Sex Hormones in the Cardiovascular System

DOI 10.1515/hmbci-2013-0048 Horm Mol Biol Clin Invest 2014; 18(2): 89–103

Roger Lyrio dos Santos, Fabrício Bragança da Silva, Rogério Faustino Ribeiro Jr. and Ivanita Stefanon* Sex hormones in the cardiovascular system

Abstract: Gender-associated differences in the develop- List of abbreviations ment of cardiovascular diseases have been described in humans and animals. These differences could explain the ACE Angiotensin converting enzyme low incidence of cardiovascular disease in women in the AR Androgen receptor reproductive period, such as stroke, hypertension, and DHT Dihydrotestosterone atherosclerosis. The cardiovascular protection observed ER Estrogen receptor in females has been attributed to the beneficial effects of ERRs Estrogen related receptors estrogen on endothelial function. Besides estrogen, sex GPER G protein–coupled estrogen receptor hormones are able to modulate blood pressure by acting HERS Heart and Estrogen/Progesterone Replace- on important systems as cardiovascular, renal, and neural. ment Study They can have complementary or antagonistic actions. For HRT Hormone replacement therapy example, testosterone can raise blood pressure by stimu- HT Hormone therapy lating the renin-angiotensin-aldosterone system, whereas mPR Membrane receptors for progesterone estrogen alone or combined with progesterone has been NHANES National Health and Nutrition Examination associated with decreased blood pressure. The effects of Survey testosterone in the development of cardiovascular disease OSH Ovarian sex hormones are contradictory. Although some researchers suggest a PLB Phospholamban positive effect, others indicate negative actions of testos- PR Progesterone receptor terone. Estrogens physiologically stimulate the release of RUTH Raloxifen Use for the Heart endothelium-derived vasodilator factors and inhibit the SERCA2a Sarcoplasmic reticulum calcium pump renin-angiotensin system. Although the cardioprotective SERMs Selective estrogen receptor modulators effects of estrogen are widely appreciated, little is known WHI Women’s Health Initiative about the effects of progesterone, which is commonly used in hormone replacement therapy. Progesterone has both vasodilatory and vasoconstrictive effects in the vasculature, depending on the location of the vessel and the Introduction level of exposure. Nevertheless, the mechanisms through Cardiovascular diseases are major contributors to morbid- which sex hormones modulate blood pressure have not ity and mortality among postmenopausal women in the been fully elucidated. Therefore, the characterization of Western world [1]. Interestingly, premenopausal women those could lead to a better understanding of hyperten- have a reduced risk of mortality from cardiovascular sion in women and men and perhaps to improved forms disease, whereas postmenopausal women have a similar of therapy. or even increased risk for cardiovascular disease as compared with men [2]. It has been suggested that ovarian sex Keywords: cardioprotection; sex hormones; vascular and hormones (OSH) have a protective effect on the cardiovas- cardiac function. cular system [2, 3]. However, the effects of estrogen replacement therapy on the cardiovascular risk only account for *Corresponding author: Ivanita Stefanon, Departamento de about 50% of the reduction seen in cardiovascular disease, Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade suggesting that there must be additional mechanisms Federal do Espírito Santo, Av. Marechal Campos, 1468, 29042-755, whereby estrogen exerts its cardioprotective effects [4]. Vitória, ES, Brazil, E-mail: [email protected] Sexual dimorphism is apparent in humans as well Roger Lyrio dos Santos, Fabrício Bragança da Silva, Rogério Faustino Ribeiro Jr.: Department of Physiological Sciences, as most, if not all, mammals. Numerous studies have Postgraduate Program in Physiological Sciences, Federal University shown that human females exhibit lower levels over of Espírito Santo, Vitória, Brazil much of their life span compared with their age-matched 90 Santos et al.: Sex hormones in the cardiovascular system male counterparts. Steroid hormones have roles in the receptor 30 (GPR30) and as the membrane ER [14, 15]. regulation of a wide variety of bodily processes includ- These receptors are present in both genders and are widely ing regulation of blood pressure. As such, they appear to distributed in many organs, including cardiovascular and be important factors in the development of hypertension reproductive systems, liver, brain, and bone. ERα and and coronary artery disease, some of the most common ERβ are found both in associations with the plasma mem- cardiovascular diseases in the industrialized world [5, 6]. brane, in the cytoplasm and in the nucleus [16]. Some of For example, after the onset of menopause blood pres- the known functions of ERα are related to the activation sure levels increase and become similar to those in men, of eNOS [17, 18], prevention of vascular smooth muscle suggesting an important role of sex hormones in the cells (VSMCs) proliferation [19], and inhibition of increase regulation of blood pressure [7, 8]. These