HORMONES 2007, 6(1):9-24
DOI: 
Review
Coronary heart disease in postmenopausal women; the role of endogenous estrogens and their receptors
Katerina Saltiki, Maria Alevizaki

Endocrine Unit, Evgenidion Hospital and Department Medical Therapeutics, Alexandra Hospital, Athens University School of Medicine, Athens 11528, Greece

Abstract

Coronary heart disease is the main cause of death in women. Women during reproductive years are at lower risk for coronary heart disease than men but this difference tends to disappear after the menopause. In this article, we briefly review the clinical and experimental data which highlight the protective role of endogenous estrogens in the pathogenesis of coronary heart disease focusing on women after the menopause. Furthermore, recent data about the molecular and biochemical mechanisms of estrogen action on the vasculature are presented.

Keywords

Atherosclerosis, Cardiovascular disease, Coronary, Estrogens, Estrogen receptor, Gene polymorphism, Menopause


Read PDF

INTRODUCTION

Coronary heart disease (CHD) is a multifactorial disease, its expression probably being influenced by the interaction of genetic and environmental risk factors.1 Especially in women, CHD is a leading cause of death, in fact more frequent than breast cancer, at all ages.2 Several epidemiological studies indicate a higher incidence of the disease in postmenopausal women when compared to women of reproductive age.3-6 In addition, postmenopausal women with CHD have more advanced coronary artery stenosis compared to premenopausal women.7

On the other hand,at a younger age and independently of differences in lifestyle, women are at lower risk for CHD than men.8 However this disparity tends to disappear after menopause.9 On this evidence, the hypothesis as to a protective effect of estrogens against atherosclerosis has been based. In accordance with this hypothesis, estrogen deprivation may play an important role in the appearance of early CHD in women.10,11 There is also evidence that higher levels of androgens during the reproductive years may contribute to a higher risk for CHD,12 the exact reverse being observed in men.13

This brief review will discuss some of the data on the importance of endogenous estrogens and their receptors for the cardiovascular system in women mostly after menopause.

EPIDEMIOLOGICAL STUDIES

In a recent review of studies which examined the influence of diverse reproductive parameters on the risk for CHD in postmenopausal women, de Kleijn et al reported that the factors which correlated with higher risk for CHD were the irregularity of the menstrual cycles, the number of abortions and, most importantly,the age at menopause.14 The importance of age at menopause, both natural and surgical, for the development of CHD has been thoroughly stud-ied during the last few decades.4-6,15-17 Most of these studies showed that women with earlier menopause have a higher risk for CHD independently of other risk factors such as blood pressure, dyslipidaemia, obesity and smoking, these risk factors themselves being associated with menopause.3,18,19 In a recent study by Saltiki et al it was found20 that age at menopause was significantly lower in women who had 2 myocardial infarctions (MI) compared to those with 0 or 1 MI.

Similarly, CHD mortality has been investigated in relation to age at menopause, but this association was not always significant.5,6,21-23 Van der Schouw et al,24 in a large prospective cohort study of 12,115 postmenopausal women, showed that each year’s delay in natural menopause results in a 2% decrease in risk of death from CHD events, and similar results were shown in another very recent study.25 Jacobsen et al reported that earlier menopause is related to an increase in total mortality, while when age at menopause is over 53 years, cardiovascular disease mortality decreases as much as 60%.21 However, an extremely delayed menopause may be associ-ated with an increased CHD risk.21 Another large epidemiological study showed an increased risk for CHD in women with earlier menopause, especially in smokers.26 In a study in which women with higher estrogen levels (either premenopausal or postmeno-pausal receiving HRT) were compared to women with lower estrogen levels (postmenopausal), it was found that estrogen status may constitute an inde-pendent prognostic factor of morbidity and mortality in women presenting for stress testing for suspected CHD.27

In the literature there are few studies which have examined the role of age at menarche and the calculated total lifetime exposure to endogenous estrogen as a predictor for the risk of CHD. Saltiki et al20 showed that postmenopausal women with a shorter lifetime exposure to endogenous estrogens are more likely to present MI (Figure 1). These findings are similar to those reported by de Kleijn et al, namely an inverse correlation of cardiovascular mortality with the length of exposure to estrogens and a 20% decrease in mortality from CHD in postmenopausal women with longer exposure to endogenous estrogens.23 Jansen et al also showed a decrease in mortality when lifetime exposure to endogenous estrogens was more than 40 years compared to less than 33 years.22





Figure 1. Total lifetime exposure to endogenous estrogens (age at menopause minus age at menarche) is inversely associated with myocardial infarctions in postmenopausal women under-going coronary angiography (p=0.03, Kruskal Wallis test). Re-printed with permission from Maturitas.

The time that has elapsed since menopause has been more rarely examined in relation to the various clinical manifestations of cardiovascular disease.16,20 This parameter combines the influence of both the age at menopause and the current age and is an index of the length of estrogen deprivation. In the study of Saltiki et al,20 significant associations between the time that elapsed since menopause and several manifestations of coronary artery disease such as history of angina and myocardial infarction were found (Figure 2). Specifically, the correlation with myocardial infarction was independent of current chronological age, which is by itself a strong predisposing factor.



Figure 2.
In 100 postmenopausal women undergoing coronary angiography, longer time interval since menopause is associated with history of angina or myocardial infarction (MI) (p<0.03 and p<0.05, respectively, t test). Reprinted with permission from Maturitas.

Finally, current estrogen levels have been examined in relation to the presence and severity of CHD in postmenopausal women,28-30 but no associations have been found in the majority of the studies. Only one longitudinal study showed that low levels of endogenous estrogens and relatively higher levels of androgens are associated with the risk for an acute myocardial infarction at menopause.31 Current estrone levels have not been associated with a higher risk for CHD.28,32 This is to be expected, as most post-menopausal women present with very low hormonal levels during menopause. It has been reported that premenopausal women with lower estrogen levels had more severe atherosclerosis in their vessels in the coronary angiography.33 This provides some evidence of an increase drisk for future coronary artery disease in women whose total exposure to estrogens has been lower during the reproductive years.

It appears that menopause by itself affects several classical predisposing factors for CHD10,18,19,26,34-36 and is associated with an increase in triglyceride, total and low-density cholesterol (LDL) levels as well as with an increase in central fat deposition and insulin resistance.18,36 The increase in central adiposity during menopausal transition is independent of the effect of total body adiposity and of age, as has been shown in several longitudinal and prospective studies.18 In the study of Saltiki et al, positive associations of several predisposing factors such as hyperlipidaemia, diabetes mellitus, positive family history of CHD as well as insulin resistance (HOMA) with the severity of CHD in the angiography in postmenopausal women were found.20 The associations of CHD severity with time since menopause remained significant when hyper-lipidemia, measures of adiposity, insulin resistance and chronological age were taken into account.

In conclusion, it seems that shorter lifetime ex-posure to endogenous estrogens is an important risk factor for the presence and the severity of CHD, whereas endogenous estrogens appear to play a protective role for the cardiovascular system. Some of the implicated mechanisms will be analysed below.


ESTROGENS DURING MENOPAUSE

The main sources of estrogen in premenopausal women are the ovaries. During steroidogenesis, the theca cells produce androgens, which are aroma-tized to estrogens with the enzyme aromatase in the granulosa cells of the ovary. During menopause the main source of estrogen production is extragonadal. Estrogens are mainly produced by the adipose tissue which expresses the steroidogenic enzymes aroma-tase and 17βHSD. In postmenopausal women the predominant estrogen is estrone, which is 50-70% less active than 17β estradiol. Estriol is another circulating estrogen. The extragonadal production of estrogens is influenced by age and weight. Aromatase is also expressed in the endothelial cells and smooth-muscle cells of blood vessels. This fact suggests a paracrine or autocrine action of estrogens.The blood concentration of estrogens does not reflect the biologically active forms at the tissue level, as these are dependent on the local enzyme activity and the bind-ing on the protein transporters such as sex hormone binding globulin (SHBG). The bio availability and the functionality of estrogen receptors also play a major role in the tissue response to estrogens.37

MECHANISMS OF ESTROGEN ACTIONS

Estrogens act on cellular function through genomic and non-genomic mechanisms; the genomic effect is slower and is better characterized. The alteration of the expression of various genes resulting from estrogen action depends upon the activation of the two nuclear estrogen receptors ERα and ERβ which act as transcription factors.38,39 The second mechanism of estrogen action is non-genomic, as it is not dependent on changes in gene expression and occurs within minutes after estrogen binding with receptors situated on the cellular membrane. For example, the vasodilatation occurring rapidly after estrogen administration is attributed to a non-genomic effect. The effect of estrogens on the changes in the metabolic profile, on the immune process and on the response to vascular injury is dependent on genomic transcriptional activity37,40 (Table 1 ).

