, these last four beneficial vascular effects were lost in ERαAF20 mice in response to E2. In line with these results, selective activation of nuclear ERα with estetrol (E4) was capable of inducing arterial protection, except for the increase in NO production and the acceleration of endothelial healing. Conclusion Altogether, these results reveal a prominent role for nuclear ERα in the vasculoprotective action of estrogens. This will have major implications in medicine, since the selective nuclear ERα agonist, estetrol, is currently developed as a new oral contraceptive, and as hormone replacement therapy in menopausal women.

GOAL: The aim was to review the literature on nitric oxide and female lower urinary tract. MATERIAL: A literature review through the PubMed library until December, 31 2012 was carried out using the following keywords: lower urinary tract, bladder, urethra, nervous central system, innervation, female, women, nitric oxide, phosphodiesterase, bladder outlet obstruction, urinary incontinence, overactive bladder, urinary tract infection. RESULTS: Two nitric oxide synthase isoforms, the neuronal (nNOS) and the endothelial (eNOS), are constitutively expressed in the lower urinary tract. Nevertheless, nNOS is mainly expressed in the bladder neck and the urethra. In the bladder, NO modulates the afferent neurons activity. In pathological condition, inducible NOS expression induces an increase in detrusor contractility and bladder wall thickness and eNOS facilitates Escherichia coli bladder wall invasion inducing recurrent urinary tract infections. In the urethra, NO play a major role in smooth muscle cells relaxation. CONCLUSION: The NO pathway plays a major role in the female lower urinary tract physiology and physiopathology. While it acts mainly on bladder outlet, in pathological condition, it is involved in bladder dysfunction occurrence.

International audience; Based on both experimental and clinical data, tamoxifen has been proposed to have cardiovascular benefits, although the mechanism(s) contributing to that protective effect are still poorly understood. In vitro experiments demonstrated that tamoxifen elicits its transcriptional effect through estrogen receptor (ER) α, but other targets can participate in its actions. However, although tamoxifen selectively activates the activating function (AF)-1 of ERα, we recently showed that this ERα subfunction is dispensable for the atheroprotective action of 17β-estradiol (E2), the main ligand of ERs. The goal of the present work is to determine to which extent ERα and its AF-1 mediate the vasculoprotective action of tamoxifen. Our data confirm that tamoxifen exerts an atheroprotective action on low density lipoprotein receptor (LDL-r(-/-)) female mice, but, in contrast to E2, it fails to accelerate reendothelialization after carotid electric injury. Tamoxifen and E2 elicit differences in gene expression profiles in the mouse aorta. Finally, the atheroprotective action of tamoxifen is abrogated in ERα(-/-)LDL-r(-/-) mice and in LDL-r(-/-) mice selectively deficient in ERαAF-1 (ERαAF-1(0/0)LDL-r(-/-)). Our results demonstrate, for the first time to our knowledge, that tamoxifen mediates its actions in vivo through the selective activation of ERαAF-1, which is sufficient to prevent atheroma, but not to accelerate endothelial healing.

Background: Statins have been shown to increase the level of circulating progenitor cells in peripheral blood supposedly due to a mobilization of progenitor cells from the bone marrow niche. Osteoclast/osteoblast interaction has been associated with progenitor cell mobilization. Here, we investigated the role of statins on progenitor cell mobilization with a focus on bone metabolism. Methods and results: FGF2−/− and wild type (wt) mice were treated with atorvastatin or placebo. In contrast to wt mice, the number of sca-1/flk-1 positive progenitor cells in peripheral blood (PB) of atorvastatin treated FGF2−/− mice did not increase, and was accompanied by a defective reendothelialization after perielectric injury of the common carotid artery. In wt, but not FGF2−/− mice, statin treatment was associated with increased levels of receptor activator of NF-κB ligand (RANKL) in bone marrow (BM) supernatant. Treatment with recombinant RANKL increased sca-1/flk-1 positive progenitors in FGF2−/− mice. Interestingly, osteoclast activation was not altered. To measure the egress of sca-1/flk-1 positive progenitor cells from the bone marrow, we performed in-situ perfusion experiments of isolated hind limbs. Mobilization was not significantly affected by atorvastatin in both wt and FGF2−/− mice. Furthermore, RANK – the specific receptor to RANKL – is expressed on progenitor cells, and RANKL stimulation increases cell proliferation in vitro and in vivo. Conclusions: Atorvastatin treatment increases RANKL levels with no measurable effect on bone metabolism and mobilization of progenitor cells from BM to PB. RANKL is essential for the statin-mediated increase of progenitor cell levels but predominantly due to a RANKL-induced stimulation of cell proliferation.

