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Cardiovascular Research 2002 56(1):4-7; doi:10.1016/S0008-6363(02)00573-4
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Clues to understanding the role of estrogen receptors in mediating cardiovascular protection

Xiao-Jun Du*

Experimental Cardiology Laboratory, Baker Heart Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia

xiaojun.du{at}baker.edu.au

* Tel.: +61-3-8530-1294; fax: +61-3-8532-1100

Received 15 July 2002; accepted 15 July 2002

See article by Molero et al. [14] (pages 43–51) in this issue.

Gender-related differences in the risk of cardiovascular diseases have been well recognized with premenopausal women exhibiting a lower risk compared to age-matched men [1]. Estrogen is implicated as the factor responsible for this gender difference due to the facts that the female advantage over male in cardiovascular morbidity disappears following menopause and that administration of 17{alpha}-estradiol (E2) is protective against cardiovascular injury. In addition to changes in lipid metabolism, E2 at physiological levels mediates direct cardiovascular actions. The mechanisms and signal pathways responsible for the cardiovascular protection by estrogen have received great attention in recent years and a wealth of literature in this area has been recently reviewed [1–3].

Nuclear and putative membrane-bound estrogen receptors (ERs) are present in cardiovascular tissues of women and men. ER {alpha} and β isoforms mainly form homodimers (ER{alpha} and ERβ) that mediate predominant effects of estrogen. Nuclear ER{alpha} and ERβ regulate gene expression in two modes. The authentic signalling mode involves binding of ERs with specific DNA estrogen-response elements (EREs) which are located within regulatory regions of targeted genes. The binding of ERs with ERE recruits some coactivators thereby modulating machinery of gene transcription [4]. The second mode of action is independent of ERE (i.e., occurs in the absence of a direct binding of ERs to DNA) and requires interactions of ERs with promoter elements that directly bind to transcription factors. These sites include AP-1 sites that bind Jun/Fos, cyclic AMP response element (CREs) that bind c-Jun/ATF-2, and SP-1 sites [4–6]. Recently, it is believed that the rapid nongenomic actions of estrogen are mediated by membrane ERs through several pathways, including tyrosine kinase, mitogen-activated protein kinase (MAPK), adenylyl cyclase/cAMP, and guanylyl cyclase/cGMP [1,7]. Estrogen is a key regulator of growth, differentiation or apoptosis, and function of vascular smooth muscle cells, endothelial cells, fibroblasts and cardiomyocytes [2]. Expression of a number of proteins, that are important in the development of cardiovascular diseases, is either up-regulated (e.g., metalloproteinases, nitric oxide synthase, prostaglandin synthase, vascular endothelial growth factor, Akt, atrial natriuretic factor, coagulation factors, tissue plasminogen activator) or down-regulated by estrogen (e.g., cytokines, adhesion molecules, angiotensin converting enzymes, endothelin-1, angiotensin receptor AT1) [1]. Estrogen also activates expression of the subtypes of ER, thereby promoting the responsiveness to estrogen. Cell biology studies indicate that estrogen modulates Ca2+ and K+ ion channels of vascular smooth muscle cells, endothelial cells and cardiomyocytes thereby reducing vascular tone and electrophysiological perturbations [7–9].

Circulating neutrophils are involved in a number of cardiovascular events including atherosclerosis, atherosclerotic plaque rupture, vascular injury following endothelial damage, and myocardial ischemia/reperfusion injury. Endothelial damage or inflammation triggers local accumulation of neutrophils. This is then followed by neutrophil adhesion, migration into the subendothelial layer and activation. Localised neutrophils (macrophages and monocytes) take up low-density lipoproteins to form foam cells and release growth factors leading to smooth muscle cell growth and migration [10]. Further, neutrophils play a pivotal role in platelet activation and myocardial injury following ischemia/reperfusion [11,12]. In the latter, neutrophils accumulate in the microcirculation and interstitial space and, via release of toxic molecules, cause endothelial dysfunction and tissue injury. Estrogen suppresses expression of several adhesion molecules and is protective against platelet aggregation and ischemic reperfusion injury [2,13] although the mechanisms involved are only partially understood.