Genomic actions and the role of estrogen receptors

Estrogen receptors (ER) are members of the superfamily of nuclear receptors; the androgen, pro-gesterone and glucocorticoid receptors also belong to the same family. There are two different genes encoding ERs, the ERα and the ERβ genes located on different chromosomes. The classical ERα was cloned two decades ago. ERβ was cloned more re-cently. Functional estrogen receptors are present in the cardiovascular system. Various mRNA splice variants have been found in normal and atherosclerotic tissues, but the proteins which are encoded and their pathophysiological role are not well established.41 They carry several functional domains characteristic for the receptors of this family. The two receptors have 53-96% homology in their different domains. These structural differences contribute to the variable affinity to various ligands and offer cellular specificity. Growth factors may also act as ligands to the ERs.37,40,42

In the absence of a ligand, the receptors are located within the cytoplasm, associated with cyto-plasmic proteins which act as chaperones, like heat shock protein 90 (hsp 90). When free estrogen diffuses into the cell-target, these proteins dissociate from the receptor and several biochemical events, such as activation of ion channels and changes in the enzymatic activity, occur.43 When the hsp dissociates from the receptor, the complex estrogen/estrogen receptor diffuses in the nucleus, where this complex is homodimerised or heterodimerised by either one ERα or one ERβ and then binds to the estrogen response element (ERE) of the DNA sequence, close to the responsive gene. The transcription process depends on the promoter of the gene and the various co-activators and co-repressors. Depending on the ligand, the complex acquires a special conformation which finally determines the binding of a certain co-activator to the promoter.37,40,42 The complex interaction of co-regulators and homo- or hetero-dimers of ERs results in a highly specific re-sponse after transcriptional activation. An example of cardiovascular co-regulator specificity is the steroid receptor co-activator 3 (SRC3), which facilitates the estrogen-mediated vasoprotection from vascular injury.44 The cellular environment and the nuclear receptor’s and co-regulator’s phosphorylations are also important for the specificity of the response of the target-tissue.45 This phenomenon has been taken advantage of in clinical practice with the design of selective estrogen receptor modulators (SERMS), which act either as agonists or antagonists in various tissues depending on the co-activators which participate in the process.46 Estrogens may regulate the transcription of genes lacking ERE by modulat-ing the activity of other transcription factors such as activating protein 1 (ΑΡ1) and Nuclear factor kappa Β (NF-κB).47

Estrogens act on the cardiovascular system40,42,48 through their receptors ERα and ERβ, which are expressed in the endothelial cells49 andonthesmooth muscle cells of vessels.50-52 ERα has been more thoroughly studied, most of the studies having shown the importance of this receptor for the atheroprotective effects of estrogens.53 In premenopausal women with more severe atherosclerotic lesions in their vessels a lower expression of ERα has been found.50 More recently there has been growing interest in the participation of ERβ in the physiology of the cardiovascular system.54,55 It seems that ERβ is the receptor which is expressed to a larger degree on the endothelial cells and the smooth muscle cells of healthy and atherosclerotic vessels in both sexes.52,54,55

Apart from the level of the expression of the receptors, the functionality of these receptors has also been investigated at the tissue level. There are differ-ent isoforms of the estrogen receptors which are all expressed, but their significance is unknown.56 Other studies have shown that each one of these receptors on its own has the ability to preserve the estrogen activity; in knockout mice for either the ERα or the ERβ gene, estrogen administration resulted in a decrease of the intima media layer thickness.57,58 Finally, higher methylation of the ERα gene may play a role in atherosclerotic vessels.59

Genetic polymorphisms of the estrogen receptors may affect the tissue response, i.e. the tissue sensitivity to estrogen. In 1997, Sudhir et al reported an extreme example of dysfunction of the receptor in a young man who carried an inactivating mutation in theERαgenecausingsevereestrogenresistance;this individual manifested premature atherosclerosis de-spite high circulating levels of estrogens.60 Similarly, polymorphisms of the ERα and ERβ genes may af-fect the sensitivity of tissues to estrogens and could be related to a higher risk for CΗD. This issue will be analyzed in the last section of this brief review.

Non-genomic actions

Apart from the genomic actions, estrogens also showrapidactionsashasalreadybeenmentioned.In most of these actions the transcriptional machinery does not participate; in the non-genomic process, membrane receptors may be involved as well as cellular signaling pathways with the participation of ion channels (Ca and Κ), G proteins and G protein-coupled receptors (GPCRs), tyrosine kinases (PI3K) and ΜΑΡ kinase cascades.61

These membrane receptors have not been well characterized, but there is evidence that they may be the same cytoplasmic receptors ERα and ERβ situated in caveolae on the cellular membrane.62,63 Recently, it has been reported that an intracellular transmembrane G protein-coupled receptor acts as estrogen receptor probably mediating some of the rapidly occurring estrogen effects.64 The non-genomic actions of estrogens are very critical for the cardiovascular system because they regulate the rapid vasodilation of coronary and other vessels. This vasodilation is achieved through the opening of Ca channels and activation of K channels, as well as through the secretiοn of vasoactive molecules like nitric oxide (NO) from the endothelium and the vascular smooth muscle cells.61 Similarly, the rapid insulin secretion by the pancreatic β cells is possibly regulated by estrogen through a non-genomic action.65 Recently, it has been reported that some non-genomic effects can be converted to genomic ones and to transcriptional activation with the mediation of cascades of cellular signaling pathways.66

DIRECT ACTIONS OF ESTROGENS ON THE VASCULATURE

Atherosclerosis is a chronic hyperplastic inflammation of the layers of the vessels and is influenced by genetic, metabolic and hormonal factors.67 During the early stage of the atherosclerotic process, the subepithelial trapping of oxidised LDL molecules that trigger the local reaction of inflammation play a major role. The result is the accumulation of macrophages, monocytes and T cells, the production of matrix and various enzymes such as metalloprotein-ases (MMPs) and the production of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin 1 & 6 (IL-1 & 6), which mediate a Τh1 response;68 the final result is a rupture in the atherosclerotic plaque. Another important step is the calcification of the coronary vessels, which has a genetic element but is also influenced by hormonal factors.54,69

It seems that estrogens protect the vasculature at different levels. Through their direct actions, estrogens influence the evolution of the atherosclerotic lesions, whereas by their indirect actions they modulate various vasoactive, proinflammatory and metabolic factors as well as factors of the coagulation system40,42,48,70 (Table 2 ).