data provide in medial thickness [20], whereas ERβ reduces pathologi- evidence that sex hormones influence the cardiovas- cal cardiac hypertrophy [21], inhibit cardiac fibrosis [22], cular system; however, these influences are still poorly and is related to the development of sustained systolic and understood. diastolic hypertension in ERβ-deficient mice as they age There are observations that suggest both estrogen defi- [14]. Both ERs are necessary and sufficient for estrogen- ciency in women and androgen deficiency in man might mediated protection against measures of vascular injury contribute to the development of cardiovascular disease in mice [14] and may activate phosphoinositide 3-kinase and hypertension [9, 10]. Most epidemiological studies (PI3K), AKT, and mitogen-activated protein kinase (MAPK) have found that there is a high prevalence of low testos- pathways [16]. The GPER was cloned in 1997 from shear- terone levels in men with coronary heart disease, and that stress-exposed human endothelial cells [23]. It can induce this association exists regardless of the age of the patient relaxation in porcine aortic rings and human coronary [9]. Additionally, considerable evidence now suggests that artery, which is dependent on the large-conductance testosterone and other androgens have protective effects calcium (BKCa) and voltage-activated potassium channels on cardiovascular and may play important roles in the (Kv) activation [24]. Additionally, GPER can activate the acute regulation of vascular function [10]. Indeed, studies nonnuclear protective response, i.e., PI3-kinase, Akt, via have demonstrated that testosterone exerts beneficial a truncated 36-kDa ERα [25] and attenuate diastolic dys- effects on cardiovascular function by inducing rapid vas- function and ventricular remodeling in ovariectomized orelaxation of vascular smooth muscle [11]. rats. Endogenous human 17β-estradiol, selective estrogen Although the exact mechanisms by which sex hor- receptor modulators (SERMs) including tamoxifen and mones contribute to the regulation of cardiovascular raloxifene, and selective estrogen receptor downregula- function and blood pressure are still being investigated, tors (SERDs) such as ICI 182,780 are all agonists of GPER, there is increasing evidence that modulating the activ- which has been implicated in the regulation of vasomotor ity of locally active hormone systems is one of the major tone and protection from myocardial ischemia/reperfu- mechanisms of sex hormone actions in target organs, sion injury. As a result, understanding the individual roles including the vasculature and kidneys [8, 12]. Estrogen, of ERα, ERβ, and GPER in cardiovascular function has progesterone, and testosterone receptors have been become increasingly complex [26]. Some estrogen’s actions identified in blood vessels where they appear to cause can be evoked by estrogen binding to other receptors, such a stimulation of endothelium-dependent mechanisms as estrogen-related receptors (ERRs). These receptors are of vascular relaxation and inhibition of the mechanisms orphan nuclear receptors, with three isoforms – β and γ. of vascular smooth muscle contraction. These potential The ERRs are involved in energy metabolism and mito- actions might contribute to the gender differences in vas- chondrial biogenesis, and their activity depends on coac- cular tone and represent potential beneficial vascular tivator proteins [27–29]. However, little is known about the effects of hormone replacement therapy (HRT) during interaction between estrogen and ERRs and other possible natural and surgically induced deficiencies of gonadal ligands for these receptors. hormones [5, 12, 13]. Estrogen diffuses through the plasma membrane and forms complexes with cytosolic and nuclear ER (Figure 1), which bind to chromatin, stimulate gene transcription, Mechanism of action of sex hormones and induce genomic effects. For example, the estrogen deficiency in young adult rats resulted in increased The estrogen receptor (ER) exists in three main forms, expression of a number of pro-inflammatory genes in the ERα, ERβ, and G-protein-coupled estrogen receptor (GPER) heart including L-selectin, calpain, tumor necrosis factor, (Figure 1), the latter being also known as G-protein-coupled iNOS, and fibronectin [30]. Santos et al.: Sex hormones in the cardiovascular system 91

Figure 1 Signaling pathways of sex hormones. The receptors of sex hormones are located at the sarcolemma, cytosol, and nucleus. Binding of sex steroids to their receptors elicits both genomic transcriptional changes and more rapid non-genomic actions through activation of associated signaling intermediates. The expression of aromatase in the some cells, which itself may be influenced by local levels of testosterone and estrogen, suggests estrogen may be synthesized within the target cell to exert autocrine/paracrine actions in the milieu of fluctuating circulating sex hormone levels. mTOR, mammalian target of rapamycin complex 1; GSK3 β, glycogen synthase kinase 3β; mPTP, mitochondrial permeability transition pore. Dotted line represents movement of sex steroids; dashed line represents translocation of ERα to nucleus.

