Note: Figures not included because of formating issues
Abstract
Significant biological sex differences exist between men and women in pathophysiology, treatment and outcome of cardiovascular disease (CVD). While cardiovascular disease is the leading cause of mortality in women, recent studies have shown that pre-menopausal women are more protected against CVD than men. Estrogen has been found in increase levels in females from adolescence to menopause and in low levels in men. Putative evidence has suggested that estrogen plays a role in protecting post- menopausal women from CVD. Several double blind controlled trials of estrogen replacement therapy have demonstrated that estrogen can play a key role in protecting women from CVD. The purpose of this paper is to highlight the role of 17ß-estradiol and its estrogen receptors in experimental models of CVD and to touch on the clinical use of exogenous E2 as a therapeutic agent for women with heart failure.
Introduction
Cardiovascular Disease is the leading cause of death in both men and women. Major biological sex differences that contribute to the manifestation and outcome of CVD have been documented. Comparing animals to humans is complex, particularly with variables related to hormonal cycles, age and disease onset mechanisms and duration. There are numerous hormones, genetic and epigenetic modifications, aging, metabolic or anatomic abnormalities that factor into differences. It is important to recognize that in comparison to males/men, females/women are more prone to diastolic dysfunction, have lower dilation and no treatment options to address these in hypertensive heart failure (8). Whereas, men by comparison are more prone to systolic dysfunction, eccentric hypertrophy and reduced pump function with established treatment options to address these (8). Moreover, there have been paradigmatic changes in male and female hearts under pressure overload (17). Both men and women undergo concentric myocardial hypertrophy (MH), but women stay more in concentric MH with maintained systolic dysfunction whereas men develop the more unfavourable type, eccentric MH (8). Sex hormones have been linked to play an important role in observed sex differences in CVD.
Particularly, the steroid hormone, 17ß-estradiol (E2), and its receptors estrogen receptor alpha (ERα) and estrogen receptor beta (ERß). These sex hormones have been implicated in sexual dimorphisms in the physiology and pathophysiology of the heart. An accumulated number of studies have revealed the role of E2 and ER in animal studies, such as ER knockout mouse models (3). There have also been various clinical trials that demonstrated the protective role of E2 in pre/postmenopausal women (12). This review will look at the biological mechanism of sex differences in CVD, including the interaction of sex hormones in intracellular signalling, and the recent use of E2 in hormone replacement therapy (12). The comparison of both sexes in cardiac pathophysiology may give rise to a more protective and accurate mechanism that give insight on new therapeutic targets in men and women.
Sexual dimorphism in the cardiac disease
Recent literature has suggested that women develop CVD, such as myocardial infarction, 10 years after men. Compared to men, women are reported to show higher rates of Heart Failure preserved ejection Fraction (HFpEF), less dilation and sex differences in calcium handling and fibrotic remodelling (5). The current challenge in this regard has been partly linked with the fact that animal models of HFpEF are not well developed and consistent with the clinical paradigm
(5). Women demonstrate more microvascular dysfunction, vasospasm, arterial dissections, though progressively less severe atheroma, mostly attributed to estrogen, since men with mutant estrogen receptors show onset coronary heart disease. Moreover, women are twice as more likely than men to have age associated onset of hypertension and so with more pronounced stiffness in the myocardium stiffness in the heart muscle and arteries. Along with these outcomes, numerous genomic pathways have dictated the process of remodelling in males and females (15). In pressure overload, female hearts adapt to induced hypertrophy by developing smaller internal cavity and larger wall thickness than men. Karigagas et al. (2011, 438-46) showed that both male and female patients undergoing aortic valve replacement, experienced similar degree of increased left ventricle morphological changes, however females were more prone to develop LV hypertrophy (15). As pressure overload, both men and women respond similar with concentric myocardial hypertrophy, but as this condition progresses, the myocardium is remodelled in a sex specific manner, with a more concentric MH, less fibrosis, and maintained diastolic dysfunction Similarly, sex differences have also been found in animal models in cardiovascular disease. Pressure overload induced myocardial hypertrophy after transverse aortic constriction (TAC), was produced more severely in male than female mice, and associated with myocyte hypertrophy and increased fibrosis (8). However, in response to volume overload, like human studies, female mice develop a concentric type of myocardial hypertrophy (13).