There is evidence that the administration of estrogens is protective for the formation of the atherosclerotic plaque when administered to either healthy or hypercholesterolemic rats. Hodgin et al have shown that this protective action of estrogens is mediated by ERα in hypercholesterolemic transgenic Apo E knockout mice, whereas when these rats were double knockout for the Apo E and for the ERα gene, this protective effect was diminished.71

Estrogen deficiency also induces the calcification of atherosclerotic plaques. Postmenopausal women not receiving hormone replacement therapy (HRT) have more calcified atherosclerotic plaques in their coronaries than premenopausal or postmenopausal HRT users.72 Aortic calcification increases when the time elapsed since menopause is longer.73 Men have twice as many calcifications on their vessels as women until the age of 60, but after this age this gender difference attenuates, indicating the role that estrogen deficiency may play in vascular health.74 It is interesting that the mechanism of vessel calcification is similar to that of bone formation. Paradoxically, the vessel calcification during menopause occurs inversely to the bone demineralization and postmenopausal osteoporosis.75 It seems that in these two processes, various molecules such as me-talloproteinases (MMPs) and osteoprotegerin may play an important role.75 In their very recent study in autopsies of arteries of pre- and postmenopausal women, Christian et al54 reported the critical role of ERβ in the atherosclerotic and calcification process; these receptors were found to a greater extent in atherosclerotic coronary vessels and were correlated with more severe lesions, independently of the chronological age of these women. One other point that should be mentioned is that estrogen receptor gene expression may be affected by DNA methyla-tion, which occurs with aging thus contributing to the atherogenic process in the cardiovascular system.59

Estrogens also modulate the response to vessel injury with the mediation of both ERs.42,53,57,58 Both ERs expression increases after vessel injury.42,76 In animal models, ERβ appear to be important for the differences in the response to vascular ischemic injury between the two sexes.77 Experiments in ERβ knockout mice show that acute myocardial infarction results in more severe cardiac dysfunction.78

Estrogens act positively on the endothelial cells. This angiogenic activity and re-endothelialization is mediated by ERα53,79 and ERβ,80 which increase the in vitro and in vivo local production of growth factors such as FGF, TGF and VEGF.81 They also inhibit the apoptosis of endothelial cells in cell cultures.82 On the other hand, they have antihyperplastic effects on vascular smooth muscle cells: they inhibit the proliferation of vascular muscle cells, thus affecting the thickness of the vessel wall.40,42 The thickness of carotids intima media layer, an index of the risk for CHD,isincreasedin45%ofpostmenopausalwomen while in only 16% of premenopausal women.83

Functional ERs are expressed in both animal and human cardiomyocytes and may play a role in gender differences concerning cardiac contractility, heart rate and myocardial hypertrophy42 as well as in the myocardial protection of ischemic insults through inhibition of mitochondrial reactive oxygen species (ROS).84

Another very important action of estrogens is their vasodilatory action. This is mediated by the increase in the production of vasodilating molecules such as NO and prostacyclines, as well as by the decrease in vasoconstricting factors such as endothelin-1, renin, angiotensinogen converting enzyme and the down-regulation of the receptor of angiotensin AT1.40,48 Transdermal estrogen administration in postmenopausal women with angina but normal vessels in the angiography increases vasodilation.85 Female animals have a higher basic rate of NO pro-duction than males.86 This increase in NO production is mediated by a rapid non-genomic as well as by a genomic mechanism through transcriptional activa-tion of the NO synthase gene (eNOS) in the vessels.87 The activation of eNOS is probably mediated by ERα, as in transgenic knockout mice for the ERα gene the NO levels are decreased.88 In parallel, other signaling pathways and factors such as MAP kinases, PI3K/Akt and hsp90 participate in the activation of eNOS, showing how complicated this mechanism is.87 The eΝΟS affects the function of other estrogen dependent tissues such as bone tissue and contributes to the prevention of bone loss.89 ΝΟ attenuates the atherogenic process by decreasing the proliferation of vascular muscle cells. It also plays a central role in the inflammatory events, influencing the production of cytokines and decreasing the adhesion and accumulation of monocytes and platelets on the wall of the affected vessels.87 Premature ovarian failure is associated with impaired endothelial function, which may contribute to the increased cardiovascular disease risk. Early initiation of hormone therapy may reverse this atherosclerotic process.90,91

What happens when atherosclerotic plaques already exist? In ovariectomized monkeys, estro-gen administration decreases the formation of new plaques but has no beneficial effect on pre-existing ones.92 Estrogen administration probably makes these plaques more destabilised as estrogens increase new vessel formation and the risk of haemorrhage. This may be one of the mechanisms through which HRT use is related to a higher risk of cardiovascular events during the first year of their use.93,94

INDIRECT SYSTEMIC ACTIONS OF ESTROGENS

Effects on metabolism of lipids and carbohydrates

During their reproductive years, women have lower levels of lipids and LDL than men while these levels increase after the menopause.3,19,95 By contrast, the difference in HDL levels between men and postmenopausal women remains the same. After menopause, the increase in LDL, total cholesterol and Lp(a) levels is reversed with oral (p.o.) estrogen administration,96 while triglycerides levels become worse.96 The estrogens effects on lipid metabolism are mediated by ERα. It has been shown that polymorphisms of the ERα gene may influence the lipid response after HRT.97,98

Estrogens participate in both lipogenesis and lipolysis. At the transcriptional level they increase the hepatic expression of apoprotein genes and the LDL receptors and decrease the transcription of the lipoprotein lipase (LPL) gene through ERα. Thus, when estrogen levels decrease after the menopause, an increase of the LPL activity is observed and this probably contributes to the increase of free fatty acids (FFA) and the accumulation of abdominal fat.99 There is also evidence that estrogen receptors regulate the expression of other non sex steroid hormone nuclear receptors such as the peroxisome proliferator-activated receptor α (PPARα) and the liver X receptors (LXRs), which mediate various metabolic pathways relevant to cardiovascular disease.42 By inhibiting lipogenesis, estrogens alter the expression of hormone-sensitive lipase.100 On the other hand, through ERα and ERβ, estrogens are involved in the proliferation of adipocytes,99 whereas their deprivation increases central obesity which is associated with a more atherogenic profile.101 Plasminogen activator inhibitor-1 (ΡΑΙ-1),102 IL6 and CRP levels70 often increase during menopause, while insulin resistance and several other components of the metabolic syndrome emerge;18 all these factors may contribute to the increase in cardiovascular morbidity. Finally, levels of adiponectin, which is produced by adipocytes and whose role is protective for the metabolic syndrome, do not change during menopause.103 Similarly, leptin levels donot correlate with menopausal status; there is, however, evidence that estrogens may act on the hypothalamus, influencing central sensitivity to leptin.104,105

Changes in proinflammatory factors and antioxidative effects

As has already been mentioned, atherosclerosis is a chronic inflammatory process. Evidence for such association is provided by several studies in animal models, reviewed by Hansson et al, in which a 10-fold higher lipoprotein accumulation was shown in the vessels of hypercholesterolemic animals compared to similar animals in which various genes important for the immune system had been knocked out.67

Some of the atheroprotective effects of estrogens are mediated through their interaction with the in-flammatory process (Figure 3). Estrogens decrease the adhesion molecules, vascular cell and intracel-lular adhesion molecules (VCAM-1 and ICAM-1) and decrease the accumulation of leucocytes on the endothelium.106



Figure 3. Effects of estradiol on the inflammatory process.

It has been found that monocytes, neutrophils and T and B lymphocytes express estro-gen receptors, mostly ERα. In estrogen deficiency, these cells of the immune system are more active.70 Ιn vitro experiments have shown that estrogens affect the release of proinflammatory cytokines by these cells probably through the cross-talk of the ERs with NF-κΒ, thus resulting in inhibition of NF-κΒ activity.70,107 It has been suggested that estrogen de-ficiency during the first years of menopause induces theproductionofvariousproinflammatorycytokines and the expression of their receptors such as ΙL6, IL1 and TNFα on the vessel wall.70 In the model of ovari-ectomized Apo-E knockout mice, the expression of the IL6 gene and IL6 production are increased, while they decrease when estrogens are administered.108 These alterations in cytokines affect the biosynthesis of several other molecules which are important for the pathogenesis of atherosclerosis, such as CRP (through IL6). CRP acts on the accumulation of monocytes in the atherosclerotic plaques109 and is an index of inflammation and risk for new cardiovascular events.110 Cytokines and CRP can stimulate NF-κB and angiotensin receptors AT1. As a result, oxidative stress is increased, smooth muscle cells proliferate and the production of metalloproteinases (MMPs) increases and participates in the destabilization and the rupture of atherosclerotic plaques.110