The physiological consequence of non-genomic been shown to bind estradiol and induce rapid signaling effects, in most cases, is a rapid vasodilatation caused events [40]. The molecular mechanisms by which estrogen by activating endothelial factor production, opening of causes a rapid vasodilation is an area of intense current potassium channels [31], and activation of numerous interest, and the contributions by the various pathways signal transduction cascades that support the favora- need to be further defined. ble effects of estrogen on vascular structure and func- Similar to estrogen, progesterone effects are mediated tion [32]. Estrogen binds to signal-generating ER on the by binding to the nuclear progesterone receptor (PR) [41]. plasma membrane of vascular cells and induces rapid Receptors for progesterone are expressed as two distinct non-genomic endothelium-dependent and -independent isoforms, PR-A and PR-B (Figure 1) that arise from a single signaling cascades [PI3K and Akt as well as ERK, extra- gene [42]. These isoforms are present in various human cellular signal-regulated kinases (ERK1/2), c-Jun NH2-ter- tissues including the uterus, mammary gland, brain, pan- minal kinase (JNK), and p38] [33, 34] and vascular effects creas, bone, ovary, testes, and tissues of the lower urinary [5]. Rapid vasodilatory effects of estrogen are produced by tract. The ubiquitous expression of PR highlights the estrogen-stimulated increases of eNOS activity [35, 36]. widespread physiological effects that progesterone can Caulin-Glaser et al. [37] demonstrated a rapid increase in exert in a variety of organs throughout the body [41]. The nitric oxide (NO) from female umbilical vein endothelial general pathway of progesterone inducible PR-mediated cells in response to 17β-estradiol. Chen et al. [38] showed gene transcription has been well characterized. Proges- that ERα can mediate the short-term effects of estrogen on terone binding induces a conformational change in PR eNOS activity within 5 min. Additionally, Chambliss et al. that promote dissociation from a multi-protein chaperone [39] demonstrated that ERβ mediates non-genomic effects complex, homodimerization, and binding to specific pro- on eNOS in plasma membrane caveolae. GPER has also gesterone response elements within the promoter of target 92 Santos et al.: Sex hormones in the cardiovascular system genes. DNA bound receptors increase or decrease rates [51]. However, although the activation of ER is associated of gene transcription by influencing recruitment of RNA with the reduction or attenuation of blood pressure, the polymerase II to the initiation site. Through protein-pro- action of male sex hormones leads to an elevation in tein interaction, hormone-activated PR recruits coactiva- blood pressure. tors that serve as essential intermediates for transmitting signals from the receptor to the transcription initiation complex. Coactivators facilitate transcription initiation through protein interactions with components of the Sex hormones in the cardiovascular general transcription machinery and by promoting local system remodeling of chromatin at specific promoters [43]. A new class of PRs located in the plasma membrane Sex hormones have a role in the regulation a wide range was found initially in spotted sea trout ovaries [44] and of physiological processes, including blood pressure. subsequently detected in human tissues [45]. Membrane Thus, they might be important factors in the development receptors for progesterone (mPR) has three isoforms of hypertension and coronary artery disease, some of the (mPRα, mPRβ, and mPRγ) that are structurally unre- most common cardiovascular diseases in the industrial- lated to nuclear steroid receptors (Figure 1), but instead ized world [12]. For example, the high levels of endogenous has features typical of G-protein-coupled receptors. The estrogens in premenopausal women have been associated non-genomic mechanisms of action of the mPRs are typi- with lower risk for a number of diseases, such as hyper- cally coupled to the inhibitory G-protein Gαi, whereby tension, diabetes mellitus, obesity, vascular disease, and progesterone stimulation decreases 3′,5′-cyclic adenosine stroke compared with age-matched men [6, 15]. In addition, monophosphate (cAMP) synthesis, which is sensitive data from the National Health and Nutrition Examination to pertussis toxin inhibition [46]. mPR stimulation also Survey indicate that age-standardized hypertension prev- causes activating phosphorylations of the MAPKs ERK1/2 alence rates are increasing in general, and there is no sub- and JNK1/2 [45]. group in which they are declining, and among each race/ All three isoforms of mPRs have been shown to bind ethnicity, these trends are more favorable in men than progesterone with high affinity, but not with either syn- in women. However, this clear female benefit no longer thetic progestins or anti-progestins [47]. The androgen exists after menopause, showing again the influence of receptor (AR) exists in two forms, ARα and ARβ, which sex hormones in the pathophysiology of cardiovascular have distinct tissue expression patterns [48]. Sex differ- disease. Nevertheless, the impact of testosterone on the ences in the distribution and abundance of AR have been cardiovascular system is controversial. Elderly men are demonstrated in a variety of tissues including VSMCs typically at higher risk for adverse cardiovascular events and endothelial cells [49]. Testosterone also binds to an than age-matched women, and one study suggested that intracellular receptor (Figure 1), and this receptor-steroid exogenous testosterone was associated with an increase complex then binds to DNA in the nucleus, facilitating in adverse cardiovascular events in this population [52]. In transcription of various genes. In some target cells, tes- contrast, other clinical studies suggest that testosterone is tosterone is converted to dihydrotestosterone (DHT) by beneficial to the cardiovascular system and that low levels 5α-reductase and DHT binds to the same intracellular of testosterone negatively affect the cardiovascular system receptor as testosterone. Testosterone-receptor complexes [53, 54]. Indeed, there is no compelling evidence that tes- are less stable than