Estetrol modulates endothelial nitric oxide synthesis in human endothelial cells

Estetrol (E4) is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO) is a key player for vascular function and disease during pregnancy and throughout aging in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVEC). E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2, and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use

Androgens Regulate T47D Cells Motility and Invasion through Actin Cytoskeleton Remodeling

The relationship between androgens and breast cancer is controversial. Androgens have complex effects on breast cancer progression and metastasis. Moreover, androgen receptor (AR) is expressed in approximately 70 to 90% of invasive breast carcinomas, which has prognostic relevance in basal-like cancers and in triple-negative breast cancers. Recent studies have associated the actin-binding proteins of the ezrin-radixin-moesin (ERM) family with metastasis in endocrine-sensitive cancers. We studied on T47D breast cancer cells whether androgens with different characteristics, such as testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) may regulate breast cancer cell motility and invasion through the control of actin remodeling. We demonstrate that androgens promote migration and invasion in T47D via Moesin activation. We show that T and DHEA exert their actions via the AR and estrogen receptor (ER), while the non-aromatizable androgen - DHT - only recruits AR. We further report that androgen induced significant changes in actin organization with pseudopodia along with membrane ruffles formation, and this process is mediated by Moesin. Our work identifies novel mechanisms of action of androgens on breast cancer cells. Through the modulation of Moesin, androgens alter the architecture of cytoskeleton in T47D breast cancer cell and promote cell migration and invasion. These results could help to understand the biological actions of androgens on breast cancer and, eventually, to develop new strategies for breast cancer treatment

Ulipristal acetate interferes with actin remodeling induced by 17β-estradiol and progesterone in human endometrial stromal cells

Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) used for emergency contraception and for the medical management of symptomatic uterine fibroids (UF). Treatment with UPA turns in amenorrhea and UF volume reduction. Treatment with UPA is associated with the frequent development of benign, transitory endometrial changes known as SPRM-associated endometrial changes (PAECs). Why PAECs develop and their biological or cellular basis is unknown. Sex steroids, including estrogen and progesterone, are established modulators of the actin cytoskeleton in various cells, including endometrial cells. This explains several morphological and functional changes in endometrial cells. We thus hypothesized that UPA may alter the appearance of the endometrium by interfering with the actions of 17β-estradiol (E2) or progesterone (P4) on actin dynamics. We isolated and cultured human endometrial stromal cells (ESC) from endometrial biopsies from healthy fertile women. Treatment with E2 or P4 stimulated visible actin rearrangements with actin remodeling toward the membrane. Activation through phosphorylation of the actin regulatory proteins, Moesin, and focal adhesion kinase (FAK), hacked actin remodeling induced by E2 and P4. Membrane re-localization of Paxillin and Vinculin were also induced by E2 and P4, showing the formation of focal adhesion complexes. All these E2 and P4 actions were inhibited by co-treatment with UPA, which was otherwise inactive if given alone. The cytoskeletal changes induced by E2 and P4 turned into increased motility of ESC, and UPA again blocked the actions E2 and P4. In conclusion, we find that UPA interferes with the cytoskeletal actions of E2 and P4 in ESC. This finding helps understanding the mode of actions of SPRMs in the endometrium and may be relevant for other potential clinical applications of UPA.Fil: Shortrede, Jorge Eduardo. Università degli Studi di Pisa; ItaliaFil: Montt Guevara, Maria Magdalena. Università degli Studi di Pisa; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pennacchio, Gisela Erika. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Finiguerra, Michele. Università degli Studi di Pisa; ItaliaFil: Giannini, Andrea. Università degli Studi di Pisa; ItaliaFil: Genazzani, Alessandro D.. Universita Degli Studi Di Modena E Reggio Emilia;Fil: Simoncini, Tommaso. Università degli Studi di Pisa; Itali

Effects of estetrol on migration and invasion inT47-D breast cancer cells through the actin cytoskeleton

Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr(558), which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-α. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast

Robot-assisted pelvic floor reconstructive surgery:an international Delphi study of expert users

Background: Robotic surgery has gained popularity for the reconstruction of pelvic floor defects. Nonetheless, there is no evidence that robot-assisted reconstructive surgery is either appropriate or superior to standard laparoscopy for the performance of pelvic floor reconstructive procedures or that it is sustainable. The aim of this project was to address the proper role of robotic pelvic floor reconstructive procedures using expert opinion. Methods: We set up an international, multidisciplinary group of 26 experts to participate in a Delphi process on robotics as applied to pelvic floor reconstructive surgery. The group comprised urogynecologists, urologists, and colorectal surgeons with long-term experience in the performance of pelvic floor reconstructive procedures and with the use of the robot, who were identified primarily based on peer-reviewed publications. Two rounds of the Delphi process were conducted. The first included 63 statements pertaining to surgeons’ characteristics, general questions, indications, surgical technique, and future-oriented questions. A second round including 20 statements was used to reassess those statements where borderline agreement was obtained during the first round. The final step consisted of a face-to-face meeting with all participants to present and discuss the results of the analysis. Results: The 26 experts agreed that robotics is a suitable indication for pelvic floor reconstructive surgery because of the significant technical advantages that it confers relative to standard laparoscopy. Experts considered these advantages particularly important for the execution of complex reconstructive procedures, although the benefits can be found also during less challenging cases. The experts considered the robot safe and effective for pelvic floor reconstruction and generally thought that the additional costs are offset by the increased surgical efficacy. Conclusion: Robotics is a suitable choice for pelvic reconstruction, but this Delphi initiative calls for more research to objectively assess the specific settings where robotic surgery would provide the most benefit.</p

