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Endothelial barrier integrity is required for maintaining vascular homeostasis and fluid balance between the circulation and surrounding tissues. In contrast, abnormalities of endothelial cell function and loss of the integrity of the endothelial monolayer constitute a key step in the onset of atherosclerosis. Endothelial erosion is directly responsible for thrombus formation and cardiovascular events in about one-third of the cases of acute coronary syndromes. Thus, after endothelial injury, the vascular repair process is crucial to restore endothelial junctions and rehabilitate a semipermeable barrier, preventing the development of vascular diseases. Endothelial healing can be modulated by several factors. In particular, 17β-estradiol (E2), the main estrogen, improves endothelial healing, reduces neointimal accumulation of smooth muscle cells and atherosclerosis in several animal models. The aim of this review is to highlight how various experimental models enabled the progress in the cellular and molecular mechanisms underlying the accelerative E2 effect on arterial endothelial healing through the estrogen receptor (ER) α, the main receptor mediating the physiological effects of estrogens. We first summarize the different experimental procedures used to reproduce vascular injury. We then provide an overview of how the combination of transgenic mouse models impacting ERα signalling with pharmacological tools demonstrated the pivotal role of non-genomic actions of ERα in E2-induced endothelial repair. Finally, we describe recent advances in the action of selective estrogen receptor modulators (SERMs) on this beneficial vascular effect, which surprisingly involves different cell types and activates different ERα subfunctions compared to E2.

Estrogen 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 H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

Estetrol (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 with 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 estrogen receptor (ER) α in the preventive effect of E4 against obesity and associated disorders such as atherosclerosis and steatosis, mice harboring 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.NEW & NOTEWORTHY Estetrol prevents obesity, steatosis, and atherosclerosis in mice fed a Western diet. Hepatic ERα is necessary for the prevention of steatosis, but not of obesity and atherosclerosis.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol 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 3 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 17β-estradiol 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 subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17β-estradiol-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 antiestrogen 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.

Objective: 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 C451A-ERα , the R264A-ERα 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 R264A-ERα 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.

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.

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.

Les effets benefiques des œstrogenes sur le systeme vasculaire sont multiples : diminution de l’atherosclerose, de la dysfonction endotheliale, de l’hyperplasie neo-intimale, mais aussi acceleration de la cicatrisation endotheliale et augmentation de la production de NO. Cependant, les actions proliferatives des œstrogenes au niveau de la glande mammaire et de l’endometre uterin induisent un risque de cancers dans ces deux tissus, ce qui limite leur utilisation prolongee a long terme. L’amelioration de ces traitements reste donc un enjeu de sante public majeur pour la sante de la femme. L’ensemble de ces effets benefiques et deleteres sont relayes par le recepteur nucleaire ER∝. La modulation selective de ce recepteur nucleaire permet d’induire des tissus specificite d’action offrant ainsi la perspective de nouveaux traitements optimises. ER∝ agit classiquement au niveau nucleaire via la transcription de ses genes cibles, mais egalement a la membrane en activant differentes voies de signalisation. Dans cette etude, nous avons evalue les roles respectifs des effets membranaires et nucleaires dans la prevention de l’atherosclerose en utilisant une approche pharmacologique permettant d’induire selectivement la voie membranaire (œstrogene couple a un dendrimere) ou l’estetrol (E4) activant selectivement la voie nucleaire. En parallele, l’effet atheroprotecteur du 17β-œstradiol (E2) a ete etudie dans des modeles de souris invalidees specifiquement pour le ER∞nucleaire (ER∝AF20) ou membranaire (ER∝C451A) croisees avec le modele de souris atheromateuses LDLR−/−. Les donnees obtenues permettent de demontrer que contrairement a la cicatrisation endotheliale qui depend exclusivement du ER∝ membranaire, l’effet atheroprotecteur repose uniquement sur l’activation du ER∝ nucleaire.

The lower incidence of cardiovascular diseases in pre-menopausal women compared to men is well-known documented. This protection has been largely attributed to the protective effect of estrogens, which exert many beneficial effects against arterial diseases, including vasodilatation, acceleration of healing in response to arterial injury, arterial collateral growth and atheroprotection. More recently, with the visualization of the lymphatic vessels, the impact of estrogens on lymphedema and lymphatic diseases started to be elucidated. These estrogenic effects are mediated not only by the classic nuclear/genomic actions via the specific estrogen receptor (ER) α and β, but also by rapid extra-nuclear membrane-initiated steroid signaling (MISS). The ERs are expressed by endothelial, lymphatic and smooth muscle cells in the different vessels. In this review, we will summarize the complex vascular effects of estrogens and selective estrogen receptor modulators (SERMs) that have been described using different transgenic mouse models with selective loss of ERα function and numerous animal models of vascular and lymphatic diseases.