However, some of the data in the literature are conflicting. Several studies have shown that estrogens induce a decrease in anti-inflammatory cytokine levels such as IL4, IL10 and IL3 and also an increase of interferon-γ, causing a Th1 immune response which promotes atherosclerosis. It seems that estrogens affect the inflammatory response through many different pathways and modulate accordingly the atherogenetic process.111 On the other hand, clini-cal studies have shown an increase in CRP and IL-6 levels during p.o. HRT use, whereas transdermal administration does not influence these inflammatory factors. There are data in the literature which demonstrate that estrogens have atheroprotective effects when administered before the vascular dam-age occurs. It is thus possible that the adverse effects of oral estrogen on thrombosis and inflammation may predominate in the presence of pre-existing atherosclerotic lesions.42,112,113

Many in vitro and in vivo studies have shown anti-oxidant effects of estrogens. For example, estrogens inhibit the oxidation of LDL through both genomic and non-genomic mechanisms.114 Inversely, estrogen deficiency induces the production of ROS, which participate in the oxidation of LDL molecules which then contribute to the formation of foam cells on the vessel wall, to the production of proinflammatory cytokines and to the increase of NO catabolism. Also during menopause, the expression of the angiotensin receptors is increased and this contributes further to ROS production.70,110

Finally, it has been speculated that the interaction of the hypothalamo-pituitary-gonadal axis with, for instance, the hypothalamic pituitary adrenal (HPA) axis might be involved in the atherosclerotic process: estrogens increase the expression of the corticotrophin releasing hormone (CRH) gene which affects the immune response.115

Estrogen effects on the coagulation system

Estrogens alter the transcription of genes coding for several proteins participating in the coagulation system. They affect fibrinogen and factors V, VII, IX X and TFPI116 and they decrease the levels of anti-thrombinIII,proteinSandPAI-1.116,117 Furthermore, estrogen receptors are expressed on platelets: they influence the migration, the adhesion and the aggre-gation of these cells and thus increase the thrombotic risk.116 These effects may offer an explanation for the increased thrombosis events related to the p.o. use of HRT, which were reported in studies such as HERS93,118 and WHI.94,119 Transdermal administra-tion carries a reduced risk.120-122 In women taking HRT, estrogen dosage, medical history about the inherited hypercoagulable states frequently caused by factor V (Leiden) and prothrombin gene mutations, as well as a history of smoking, are well recognized factors influencing the thrombotic risk associated with HRT.122,123



POLYMORPHISMS IN THE ESTROGEN RECEPTOR GENES, ERα AND ERβ

ERα polymorphisms

Recently, there have been many studies per-formed in the general population about the effect of genetic variants of ERs, which may influence the tissue sensitivity to estrogens. The case of a young man who had an inactivating mutation in the ERa gene causing resistance to estrogen and also had premature atherosclerosis has already been mentioned.60 Since this report, several studies have investigated possible associations between ERα single nucleotide polymorphisms and a variety of clinical and biochemical parameters predisposing to heart disease, especially in men.124-133 Most studies concern the PvuII c.454-397 T>C and XbαI c.454-351 A>G polymorphic sites in intron 1 of the ERα gene (Figure 4) which may be of functional importance (see below). Shearman et al in a subpopulation of the Framingham study showed that men with the PvuII C variant had a 3-fold increased risk for cardiovascu-lar disease125 and stroke.134 In a different study, men carriers of the PvuΙΙ variant had more generalized atherosclerotic lesions and extensive calcification of the atherosclerotic plaques at autopsies,135 while ap-parently healthy men with the polymorphism present premature coronary artery dysfunction.133



Figure 4. ERα gene and the two common polymorphic sites in intron 1: PvuII c.454-397 T>C and XbαI c.454-351 A>G.

So far as women are concerned, we have recently found136 similar associations to those shown in men, namely a positive correlation of two ERα variants of PvuII T>C and XbαI A>G with the severity of coronary artery disease, while the Rotterdam study showed conflicting results.129 Finally, there are sev-eral studies which found no significant correlations between these ERα polymorphisms and the presence132,137 or severity138 of cardiovascular disease in either gender.

In the literature there are several studies which suggest that these polymorphisms of ERs may modify the sensitivity of various tissues to estrogens38 and affect the clinical phenotype in other diseases such as breast cancer,139 endometrial cancer,140 endometriosis141 and osteoporosis.142 It is also possible that these variants may influence clinical parameters such as age of menarche and menopause,143,144 blood pressure145 and lipid levels146 as well as their response to the HRT98 or their association to tobacco use.147

There are several studies in the literature supporting the functional importance of these ERα polymorphisms on tissue sensitivity to estrogens.98,148 It has been speculated that this intronic site of the variants, which is situated at a distance between 397 and 351 nucleotides from exon 2, might result in alternative splicing, thus modifying the gene’s function, as has been reported for other genes.149,150 It is also possible that this site might be the locus of attachment of a transcription factor, B-myb, which is nullified when nucleotide T is present, thus affecting the speed of transcription of the receptor gene.98,148 Finally, it is possible that this polymorphic site is linked to some other locus, which could have a role in cardiovascular disease. It has further been reported that the PvuII intronic polymorphism is linked to the polymorphic TATA repeat site in the ER α promoter region.151

ΕRβ polymorphisms

Recently, a growing number of studies discuss the clinical significance of ERβ polymorphisms. Especially as regards the cardiovascular system, these variants appear to be associated with earlier presence of atherosclerosis,137 as well as with lower LDL levels in women taking HRT152 and higher HDL levels in women taking isoflavones.153 They have also been associated with left ventricular hypertrophy in women154 and with a history of arterial hypertension.145,155 Pertinent experiments in animals showed analogous results; knockout mice for the ERβ gene develop hypertension, arterial dysfunction and chronic heart failure.156,157 Also, polymorphisms of the androgen receptor (ΑR) and ERβ may influence circulating androgen levels in women158 and thus affect in directly the cardiovascular system. Finally, as is the case with ERα,ERβ may also affect clinical parameters such as age at menarche159 or estrogen related diseases such as breast cancer160 and osteoporosis.161

CONCLUSIONS

The in vivo and in vitro data that were presented in this shor treview show that estrogen is of particular importance for vascular health. Data from experimental studies or observations from the natural lack of endogenous estrogen occurring during menopause show that estrogen deprivation in women is associated with adverse effects on the cardiovascular system and with acceleration of atherosclerosis. On the otherhand, HRT use was associated with are duction of CHD risk by as much as 40-50%162-165 in large epidemiological observational studies. This protective effect on the cardiovascular system was attributed mostly to the favorable effect on the lipid profile. Despite all these favorable effects, and despite the results of the large observational studies, the recent randomized prospective studies did not prove that exogenous estrogen has the expected protective effect. In HERS,93 where HRT was administered to women with pre-existing coronary artery disease, an increased incidence of fatal cardiovascular events and an increase of thromboembolic incidents were observed.118 In the WHI,94,119,166 the administration of a combination of conjugated estrogen with a progestogen was associated with an increased incidence of cardiovascular events as well as with an increase in the incidence of breast cancer; the administration of estrogens only in women with hysterectomy was also associated with an increase in thromboembolic incidents. It has been reported that the design of this study had intrinsic problems associated with the population group, their age, the distance since menopause, the presence of further risk factors for CHD as well as the presence of already established CHD.

How is it then possible to use the information from the experimental and epidemiological data showing a protective effect of estrogen in clinical practice? There are several questions that have to be answered. The first one is whether younger women might benefit from the use of HRT immediately after menopause. The second question concerns the compound, the dosage, the duration and the ideal and safest route of administration. Clearly, the careful design of further studies which will try to answer these questions is warranted, so that the apparent cardioprotective effects of endogenous estrogen may find some safe application in clinical practice for the benefit of women’s health.

