Stefania Spina

Actin Cytoskeleton Remodelling by Sex Steroids in Neurones

Cell morphology and its interaction with the extracellular environment are integrated processes involving a number of intracellular controllers orchestrating cytoskeletal proteins and their interaction with the cell membrane and anchorage proteins. Sex steroids are effective regulators of cell morphology and tissue organisation, and recent evidence indicates that this is obtained through the regulation of the actin cytoskeleton. Intriguingly, many of these regulatory actions related to cell morphology are achieved through the rapid, nonclassical signalling of sex steroid receptors to kinase cascades, independently from nuclear alteration of gene expression or protein synthesis. The identification of the mechanistic basis for these rapid actions on cell cytoskeleton has special relevance for the characterisation of the effects of sex steroids under physiological conditions, such as for the development of neurone/neurone interconnections and dendritic spine density. This is considered to be critical for gender‐specific differences in brain function and dysfunction. Recent advancements in the characterisation of the molecular basis of the extranuclear signalling of sex steroids help to clarify the role of oestrogen and progesterone in the brain, and may turn out to be of relevance for clinical purposes. This review highlights the regulatory effects of oestrogens and progesterone on actin cytoskeleton and neurone morphology, as well as recent progresses in the characterisation of these mechanisms, providing insights and working hypotheses on possible clinical applications for the modulation of these pathways in the central nervous system.

Vasodilatory effects of the selective GPER agonist G-1 is maximal in arteries of postmenopausal women

G-protein-coupled estrogen receptors (GPERs) have been proposed to mediate estrogen-mediated vasodilation. The presence of GPER-dependent vasodilation in human resistance-sized arteries (HRAs) or its signal transduction pathways have not been investigated. HRAs in subcutaneous fat tissues (biopsies from postmenopausal women (PMW), age-matched men (M) and pregnant women (PGW)) were mounted for in vitro isometric force recording. Vasodilation induced by G-1 (selective GPER-agonist, 3 μM) from HRAs pre-contracted with norepinephrine amounted to 40±5% in PMW, significantly larger than those obtained from M (20±5%) or PGW (20±5%). L-NAME (nitric oxide (NO) synthase inhibitor) abolished these relaxations in PGW, attenuated them in PMW and had no effect in M. Immunohistochemical analysis confirmed the presence of GPER in both smooth muscle and endothelial cells of HRA with maximum expression in PGW. In cultured human umbilical vein endothelial cells (HUVECs), G-1 increased NO-synthesis concentration-dependently through higher expressions of endothelial NO-synthase (eNOS) and through enhanced phosphorylation of eNOS on Ser(1177). In conclusion, GPER vasodilates human resistance arteries through various activating mechanisms of the eNOS-signaling pathway.

Estrogen regulates endothelial migration via plasminogen activator inhibitor (PAI-1)

Endothelial plasminogen activator inhibitor (PAI-1) controls vascular remodeling, angiogenesis and fibrinolysis. PAI-1 blood levels in women are related to estrogen. The aim of this study was to characterize the signaling pathways through which estrogen regulates PAI-1 in endothelial cells. Furthermore, we aimed to investigate whether PAI-1 is implicated in the control of endothelial migration by estrogen. Cultured human umbilical vein endothelial cells (HUVECs) and ovariectomized rats were used to test the effects of 17β-estradiol (E(2)) on PAI-1 expression and its role on endothelial migration. At physiological concentrations, E(2) increases the expression of PAI-1 in HUVEC within 6-12 h through activation of a signaling cascade initiated by estrogen receptor α and involving G proteins, phosphatidylinositol-3-OH kinase and Rho-associated kinase II. ROCK-II activation turns into an over-expression of c-Jun and c-Fos that is required for E(2)-induced expression of PAI-1. Estrogen-induced PAI-1 expression is implicated in HUVEC horizontal migration. PAI-1 regulation is found also in vivo, in female rats, where ovariectomy is associated with reduced PAI-1 expression, while estrogen replacement counteracts this change. In conclusion, E(2) increases PAI-1 synthesis in human endothelial cells and in rodent aorta through a G protein-initiated signaling that targets early-immediate gene expression. This regulatory pathway is implicated in endothelial cell migration. These findings describe new mechanisms of action of estrogens in the vessels, which may be important for vascular remodeling and hemostasis.

Effects of Estetrol on Migration and Invasion in T47-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 Thr558, 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.

Dydrogesterone exerts endothelial anti-inflammatory actions decreasing expression of leukocyte adhesion molecules

Clinical observations and basic studies show that progesterone and progestins have a variable influence on endothelial function. Dydrogesterone (DG) is a widely used progestin, but its endothelial actions have not been thoroughly assessed. In this study, we investigated the effects of DG and its metabolite 20-α-dihydro-dydrogesterone (DHD), natural progesterone as well as medroxyprogesterone acetate, on the expression of leukocyte adhesion molecules in human endothelial cells using an in vitro experimental endothelial inflammation system. Our findings show that all progestins significantly suppress endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule-1 (ICAM-1) induced by bacterial lypopolysaccharide (LPS). These inhibitory effects of DG and DHD require activation of progesterone receptor. DG and DHD decrease adhesion molecule expression associated with LPS administration by preventing nuclear translocation of the pro-inflammatory transcription factor nuclear factor-κB. In addition, DG and DHD do not alter the anti-inflammatory effects of 17β-estradiol. In conclusion, DG and DHD decrease endothelial inflammatory responses induced by LPS, via reduced expression of the pro-atherogenic adhesion molecules VCAM-1 and ICAM-1. These actions may be relevant for the vascular effects of DG.