International audience; Here, the authors report the fabrication of lotus-leaf-like tailored SU8 micropillars and their application in the context of a multitechnique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, as it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) is also expressed in ERα positive breast cancers and breast cancer cell lines. The superhydrophobic supports were developed by using a two-step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the biological samples resulted in stretched fibers of different diameters which were then characterized by synchrotron x-ray diffraction (XRD), Raman and Fourier-transform infrared spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biomarkers by exploiting a multitechnique in situ characterization approach.

Resistance arteries play a key role in the control of local blood flow. They undergo outward remodeling in response to a chronic increase in blood flow as seen in collateral artery growth in ischemic disorders. We have previously shown that mesenteric artery outward remodeling depends on the endothelial estrogen receptor alpha. As outward arterial remodeling is associated with improved endothelium-dependent dilation, we hypothesized that estrogens might also play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in first order mesenteric arteries was obtained after ligation of adjacent arteries in three-month old ovariectomized female rats treated with 17-beta-estradiol (OVX+E2) or vehicle (OVX). After 2 weeks, diameter was equivalent in high flow (HF) than in normal flow (NF) arteries with a greater wall to lumen ratio in HF vessels in OVX rats. Acetylcholine-mediated relaxation was lower in HF than in NF vessels. eNOS and caveolin-1 expression level was equivalent in HF and NF arteries. By contrast, arterial diameter was 30% greater in HF than in NF arteries and the wall to lumen ratio was not changed in OVX+E2 rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries. The expression level of eNOS was higher and that of caveolin-1 was lower in HF than in NF arteries. Acetylcholine (NO-dependent)-mediated relaxation was partly inhibited by the NO-synthesis blocker L-NAME in OVX rats whereas L-NAME blocked totally the relaxation in OVX+E2 rats. Endothelium-independent relaxation (sodium nitroprusside) was equivalent in OXV and OVX+E2 rats. Similarly, serotonin- and phenylephrine-mediated contractions were higher in HF than in NF arteries in both OVX and OVX+E2 rats in association with high ratio of phosphorylated ERK1/2 to ERK1/2. Thus, we demonstrated the essential role of endogenous E2 in flow-mediated improvement of endothelium (NO)-mediated dilatation in rat mesenteric arteries.

Estrogen receptor-α (ERα) acts primarily in the nucleus as a transcription factor involving two activation functions, AF1 and AF2, but it can also induce membrane-initiated steroid signaling (MISS) through the modulation of various kinase activities and/or secondary messenger levels. Previous work has demonstrated that nuclear ERα is required for the protective effect of the estrogen 17β-estradiol (E2), whereas the selective activation of ERαMISS is sufficient to confer protection in cortical but not cancellous bone. The aim of this study was to define whether ERαMISS is necessary for the beneficial actions of chronic E2 exposure on bone. We used a mouse model in which ERα membrane localization had been abrogated due to a point mutation of the palmitoylation site of ERα (ERα-C451A). Alterations of the sex hormones in ERα-C451A precluded the interpretation of bone parameters that were thus analyzed on ovariectomized and supplemented or not with E2 (8 μg/kg/d) to circumvent this bias. We found the beneficial action of E2 on femoral bone mineral density as well as in both cortical and cancellous bone was decreased in ERα-C451A mice compared with their wild-type littermates. Histological and biochemical approaches concurred with the results from bone marrow chimeras to demonstrate that ERαMISS signaling affects the osteoblast but not the osteoclast lineage in response to E2. Thus, in contrast to the uterine and endothelial effects of E2 that are specifically mediated by nuclear ERα and ERαMISS effects, respectively, bone protection is dependent on both, underlining the exquisite tissue-specific actions and interactions of membrane and nuclear ERα.