REFERENCES

1. Yamada Y, Izawa H, Ichihara S, et al, 2002 Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 347: 1916-1923.
2. Wingo PA, Calle EE, McTiernan A, 2000 How does breast cancer mortalityc ompare with that of other cancers and selected cardiovascular diseases at different ages in U.S. women? J Womens Health Gend Based Med 9: 999-1006.
3. Matthews KA, Meilahn E, Kuller LH, et al, 1989 Meno-pause and risk factors for coronary heart disease. N Engl J Med 321: 641-646.
4. Rosenberg L, Hennekens CH, Rosner B, et al, 1981 Early menopause and the risk of myocardial infarction. Am J Obstet Gynecol 139: 47-51.
5. Colditz GA, Willett WC, Stampfer MJ, et al, 1987 Menopause and the risk of coronary heart disease in women. N Engl J Med 316: 1105-1110.
6. Gordon T, Kannel WB, Hjortland MC, McNamara PM, 1978 Menopause and coronary heart disease. The Framingham Study. Ann Intern Med 89: 157-161.
7. Gurevitz O, Jonas M, Boyko V, Rabinowitz B, Reicher-Reiss H, 2000 Clinical profile and long-term prognosis of women < or = 50 years of age referred for coronary angiography for evaluation of chest pain. Am J Cardiol
85: 806-809.
8. Isles CG, Hole DJ, Hawthorne VM, Lever AF, 1992 Relation between coronary risk and coronary mortality in women of the Renfrew and Paisley survey: comparison with men. Lancet 339: 702-706.
9. Barrett-Connor E, 2003 Clinical review 162: cardiovascular endocrinology 3: an epidemiologist looks at hormones and heart disease in women. J Clin Endocrinol Metab 88: 4031-4042.
10. Barrett-Connor E, Bush TL, 1991 Estrogen and coronary heart disease in women. JAMA 265: 1861-1867.
11. Davis CE, Pajak A, Rywik S, et al, 1994 Natural menopause andc ardiovascular disease risk factors. The Poland and US Collaborative Study on Cardiovascular Disease Epidemiology. Ann Epidemiol 4: 445-448.
12. Liu Y, Ding J, Bush TL, et al, 2001 Relative androgen excess and increased cardiovascular risk after menopause: a hypothesized relation. Am J Epidemiol 154: 489-494.
13. Phillips GB, 2005 Is atherosclerotic cardiovascular disease an endocrinological disorder? The estrogen-androgen paradox. J Clin Endocrinol Metab 90: 2708-2711.
14. de Kleijn MJ, van der Schouw YT, van der Graaf Y, 1999 Reproductive history and cardiovascular disease risk in postmenopausal women: a review of the literature. Maturitas 33: 7-36.
15. Palmer JR, Rosenberg L, Shapiro S, 1992 Reproductive factors and risk of myocardial infarction. Am J Epidemiol 136: 408-416.
16. Fioretti F, Tavani A, Gallus S, Franceschi S, La Vecchia C, 2000 Menopause and risk of non-fatal acute myocardial infarction: an Italian case-control study and a review of the literature. Hum Reprod 15: 599-603.
17. Cooper GS, Ephross SA, Weinberg CR, Baird DD, Whelan EA, Sandler DP, 1999 Menstrual and reproductive risk factors for ischemic heart disease. Epidemiology 10: 255-259.
18. Carr MC, 2003 The emergence of the metabolic syn-drome with menopause. J Clin Endocrinol Metab 88: 2404-2411.
19. Mudali S, Dobs AS, Ding J, et al, 2005 Endogenous postmenopausal hormones and serum lipids: the atherosclerosis risk in communities study. J Clin Endocrinol Metab 90: 1202-1209.
20. Saltiki K, Doukas C, Kanakakis J, Anastasiou E, Mantzou E, Alevizaki M, 2006 Severity of cardiovascular disease in women: Relation with exposure to endogenous estrogen. Maturitas 55: 51-57.
21. Jacobsen BK, Knutsen SF, Fraser GE, 1999 Age at natu-ral menopause and total mortality and mortality from ischemic heart disease: the Adventist Health Study. J Clin Epidemiol 52: 303-307.
22. Jansen SC, Temme EH, Schouten EG, 2002 Lifetime estrogen exposure versus age at menopause as mortality predictor. Maturitas 43: 105-112.
23. de Kleijn MJ, van der Schouw YT, Verbeek AL, Peeters PH, Banga JD, van der Graaf Y, 2002 Endogenous estrogen exposure and cardiovascular mortality risk in postmenopausal women. Am J Epidemiol 155: 339-345.
24. van der Schouw YT, van der Graaf Y, Steyerberg EW, Eijkemans JC, Banga JD, 1996 Age at menopause as a risk factor for cardiovascular mortality. Lancet 347: 714-718.
25. Ossewaarde ME, Bots ML, Verbeek AL, et al, 2005 Age at menopause, cause-specific mortality and total life expectancy. Epidemiology 16: 556-562.
26. Hu FB, Grodstein F, Hennekens CH, et al, 1999 Age at natural menopause and risk of cardiovascular disease. Arch Intern Med 159: 1061-1066.
27. Morise AP, 2006 Assessment of estrogen status as a marke of prognosis in women with symptoms of suspected coronary artery disease presenting for stress testing. Am J Cardiol 97: 367-371.
28. Barrett-Connor E, Goodman-Gruen D, 1995 Prospective study of endogenous sex hormones and fatal cardiovascular disease in postmenopausal women. BMJ 311: 1193-1196.
29. Phillips GB, Pinkernell BH, Jing TY, 1997 Relationship between serum sex hormones and coronary arteryd isease in postmenopausal women. Arterioscler Thromb Vasc Biol 17: 695-701.
30. Rexrode KM, Manson JE, Lee IM, et al, 2003 Sex hormone levelsand risk of cardiovascular events in postmenopausal women. Circulation 108: 1688-1693.
31. Guthrie JR, Taffe JR, Lehert P, Burger HG, Denner-stein L, 2004 Association between hormonal changes at menopause and the risk of a coronary event: alongitudinal study. Menopause 11: 315-322.
32. Cauley JA, Gutai JP, Glynn NW, Paternostro-Bayles M, Cottington E, Kuller LH, 1994 Serum estrone concentrations and coronary artery disease in postmenopausal Women. Arterioscler Thromb 14: 14-18.
33. Bairey Merz CN, Johnson BD, Sharaf BL, et al, WISE Study Group, 2003 Hypoestrogenemia of hypothalamic origin and coronary artery disease in premenopausal women: a report from the NHLBI-sponsored WISE study. J Am Coll Cardiol 41: 413-419.
34. Bolibar I, Thompson SG, von Eckardstein A, Sandkamp M, Assmann G, 1995 Dose-response relationships of serum lipid measurements with the extent of coronary stenosis.Strong, independent, and comprehensive. ECAT Angina Pectoris Study Group. Arterioscler Thromb Vasc Biol 15: 1035-1042.
35. Jensen J, Nilas L, Christiansen C, 1990 Influence of menopause on serum lipids and lipoproteins. Maturitas 12: 321-331.
36. Morita Y, Iwamoto I, Mizuma N, et al, 2006 Precedence of the shift of body-fat distribution over the change in body composition after menopause. J Obstet Gynaecol Res 32: 513-516.
37. Gruber CJ, Tschugguel W, Schneeberger C, Huber JC, 2002 Production and actions of estrogens. N Engl J Med 346: 340-352.
38. Deroo BJ, Korach KS, 2006 Estrogen receptors and human disease. J Clin Invest 116: 561-570.
39. Koehler KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA, 2005 Reflections on the discovery and significance of estrogen receptor beta. Endocr Rev 26: 465-478.
40. Mendelsohn ME, Karas RH, 1999 The protective effects of estrogen on the cardiovascular system. N Engl J Med 340: 1801-1811.
41. Herynk MH, Fuqua SA, 2004 Estrogen receptor mutations in human disease. Endocr Rev 25: 869-898.
42. Turgeon JL, Carr MC, Maki PM, Mendelsohn ME, Wise PM, 2006 Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies. Endocr Rev 27: 575-605.
43. Kimmins S, MacRae TH, 2000 Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins. Cell Stress Chaperones 5: 76-86.
44. Yuan Y, Liao L, Tulis DA, Xu J, 2002 Steroid receptor coactivator-3 is required for inhibition of neointima formation by estrogen. Circulation 105: 2653-2659.