In this issue of Cardiovascular Research, Molero et al. have reported some interesting findings on the bioactivity of estrogen on neutrophils harvested from pre-menopausal women and age-matched men [14]. They measured, using Western blotting, the expression levels of the subtypes of ER and nitric oxide synthase (NOS) in neutrophils and explored signalling mechanisms. In vitro experiments were also performed to test the effect of incubation with 10 nM E2. Several highlights from this study worth particular attention. Firstly, autoregulation of ER transcription by E2 was observed in vivo (ovulation versus follicular phases in women) and in vitro (incubation with E2). Interestingly, neutrophils obtained from men and women differ in that while cells from women showed markedly increased expression of both ER{alpha} and ERβ upon exposure to E2, E2 upregulated ER{alpha} but not ERβ in neutrophils from men. They also observed a background expression of both ER{alpha} and ERβ in neutrophils from women and men [14]. This finding differs from a recent study by Stefano et al. showing that human neutrophils express only ER{alpha} but not ERβ [15]. Differences in expression of ER{alpha} and ERβ may imply different physiological roles of the subtypes of ERs. Secondly, neuronal NOS (nNOS or NOS1), a constitutively expressed NOS, is upregulated by estrogen via ERs. Previous works by this and other groups have shown that enhanced nitric oxide (NO) production by neutrophils potently inhibits platelet aggregation and neutrophil adhesion to the vessel wall [16,17]. Considering that this upregulation of ER{alpha} and nNOS by estrogen was similar in neutrophils harvested from women and men, it appears reasonable to postulate that the induction of nNOS is mediated by ER{alpha} alone. The effects of E2 were partially or completely inhibited by ER antagonists tamoxifen and ICI-182780, respectively. In this regard, it is interesting to know that the affinity for AP-1 binding sites is found fourfold higher for ER{alpha} than ERβ [18]. Thirdly, their data showed that ER signal pathway involves binding of ERs to AP-1 and SP-1 sites as specific inhibitors completely abolished this action. This finding is indicative of nuclear ERs as the responsible ERs.

The role of subtypes of nuclear and membrane ERs on the cardiovascular system is a key but largely unknown issue. A major limitation for research on the ER subtypes is the lack of specific ER antagonists. The current research tools involve the use of ER subtype null mouse strains and gene transfection. Whereas Molero et al. claimed that in the human neutrophils, ER{alpha} mediated the activation of nNOS [14], this subtype-dependency is likely to vary in different cardiovascular cells. Several recent studies provide evidence for this argument [19–21]. Zhu et al. recently reported that ERβ null mice exhibited profound functional and biochemical abnormalities, including enhanced vasoconstriction, development of hypertension with aging and loss of outward K+ currents in vascular smooth muscle cells [19]. They also provided convincing evidence that estrogen activates inducible NOS (iNOS or NOS2) expression in endothelium-denuded vascular rings, an effect that is mediated by ERβ rather than ER{alpha}. Thus, it seems that in normal blood vessels, estrogen-activated iNOS expression is mediated by ERβ leading to attenuated vasoconstriction. Interestingly, this effect of ERβ is antagonized by ER{alpha}. As showed by Zhu et al., E2 treatment of cells transfected with ER{alpha} suppressed iNOS expression and that in vessel rings from ERβ null mice infusion with E2 enhanced {alpha}-adrenergic agonist mediated vasoconstriction, effect apparently mediated by ER{alpha} [19]. In another recent study, Pare et al. investigated the role of ER subtypes in mediating vascular protection using single and duel ER knockout mouse strains [20]. After ovariectomy and induction of carotid artery injury (medial thickening and proliferation of vascular smooth muscle cells), treatment with E2 inhibited vascular injury in ERβ knockout strain but had no such effect in ER{alpha} knockout (ER{alpha}KOst) or double ER{alpha}/ERβ knockout strains. These observations justify the conclusion that ER{alpha} mediates the protective effects of estrogen on the response to vascular injury. In cardiomyocytes and vascular smooth vascular cells, estrogen activates expression of both iNOS and endothelial NOS (eNOS or NOS3), effect that was prevented by an ER antagonist R,R-THC [21]. Neudling et al. further addressed the subtypes of ERs in mediating this effect using COS7 cells transfected with relevant genes. E2-stimulated NOS expression was inhibited by R,R-THC in COS7 cells transfected with ERβ, but not with ER{alpha}, implying a role of ERβ in promoting NOS expression [21]. Modulation of NO production by estrogen is one of important biochemical mechanisms upon which gender differences and beneficial effects of estrogen in cardiovascular disease are based. Thus, taking NOS expression as an endpoint, we have seen the diversity or even opposite actions by ER{alpha} and ERβ, largely depending on the cell type studied. Inhibitory interactions between ER{alpha} and ERβ have also been reported in various models [22,23]. Furthermore, roles of the subtypes of ERs on vascular pathophysiology may vary depending on the etiology considering selective activation of ERβ expression following vascular injuries [24,25] and attenuated expression of ER{alpha}, but not ERβ, by estrogen deficiency [26]. In cardiac fibroblasts, hypoxia upregulates expression of ERβ and ERE [27].