DHT-receptor complexes in target tosterone replacement to levels within the normal healthy cells, and they conform less well to the DNA-binding state. range contributes adversely to the pathogenesis of cardio- Thus, DHT formation is a way of amplifying the action vascular disease [55]. Obviously, the use of testosterone of testosterone in target tissues. In addition to genomic therapy that is beneficial, or at least safe, should be based effect, testosterone can induce non-genomic effect by both on thorough understanding of relationship between membrane receptors. The non-genomic effects are rapidly endogenous androgens and cardiovascular disease in produced and do not require the association of AR to DNA. conditions of health and disease and on findings of con- These effects involve the activation of various signaling trolled trials with relevant end-points, with appropriate pathways, including calcium, protein kinase A, protein design, and of sufficient duration. Unfortunately, experi- kinase C, and MAPK [50]. mental evidence for potential beneficial or adverse effects Thus, both the male and female sex hormones are of testosterone on the cardiovascular system is rather able to act through genomic and non-genomic mecha- limited. There are no published reports of randomized nisms. These hormones seem to modulate blood pressure clinical trials with the primary goal to evaluate the effects Santos et al.: Sex hormones in the cardiovascular system 93 of testosterone on the incidence of cardiovascular events. “functional stoichiometry” of PLB/SERCA2 is > 1:1 in native Many of the trials to evaluate the effects of testosterone cardiac sarcoplasmic reticulum membranes [75]. In these were conducted in disease men [56–58], thereby limiting transgenic mice, there was a 2-fold higher level of PLB as interpretation and generalization of results. In the fol- compared with non-transgenic mice that resulted in a lowing sections, we will discuss the cardiac and vascular greater inhibition of Ca2+-ATPase affinity for Ca2+, which was functions of sex hormones in detail. associated with decreases in contractility and Ca2+ transport in to cardiomyocytes. Because 17β-estradiol replacement restored sarcoplasmic reticulum protein expression Sex hormones and cardiac function and myocardial contractility, it is plausible that OSH partic- ipate in the long-term regulation of cardiac contractility. In Several studies have also indicated that estrogens might fact, a previous study observed thyroid hormone regulation have cardioprotective actions on myocardial contractil- of sarcoplasmic reticulum protein expression and cardiac ity [59, 60]. Estrogens alters the myocardial expression contractility [76]. These authors also observed enhanced of the following mediators of cardiac contractility: ven- cardiac PLB expression that was associated with decreased 2+ tricular β1-adrenoreceptor [61, 62], intracellular calcium rates of cardiac sarcoplasmic reticulum Ca uptake, which homeostasis related to the L-type Ca2+ channel (LTCC) is consistent with increased inhibition of SERCA2a and [63], cardiac sarcoplasmic reticulum Ca2+ uptake [64], and decreased contractility in hypothyroid rats. Ca2+ sensitivity of cardiac myofilaments in ovariectomized The results of long-term studies demonstrated the rats [65]. Although a cardiac transcriptional regulation of influence of OSH on rat cardiac contractility [69] through estrogen is well described, there is little direct evidence of impaired left ventricular function. Impaired function was membrane cardiac ERs. Data on the non-genomic signal characterized by decreases in cardiac output, peak sys- transduction in cardiomyocytes have demonstrated that tolic pressure, and ejection fraction at all preloads in the 17β-estradiol has a negative inotropic effect on guinea pig ovariectomized rat hearts before puberty. These contrac- single ventricular myocytes produced by inhibiting ICa2+ tile changes were further associated with a decrease in and thus reducing systolic Ca2+ [66]. Furthermore, in iso- myosin ATPase activity. Another study [70] demonstrated lated rat ventricular cardiomyocytes, it was demonstrated that the same changes in cardiac function were reversed that estrogens exerts opposite effects on the intracellular by 17β-estradiol replacement. Changes in cardiomyo- pH of myocytes, which is associated with both pro-and cyte intracellular Ca2+ homeostasis suggested a possible anti-hypertrophic effects [67]. One important question is myocardial dysfunction resulting from a deficiency of whether OSH affect the myocardium directly or by second- OSH [64, 65]. The regulatory role of OSH in the calcium ary mechanisms involving other hormones modulated by uptake activity of cardiac sarcoplasmic reticulum was estrogens. also demonstrated in 10-week ovariectomized rat [64]. Studies have indicated that OSH therapy restores con- These authors demonstrated that estrogen and progester- tractile performance in postmenopausal women [68–70]. one supplementation were equally effective in preventing Recent studies further suggest that mechanical functioning changes in hearts from ovariectomized animals. and proteomic profiles in ventricular myocytes are directly Proudler et al. [77] demonstrated that estrogen regulated by estrogens [61, 64, 71, 72]. The long-term defi- replacement therapy is able to decrease angiotensin- ciency of OSH reduces cardiac contractility associated converting enzyme (ACE) activity and the risk of coronary with changes in the expression of key contractile pro- artery disease in women. Results of animal studies also teins, sarcoplasmic reticulum calcium pump (SERCA2a), showed that ACE activity and levels of type 1 angioten- and phospholamban (PLB) [61, 64, 71–73]. Paigel et al. [73] sin II receptor (AT1) are reduced by estrogen therapy [78]. demonstrated that in rats at 60 days after ovariectomy, the According to Gallagher et al. [79], the reduction in ACE PLB/SERCA2a ratio was increased by approximately 1.6- activity was not due to a direct interaction of estrogen with fold and it was restored to control values after 17β-estradiol the enzyme; rather, it seems that estrogen regulates ACE treatment. No changes in the expression of sodium-cal- mRNA synthesis at the tissue level. Thus, estrogen replace- cium exchanger protein were observed following ovariec- ment therapy may contribute to enhanced cardiovascular tomy. The results of transgenic and gene-targeted studies protection by down regulating ACE, thereby reducing in mice suggested that changes in the PLB/SERCA2a ratio angiotensin II levels. Angiotensin II is an important factor might be a major factor in altering cardiac contractility [74, in cardiac remodeling and left ventricular dysfunction, 75]. In vivo studies using a strain of transgenic mice that and tissue angiotensin II is increased in failing hearts over expresses cardiac specific PLB suggested that the [80, 81]. In fact, Ribeiro et al. [82] demonstrated that daily 94 Santos et al.: Sex hormones in the cardiovascular system

