Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

Rationale: 17ß-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors.

Membrane estrogen receptor-α contributes to female protection against high-fat diet-induced metabolic disorders.

Background: Estrogen Receptor α (ERα) is a significant modulator of energy balance and lipid/glucose metabolisms. Beyond the classical nuclear actions of the receptor, rapid activation of intracellular signaling pathways is mediated by a sub-fraction of ERα localized to the plasma membrane, known as Membrane Initiated Steroid Signaling (MISS). However, whether membrane ERα is involved in the protective metabolic actions of endogenous estrogens in conditions of nutritional challenge, and thus contributes to sex differences in the susceptibility to metabolic diseases, remains to be clarified. Methods: Male and female C451A-ERα mice, harboring a point mutation which results in the abolition of membrane localization and MISS-related effects of the receptor, and their wild-type littermates (WT-ERα) were maintained on a normal chow diet (NCD) or fed a high-fat diet (HFD). Body weight gain, body composition and glucose tolerance were monitored. Insulin sensitivity and energy balance regulation were further investigated in HFD-fed female mice. Results: C451A-ERα genotype had no influence on body weight gain, adipose tissue accumulation and glucose tolerance in NCD-fed mice of both sexes followed up to 7 months of age, nor male mice fed a HFD for 12 weeks. In contrast, compared to WT-ERα littermates, HFD-fed C451A-ERα female mice exhibited: 1) accelerated fat mass accumulation, liver steatosis and impaired glucose tolerance; 2) whole-body insulin resistance, assessed by hyperinsulinemic-euglycemic clamps, and altered insulin-induced signaling in skeletal muscle and liver; 3) significant decrease in energy expenditure associated with histological and functional abnormalities of brown adipose tissue and a defect in thermogenesis regulation in response to cold exposure. Conclusion: Besides the well-characterized role of ERα nuclear actions, membrane-initiated ERα extra-nuclear signaling contributes to female, but not to male, protection against HFD-induced obesity and associated metabolic disorders in mouse.

The beneficial effects of tamoxifen on arteries: A key player for cardiovascular health of breast cancer patient.

Breast cancer is the most common cancer in women. Over the past few decades, advances in cancer detection and treatment have significantly improved survival rate of breast cancer patients. However, due to the cardiovascular toxicity of cancer treatments (chemotherapy, anti-HER2 antibodies and radiotherapy), cardiovascular diseases (CVD) have become an increasingly important cause of long-term morbidity and mortality in breast cancer survivors. Endocrine therapies are prescribed to reduce the risk of recurrence and specific death in estrogen receptor-positive (ER +) early breast cancer patients, but their impact on CVD is a matter of debate. Whereas aromatase inhibitors and luteinizing hormone-releasing hormone (LHRH) analogs inhibit estrogen synthesis, tamoxifen acts as a selective estrogen receptor modulator (SERM), opposing estrogen action in the breast but mimicking their actions in other tissues, including arteries. This review aims to summarize the main clinical and experimental studies reporting the effects of tamoxifen on CVD. In addition, we will discuss how recent findings on the mechanisms of action of these therapies may contribute to a better understanding and anticipation of CVD risk in breast cancer patients.

The different natural estrogens promote endothelial healing through distinct cell targets.

The main estrogen, 17ß-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.

Profile of estetrol, a promising native estrogen for oral contraception and the relief of climacteric symptoms of menopause.

INTRODUCTION: Estrogens used in women's healthcare have been associated with increased risks of venous thromboembolism (VTE) and breast cancer. Estetrol (E4), an estrogen produced by the human fetal liver, has recently been approved for the first time as a new estrogenic component of a novel combined oral contraceptive (E4/drospirenone [DRSP]) for over a decade. In phase 3 studies, E4/DRSP showed good contraceptive efficacy, a predictable bleeding pattern, and a favorable safety and tolerability profile. AREAS COVERED: This narrative review discusses E4's pharmacological characteristics, mode of action, and the results of preclinical and clinical studies for contraception, as well as for menopause and oncology. EXPERT OPINION: Extensive studies have elucidated the properties of E4 that underlie its favorable safety profile. While classical estrogens (such as estradiol) exert their actions via both activation of nuclear and membrane estrogen receptor α (ERα), E4 presents a specific profile of ERα activation: E4 binds and activates nuclear ERα but does not induce the activation of membrane ERα signaling pathways in specific tissues. E4 has a small effect on normal breast tissue proliferation and minimally affects hepatic parameters. This distinct profile of ERα activation, uncoupling nuclear and membrane activation, is unique.