Background Cardiovascular diseases (CVD) incidence increases with aging and numerous risk factors like arterial hypertension. Estrogens reduce vascular damaging triggered by CVD like hypertension, mainly through estrogen receptor alpha (ERα). However, recent studies have shown that a fraction of ERα is located at the cell membrane where it initiates a Membrane Initiated Steroid Signaling (MISS). Both membrane and nuclear pathways of ERα seem to participate to the protective effect of estrogens but their respective role in hypertension is unknown. Objective This study aims to better understand the protective role of ERα in hypertension and its vascular consequences. Method Five groups of female mice were used in this study, each one lacking either estrogens (WT-OVX), ERα (ERα-KO), membrane-ERα (C451A-ERα) or ligand-dependent-transcriptional function of ERα (AF2°). Half of the mice received a chronic infusion of angiotensin II (0.5 mg/kg/d during 1 month) to induce moderate hypertension. Systolic blood pressure (SBP) and vascular structure were measured in KO mice and compared to wild-type mice (WT). Studies were performed in 5 months mice and 16 months mice in order to evaluate the impact of aging in the estrogens protection. Results SBP increased by 14 mmHg (ns) in WT mice. A similar increased was observed in C451A mice (20 mmHg, ns). By contrast, SBP increased significantly more in WT-OVX mice (31 mmHg, p=0,0007), ERα-KO (45 mmHg, p Conclusion Taken together, these data suggest that the protective effect of ERα against angiotensin II-induced hypertension depend on ERα gene transcription through AF2 and not membrane ERα signaling by contrast with most described vasculoprotective effects of estrogens. The author hereby declares no conflict of interest

Resistance arteries are sensitive to mechanical forces exerted on the vessel wall. While endothelial shear stress triggers flow-mediated dilation (FMD), chronic increase in shear forces drives expansive arterial remodeling. We showed previously that endothelial estrogen receptor alpha (ER‹) controlled this adaptive remodeling. We aimed to evaluate ER‹contribution in acute response to flow, by using various genetic models of ER‹deficiency in male mice, reducing estrogen hormonal influence. FMD was evaluated following step increase in flow applied to pressurized mesenteric arteries (arteriography). Arteries were isolated from wild-type (WT), (i) ER‹KO, or mice invalidated for (ii) the ligand-dependent transactivation function AF2 of ERa (AF2°) and (iii) the plasma membrane-located ER‹(mutated for the palmitoylation site C451A). In mice deficient in ER‹, FMD was attenuated (dilation 50μl/min WT: 59+/–4% vs. ER‹KO: 41+/–4% p The author hereby declares no conflict of interest

International audience; OBJECTIVES:Chronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats.METHODS:Blood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat.RESULTS:Arterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups.CONCLUSION:Resveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.

Clinical parameters of the breast tumor samples. IDC invasive ductal carcinoma, ILC invasive lobular carcinoma. Figure S2. Expression of Flag-ERα36 in transiently transfected MDA-MB231 as detected by immunocytochemistry using an anti-Flag antibody. Figure S3. Analysis of potential correlation between the clinical parameters of the breast tumor samples and the expression of ERα46 isoform. A significant P value (indicated in red) was only found between ERα46 expression and HER-2 positive breast tumors. IDC invasive ductal carcinoma, ILC invasive Lobular Carcinoma. Figure S4. Results of the proteomic analysis of the ERα46 protein detected in tumor samples. A) Western blot with the SP1 antibody obtained after immunoprecipitation of ERα with HC20 antibody in two human tumors overexpressing the putative ERα46 isoform. B and C) Sequence coverage obtained from the peptides identified by proteomic analysis shown in bold red on ERα66 and on ERα46 isoforms M (methionine): putative translational start sites generating the ERα46 isoform. Figure S5. The stress-induced increase in LucF activity is reproducible in MCF7 cells (A) and is not due to the generation of mono-cistronic LucF transcripts via an internal promoter or cryptic splicing as observed in MDA-Lenti-AB exposed to two siRNAs-lucR (B and C). Figure S6. Modulation profiles of the interaction of ERα46 (red) and ERα66 (blue) with coregulators in A) Apo proteins and B) in response to E2 binding. C) Profile of EC50 values of 4-OH-tamoxifen- (red) and fulvestrant- (blue) induced modulation of ERα46 and ERα66 coregulators interaction when use in antagonists mode with 6.3 nM E2. Figure S7. List of primers used in the expression profiling of target genes. Figure S8. Fold-changes (FC) in gene expression± SEM in MDA-ERα46 and MDA-ERα66 cells in response to 4-h treatment with E2 (10–8 M). P values were determined by Mann-Whitney test. Significant P values are indicated in red. (PPTX 939 kb)

Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18−/−) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18−/− mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

The ancestral status of estrogen receptor (ER) in the family of the steroid receptors has probably contributed to the pleiotropic actions of estrogens, and in particular of 17β-estradiol (E2). Indeed, in addition to their well described role in sexual development and reproduction, they influence most of the physiological processes. The pathophysiological counterpart of these actions includes several highly beneficial effects such as prevention of osteoporosis, atheroma and type 2 diabetes,… However, estrogens also promotes two deleterious actions : the stimulation of the proliferation of the epithelium of two sex targets : uterus and breast, favoring an increase in risk of epithelial cancer in these two tissues. These actions are mediated by the activation of ER alpha (ERα) and beta (ERβ), which regulate target gene transcription (genomic action) through two independent activation functions AF-1 and AF-2, but can also elicit rapid membrane initiated steroid signals. Although ERβ plays an important role in the central nervous system and in the heart, ERα appears to play a prominent role in most of the other tissues. One major challenge consists in uncoupling some beneficial actions from other deleterious ones, i.e. selective ER modulation. Tamoxifen and raloxifen are beneficial to prevent the recurrence of breast cancer, and mimic estrogen action mainly on bone, but their effets on atheroma and on type 2 diabetes are if any marginal. These last years, several labs, and in particular our lab, have attempted: 1)To perform an in vivo molecular "dissection" of ERα, allowing the uncoupling of some of its actions, and potentially paving the way to optimized selective ER modulators. (reviewed in Arnal JF, et al. Br J Pharmacol. 2012;165:57-66). 2)To describe an unexpected action of E2 treatment at the level of platelet responses in mice, that protects the animals from thromboembolism through the haematopoietic ERα. (Valera MC et al. Blood. 2012, in press).

Although estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or β were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including β1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies.

The ancestral status of oestrogen receptor (ER) in the family of the steroid receptors has probably contributed to the pleiotropic actions of oestrogens, and in particular, that of 17β-oestradiol (E2). Indeed, in addition to their well-described role in sexual development and reproduction, they influence most of the physiological processes. The pathophysiological counterpart of these actions includes prevention of osteoporosis, atheroma and type 2 diabetes, and also the promotion of uterus and breast cancer growth. Thus, the major challenge consists in uncoupling some beneficial actions from other deleterious ones, that is, selective ER modulation. Tamoxifen and raloxifene are already used, as they prevent the recurrence of breast cancer and mimic oestrogen action mainly on bone. Both E2 and tamoxifen exhibit a proliferative and, thus, a protumoural action on the endometrium. Activation of ERα and ERβ regulates target gene transcription (genomic action) through two independent activation functions, AF-1 and AF-2, but can also elicit rapid membrane-initiated steroid signals. In the present review, we attempted to summarize recent advances provided by the in vivo molecular ‘dissection’ of ERα, allowing the uncoupling of some of its actions and potentially paving the way to optimized selective ER modulators.

International audience; Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

Although the accelerative effect of 17β-estradiol (E2) on endothelial regrowth has been clearly demonstrated, the local cellular events accounting for this beneficial vascular action are still uncertain. In the present work, we compared the kinetics of endothelial healing of mouse carotid arteries after endovascular and perivascular injury. Both basal reendothelialization as well as the accelerative effect of E2 were similar in the two models. Three days after endothelial denudation, a regenerative area was observed in both models, characterized by similar changes in gene expression after injury, visualized by en face confocal microscopy (EFCM). A precise definition of the injury limits was only possible with the perivascular model, since it causes a complete and lasting decellularization of the media. Using this model, we demonstrated that the migration of uninjured endothelial cells precedes proliferation (bromodeoxyuridine incorporation) and that these events occur at earlier time points with E2 treatment. We have also identified an uninjured retrograde zone as an intimate component of the endothelial regeneration process. Thus, in the perivascular model, the regenerative area can be subdivided into a retrograde zone and a reendothelialized area. Importantly, both areas are significantly enlarged by E2. In conclusion, the combination of the electric perivascular injury model and EFCM is well adapted to the visualization of the endothelial monolayer and to investigate cellular events involved in reendothelialization. This process is accelerated by E2 as a consequence of the retrograde commitment of an uninjured endothelial zone to migrate and proliferate, contributing to an enlargement of the regenerative area.