45. Smith CL, O’Malley BW, 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25: 45-71.
46. Shang Y, Brown M, 2002 Molecular determinants for the tissue specificity of SERMs. Science 295: 2465-2468.
47. McKay LI, Cidlowski JA, 1999 Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr Rev 20: 435-459.
48. Mendelsohn ME, 2002 Genomic and nongenomic effects of estrogen in the vasculature. Am J Cardiol 90: 3F-6F.
49.
Venkov CD, Rankin AB, Vaughan DE, 1996 Identification o authentic estrogen receptor incultured endothelial cells. A potential mechanism for steroid hormone regulation of endothelial function. Circulation 94: 727-733.
50. Losordo DW, Kearney M, Kim EA, Jekanowski J, Isner JM, 1994 Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenopausal women. Circulation 89: 1501-1510.
51. Karas RH, Patterson BL, Mendelsohn ME, 1994 Human vascular smooth muscle cells contain functional estrogen receptor. Circulation 89: 1943-1950.
52. Liu PY, Christian RC, Ruan M, Miller VM, Fitzpatrick LA, 2005 Correlating androgen and estrogen steroid receptor expression with coronary calcification and atherosclerosis in men without known coronary artery disease. J Clin Endocrinol Metab 90: 1041-1046.
53. Pare G, Krust A, Karas RH, et al, 2002 Estrogen recep-tor-alpha mediates the protective effects of estrogen against vascular injury. Circ Res 90: 1087-1092.
54. Christian RC, Liu PY, Harrington S, Ruan M, Miller VM, Fitzpatrick LA, 2006 Intimal ER{beta}, but not ER{alpha} Expression is Correlated with Coronary Calcification and Atherosclerosis in Pre-and Postmenopausal Women. J Clin Endocrinol Metab 91: 2713-2720.
55. Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD, 2000 Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects. Circulation 101: 1792-1798.
56. Inoue S, Hoshino S, Miyoshi H, et al, 1996 Identification of a novel isoform of estrogen receptor, a potential inhibitor of estrogen action, in vascular smooth muscle cells. Biochem Biophys Res Commun 219: 766-772.
57. Karas RH, Hodgin JB, Kwoun M, et al, 1999 Estrogen inhibits the vascular injury response in estrogen receptor beta-deficient female mice. Proc Natl Acad Sci USA 96: 15133-15136.
58. Iafrati MD, Karas RH, Aronovitz M, et al, 1997 Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat Med 3: 545-548.
59. Post WS, Goldschmidt-Clermont PJ, Wilhide CC, et al, 1999 Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res 43: 985-991.
60. Sudhir K, Chou TM, Chatterjee K, et al, 1997 Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation 96: 3774-3777.
61. Simoncini T, Genazzani AR, 2003 Non-genomic actions of sex steroid hormones. Eur J Endocrinol 148: 281-292.
62. Chambliss KL, Yuhanna IS, Anderson RG, et al, 2002 ERbeta has nongenomic action in caveolae. Mol Endocrinol 16: 938-946.
63. Kim HP, Lee JY, Jeong JK, Bae SW, Lee HK, Jo I, 1999 Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae. Biochem Biophys Res Commun 263: 257-262.
64. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER, 2005 A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307: 1625-1630.
65. Nadal A, Rovira JM, Laribi O, et al, 1998 Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor. FASEB J 12: 1341-1348.
66. Bjornstrom L, Sjoberg M, 2002 Signal transducers and activators of transcription as downstream targets of nongenomic estrogen receptor actions. Mol Endocrinol 16: 2202-2214.
67. Hansson GK, 2005 Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352: 1685-1695.
68. Szabo SJ, Sullivan BM, Peng SL, Glimcher LH, 2003 MolecularmechanismsregulatingTh1immuneresponses. Annu Rev Immunol 21: 713-758.
69. Doherty TM, Fitzpatrick LA, Inoue D, et al, 2004 Molecular, endocrine, and genetic mechanisms of arterial calcification. Endocr Rev 25: 629-672.
70. Pfeilschifter J, Koditz R, Pfohl M, Schatz H, 2002 Changes in proinflammatory cytokine activity after menopause. Endocr Rev 23: 90-119.
71. Hodgin JB, Krege JH, Reddick RL, Korach KS, Smithies O, Maeda N, 2001 Estrogen receptor alpha is a major mediator of 17beta-estradiol’s atheroprotective effects on lesion size in Apoe-/- mice. J Clin Invest 107: 333-340.
72. Christian RC, Harrington S, Edwards WD, Oberg AL, Fitzpatrick LA, 2002 Estrogen status correlates with the calcium content of coronary atherosclerotic plaques in women. J Clin Endocrinol Metab 87: 1062-1067.
73. Witteman JC, Grobbee DE, Kok FJ, Hofman A, Valken-burg HA, 1989 Increased risk of atherosclerosis in women after the menopause. BMJ 298: 642-644.
74. Janowitz WR, Agatston AS, Kaplan G, Viamonte M Jr, 1993 Differences in prevalence and extent of coronary artery calcium detected by ultrafast computed tomogra-phy in asymptomatic men and women. Am J Cardiol 72: 247-254.
75. Hofbauer LC, Schoppet M, 2001 Osteoprotegerin: a link between osteoporosis and arterial calcification? Lancet 358: 257-159.
76. Lindner V, Kim SK, Karas RH, Kuiper GG, Gustafsson JA, Mendelsohn ME, 1998 Increased expression of estrogen receptor-beta mRNA in male blood vessels after vascular injury. Circ Res 83: 224-229.
77. Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E, 2005 Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury. J Mol Cell Cardiol 38: 289-297.
78. Pelzer T, Loza PA, Hu K, et al, 2005 Increased mortality and aggravation of heart failurein estrogen receptor-beta knockout mice after myocardial infarction. Circulation 111: 1492-1498.
79. Brouchet L, Krust A, Dupont S, Chambon P, Bayard F, Arnal JF, 2001 Estradiol accelerate sreen do the lialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta. Circulation 103: 423-428.
80. Evans MJ, Harris HA, Miller CP, Karathanasis SK, Adelman SJ, 2002 Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways. Endocrinology 143: 3785-3795.
81. Cid MC, Schnaper HW, Kleinman HK, 2002 Estrogens and the vascular endothelium. Ann N Y Acad Sci 966: 143-157.
82. Spyridopoulos I, Sullivan AB, Kearney M, Isner JM, Losordo DW, 1997 Estrogen-receptor-mediated inhibition of human endothelial cell apoptosis. Estradiol as a survival factor. Circulation 95: 1505-1514.
83. Sutton-Tyrrell K, Lassila HC, Meilahn E, Bunker C, Matthews KA, Kuller LH, 1998 Carotid atherosclerosis in premenopausal and postmenopausal women and its association with risk factors measured after menopause. Stroke 29: 1116-1121.
84. Kim JK, Pedram A, Razandi M, Levin ER, 2006 Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms. J Biol Chem 281: 6760-6767.
85. Roque M, Heras M, Roig E, et al, 1998 Short-term effects of transdermal estrogen replacement therapy on coronary vascular reactivity in post menopausal women with angina pectoris and normal results on coronary angiograms. J Am Coll Cardiol 31: 139-143.
86. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G, 1992 Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci USA 89: 1125911263.
87. Chambliss KL, Shaul PW, 2002 Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 23: 665686.
88. Rubanyi GM, Freay AD, Kauser K, et al, 1997 Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption. J Clin Invest 99: 2429-2437.
89. Armour KE, Armour KJ, Gallagher ME, et al, 2001 Defec-tive bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase. Endocrinology 142: 760-766.
90. Kalantaridou SN, Naka KK, Papanikolaou E, et al, 2004 Impaired endothelial function in young women with premature ovarian failure: normalization with hormone therapy. J Clin Endocrinol Metab 89: 3907-3913.