administration of losartan to block AT1 receptors initiated Many studies have disclosed that arterial relaxation immediately following ovariectomy prevented cardiac mediated by estrogen could result from endothelial cell contractile dysfunction and oxidative stress observed at activation, which releases NO and leads to 3′,5′-cyclic 58 days after ovariectomy. This response was associated guanosine monophosphate (cGMP) pathway activation with changes in expression levels of two key proteins in VSMC, which could activate calcium-activated potas- involved in calcium homeostasis, SERCA2a and PLB. sium channels. However, this pathway is probably not the Losartan treatment reversed the ovariectomy-induced main route through which estrogen elicits vasorelaxation contractile dysfunction, restored the SERCA2a and PLB because it has been demonstrated several times that estro- levels to control values, and also diminished reactive gen can relax endothelium denuded arteries [92]. Vascular oxygen species formation. Furthermore, AT1 receptor relaxation induced by estrogen in vascular smooth muscle blockade attenuated the increase in p22phox expression but was correlated with decreased Ca2+ influx [93, 94] and did not affect the increased ACE activity in the ovariec- increased K+ efflux [95, 96]. tomized group. It has been shown that testosterone also The hormone 17β-estradiol is a potent stimulus for has effects on the cardiac muscle. It should be noted that eNOS activation and NO release [92]. NO is a potent vaso- cardiac muscle has AR [83]. In experimental myocardial dilator and inhibitor of platelet aggregation and adhe- infarction in rats and mice, testosterone treatment caused sion and proliferation of VSMCs. In addition, it prevents maladaptive myocardial hypertrophy, with an increase in the development of atherosclerosis. Estrogen also has a the β/α-MHC ratio and in IGF-1 expression [84, 85]. Mean- modulating effect on constrictive factors and positively while, Chen et al. [86] showed that testosterone insuf- upregulates the production of endothelium-derived relax- + ficiency impaired the mobilization and homing of CD34 ing factors such as prostacyclin (PGI2) and the endothe- stem cells and expression of hypoxia-inducible factor 1a, lium-derived hyperpolarizing factors (EDHFs) [31], both stromal cell-derived factor 1a, and vascular endothelium of which are important mediators of vascular relaxation growth factor in ischemic myocardium in the early stage in resistance arteries (Figure 2). In addition, estrogen of myocardial infarction. Chen et al. [86] also found that increases NO bioavailability by mechanisms that either testosterone replacement therapy reversed these changes, directly increases NO generation or decrease superoxide – – and resulted in a concomitant increase in neovasculariza- anion (O2 ) concentration, thereby attenuating O2 -me- tion. In addition, Kang et al. [87] demonstrated that the diated NO inactivation. Estrogens such as 17β-estradiol, castration increased type 2 angiotensin II receptor (AT2) estrone, and estriol have been described to act as reac- expression, cardiac fibrosis, and cardiac cell apoptosis in tive oxygen species scavengers by virtue of the hydrogen- rats with heart failure, which were reduced following tes- donating capacity of their phenolic molecular structure. tosterone replacement. The peripheral actions of estrogen could reduce the Regarding the electrical activity of the heart, both development of hypertension through upregulation of acute intravenous and chronic transdermal testosterone endothelium-derived vasodilator factors with simultane- therapy delay the onset of exercise-induced S-T segment ous downregulation of vasoconstrictor factors such as depression in men with chronic, stable angina [56, 88]. endothelin 1 [97] and inhibition of the renin-angiotensin The steroid also extends total exercise time before devel- system by reducing transcription of ACE [98]. Depletion of opment of myocardial ischemia and shortens return of the estrogen with a concomitant increase in androgens would, S-T segment to the isoelectric line [88]. therefore, reduce local inhibitory signals while increasing pro-contractile signals at the vascular wall, leading to increased peripheral resistance and blood pressure in the Sex hormones and vascular function absence of concomitant decreases in sympathetic tone. Little is known about progesterone effects on vascu- Numerous vascular effects of sex hormones including lar bed [99]. Progesterone is a vasoactive hormone that modulation of vascular function, inflammatory response, predominantly has vasodilatory actions on several vas- metabolic, and hemodynamic effects are attributed to cular beds, although some studies have reported conflict- female sex hormones. The vascular protection conferred ing effects. Indeed, some studies have shown beneficial by estrogen may be mediated indirectly by its influence effects of progesterone [99, 100], whereas others show on the metabolism of lipoproteins [89] and extraneuronal negative effects [101]. Similar to estrogen, progesterone uptake of noradrenalin [90] or by a direct action on the has anti-atherogenic effects, decreasing low-density modulation of molecular pathways in the vessel wall and lipoprotein cholesterol (LDL-C) and increasing high-den- more specifically on endothelial cells [91]. sity lipoprotein cholesterol (HDL-C) [102]. Meanwhile, Santos et al.: Sex hormones in the cardiovascular system 95

Figure 2 Vascular actions of estrogen and testosterone.