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries.

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1-/- (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1-/- and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.

Segregation of nuclear and membrane-initiated actions of estrogen receptor using genetically modified animals and pharmacological tools.

Estrogen receptor alpha (ERα) and beta (ERß) are members of the nuclear receptor superfamily, playing widespread functions in reproductive and non-reproductive tissues. Beside the canonical function of ERs as nuclear receptors, in this review, we summarize our current understanding of extra-nuclear, membrane-initiated functions of ERs with a specific focus on ERα. Over the last decade, in vivo evidence has accumulated to demonstrate the physiological relevance of this ERα membrane-initiated-signaling from mouse models to selective pharmacological tools. Finally, we discuss the perspectives and future challenges opened by the integration of extra-nuclear ERα signaling in physiology and pathology of estrogens.

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

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.

A historical view of estrogen effect on arterial endothelial healing: From animal models to medical implication.

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, 17ß-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.

Nuclear translocation of MRTFA in MCF7 breast cancer cells shifts ERα nuclear/genomic to extra-nuclear/non genomic actions.

The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor's activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling.

Estrogen Receptor and Vascular Aging.

Cardiovascular diseases remain an age-related pathology in both men and women. These pathologies are 3-fold more frequent in men than in women before menopause, although this difference progressively decreases after menopause. The vasculoprotective role of estrogens are well established before menopause, but the consequences of their abrupt decline on the cardiovascular risk at menopause remain debated. In this review, we will attempt to summarize the main clinical and experimental studies reporting the protective effects of estrogens against cardiovascular diseases, with a particular focus on atherosclerosis, and the impact of aging and estrogen deprivation on their endothelial actions. The arterial actions of estrogens, but also part of that of androgens through their aromatization into estrogens, are mediated by the estrogen receptor (ER)α and ERß. ERs belong to the nuclear receptor family and act by transcriptional regulation in the nucleus, but also exert non-genomic/extranuclear actions. Beside the decline of estrogens at menopause, abnormalities in the expression and/or function of ERs in the tissues, and particularly in arteries, could contribute to the failure of classic estrogens to protect arteries during aging. Finally, we will discuss how recent insights in the mechanisms of action of ERα could contribute to optimize the hormonal treatment of the menopause.

Estetrol prevents Western diet-induced obesity and atheroma independently of hepatic estrogen receptor α.

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.

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

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 17ß-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 17ß-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 17ß-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.

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

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 17ß-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.

The Impact of Estrogen Receptor in Arterial and Lymphatic Vascular Diseases.

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 ß, 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.

Estrogen Receptors and Endometriosis.

Endometriosis is a frequent and chronic inflammatory disease with impacts on reproduction, health and quality of life. This disorder is highly estrogen-dependent and the purpose of hormonal treatments is to decrease the endogenous ovarian production of estrogens. High estrogen production is a consistently observed endocrine feature of endometriosis. mRNA and protein levels of estrogen receptors (ER) are different between a normal healthy endometrium and ectopic/eutopic endometrial lesions: endometriotic stromal cells express extraordinarily higher ERß and significantly lower ERα levels compared with endometrial stromal cells. Aberrant epigenetic regulation such as DNA methylation in endometriotic cells is associated with the pathogenesis and development of endometriosis. Although there is a large body of data regarding ERs in endometriosis, our understanding of the roles of ERα and ERß in the pathogenesis of endometriosis remains incomplete. The goal of this review is to provide an overview of the links between endometriosis, ERs and the recent advances of treatment strategies based on ERs modulation. We will also attempt to summarize the current understanding of the molecular and cellular mechanisms of action of ERs and how this could pave the way to new therapeutic strategies.

Growth and differentiation factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans.