Several issues in the paper by Molero et al. [14] warrant further comment. A critical matter is to determine the functional implication of the difference in ER expression in neutrophils. In particular, we need to know whether neutrophils from women and men behaviour differently under the setting of healthy, aging and cardiovascular disease. Another issue is on the mechanism responsible for the increased NO production by estrogen. Stefano and coworkers have reported an enhanced NO production in human neutrophils upon E2 stimulation [15,28]. This response occurred within seconds, was short lasting and was mediated by membrane ERs as E2 conjugated to bovine serum albumin (which is membrane impermeable) was also able to elicit NO production [15]. Their data imply that a rise in intracellular Ca2+ is necessary for this action. Interestingly, this group has reported a similar action by E2 in vascular endothelia [7]. In the study by Molero et al., the involvement of nuclear ER{alpha} in the enhanced nNOS expression is supported by the effectiveness of specific inhibitors for AP-1 (curcumin) and SP-1 (mithramycin A) [14]. Thus, it is apparent that in neutrophils, E2-stimulated NO production has rapid and slow phases mediated by distinct ERs and signal mechanisms. A further point is on the finding by Molero et al. that inhibition of either SP-1 or AP-1 completely prevented the up-regulation of ER and nNOS [14]. SP-1 and AP-1 are well documented transcriptional factors [4,6,22]. There has been no evidence to date to indicate interaction or synergy by these two DNA binding sites [14]. Actually Saville et al. have shown that only ER{alpha} is able to activate SP-1 in several cell lines [6]. Addressing this question further using neutrophils requires techniques such as transfection of genes with recessive or dominant mutations. Another interesting finding that was only briefly commented on by Molero et al. is that at equal molar concentration, ICI-182780 was much more potent than tamoxifen in the inhibition of E2-induced expression of ER{alpha} and nNOS [14]. In contrast, Stefano et al. reported that ICI 182780 failed to block E2-stimulated NO production (rapid phase) in neutrophils but tamoxifen was effective [15]. In vascular endothelial cells, however, several groups have found that, ICI-182780, but not tamoxifen, is able to block E2 activated rapid NO production and eNOS expression [29–31] and there is indeed evidence that ICI-182780 can also block estrogen-stimulated NO release via a nongenomic mechanism [31]. The difference between the two ER antagonists is interesting and warrants further investigation. Finally, Molero et al. studied only pre-menopausal women and age-matched men with an averaged age of 30 years. It remains to be seen whether the findings on ER{alpha} autoregulation and induction of nNOS in the present study are replicable in aged women and men, a sub-population at much higher cardiovascular risk. It should be noticed that one of their previous studies did show a threefold upregulation of nNOS by estrogen replacement therapy in neutrophils from postmenopausal women aged 45–65 years [16]. However, age-matched men were not studied.

A much less understood area is the mechanism by which the putative membrane ERs mediate rapid and nongenomic bioactions of estrogen, including vasodilatation and alterations in ion channel activities [32]. It should be emphasized that the findings on the signalling of estrogen via classic ERs, like the study by Molero et al. [14], have been made with complete unknown of possible involvement by membrane ERs. There is evidence that membrane ER can also alter gene expression [4,29]. Although the density of membrane ERs is only 2–3% of the density of nuclear ERs indicated by a cDNA transfection study [33], membrane ERs (ER{alpha} subtype) are rich in plasmalemmal caveolae where they are coupled with signal transduction molecules, including NOS, to form an efficient signalling module [29,30]. Whether this mechanism is responsible for the membrane ER-mediated rapid nongenomic actions in neutrophils is worth further investigation. Note that the slow and genomic actions and rapid membrane actions can synergize [34,35], probably via phosphorylation of nuclear ERs by protein kinases [34].

Thus, the study by Molero and colleagues should attract more interest to this area but, as any good article, has raised key questions. Their previous and present works indicate human neutrophil as a useful model for further studies on ERs signal transduction [14,16], in particular the gender-related difference in ER autoregulation. The nature of cell-dependent differences in ER-mediated bioactivity in cardiovascular tissues and neutrophils and its pathophysiological or therapeutical relevance remain to be determined. Similar studies need to be undertaken on other cardiovascular cells such as vascular endothelium, vascular smooth muscle cells and cardiomyocytes. Further research requires the use of models such as gene-targeted animals and gene transfected cells. Studies need to include aged or diseased subjects and the effect of estrogen replacement therapy. Finally, we should also consider the necessity of examining, in a similar way, the potential action of the male steroid, testosterone.


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