91. Kalantaridou SN, Naka KK, Bechlioulis A, Makrigiannakis A, Michalis L, Chrousos GP, 2006 Premature ovarian failure, endothelial dysfunction ande strogen-progestogen replacement. Trends Endocrinol Metab 17: 101-109.
92. Mikkola TS, Clarkson TB, 2002 Estrogen replacement therapy,atherosclerosis,andvascularfunction.Cardiovasc Res 53: 605-619.
93. Hulley S, Grady D, Bush T, et al, 1998 Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 280: 605-613.
94. Pradhan AD, Manson JE, Rossouw JE, et al, 2002 Inflammatory biomarkers, hormone replacement therapy, and incident coronary heart disease: prospective analysis from the Women’s Health Initiative observational study. JAMA 288: 980-987.
95. Campos H, Walsh BW, Judge H, Sacks FM, 1997 Effect of estrogen on very low density lipoprotein and low density lipoprotein subclass metabolism in postmenopausal women. J Clin Endocrinol Metab 82: 3955-3963.
96. Bruschi F, Meschia M, Soma M, Perotti D, Paoletti R, Crosignani PG, 1996 Lipoprotein(a) and other lipids after oophorectomy and estrogen replacement therapy. Obstet Gynecol 88: 950-954.
97. Demissie S, Cupples LA, Shearman AM, et al, 2006 Estrogen receptor-alpha variants are associated with lipoprotein size distribution and particle levels in women: the Framingham Heart Study. Atherosclerosis 185: 210-218.
98. Herrington DM, Howard TD, Hawkins GA, et al, 2002 Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Engl J Med 346: 967-974.
99. Mayes JS, Watson GH, 2004 Direct effects of sex steroid hormones on adipose tissues and obesity. Obes Rev 5: 197-216.
100. Palin SL, McTernan PG, Anderson LA, Sturdee DW, Barnett AH, Kumar S, 2003 17Beta-estradiol and anti-estrogen ICI: compound 182,780 regulate expression of lipoprotein lipase and hormone-sensitive lipasein isolated subcutaneous abdominal adipocytes. Metabolism 52: 383-388.
101. Pascot A, Despres JP, Lemieux I, et al, 2001 Deteriora-tion of the metabolic risk profile in women. Respective contributions of impaired glucose tolerance and visceral fat accumulation. Diabetes Care 24: 902-908.
102. Gebara OC, Mittleman MA, Sutherland P, et al, 1995 Association between increased estrogen status and increased fibrinolytic potential in the Framingham Offspring Study. Circulation 91: 1952-1958.
103. Sieminska L, Wojciechowska C, Niedziolka D, et al, 2005 Effect of postmenopause and hormone replacement therapy on serum adiponectin levels. Metabolism 54: 1610-1614.
104.Bednarek-Tupikowska G, Filus A, Kuliczkowska-Plaksej J, Tupikowski K, Bohdanowicz-Pawlak A, MilewiczA,2 006 Serum leptin concentrations in pre-and postmenopausal women on sex hormone therapy. Gynecol Endocrinol 22: 207-212.
105. Clegg DJ, Brown LM, Woods SC, Benoit SC, 2006 Gonadal hormones determine sensitivity to central leptin and insulin. Diabetes 55: 978-987.
106. Sumino H, Ichikawa S, Kasama S, et al, 2006 Different effects of oral conjugated estrogen and transdermal estradiol on arterial stiffness and vascular inflammatory markers in postmenopausal women. Atherosclerosis 189: 436-442.
107. Kalaitzidis D, Gilmore TD, 2005 Transcription factor cross-talk: the estrogen receptor and NF-kappa B.Trends Endocrinol Metab 16: 46-52.
108.Sukovich DA, Kauser K,Shirley FD, DelVecchio V, Halks-Miller M, Rubanyi GM, 1998 Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol. Arterioscler Thromb Vasc Biol 18: 1498-1505.
109. Torzewski M, Rist C, Mortensen RF, et al, 2000 C-reactive protein in the arterial intima: role of C-reactive protein receptor-dependent monocyte recruitment in atherogenesis. Arterioscler Thromb Vasc Biol 20: 2094-2099.
110. Jialal I, Devaraj S,Venugopal SK, 2004 C-reactiveprotein: risk marker or mediator in atherothrombosis? Hypertension 44: 6-11.
111. Arnal JF,Gourdy P, ElhageR, et al, 2004 Estrogens and atherosclerosis. Eur J Endocrinol 150: 113-117.
112. Manson JE, Bassuk SS, Harman SM, et al, 2006 Post-menopausal hormone therapy: new questions and the case for new clinical trials. Menopause 13: 139-147.
113. Davison S, Davis SR,2003 New markers for cardiovascular disease risk in women: impact of endogenous estrogen status and exogenous postmenopausal hormone therapy. J Clin Endocrinol Metab 88: 2470-2478.
114. Kuohung W, Shwaery GT, Keaney JF Jr, 2001 Tamoxifen, esterified estradiol, and physiologic concentrations of estradiol inhibit oxidation of low-density lipoprotein by endothelial cells. Am J Obstet Gynecol 184: 1060-1063.
115. Chrousos GP, Torpy DJ, Gold PW, et al, 1998 Interactions between the hypothalamic-pituitary-adrenal axis and the female reproductive system: clinical implications. Ann Intern Med 129: 229-240.
116. Bracamonte MP, Miller VM, 2001 Vascular effects of estrogens: arterial protection versus venous thrombotic risk. Trends Endocrinol Metab 12: 204-209.
117. Koh KK, 2002 Effects of hormone replacement therapy oncoagulation and fibrinolysis in post menopausal women. Int J Hematol 76: 44-46.
118. Grady D, Wenger NK, Herrington D, et al, 2000 Post-menopausal hormone therapy increases risk for venous thromboembolic disease. The Heart and Estrogen/progestin Replacement Study. Ann Intern Med 132: 689-696.
119. Anderson GL, Limacher M, Assaf AR, et al, Women’s Health Initiative Steering Committee 2004 Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA 291: 1701-1712.
120. ScarabinPY, OgerE, Plu-Bureau G;EStrogen, THrom-boEmbolism Risk Study Group, 2003 Differential asso-ciation of oral and transdermal oestrogen-replacement therapy with venous thromboembolism risk. Lancet 362: 428-432.
121. EilertsenAL,HoibraatenE,OsI,AndersenTO,Sandvik L, Sandset PM, 2005 The effects of oral and transdermal hormone replacement therapy on C-reactive protein levels and other inflammatory markers in women with high risk of thrombosis. Maturitas 52: 111-118.
122. GomesMP, Deitcher SR, 2004Riskof venousthrombo-embolicdiseaseassociatedwithhormonalcontraceptives andhormonereplacementtherapy:aclinicalreview.Arch Intern Med 164: 1965-1976.
123. Straczek C, Oger E,Yon de Jonage-Canonico MB, et al, Estrogen and Thromboembolism Risk (ESTHER) Study Group, 2005 Prothromboticmutations, hormone therapy, and venous thromboembolism among postmenopausal women: impact of the route of estrogen administration. Circulation 112: 3495-3500.
124. Evangelopoulos D, Alevizaki M, Lekakis J, et al, 2003 Molecular analysis of the estrogen receptor alpha gene in men with coronary artery disease: association with disease status. Clin Chim Acta 331: 37-44.
125. Shearman AM, Cupples LA, Demissie S, et al, 2003 Association between estrogen receptoral phagene variation and cardiovascular disease. JAMA 290: 2263-2270.
126. Petrovic D, Peterlin B,2003 Estrogen receptor dinucleotide (TA) polymorphism does not predict premature myocardial infarction in Caucasian women. Cardiology 99: 163-165.
127. Pollak A, Rokach A, Blumenfeld A, Rosen LJ, Resnik L, Dresner Pollak R, 2004 Association of oestrogen receptor alpha gene polymorphism with the angiographic extent of coronary artery disease. Eur Heart J 25: 240-245.
128. Rokach Α, Pollak Α, Rosen L, et al, 2005 Estrogen Receptor {alpha} Gene Polymorphisms are Associated with the Angiographic Extent of Coronary Artery Disease. J Clin Endocrinol Metab 90: 6556-6560.
129. Schuit SC, Oei HH, Witteman JC, et al, 2004 Estrogen receptoral phagene polymorphisms and risk of myocardial infarction. JAMA 291: 2969-2977.