Molecular actions of Estetrol in human cells

Some of the major unwanted effects associated with the use of estrogen-progestin scheduling include its strong hepatic impact, the increase of thromboembolic events and the higher risk of developing breast cancer. Steroid hormones have an influence on the cardiovascular system through modifications of the lipid profile and through lipid-independent mechanisms at the vascular level. Nowadays, there is strong evidence that estradiol metabolites are not merely waste products but may play physiological and pathophysiological roles. Estetrol (E4) is a natural human estrogen produced exclusively by the human fetal liver during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications including contraception and menopausal hormone therapy. The aim of this thesis was to analyses the impact of E4 versus estradiol (E2). First, experiments were conducted to analyses the effect of E4 on the migration and invasion in breast cancer cells. Second, we studied the impact of E4 treatment on endothelial cells functions which are recognized to be dependent on membrane initiated steroid signaling, the activation of endothelial NO synthase and the plasminogen activation pathway. This work report a dual effect of E4 on the migration and invasion of breast cancer cells, showing that E4 moderately stimulates these processes via cytoskeleton remodeling while antagonizing, at the same time, the effect of E2 thereby reducing the tumor invasiveness elicited by E2. The results for endothelial cells also highlight that E4 is less potent than E2 and exhibits antagonistic properties towards the activation of the endothelial NO synthase and the plasminogen activation pathway as well as cell migration elicited by E2. In summary, is tempting to speculate that E4 may represent a compound designed by nature to control the maternal and fetal vascular actions of estrogens during gestation. While the exclusive dual weak estrogenic/anti-estrogenic activity of E4 paves the way for several potential clinical applications, experimental and clinical studies are now needed to confirm the efficacy and the safety of E4

Estetrol: A New Choice for Contraception

Estetrol (E4) is a natural estrogenic steroid that is normally produced by human fetal liver. Recent research has demonstrated that it is a potent, orally bioavailable, natural selective estrogen receptor modulator; it has a moderate affinity for both human estrogen receptor alpha (ER&alpha;) and ER&beta;, with a preference for ER&alpha;. Clinical studies have demonstrated possible use as an estrogen in combined oral contraceptives (COC). COCs containing E4 and drospirenone (DRSP) showed a high acceptability, tolerability, and user satisfaction also when compared to COCs containing ethinylestradiol (EE). E4/DRSP effectively inhibits ovulation, with a similar effect on endometrium thickness than that of EE-containing COCs. Low doses (15 mg) of E4 with DRSP (3 mg) showed promising results in term of bleeding pattern and cycle control, also when compared to other COCs containing synthetic estrogens. Moreover, the association has limited effects on serum lipids, liver, SHBG levels, and carbohydrate metabolism. This combination also could drive a lower risk of venous thromboembolism than EE-containing COCs. In this review, we will summarize the actual knowledge about the new E4-containing contraceptive. Further large-scale studies in the full target population are needed to provide more insights into the cardiovascular safety profile and user satisfaction of E4/DRSP

Efectos vasculares del andrógeno dehidroepiandrosteediona

En los últimos años se ha situado a la dehidroepiandrostenediona (DHEA) como una alternativa para el tratamiento de condiciones hipoandrogénicas, promoviendo la formación local de esteroides activos a partir de este precursor. Se estudió el efecto vascular de DHEA, comparándolo con el de testosterona (T). Se evaluó la migración y proliferación de células endoteliales (CE) de cordón umbilical y la producción endotelial de PAI-1 y uPA, factores que regulan la reendotelización vascular modulando la adhesión, proliferación y migración de CE. Mediante Western Blot observamos que mientras que T produjo un aumento significativo de la expresión de PAI-1 a todas las concentraciones (0,1nM; 1nM y 10nM) y tiempos ensayados (12, 24, 36 y 48h) con un estímulo del 60-80% s/Cont (p<0,05), su expresión no se afectó por DHEA (2nM; 20nM y 200nM). Demostramos que el efecto de T es dependiente de la participación del receptor de andrógenos e independiente de aromatización a estradiol. En cuanto a la expresión de uPA, 24h de tratamiento con DHEA incrementaron su expresión un 30% s/Cont, no observándose cambios significativos al ser expuestas las CE a T. A continuación procedimos a estudiar el efecto de los andrógenos sobre la proliferación de CE (ensayo de MTT) y la migración celular (ensayos de reparación de la herida). Los ensayos tiempo respuesta (12, 24, 36 y 48h) demostraron que a 24h de tratamiento, tanto T como DHEA estimulan el crecimiento celular (32% y 12% s/cont T 1nM y DHEA 20nM p<0,05). Se demostró también que ambos andrógenos promueven la movilidad celular (2±2; 16±4; 90±15 Cont; T 1nM; DHEA 20nM CE migrantes/campo p<0,01), siendo el efecto de DHEA marcadamente superior al de T (5,6 veces mayor). Los resultados presentados sugieren que DHEA exhibe una acción más favorable en cuanto a la reendotelización al disminuir la expresión de PAI-1 y estimular la de uPA, efectos que se corresponden con su mayor acción estimulatoria de la migración celular.Fil: Campelo, Adrián Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Montt Guevara, Maria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Simoncini, Tommaso. Università degli Studi di Pisa; ItaliaFil: Massheimer, Virginia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaLIX Sociedad Argentina de Investigación Clínica y LXII Sociedad Argentina de InmunologíaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de Inmunologí