progesterone antagonizes the antioxidant effects of estro- dependent and the vasorelaxation induced by higher con- gen and enhances NADPH oxidase activity and production centrations of testosterone appears to be largely endothe- of reactive oxygen species in ovariectomized mice [101]. lium independent [104–106]. The differing results of in Nevertheless, most studies have shown the beneficial vitro and in vivo studies on the role and/or the extent of effects of progesterone on blood vessels. Experimental the endothelium in testosterone-induced vasorelaxation studies have associated these vascular effects to a depend- appear to be the consequences of differences in individual ent interaction progestogen type that binds to receptor. For study designs with respect to the concentration of testos- example, natural progesterone in cultured human umbili- terone used, acute or chronic exposure, the vascular bed cal vein endothelial cells has been shown to increase the investigated, and precontractile agent [107, 108]. synthesis of NO, protein expression of eNOS, and activity Although some studies show a role for the endothe- eNOS, although it was not able to potentiate these effects lium, testosterone acts predominantly on the vascular in the presence of estrogen. In addition, medroxyproges- smooth muscle. Indeed, a variety of studies have dem- terone acetate does not change any of these parameters onstrated that the key mechanism underlying the vas- – it inhibited estrogen effects on NO synthesis [103]. orelaxing action of testosterone is associated with the The vascular action of testosterone in turn seems to be modulation of VSMC membrane ion channels for Ca2+ mediated by both endothelial- dependent and -independ- and K+. These actions include an inactivation of voltage- ent mechanisms. These are concentration dependent. operated LTCC [109, 110] and an activation of K+ chan- The results of in vitro studies show that vasorelaxation nels [111, 112] (Figure 2). These ion channel modulations induced at the lower end of the concentration response produced by testosterone that results to vasorelaxation curve of testosterone appears to be partially endothelium involve intracellular signal transduction pathways that 96 Santos et al.: Sex hormones in the cardiovascular system increase the levels of cGMP [113] and cAMP [114]. Deena- cardiovascular disease and/or risk, when HRT was initi- dayalu et al. [113] showed that testosterone-induced ated, the type of HRT given (conjugated equine estrogen relaxation in endothelium-denuded coronary arteries is with progestin), dosage, and the thromboembolic proper- mediated, in part, by enhanced NO production that leads ties of estrogen and progestin [119]. to an increase in cGMP synthesis and protein kinase G In fact, in most observational studies, women started activation, which in turn opens BKCa channels. Testos- HRT around the time of menopause, which occurs on terone stimulates NO production by nNOS, which in turn average at 51 years, whereas in others, the average age evokes the formation of cGMP (guanylyl cyclase) to induce of women entering in clinical trials, such as WHI, was vasorelaxation [113]. Montaño et al [114] showed that tes- 65 years and older. The women in the late-onset studies tosterone stimulates cAMP production in single rat aortic represent study populations that have some degree of myocytes. The possible path leading to vasorelaxation fol- aging associated vascular damage. In addition, par- lowing an increased level of cAMP is an increase in the ticipants had been estrogen deficient for an average of phosphorylation of protein kinase A [115]. The modula- 10 years before starting HRT, a relatively late start that tion of intracellular cAMP by testosterone can be caused could modify the status of ER and molecular signaling so by the inhibition of cAMP-phosphodiesterase activity, and as to attenuate the benefits of estrogen. this inhibition is associated with an increase in intracellu- Despite the controversy about the benefits of estrogen lar levels of cAMP [116]. Therefore, the underlying cellular replacement therapy in postmenopausal women, much and molecular mechanisms by which testosterone modu- recent interest has focused on whether replacing ovarian lates vascular reactivity require further elucidation [117]. hormones can change the pattern of blood pressure alterations and affect the natural history of hypertension in postmenopausal women. There is experimental evidence New perspectives on the treatment that HRT reduces the incidence of hypertension in postmenopausal women by exerting a direct effect on blood of cardiovascular disease by sex vessels through modulation of endogenous vasoconstric- hormones tors and vasodilators [123]. The cardiovascular benefits of estrogen have been The years proximate to the menopause are accompanied attributed to its modulator effect on NO generation by nitric by a rise in blood pressure and an increasing prevalence oxide synthase (NOS) isoforms [124]. Estrogen deficiency, of hypertension [118]. Among the known cardiovascular induced by ovariectomy, further increased aging-associated risk factors, the incidence of both dyslipidemia and hyper- vasoconstriction and impaired NO signaling [125]. Estrogen tension are prevalent. The prevention of cardiovascular replacement also improved flow-induced vasodilation in disease and its management in postmenopausal women coronary arterioles of aged and ovariectomized rats and are therefore a major health issue [119]. restored eNOS phosphorylation (Ser1176), suggesting a pos- Hormone therapy (HT) in postmenopausal women has itive regulation of estrogen on eNOS activity in old females been thought to provide an efficient protection against the [126]. In addition, untreated postmenopausal women progression of arterial disease by correcting the estrogen present a reduction in circulating levels of NO increment on deficiency [119]. Observational studies show that post- endothelin-1 levels and impairment of endothelial function menopausal women who receive HRT have a lower rate of compared