130. Shearman AM, Cooper JA, Kotwinski PJ, et al, 2006 Estrogen Receptor {alpha} Gene Variation Is Associated with Risk of Myocardial Infarction in more than Seven Thousand Men from Five Cohorts. Circ Res 98: 590-592.
131. Lu H, Higashikata T, Inazu A, et al, 2002 Association of estrogen receptor-alpha gene polymorphisms with coronary artery disease in patients with familial hyper-cholesterolemia. Arterioscler Thromb Vasc Biol 22: 817-823.
132. Koch W, Hoppmann P, Pfeufer A, Mueller JC, Schomig A, Kastrati A, 2005 No replication of association between estrogen receptor alpha gene polymorphisms and susceptibility to myocardial infarction in a large sample of patients of European descent. Circulation 112: 2138-2142.
133.Lehtimaki T, Laaksonen R, Mattila KM, et al, 2002 Oestrogen receptor gene variation is a determinant of coronary reactivity in healthy young men. Eur J Clin Invest 32: 400-404.
134.Shearman AM, Cooper JA, Kotwinski PJ, et al, 2005 Estrogen receptor alpha gene variation and the risk of stroke. Stroke 36: 2281-2282.
135. Lehtimaki T, Kunnas TA, Mattila KM, et al, 2002 Coronary artery wall atherosclerosis in relation to the estrogen receptor 1 gene polymorphism: an autopsy study. J Mol Med 80: 176-180.
136. Alevizaki M, Saltiki K, Kanakakis I, et al, 2006 The severity of cardiovascular disease in women is associated with estrogen receptor alpha polymorphic variants. 8th European Congress of Endocrinology; vol 11, p 332.
137. Mansur Ade P, Nogueira CC, Strunz CM, Aldrighi JM, Ramires JA, 2005 Genetic polymorphisms of estrogen receptors in patients with premature coronary artery disease. Arch Med Res 36: 511-517.
138. Matsubara Y, Murata M, Kawano K, et al, 1997 Genotype distribution of estrogen receptor polymorphisms in men and postmenopausal women from healthy and coronary populations and its relation to serum lipid levels. Arterioscler Thromb Vasc Biol 17: 3006-3012.
139. Cai Q, Shu XO, Jin F,etal, 2003 Genetic polymorphisms in the estrogen receptor alpha gene and risk of breast cancer: results from the Shanghai Breast Cancer Study. Cancer Epidemiol Biomarkers Prev 12: 853-859.
140. Weiderpass E, Persson I, Melhus H, Wedren S, Kindmark A, Baron JA, 2000 Estrogen receptor alpha gene polymorphisms and endometrial cancer risk. Carcinogenesis 21: 623-627.
141. Luisi S, Galleri L, Marini F, Ambrosini G, Brandi ML, Petraglia F, 2006 Estrogen receptor gene polymorphisms are associated with recurrence of endometriosis. Fertil Steril 85: 764-766.
142. Ioannidis JP, Ralston SH, Bennett ST, et al,GENOMOS Study, 2004 Differential genetic effects of ESR1 gene polymorphisms on osteoporosis outcomes. JAMA 292: 2105-2114.
143. Stavrou I, Zois C, Ioannidis JP, Tsatsoulis A, 2002 Association of polymorphisms of the oestrogen receptor alpha gene with the age of menarche. Hum Reprod 17: 1101-1105.
144.Weel AE, Uitterlinden AG, Westendorp IC, et al, 1999 Estrogen receptor polymorphism predicts the onset of natural and surgical menopause. J Clin Endocrinol Metab 84: 3146-3150.
145. Ellis JA, Infantino T, Harrap SB, 2004 Sex-dependent association of blood pressure estrogen receptor genes ERalpha and ERbeta. J Hypertens 22: 1127-1131.
146. Kikuchi T, Hashimoto N, Kawasaki T, Uchiyama M, 2000 Association of serum low-densityl ipoprotein metabolism with oestrogen receptor gene polymorphisms in healthy children. Acta Paediatr 89: 42-45.
147. Shearman AM, Demissie S, Cupples LA, et al, 2005 Tobacco smoking, estrogen receptor alpha gene variation and small low density lipoprotein level. Hum Mol Genet 14: 2405-2413.
148. Herrington DM, Howard TD, Brosnihan KB, et al, 2002 Common estrogen receptor polymorphism augments effects of hormone replacement therapy on E-selectin but not C-reactive protein. Circulation 105: 1879-1882.
149. Gotoda T, Kinoshita M, Ishibashi S, et al, 1997 Skipping of exon 14 and possible instability of both the mRNA and the resultant truncated protein underlie a common cholesteryl ester transfer protein deficiency in Japan. Arterioscler Thromb Vasc Biol 17: 1376-1381.
150. O’Neill JP, Rogan PK, Cariello N, Nicklas JA, 1998 Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum. Mutat Res 411: 179-214.
151. Becherini L, Gennari L, Masi L, et al, 2000 Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor alpha gene and their relationship to bone mass variation in postmenopausal Italian women. Hum Mol Genet 9: 2043-2050.
152. Almeida S, Franken N, Zandona MR, Osorio-Wender MC, Hutz MH, 2005 Estrogen receptor 2 and progesterone receptor gene polymorphisms and lipid levels in women with different hormonal status. Pharmacogenomics J 5: 30-34.
153. Hall WL, Vafeiadou K, Hallund J, et al, 2006 Soy-isoflavone-enriched foods and markers of lipid and glucose metabolism in postmenopausal women: interactions with genotype and equol production. Am J Clin Nutr 83: 592-600.
154. Peter I, Shearman AM, Vasan RS, et al, 2005 Association of estrogen receptor beta gene polymorphisms with left ventricular mass and wall thickness in women. Am J Hypertens 18: 1388-1395.
155. Ogawa S, Emi M, Shiraki M, Hosoi T, Ouchi Y, Inoue S, 2000 Association of estrogen receptor beta (ESR2) gene polymorphism with blood pressure. J Hum Genet 45: 327-330.
156. Zhu Y, Bian Z, Lu P, et al, 2002 Abnormal vascular function and hypertension in mice deficient in estrogen receptor beta. Science 295: 505-508.
157. Pelzer T, Loza PA, Hu K, et al, 2005 Increased mortality and aggravation of heart failure in estrogen receptor-beta knockout mice after myocardial infarction. Circulation 111: 1492-1498.
158. Westberg L, Baghaei F, Rosmond R, et al, 2001 Polymorphisms of the androgen receptor gene and the estrogen receptor beta gene are associated with androgen levels in women. J Clin Endocrinol Metab 86: 2562-2568.
159. Stavrou I, Zois C, Chatzikyriakidou A, Georgiou I, Tsatsoulis A, 2006 Combined estrogen receptor alpha and estrogen receptor beta genotypes influence the age of menarche. Hum Reprod 21: 554-557.
160. Iobagiu C, Lambert C, Normand M, Genin C, 2006 Microsatellite profile in hormonal receptor genes associated with breast cancer. Breast Cancer Res Treat 95: 153-159.
161. Shearman AM, Karasik D, Gruenthal KM, et al, 2004 Estrogen receptor beta polymorphisms are associated with bone mass in women and men: the Framingham Study. J Bone Miner Res 19: 773-781.
162. Stampfer MJ, Colditz GA, 1991 Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 20: 47-63.
163. Grodstein F, Manson JE, Colditz GA, Willett WC, Speizer FE, Stampfer MJ, 2000 A prospective, observational study of postmenopausal hormone therapy and primary prevention of cardiovascular disease. Ann Intern Med 133: 933-941.
164. Grodstein F, Stampfer MJ, Colditz GA, et al, 1997 Post-menopausal hormone therapy and mortality. N Engl J Med 336: 1769-1775.
165. Grodstein F, Stampfer M, 1995 The epidemiology of coronary heart disease and estrogen replacement in postmenopausal women. Prog Cardiovasc Dis 38: 199-210.
166. Gouva L, Tsatsoulis A, 2004 The role of estrogens in cardiovascular disease in the aftermath of clinical trials. Hormones (Athens) 3: 171-183.

Address for correspondence:
Maria Alevizaki, 18 Papadiamantopoulou Str., 115 28, Athens, Greece,
Tel No: 210-7208181, e-mail: mani@otenet.gr

Received 20-09-06, Revised 21-11-06, Accepted 05-12-06