Agonist effect of tamoxifen on the arterial wall : towards a better understanding of the selective modulation of ERα

Les patientes atteintes d'un cancer du sein sont exposées à un risque accru de maladies cardiovasculaires. L’hormonothérapie est un élément clé dans l’approche thérapeutique chez la grande majorité de ces patientes (tumeurs positives pour le récepteur des œstrogènes (ERα+), 70-75% des cancers du sein) et dans cet arsenal thérapeutique le tamoxifène est une molécule de référence. Le tamoxifène (TAM) est un modulateur sélectif d’ERα (SERM) qui bloque l’action proliférative des œstrogènes dans la cellule cancéreuse (action antagoniste). Contrairement à leurs effets délétères vis-vis a du risque tumoral, les œstrogènes ont un effet protecteur sur le plan cardiovasculaire chez la femme. Dans ce contexte, il parait crucial de mieux définir l’impact et les mécanismes d’action du TAM sur le plan vasculaire. En accord avec les études cliniques en faveur d’un effet protecteur du TAM vis-à-vis du risque cardiovasculaire, mon équipe d’accueil a contribué à démontrer que le TAM pouvait mimer les effets bénéfiques du 17β-œstradiol (E2) (action agoniste) sur le plan artériel et protéger i) du développement de l’athérome et ii) de l’hyperplasie néointimale dans des modèles expérimentaux de souris. Alors que les mécanismes moléculaires à l’origine de l’action antagoniste mammaire ont été bien décrits, la compréhension des mécanismes des effets agonistes du TAM reste limitée. L’objectif de mon travail de thèse a été de progresser dans les mécanismes d’action du TAM sur le plan vasculaire. L'analyse de la vitesse de cicatrisation endothéliale à l'aide de trois modèles complémentaires de lésion de l'artère carotide a permis de démontrer que le TAM accélérait la cicatrisation endothéliale mais, contrairement à l'action de l’E2, le TAM nécessitait la présence de muscles lisses. Les analyses de séquençage à large échelle (RNASeq) des carotides ont révélé des différences majeures dans l’expression génique en réponse au traitement chronique avec l’E2 et le TAM. Enfin, en utilisant des modèles de souris transgéniques ciblant ERα, nous avons démontré que l'accélération de la cicatrisation endothéliale requiert l'activation du i) ERα nucléaire dans les cellules musculaires lisses en réponse au TAM, et ii) du ERα membranaire dans les cellules endothéliales en réponse à l’E2. Ces résultats apportent une nouvelle vision dans la modulation sélective du ERα qui jusque-là se reposait essentiellement sur un recrutement différentiel de cofacteurs au sein d’un même type cellulaire. En effet, nous montrons que dans l'artère de souris, les cibles moléculaires et cellulaires de l’E2 et du TAM diffèrent profondément, bien que les deux activent ERα pour accélérer directement ou indirectement la cicatrisation endothéliale.With the improvement of survival after breast cancer and the increase in duration of life expectation, cardiovascular diseases (CVD) have become an increasingly important source of long term morbidity and mortality among breast cancer survivors. Tamoxifen is a key element in the therapeutic approach to treat patients with estrogen receptor-positive breast cancers (ERα +; 70-75% of breast cancers) and to prevent risk of recurrence. Tamoxifen is a selective estrogen receptor modulator (SERM), which acts as an Estrogen Receptor (ER) α antagonist in ER-positive breast cancers, whereas it mimics protective action of 17β-estradiol (E2) in arteries. In accordance with clinical studies in favor of a protective effect of TAM against cardiovascular risk, my team contributed to demonstrate that as E2, TAM induced protection against atherosclerosis and neointimal hyperplasia in experimental mouse model. During my PhD I have shown that: accordance with clinical studies in favor of a protective effect of TAM against cardiovascular risk, my team contributed to demonstrate that as E2, TAM induced protection against atherosclerosis and neointimal hyperplasia in experimental mouse model. During my PhD I have shown that: 1) TAM accelerated endothelial healing but, by contrast to E2 action, TAM required the presence of smooth muscle, using three complementary models of carotid artery injury. 2) E2 and TAM induced major gene signature differences as revealed by RNA sequencing analysis on the carotid artery. 3) Acceleration of endothelial healing is mediated by the activation of i) nuclear ERα in smooth muscle cells by TAM, and ii) of membrane ERα in endothelial cells by E2. These results suggest a new vision in the selective modulation of ERα which is till now is mainly interpreted as differential cofactor recruitment within the same cell type. Indeed, here we show that in the mouse artery, molecular and cellular targets of E2 and TAM differ profoundly, although both activate ERα and converge to directly or indirectly accelerate endothelial healing

Autonomic Nervous System Adaptation and Circadian Rhythm Disturbances of the Cardiovascular System in a Ground-Based Murine Model of Spaceflight

Whether in real or simulated microgravity, Humans or animals, the kinetics of cardiovascular adaptation and its regulation by the autonomic nervous system (ANS) remain controversial. In this study, we used hindlimb unloading (HU) in 10 conscious mice. Blood pressure (BP), heart rate (HR), temperature, and locomotor activity were continuously monitored with radio-telemetry, during 3 days of control, 5 days of HU, and 2 days of recovery. Six additional mice were used to assess core temperature. ANS activity was indirectly determined by analyzing both heart rate variability (HRV) and baroreflex sensitivity (BRS). Our study showed that HU induced an initial bradycardia, accompanied by an increase in vagal activity markers of HRV and BRS, together with a decrease in water intake, indicating the early adaptation to fluid redistribution. During HU, BRS was reduced; temperature and BP circadian rhythms were altered, showing a loss in day/night differences, a decrease in cycle amplitude, a drop in core body temperature, and an increase in day BP suggestive of a rise in sympathetic activity. Reloading induced resting tachycardia and a decrease in BP, vagal activity, and BRS. In addition to cardiovascular deconditioning, HU induces disruption in day/night rhythmicity of locomotor activity, temperature, and BP