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17β-estradiol (E2) has been shown to promote the differentiation of DCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERα- or ERβ-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERα, but not ERβ, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERα activation and provide evidence for a cell-autonomous function of ERα signaling in DC differentiation. ERα-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERα-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4+ T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERα-deficient DCs were defective in their ability to secrete such cytokines in response to CD40–CD40L interactions. Taken together, these results provide the first genetic evidence that ERα is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.

Aims We analysed the possible protective role of human endothelial (EPCs) and smooth muscle (SPCs) progenitor cells on atherosclerosis development in apoE−/−RAG2−/− mice. We determined plasma levels of SPCs in coronary patients. Methods and results ApoE−/−RAG2−/− mice received four intravenous injections of saline, 5 × 105 SPCs, or 5 × 105 EPCs every other week, one (preventive approach) or 12(curative approach) weeks after starting a high fat diet. Derived-SPC levels were quantified from blood mononuclear cells of patients with stable angina ( n = 10) and acute coronary syndromes (ACS, n = 9). SPCs reduced atherosclerosis development by 42% ( P < 0.001), but had no effect on lesion progression. In the SPC group, collagen and smooth muscle cell content were increased (+80%, P < 0.001, +46%, P < 0.05, respectively), and macrophage content was decreased (−41%, P < 0.05). In the curative approach, macrophage content decreased by 40.5% ( P < 0.05) after SPC injection. EPC injection had no effect on atherosclerosis development or progression. Peripheral blood-derived SPC levels were reduced in patients with ACS compared with stable angina patients ( P < 0.05). Conclusion We demonstrate that SPCs limit plaque development and promote changes in plaque composition towards a stable phenotype in mice. Our finding in patients suggests that reduced peripheral blood-derived SPC levels might represent a mechanism contributing to plaque destabilization.

Objective— Whereas estradiol prevents fatty streak deposit in immunocompetent apoE −/− or LDLr −/− mice, it is totally ineffective in immunodeficient mice, underlining the key role of immunoinflammation in this effect. In the present work, the role of several major pro- and antiinflammatory cytokines involved in the atheromatous process was evaluated in the effect of estradiol on fatty streak constitution. Methods and Results— The preventive effect of estradiol was fully maintained in LDLr −/− mice grafted with bone marrow from either IFN-γ or interleukin (IL)-12–deficient mice, showing that this beneficial effect was not mediated through a specific decrease in the production of these 2 proinflammatory cytokines. Furthermore, IL-10 −/− apoE −/− mice remained protected by estradiol, excluding a significant contribution of this antiinflammatory cytokine. In contrast, the protective effect of estradiol was (1) associated with enhanced aortic expression of TGF-β1 in apoE −/− mice during early steps of atherogenesis; (2) abolished and even reversed in apoE −/− mice administered with a neutralizing anti–TGF-β antibody; (3) abolished in LDLr −/− mice grafted with bone marrow from Smad3-deficient mice. Conclusions— The status of the TGF-β pathway crucially determines the antiatherogenic effect of estradiol in hypercholesterolemic mice, whereas neither IFN-γ, IL-12, nor IL-10 are specifically involved in this protection.

International audience; PURPOSE OF REVIEW: Whereas hormone therapy may increase the risk of coronary heart disease and stroke in menopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol. There is also evidence for a thrombogenic effect of oral estrogens. An understanding of the deleterious and beneficial effects of estrogens is thus required. RECENT FINDINGS: The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas estradiol favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo involving several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for estrogens, since estradiol potentiates endothelial nitric oxide and prostacyclin production. The respective actions of estrogens on these cell populations may be influenced by the timing of hormonal therapy initiation, hormone regimens, status of the vessel wall and expression of isoforms of estrogen receptors alpha and beta. SUMMARY: A better understanding of the balance between the deleterious and beneficial effects of estrogens is required and should help to improve hormonal therapy safety and to optimize the prevention of cardiovascular disease after menopause.