Contribution of sex hormones to cardiac pathophysiology
Sex hormones are produced early on in the embryonic development. Different epigenetic and genetic factors play a role in the modification and organization of sex hormones during development. Sex hormones are part of the endogenous signaling molecules that form the cellular pathways through gene regulation and synthesis. Sex hormones such as estrogen, progesterone, androgen, and testosterone together or individually play a role in the cardiovascular system. The most prominent type of sex hormones found in cardiomyocytes is estrogen and androgen along with their corresponding receptors. Testosterone is a natural androgen synthesised in the gonads (15). The synthesis of testosterone results in an active metabolite called dihydrotestosterone (DHT), which binds to the androgen receptor (AR). When this binding occurs, DHT becomes the most prominent type of androgen in the body and its main role becomes to amplify testosterone-dependent actions and form reproductive organs (15). DHT is converted from different types of testosterone isoforms, 1-3 of the enzyme 5-α-reductase, which are equally expressed in hearts of female and male mice (15). It has been recently demonstrated that 3 of the enzyme 5-α-reductase (Srd5a3) is the most prominent type of enzyme found in the heart (15). The expression of all the 5- α -reductase is found to be increased in human and mouse hypertrophic hearts, therefore leading to increased levels of DHT (15).
In addition to DHT, the most abundant and circulating estrogen is 17β-estradiol (E2). E2 binds equally to the receptors, ERα and ERβ and is strongly identified among other types of estrogen, such as estrone and estriol (11). E2 is produced as a result of testosterone converting by the enzyme aromatase (2). The enzyme aromatase is found in a number of extra gonadal tissues including the brain, heart, bones and vasculature in both of the sexes (2). There has been a correlation between the increase level of aromatase conversion in adipose tissue that has resulted in significant increase in circulating E2. Naturally in men, E2 is produced in large quantities by aromatization of the androgenic precursor from the testes and adrenal glands (2). In recent literature, it has been found that E2 is abundant in elderly men than postmenopausal women (7).
Estrogen and its receptors in the heart
Sex hormones and their activated receptors include estrogen, estrogen alpha (ERα) and estrogen beta (ERβ), and the G protein-coupled receptor (GPR30) (15). These receptors, at the cellular level, affect both genomic and non-genomic pathways. The regulation of these sex hormones in cardiovascular disease is not very well studied, but there is report that these receptors have an positive effect against cardiovascular disease.
ERα and ERβ belong to the nuclear steroid hormone receptor family (15). When activated, these receptors play a role in gene expression in a hormone-dependent fashion. They are constricted of a “Zinc- finger” based DNA-binding domain (DBD) in the C region, which is a region that mediates dimerization and a COOH terminal ligand binding domain (LBD) located in the E region that enhances the binding to specific nucleotide sequences (15). Moreover, these receptors contain an NH2-terminal A/B domain with component activation function (AF-1) and a hinge domain (D region) (Figure 2) (15). Numerous polymorphisms of ERα and ERβ have also been identified and localized to specific diseases in the sexes (15). For example, ERβ polymorphisms is associated with increased LV mass and wall thickness, while ERα is correlated with increased risk in MI.
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ERα and ERβ localization
ERα and ERβ have been localized in vascular smooth muscle cells, cardiac fibroblasts and cardiomyocytes. ERα mRNA are similarly expressed in the hearts of male and females, while ERβ mRNA levels are higher in
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the male heart than that of the female. Dworatzek, et al. (2017,
27-35) demonstrated the presence of ERα and ERβ through the upregulation of mRNA levels in patients with aortic stenosis (3). They also observed a significant increase in the intracellular localization of ERα in end-stage
failing hearts. The cellular and molecular mechanisms of sex differences are complex and may involve influences that are hidden from development, hormonal cycling changes on menstrual, circadian rhythms or differential effects through estrogen and it receptors. An accumulative number of studies from animals and humans have shown that the sex hormone estrogen, in particular 17β-estradiol (E2), plays an important role in observed sex differences in CVD (3). E2 is synthesized and secreted in the ovaries and modulated predominantly via ERα and ERβ. As levels of aromatase are increased, more estrogen is produced and in turn becomes more bioavailable to the estrogen receptors (3). Depending on the type of pathway, genomic or non-genomic, E2 binds to ER and creates a heterodimer complex in which it is activated (Figure 3). The complex binds to estrogen receptor elements (ERE) and regulates gene transcription by interacting with other transcription factors (TF) (3). Moreover, the ER/E2 complex can also induce signalling transduction pathways that lead to the phosphorylation of ER or other TF, consequently alternating gene expression (3). Additionally, E2 regulates a number of genes, including the progesterone receptor (PGR), which in turn contributes to cardiac remodelling (7). Furthermore, the E2/ER complex can activate many signal transduction pathways, such as ERK/MAPK and PI3K/AKT reference (6). Et al have shown that Erk1/2 is the most canonical (6), causal molecular node regulating cardiac hypertrophy in the adult heart (14) and that ERα and ERβ activation regulate AKT and Erk1/2 to reduce inflammation and hypertrophy in the heart (14). Another study by reference (10) demonstrated that estrogen attenuates right ventricular hypertrophy and fibrosis through ADAM15/ADAM17 by AKT, not Erk1/2, via the ERβ pathway (10). Together these findings suggest that, not only does the ER/E2 complex activate the signalling transduction pathways ERK1/2 and AKT, but they do so through either the ERα and ERβ receptors (10). This stressing the importance of the interaction of estrogen with its estrogen receptors and their connection between the proposed cell and molecular mechanisms. Moreover, through non- genomic actions, the activation of E2 is mediated by the classic ER that is linked to signal transduction proteins in the plasma membrane, leading to rapid tissue response via phosphorylation of extracellular signals (3). The E2 pathway is summarized in Figure 3 (3).