Estetrol prevents western diet-induced obesity and atheroma independently of hepatic estrogen receptor (ER)α

International audienceEstetrol (E4), a natural estrogen synthesized by the human fetal liver, is currently evaluated in phase III clinical studies as a new menopause hormone therapy. Indeed, E4 significantly improves vasomotor and genito-urinary menopausal symptoms and prevents bone demineralization. Compared to other estrogens, E4 was found to have limited effects on coagulation factors in the liver of women allowing to expect less thrombotic events. To fully delineate its clinical potential, the aim of this study was to assess the effect of E4 on metabolic disorders. Here, we studied the pathophysiological consequences of a western diet (42% kcal fat, 0.2% cholesterol) in ovariectomized female mice under chronic E4 treatment. We showed that E4 reduces body weight gain and improves glucose tolerance in both C57Bl/6 and LDLR(-/-) mice. To evaluate the role of hepatic ERα in the preventive effect of E4 against obesity and associated disorders such as atherosclerosis and steatosis, mice harbouring a hepatocyte-specific ERα deletion (LERKO) were crossed with LDLR(-/-) mice. Our results demonstrated that, whereas liver ERα is dispensable for the E4 beneficial actions on obesity and atheroma, it is necessary to prevent steatosis in mice. Overall, these findings suggest that E4 could prevent metabolic, hepatic and vascular disorders occurring at menopause, extending the potential medical interest of this natural estrogen as a new hormonal treatment

The different natural estrogens promote endothelial healing through distinct cell targets

International audienceThe 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

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cell.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an Estrogen Receptor (ER) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol (E2) in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear.Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. Methods and Results: Using three complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with E2 and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα sub-functions (membrane/extra-nuclear versus genomic/transcriptional) demonstrated that E2-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. Conclusions: Whereas tamoxifen acts as an anti-estrogen and ERα antagonist in breast cancer, but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα

Selective Liver Estrogen Receptor Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice

International audienceHepatocyte estrogen receptor α (ERα) was recently recognized as a relevant molecular target for nonalcoholic fatty liver disease (NAFLD) prevention. The present study defined to what extent hepatocyte ERα could be involved in preserving metabolic homeostasis in response to a full (17β-estradiol [E2]) or selective (selective estrogen receptor modulator [SERM]) activation. Ovariectomized mice harboring a hepatocyte-specific deletion ( mice) and their wild-type (WT) littermates were fed a high-fat diet (HFD) and concomitantly treated with E2, tamoxifen (TAM; the most used SERM), or vehicle. As expected, both E2 and TAM prevented all HFD-induced metabolic disorders in WT mice, and their protective effects against steatosis were abolished in mice. However, while E2 still prevented obesity and glucose intolerance in mice, hepatocyte deletion also abrogated TAM-mediated control of food intake as well as its beneficial actions on adiposity, insulin sensitivity, and glucose homeostasis, suggesting a whole-body protective role for liver-derived circulating factors. Moreover, unlike E2, TAM induced a rise in plasma concentration of the anorectic hepatokine growth differentiation factor 15 (Gdf15) through a transcriptional mechanism dependent on hepatocyte ERα activation. Accordingly, ERα was associated with specific binding sites in the regulatory region in hepatocytes from TAM-treated mice but not under E2 treatment due to specific epigenetic modifications. Finally, all the protective effects of TAM were abolished in HFD-fed knockout mice. We identified the selective modulation of hepatocyte ERα as a pharmacologic strategy to induce sufficient anorectic hepatokine Gdf15 to prevent experimental obesity, type 2 diabetes, and NAFLD

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner

International audienceEstrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα ( Esr1 -/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H 2 O 2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner

Mutation of Arginine 264 on ERα (Estrogen Receptor Alpha) Selectively Abrogates the Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility

International audienceObjective - ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17β-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the , the females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. Conclusions - These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions

Clinical efficacy and safety of angiogenesis inhibitors: sex differences and current challenges

Vasoactive molecules, such as vascular endothelial growth factor (VEGF) and endothelins, share cytokine-like activities and regulate endothelial cell (EC) growth, migration and inflammation. Some endothelial mediators and their receptors are targets for currently approved angiogenesis inhibitors, drugs that are either monoclonal antibodies raised towards VEGF, or inhibitors of vascular receptor protein kinases and signaling pathways. Pharmacological interference with the protective functions of ECs results in a similar spectrum of adverse effects. Clinically, the most common side effects of VEGF signaling pathway inhibition include an increase in arterial pressure, left ventricular (LV) dysfunction ultimately causing heart failure, and thromboembolic events, including pulmonary embolism, stroke, and myocardial infarction. Sex steroids such as androgens, progestins, and estrogen and their receptors (ERα, ERβ, GPER; PR-A, PR-B; AR) have been identified as important modifiers of angiogenesis, and sex differences have been reported for anti-angiogenic drugs. This review article discusses the current challenges clinicians are facing with regard to angiogenesis inhibitor treatments, including the need to consider sex differences affecting clinical efficacy and safety. We also propose areas for future research taking into account the role of sex hormone receptors and sex chromosomes. Development of new sex-specific drugs with improved target and cell-type selectivity likely will open the way personalized medicine in men and women requiring antiangiogenic therapy and result in reduced adverse effects and improved therapeutic efficacy