Regulatory effects of estetrol on the endothelial plasminogen pathway and endothelial cell migration

BACKGROUND Estetrol (E4) is a natural estrogen produced solely during human pregnancy. E4 is suitable for clinical use since it acts as a selective estrogen receptor modulator. In clinical trials E4 has been seen to have little or no effect on coagulation. Hence, it is interesting to investigate whether E4 alters endothelial-dependent fibrinolysis. OBJECTIVES We studied the effects of E4 on the fibrinolytic system and whether this could influence the ability of endothelial cells to migrate. In addition, we compared the effects of E4 with those of 17β-estradiol (E2). STUDY DESIGN Human umbilical vein endothelial cells (HUVEC) were obtained from healthy women. Expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) proteins was evaluated by Western blot analysis. Endothelial cell migration was studied by razor-scrape horizontal and multiwell insert systems assays. RESULTS E4 increased the expression of t-PA, u-PA and PAI-1 in HUVEC, but less so than did equimolar amounts of E2. The effects of E4 on t-PA, u-PA and PAI-1 were mediated by the induction of the early-immediate genes c-Jun and c-Fos. E4 in combination with E2 antagonized the effects induced by pregnancy-like E2 concentrations but did not impair the effects of postmenopausal-like E2 levels. We also found that the increased synthesis of PAI-1, u-PA and t-PA induced by E2 and E4 is important for horizontal and three-dimensional migration of HUVEC. CONCLUSIONS These results support the hypothesis that E4 acts as an endogenous selective estrogen receptor modulator (SERM), controlling the fibrinolytic system and endothelial cell migration.

Menopause and HRT: Doubts and Certainties

Menopausal transition results from declining ovarian function and leads to dramatic decreases in estrogens, which turns into a number of disturbances. Because climacteric symptoms result from a decrease in estrogens, it naturally follows that replacing estrogens with hormone replacement therapy (HRT) is effective against menopausal symptoms. HRT is the most effective treatment to reduce the frequency and intensity of hot flushes and vaginal atrophy and positively affects bone loss and risk of fractures. Doubts still linger on the impact of HRT on cardiovascular disease, breast cancer, cognitive impairment, and dementia. Recent data supports the initiation of HRT around the time of menopause to treat menopause-related symptoms and to prevent osteoporosis in women at high risk of fracture. Evidence of a direct link between time since menopause and initiation of HRT has also emerged, which may affect the impact on cardiovascular disease and brain deterioration.

Cardiovascular Prevention at the Menopausal Transition: Role of Hormonal Therapies

Cardiovascular disease (CVD) has become the leading cause of death for women. The incidence of CVD in women increases substantially with aging, probably because menopause diminishes the gender protection contributing to an adverse impact on cardiovascular risk variables. In this setting, the menopausal transition (MT) must be considered a critical period in women’s life because it leads to several adverse changes, especially with regard to lipid profile and modifies cardiovascular risk factors. Thus, it may be necessary to encourage lifestyle measures and therapeutic interventions such as hormone replacement therapy (HRT) throughout the MT to counteract or prevent these events. HRT for postmenopausal women has been available for more than 60 years. Ever since, HRT has been the subject of discussion and debate regarding its safety and efficacy; nowadays, it is well established that timing in the initiation as well as the type, dose and route of HRT are crucial for the success and benefit of HRT. Furthermore, a new study, published by the British Medical Journal, recently added interesting evidence to this debate, proving that menopausal hormone therapy saves lives and reduces disability.

Postmenopause and Ageing: The Concept of Personalized Therapy

The climacteric syndrome is a complex condition characterized by a set of symptoms and degenerative changes that ensue due to the decline in production of sex steroids by the ovaries. This set of changes identifies women at additional risk of degenerative changes during ageing and calls for personalized interventions. Hence, there is a need for safe methods for the short- and long-term management of postmenopausal women. Hormone replacement therapy (HRT) is widely used for the relief of menopausal symptoms and for the prevention of diseases linked to long-term hormonal deprivation. The use of HRT has increased rapidly in the past decade but is currently a subject of debate because of the possible negative effects on the breast. Other molecules have been studied as alternatives to standard hormonal replacement therapy to relief the symptoms also in women with contraindications to hormones or who refuse sex steroids for personal reasons. The aim of this chapter is to discuss the results of the principal studies performed in these last few years on the safety of HRT and of other climacteric therapies and to suggest how to personalize therapies in climacteric women.

Therapeutic Management of the Menopausal Transition

Reproductive age in women is characterized by several different phases, which constitute a unique endocrinologic continuum. It starts with the beginning of fertile life, with regular, cyclic menses, typical of ovulatory cycles, and it ends with a concluding menstrual period, due to final ovarian senescence, indicated as the menopause.