We hypothesized that skeletal muscle contraction produces a cellular stress signal, triggering adipose tissue lipolysis to sustain fuel availability during exercise. The present study aimed at identifying exercise-regulated myokines, also known as exerkines, able to promote lipolysis. Human primary myotubes from lean healthy volunteers were submitted to electrical pulse stimulation (EPS) to mimic either acute intense or chronic moderate exercise. Conditioned media (CM) experiments with human adipocytes were performed. CM and human plasma samples were analyzed using unbiased proteomic screening and/or ELISA. Real-time qPCR was performed in cultured myotubes and muscle biopsy samples. CM from both acute intense and chronic moderate exercise increased basal lipolysis in human adipocytes. Growth and differentiation factor 15 (GDF15) gene expression and secretion increased rapidly upon skeletal muscle contraction. GDF15 protein was upregulated in CM from both acute and chronic exercise-stimulated myotubes. We further showed that physiological concentrations of recombinant GDF15 protein increased lipolysis in human adipose tissue, while blocking GDF15 with a neutralizing antibody abrogated EPS CM-mediated lipolysis. We herein provide the first evidence to our knowledge that GDF15 is a potentially novel exerkine produced by skeletal muscle contraction and able to target human adipose tissue to promote lipolysis.

The tissue-specific effects of different 17ß-estradiol doses reveal the key sensitizing role of AF1 domain in ERα activity.

17ß-Estradiol (E2) action can be mediated by the full-length estrogen receptor alpha (ERα66), and also by the AF1 domain-deficient ERα (ERα46) isoform, but their respective sensitivity to E2 is essentially unknown. We first performed a dose response study using subcutaneous home-made pellets mimicking either metestrus, proestrus or a pharmacological doses of E2, which resulted in plasma concentrations around 3, 30 and 600 pM, respectively. Analysis of the uterus, vagina and bone after chronic exposure to E2 demonstrated dose-dependent effects, with a maximal response reached at the proestrus-dose in wild type mice expressing mainly ERα66. In contrast, in transgenic mice harbouring only an ERα deleted in AF1, these effects of E2 were either strongly shifted rightward (10-100-fold) and/or attenuated, depending on the tissue studied. Finally, experiments in different cell lines transfected with ERα66 or ERα46 also delineated varying profiles of ERα AF1 sensitivity to E2. Altogether, this work emphasizes the importance of dose in the tissue-specific actions of E2 and demonstrates the key sensitizing role of AF1 in ERα activity.

Nuclear Activation Function 2 Estrogen Receptor α Attenuates Arterial and Renal Alterations Due to Aging and Hypertension in Female Mice.

Background The cardiovascular protective effects of estrogens in premenopausal women depend mainly on estrogen receptor α (ERα). ERα activates nuclear gene transcription regulation and membrane-initiated signaling. The latter plays a key role in estrogen-dependent activation of endothelial NO synthase. The goal of the present work was to determine the respective roles of the 2 ERα activities in endothelial function and cardiac and kidney damage in young and old female mice with hypertension, which is a major risk factor in postmenopausal women. Methods and Results Five- and 18-month-old female mice lacking either ERα (ERα-/-), the nuclear activating function AF2 of ERα (AF2°), or membrane-located ERα (C451A) were treated with angiotensin II (0.5 mg/kg per day) for 1 month. Systolic blood pressure, left ventricle weight, vascular reactivity, and kidney function were then assessed. Angiotensin II increased systolic blood pressure, ventricle weight, and vascular contractility in ERα-/- and AF2° mice more than in wild-type and C451A mice, independent of age. In both the aorta and mesenteric resistance arteries, angiotensin II and aging reduced endothelium-dependent relaxation in all groups, but this effect was more pronounced in ERα-/- and AF2° than in the wild-type and C451A mice. Kidney inflammation and oxidative stress, as well as blood urea and creatinine levels, were also more pronounced in old hypertensive ERα-/- and AF2° than in old hypertensive wild-type and C451A mice. Conclusions The nuclear ERα-AF2 dependent function attenuates angiotensin II-dependent hypertension and protects target organs in aging mice, whereas membrane ERα signaling does not seem to play a role.