with HRT-treated postmenopausal women [123]. cardiovascular disease and cardiac death than those not Prospective studies in humans have shown variable receiving HRT [120]. Nevertheless, these studies contrast effects of HRT on blood pressure in normotensive and with the large prospective clinical trials, Heart and Estro- hypertensive postmenopausal women [123]. The Postmen- gen/Progestin Replacement Study (I and II) and Women’s opausal Estrogen/Progestin Interventions trial found no Health Initiative (WHI), where no reduction in cardiovascu- significant difference in blood pressure as a function of lar events were shown in postmenopausal women on HRT HRT (conjugated equine estrogen with or without native or [121]. In fact, these trials suggested that HRT was associated synthetic progestin) usage. Meanwhile, the WHI found an with increased risks in the development of stroke, venous increase in blood pressure with HRT (conjugated equine thromboembolic disease, and deep vein thrombosis [122]. estrogen and medroxyprogesterone acetate). Furthermore, The reasons for this paradoxical characteriza- several recent clinical trials have uniformly demonstrated tion of HRT have been extensively discussed. Many that HRT in combination with drospirenone/17β-estradiol potential factors might have contributed to the adverse reduces blood pressure in hypertensive postmenopausal outcome, among them the age of patients, preexisting women. In addition, it has an additive lowering effect on Santos et al.: Sex hormones in the cardiovascular system 97 blood pressure when administered in combination with Tamoxifen is widely used and highly effective as antihypertensive therapy with ACE inhibitors, angiotensin adjuvant therapy for the management of breast cancer receptor antagonists, and hydrochlorothiazide [118]. patients with ER-positive tumors [132]. Originally devel- Currently, data concerning the influence of HRT on oped in the 1960s as a contraceptive agent, tamoxifen blood pressure in postmenopausal women are inconclu- was later found to stimulate ovulation as well as inhibit sive because of the limitations of published studies, as the formation of mammary tumors in rats exposed to car- discussed previously. cinogens. Although tamoxifen itself is a poor ER ligand, its metabolite 4-hydroxytamoxifen is a potent antagonist with high affinity for the ER. Tamoxifen and its metabo- HRT alternative – SERMs lites exert anti-estrogenic effects in breast tissue, estrogenic effects on bones (increased density) and plasma SERMs are nonsteroidal molecules with tissue-selective cholesterol (decreased levels), and mixed effects in the estrogen agonist and antagonist effects. These com- uterus. In addition to the beneficial effects of tamox- pounds interact with the ER at the ligand-binding domain ifen, its complex, tissue-specific action can also result and differentially recruit coactivators or co-repressors to in adverse effects in women. Perimenopausal symptoms produce their effects [127]. The SERMs chosen for this of hot flushes and night sweats are frequently reported study are the most widely prescribed, tamoxifen and side effects in both premenopausal and postmenopausal raloxifene. Tamoxifen has an estrogen antagonist activity women on tamoxifen therapy. In premenopausal women, in the breast but an agonist action in the endometrium tamoxifen has also been reported to cause oligomenorrhea [128] and is used for the treatment of estrogen-sensitive or amenorrhea with a return to normal menses after dis- breast cancer. Raloxifene has antagonist actions in the continuing tamoxifen. Yet the greatest concern has been breast and uterus but agonist actions in the bone [129] focused on the effects of tamoxifen in the uterus. Tamox- and is widely used for the prevention and treatment of ifen treatment increases the relative risk for endometrial osteoporosis [121]. Evidence has shown that raloxifene, cancer and may increase the incidence of endometriosis like estrogen, induces endothelium-dependent acute and uterine mesenchymal (i.e., myometrial) tumors. vasodilation [130, 131] and directly activates NO synthesis Tamoxifen can also alter vascular reactivity [133] by in endothelial cells, resulting in dilation of blood vessels. various pathways including antagonism of several ion It is well known that the physiological effects of channels and calmodulin and inhibition of myosin light estrogen on gene transcription are determined by a char- chain kinase. These actions are consistent with studies acteristic combination of an activator and co-repressor speculating that several potentially important actions proteins that interact with any given cell. Manipulation of of tamoxifen are ER independent [134]. Clinical observa- these interactions may provide a rational basis for selec- tions have shown that treatment with tamoxifen reduces tively targeting the cardiovascular system. Raloxifene is the incidence of ischemic heart disease and coronary ath- approved for the treatment and prevention of osteoporo- erosclerosis. Other studies have also reported decreases sis in postmenopausal women. Raloxifene has properties in total serum cholesterol and HDL-C with tamoxifen similar to estrogen on the cardiovascular system, such as treatment [135]. Additionally, in experimental studies, reduction of the concentrations of serum cholesterol and tamoxifen exerted effects similar to those of estrogen on LDL, improvement of endothelial function, and antago- vascular relaxation due to several mechanisms, including nist actions to the proliferative effects of estrogen on the antagonism of various ion channels [136]. uterus and breast. These properties of raloxifene provided the basis for the Raloxifene Use for the Heart (RUTH) randomized, controlled trial that studied its ability to decrease Expert opinion fatal myocardial infarction, fatal coronary disease, and hospitalization for acute coronary syndrome. The RUTH In this work, we tried to provide a literature update on trial commenced in 2001, with an average 5-year follow- the role of endogenous sex steroids on the