L’objectif de cette etude est de decrire l’impact des estrogenes sur la fonction vesicale. Les recepteurs aux estrogenes sont exprimes au niveau du dome et du trigone vesical et au niveau du systeme nerveux central et peripherique innervant la vessie. En cas de deprivation estrogenique, il apparait, au niveau de la paroi vesicale, une atrophie musculaire lisse, une augmentation du collagene et une augmentation du ratio collagene/muscle lisse. L’estrogenotherapie entraine une hypertrophie musculaire, une augmentation du poids de la vessie, une neo-angiogenese et une modification de la densite des recepteurs α-adrenergiques, muscariniques et purinergiques. Au niveau fonctionnel, ces modifications de structure sont responsables lors d’une deprivation estrogenique d’une hypocontractilite vesicale, et en cas de traitement estrogenique, d’une augmentation de la contractilite vesicale. Au niveau de l’innervation vesicale, les estrogenes semblent avoir une action predominante au niveau de l’innervation afferente et en particulier au niveau des neurones vanilloides.

Estrogen receptors (ER) are used as therapeutic targets, either for contraception or for the hormonal replacement therapy in menopausal women, but also in physiopathology for breast cancer treatment. It is therefore important to understand the tissue-specificity of the actions of ERα to optimize the benefits/risks ratio in each tissue. Besides the conventional nuclear ERα acting as a transcription factor, many studies have demonstrated that ERα is also able to mediate extra nuclear signaling, enabling rapid actions of estrogen. Recently, new transgenic mouse models were used to study these effects, and allowed to genetically segregate membrane versus nuclear actions of a steroid hormone receptor, demonstrating their in vivo tissue-specific roles.

We recently reported that chronic 17β-estradiol (E2) treatment in mice decreases platelet responsiveness, prolongs the tail-bleeding time and protects against acute thromboembolism via the hematopoietic estrogen receptor alpha (ERα), and independently of ERβ. Here, we have explored the respective roles of membrane vs nuclear actions of ERα in this process, using: 1) the selective activator of membrane ERα: estrogen dendrimer conjugate, and 2) mouse models with mutations in ERα. The selective targeting of activation function 2 of ERα provides a model of nuclear ERα loss-of-function, whereas mutation of the ERα palmitoylation site leads to a model of membrane ERα deficiency. The combination of pharmacological and genetic approaches including hematopoietic chimera mice demonstrated that absence of either membrane or nuclear ERα activation in bone marrow does not prevent the prolongation of the tail-bleeding time, suggesting a redundancy of these two functions for this E2 effect. In addition, although hematopoietic membrane ERα is neither sufficient nor necessary to protect E2-treated mice from collagen/epinephrine-induced thromboembolism, the protection against death-induced thromboembolism is significantly reduced in the absence of hematopoietic nuclear ERα activation. Overall, this study emphasizes that hematopoietic cells (likely megakaryocytes and possibly immune cells) constitute an important target in the antithrombotic effects of estrogens, and delineate for the first time in vivo the respective roles of membrane vs nuclear ERα effects, with a prominent role of the latter.

The female life expectancy rose from an average of 48 years to over 80 years in a century. The decline in the endogenous production of estrogen (especially the main circulating physiological hormone, 17β-estradiol, E2) at menopause (51 years on average) often leads to functional disorders affecting the quality of life. Estrogen deficiency impacts different tissues and results in an increase of various diseases, such as osteoporosis or cardiovascular diseases. Hormone therapy (HT) for menopause is a rather new challenge which experienced vagaries following the women's health initiative (WHI) study conducted in largely post-menopausal women. In the first part of this review, we will try to summarize the main conclusions of the WHI trials, in particular the timing effect as well as the deleterious impact of the associated progestin, medroxyprogesterone acetate (MPA). Hormone therapy, particularly the conjugated equine estrogen (CEE) combined with the MPA favor the occurrence of breast cancer, whereas conversely selective estrogen receptor modulators (SERMs, such as tamoxifen or raloxifene) that block the activity of estrogen receptor alpha (ERa) prevent the risk of recurrence of ERa-positive breast cancers. A new strategy of ERa modulation called tissue selective estrogen complex (TSEC), combines (1) CEE to maintain the benefits of estrogen (climacteric symptoms and prevention of osteoporosis) and (2) bazedoxifene, which is not only a SERM, but which also induces a rapid degradation of ERa in the uterus and in the breast, thereby prevents the stimulatory effects of estrogens on epithelial proliferation of these two sex targets. In the second part of this review, we will summarize the recognized benefits of the TSEC approach, and our current knowledge of its potential benefits and risks.