ER knock-out mouse models
To demonstrate the molecular and cellular mechanism of E2 and ER actions in the heart, a number of studies have used ER knockout mouse models. To assess the importance of the receptors in the presence of E2, Dworatzek, et al. (2017: 27-35) experimented with female and mice mouse models with deficiency ERα (ERKO), ERβ (BERKO) and ovariectomized (OVX) wild type (WT). The mice were subjected to TAC injury (3). After TAC, a significant increase of LV hypertrophy was found in WT, ERKO and BERKO mice compared to the sham-operated mice (control). When researchers induced E2, a significant reduction of LV hypertrophy was observed in WT and ERKO mice but not BERKO (3). However, female ERKO mice developed hypertrophy similarly to the female WT mice. This suggests that ERβ, not ERα, is important to reduce hypertrophy in females not males (3). Furthermore, when chronic myocardial infarction was induced in similar ER knock out models and increase level of cardiac fibrosis was observed in male ERKO models, whereas, no significant difference in cardiac fibrosis was found in female ERKO (3). In contrast, female BERKO models presented a significant increase infarct size (3). In another study correlated the deletion of ERα with more sever cardiac damage following infarction in male mice, demonstrating a protective role of ERα male mice following ischemia/reperfusion (I/R) injury (4). E2 treatment in OVX BEKO and BERKO that were subjected to MI, resulted in smaller infarction size in ERKO than BERKO (14). In addition, mortality rates and heart failure are observed in much higher rates in BERKO mice subjected to chronic MI, demonstrating the role of ERβ in reducing MI in female mice (14). Although, the ERs lack specificity in their protective mechanism in the sexes, the data illustrates that the loss of the ERS in either of the sexes can be detrimental.
E2 regulates the pro-inflammatory pathway
Alongside estrogen’s contribution to cardiac morphology in the sexes, men and women adapt differently to biological immune responses. Estrogen exhibits a role in regulating pro-inflammatory cytokines through monocyte polarization and macrophage regulation, altering gene expression (16).
A recent study showed that high levels of serum estrogen correlate strongly with M-2 monocytes/macrophages and T-helper cells (16). The deletion of ERα in vitro is shown to reduce M-2 polarization, and interestingly, the use of Bisephenol-A (xeno-estrogen) augments M-1 accumulation in the heart (8), reducing myo-fibroblasts via ERβ (9). Another study stressed the importance of ERβ in cardio protection by demonstrating its effects on E2 neutrophil infiltration, oxidative stress and necrosis following IR injury (9). The subjection of OVX mice to I/R injury induced pro-inflammatory cytokine TNF-α release in the myocardium, which played a role in reducing tissue injury and apoptosis (2). Regitz et al. (2016, 1-37) postulated that E2- ER activational effects may be mediated by inflammatory cytokines, including mast cells and macrophages and T-cells (Figure 4) (15). Overall, the data presented in response to inflammation demonstrates that the presence of the ERs also attribute to cardio protective effects in the inflammation of the heart E2 regulates pro-fibrotic pathways
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The activation of pro-fibrotic pathways leads to severe cardiac remodelling and dysfunction. In response to hypertrophic response, cardiac remodelling is a product of an accumulation of abnormal fibroblast proliferation, resulting in cardiac fibrosis (7). Females undergo better cardiac remodelling outcomes then men, however the molecular mechanism behind this is not well understood. Recent studies have demonstrated that men suffering from aortic stenosis (AS) are found to have increased levels of pro-fibrotic pathways compared with female patients (2). E2, ERα and ERβ control the fibrotic pathway and the synthesis of collagen in a sex-specific manner and are expected to limit fibrotic remodelling (7).