cardiovascular up, and included 10,000 postmenopausal women who system. The sex hormones can act through genomic and were 55 years or older. In RUTH, a reduced risk of invasive non-genomic mechanisms and appear to modulate blood breast cancer was observed, but no effects on primary cor- pressure. However, although the activation of ERs is asso- onary events were seen. Moreover, there was an increased ciated with the reduction or attenuation of blood pressure, risk of venous thromboembolism and fatal stroke. Thus, the action of male sex hormones leads to bidirectional further studies are needed to investigate its use in HT. actions on blood pressure that depend on its dose. The use 98 Santos et al.: Sex hormones in the cardiovascular system of testosterone therapy that is beneficial, or at least safe, will improve the quality of life of female users. Finally, we should be based both on thorough understanding of rela- believe more research on G1, a GPER agonist, will advance tionship between endogenous androgens and cardiovas- and show a number of benefits to users without the risk cular disease in conditions of health and disease and on of breast and uterine cancer. The knowledge gained with findings of controlled trials with relevant end-points, with the study of hormonal therapy can contribute to a better appropriate design, and of sufficient duration. Unfortu- quality of life for women in the future. In addition, it will nately, experimental evidence for potential beneficial be necessary to study the involvement of mitochondrial or adverse effects of testosterone on the cardiovascular metabolism on cardiac dysfunction observed in estrogen- system is rather limited. deficient animals. Future studies involving the improve- Female sex hormones are able to modulate blood ment of heart function and morbidity and mortality rates pressure by acting on important systems as cardiovas- of chronic heart failure should focus on the high antioxi- cular, renal, and neural. Female and male sex hormones dant activity. can have complementary or antagonistic actions. For example, testosterone can raise blood pressure by stimu- lating the renin-angiotensin-aldosterone system, whereas Highlights estrogen alone or combined with progesterone has been associated with decreased blood pressure. Progesterone –– G1, the agonist of the GPER on the cardiovascular has both vasodilatory and vasoconstrictive effects in the system, lipid profile, oxidative stress, atherosclerosis, vasculature depending on location of the vessel and level and diabetes could represent a new kind of alternative of exposure. The knowledge of the actions of sex hor- therapy for postmenopausal women without the risk mones on the cardiovascular system could contribute to of breast and uterine cancer. the development of more appropriate therapies with sex –– The effects of SERMs on the cardiovascular system hormones. could confirm it as an effective alternative form of therapy in postmenopausal women without the risk of development of breast and uterine cancer. Outlook –– Identification of the mechanisms and intracellular pathways that activate GPER may contribute to the The use of sex hormones, especially estrogen, in the development of better forms of therapy. cardiovascular system is a complicated clinical issue –– New information about testosterone, i.e., whether it that requires an in-depth risk/benefit assessment. Much has beneficial action on the cardiovascular system, research has been done with the estrogen, but additional will help physicians and patients when they decide research is still needed. After menopause, the develop- which form of HT they will adopt. ment of cardiovascular disease is due not only to estro- –– It is important to identify the mechanisms of rapid gen decline but also to testosterone decline. Each of the (non-genomic) and chronic (genomic) actions various clinical trials that have focused on sex hormones of testosterone, which may contribute to the has the potential to contribute to our overall understand- development of better forms of therapy. ing but cannot be viewed as the single source of informa- –– Identification of new forms of alternative therapies tion. This mistake is often made by the public largely, if will help relieve not only short-term symptoms of not solely, due to the media’s sensationalist tendencies. menopause but also contribute to the protection of Nonetheless, according to the US Census Bureau, in 2020, important organ systems such as the cardiovascular, 25 million women will be over the age of 65 years. As life nervous, and renal systems. expectancy increases, women can expect to live a consid- –– It is necessary to confirm if rapid actions of estrogen erable portion of their lives after menopause. How well are mediated by GPER or by the activation of non- they live may depend on how their physicians manage genomic intracellular pathways during the passage of their hormone deficiency. We hope that the results of many the hormone trough the plasmatic membrane. experimental studies conducted in vitro and in vivo will –– Clarifying the responses of sex hormones on the be confirmed in prospective studies that will show that cardiovascular system could provide new information sex hormones, when administered properly, exert a pro- that may contribute to the development of new drugs tective effect on many systems, including the cardiovas- that can be used in hormonal therapies aimed at cular system. Often, alternative therapies to maintain the enhancing the beneficial actions of sex hormones on well-being after the fall of sex hormones with menopause the various systems of the human body. Santos et al.: Sex hormones in the cardiovascular system 99

–– Cardiac contractile dysfunction seems to depend on LSUHSC-New Orleans, for his valuable comments and edi- the increment on myocyte oxidative stress. torial assistance.

Acknowledgments: The authors are grateful to Dr. L. A. Barker, Emeritus Professor of Pharmacology, Received September 1, 2013; accepted April 29, 2014

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