Sex-specific PGR regulation by E2
It is reported that E2 regulates a number of genes, including the progesterone receptor (PGR), which regulates gene expression though genomic and non-genomic actions of progestin (1). E2 regulates the PGR in a sex-dependent fashion, contributing to the alternation of cardiac fibrosis (7). Dworatzek, et al. (2017: 27-35) induced the expression of E2 directly on cardiac tissues using an ex vivo tissue culture system. They provided evidence revealing that E2 induces the regulation of PGR and limits cardiac fibrosis in females. The treatment of E2, compared to fresh and damaged cardiac tissues, resulted in retained metabolic and viable cell activity (7). Moreover, the expression of PGR mRNA levels in both female and male cardiac tissues were assessed (Figure 5) (7). E2 increased the expression level of the PGR mRNA levels in comparison to the male cardiac tissues, which showed no effect (7). Similarly, the same trend is observed when in E2 exposed PGR protein levels. Female cardiac tissues treated with E2 exhibited an increase protein level of PGR, while there was a decreased trend in PGR protein expression E2-treated tissues of male individuals (7). These findings demonstrate that treating human cardiac tissues with E2, showed positive effects in females but not in males, suggesting that through the regulation of PGR, E2 inhibits cardiac fibroblast growth, and contributes to the inhibitory effects of E2 through the pro-fibrotic pathway.
Hormone replacement therapy
CVD is the leading cause of death in both women and men. However, women are unnecessarily suffering and dying from heart disease. Low estrogen levels due to the disruption of ovulatory cycling in premenopausal women contribute to greater risk in cardiac disease. Often estrogenic effects are attributable to sex differences and accounted for by ovariectomized and replacement therapy. Estrogen is one of the main naturally occurring hormones in women and is shown to exhibit cytoprotective effects on the heart. However, there are multiple pathways in which estrogen can be cytoprotective. Studies have shown that, compared to men, premenopausal women are protected against cardiovascular disease. Lopez-Pier et al. (2018,1-18) demonstrated that after menopause, the mortality and morbidity rate of women with CVD significantly
increases. Suggesting that women lose protection against CVD after entering menopause. The study group reviewed clinical trials that addressed the susceptibility of pre-menopausal women to CVD by using hormone replacement therapy.
Clinical Trials
Women's international study of long duration estrogen after menopause (WISDOM) proposed a clinical trial in which they induced E2 in females suffering from stroke and heart attacks (12). They first hypothesized that administering estrogen was only to decrease the risk of coronary heart disease, during early menopause (12). The trial concluded the importance of administrating estrogen in early post-menopausal women, as it contributes to reduced fracture risk (12). They also pointed out that the administration of estrogen in near menopause may reduce coronary heart disease. A similar study by Early and Late Intervention Trial with Estradiol (ELITE) induced E2 in transitioning into menopause women: both early and late; 55 and 65 years of age, respectively. Women were given a small dose of E2 daily and monitored for 6 years (12). They showed reduction of the carotid artery wall thickness compared to the patients that were given placebo pills and the late group (12). Thus, the results of these trials propose that the timing and physiological adaptation are key things to consider in translational medicine for women.
Future Medicine: a sex-specific one?
When considering major outcomes, such as mortality, there are major observed sex differences in both men and women with heart failure. Both human and animal models have stressed the role of sex hormones in cardiac pathology. The type of injury model, sex hormones and growth hormone profiles are the main attributes to these observed sex differences. In some animal models, the deletion of ERα or ERβ can result in sever cardiac morphology and be detrimental. ER specific localization and roles in regulating the activity of E2, can ease our understating in sex-specific CVD in both mouse and human models. E2 has also been shown to effect both pro-inflammatory/fibrotic pathways that overall contribute to the modelling of the heart. Exploring what pathways sex hormones and receptors are involved in, allows the manipulation of hormones in animal models and provides a better understanding of CVD. Hormonal replacement therapy trials have demonstrated the use of E2 mediated ER activities may be a new mechanism by which premenopausal women are protected from cardiovascular disease. The effect of sex on cardiac physiology have been underestimated and a better understanding of these effects in the pathophysiology of the heart should be brought forward. A more detailed characterization of sex differences may help identify specific therapeutic targets and contribute towards a more accurate and personalized health care towards men and women.
Conclusion
There are many variables that should be taken into consideration when looking at sex differences in both animal and human CVD models. Sex differences are a priority that we must be unravelled so as to address the large gap in our treatment options for females and males for whom their cardiac pathophysiology is unique. However, the cellular and molecular mechanism are complex and reticent influences such as age, hormonal cycles, and disease onset, can vary through the estrogen receptors. E2 cardio protective affects are attributed to sex differences and accounted for in OVX and hormone replacement therapy. It is important to realize that through time in development, sexual maturation and reproductive senescence can influence the effects of estrogen. Taken together, the findings in this review support the connection between estrogen and the putative molecular and cellular mechanism discovered using ER knock out models,human cardiac tissues, and clinical trials contributing to hormonal therapy, even though it has not
been identified how these mechanisms progressively interact between the sexes.
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