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Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies

Division of Endocrinology, Clinical Nutrition, and Vascular Medicine, Department of Internal Medicine (J.L.T.), University of California, Davis, Davis, California 95616; Division of Endocrinology, Metabolism, and Molecular Medicine (M.C.C.), Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611; Department of Psychiatry and Psychology (P.M.M.), University of Illinois at Chicago, Chicago, Illinois 60612; Molecular Cardiology Research Institute, Department of Medicine, and Division of Cardiology (M.E.M.), New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts 02111; and Departments of Physiology and Biophysics, and Biology (P.M.W.), University of Washington, Seattle, Washington 98195

Correspondence: Address all correspondence and requests for reprints to: Judith Turgeon, Ph.D., Department of Internal Medicine, Division of Endocrinology, Clinical Nutrition, and Vascular Medicine, University of California Davis, Davis, California 95616. E-mail: jlturgeon{at}ucdavis.edu

	Abstract

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
Recent publications describing the results of the Women’s Health Initiative (WHI) and other studies reporting the impact of hormone therapy on aging women have spurred reexamination of the broad use of estrogens and progestins during the postmenopausal years. Here, we review the complex pharmacology of these hormones, the diverse and sometimes opposite effects that result from the use of different estrogenic and progestinic compounds, given via different delivery routes in different concentrations and treatment sequence, and to women of different ages and health status. We examine our new and growing appreciation of the role of estrogens in the immune system and the inflammatory response, and we pose the concept that estrogen’s interface with this system may be at the core of some of the effects on multiple physiological systems, such as the adipose/metabolic system, the cardiovascular system, and the central nervous system. We compare and contrast clinical and basic science studies as we focus on the actions of estrogens in these systems because the untoward effects of hormone therapy reported in the WHI were not expected. The broad interpretation and publicity of the results of the WHI have resulted in a general condemnation of all hormone replacement in postmenopausal women. In fact, careful review of the extensive literature suggests that data resulting from the WHI and other recent studies should be interpreted within the narrow context of the study design. We argue that these results should encourage us to perform new studies that take advantage of a dialogue between basic scientists and clinician scientists to ensure appropriate design, incorporation of current knowledge, and proper interpretation of results. Only then will we have a better understanding of what hormonal compounds should be used in which populations of women and at what stages of menopausal/postmenopausal life.

I. Introduction

II. Pharmacology of Estrogens and Progestins

A. Menopausal hormone therapy (HT)

B. Estrogen/progestin therapy before menopause

III. The Inflammatory Process

A. Inflammation in brief

B. Estrogen and the inflammatory process

IV. Adipose/Metabolic System and Estrogens

A. Visceral adipose tissue

B. Influence of estrogen on adipose tissue

C. The influence of estrogen on markers of inflammation

V. Cardiovascular System and HT

A. Sex steroid hormone receptor action and cardiovascular physiology

B. Vascular effects of sex steroid hormones, atherosclerosis and ischemic cardiovascular diseases

C. Sex steroid hormones and the myocardium in health and disease

VI. Central Nervous System and Sex Steroids

A. Comparisons between basic science studies and the WHI

B. Studies performed using animal models

C. Mechanisms of estrogen action in the central nervous system

D. Evaluation of the Women’s Health Initiative Memory Study (WHIMS): design and findings

E. WHIMS validity: internal and external

F. Differences with previous clinical and observational studies: age of initiation of treatment

G. Potential of SERMS as HT: efficacy on central nervous system parameters

H. Effect of progestins on cognition

VII. Summary/Conclusions

	I. Introduction

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
WOMEN TYPICALLY UNDERGO the transition to postmenopause at approximately 51 yr of age. This dramatic physiological transition, which can be abrupt or can occur over several years, is associated with decreases in ovarian hormone secretion and secondary changes in multiple endocrine and metabolic parameters. Many women suffer from menopausal symptoms that have been attributed to the lack of ovarian steroids, including hot flushes, sleep disturbance, vaginal dryness, urinary symptoms, and emotional instability. In addition, postmenopausal women are thought to be at increased risk for several diseases compared with age-matched cycling women such as cardiovascular disease (CVD), cerebrovascular stroke, osteoporosis, hip fracture, dementia, and Alzheimer’s disease (AD).

Over the past 20 yr, numerous observational, retrospective, interventional, and meta-analytic studies (reviewed in Refs. 1, 2, 3) as well as studies using animal models (reviewed in Refs. 4, 5, 6, 7, 8, 9) have supported the hypothesis that ovarian steroids exert important protective actions in women and the absence of these hormones after the menopause makes postmenopausal women more vulnerable than younger premenopausal women to CVD, central nervous system imbalances, neurodegenerative diseases, osteoporosis, and immune dysfunction. However, the recent release of several studies (10, 11, 12, 13, 14, 15, 16, 17) has forced us to reassess these conclusions and reevaluate the benefits and risks of hormone therapy (HT) in older women. The study of the Women’s Health Initiative (WHI) (11) (see http://www.nih.gov/PHTindex.htm for a complete list of publications) has caught the attention of researchers, physicians, and the lay public. The WHI was comprised of two large, randomized, placebo-controlled clinical trials for HT in postmenopausal women and had three significant design characteristics: 1) only one type of hormone regimen was studied, i.e., oral conjugated equine estrogens (CEE) at a single concentration without or with continuous oral medroxyprogesterone acetate (MPA); 2) the average age of the participants at entry was 63 yr, and, for a majority of the women, estrogen deficiency had been present for more than a decade; and 3) nearly 70% of the subjects were either overweight or obese. These studies were expected to settle the question of whether postmenopausal women would benefit from HT and would solve the dilemma of what women should do after the menopause once and for all. Instead, they have raised many questions and caused much reanalysis and reinterpretation of the results of previous studies.

Fallout from the WHI has been an indictment of all menopausal HT. The unfortunate consequence is that women and their physicians are left with, at best, unsettling choices. In attempting to reconcile the disparate conclusions derived from the WHI and observational studies/basic research, three points emerge that may account for the disparities: 1) different estrogens (or progestins) are not recognized in the same way in all cells and do not have equivalent functions; 2) hormone delivery regimens have a major impact on outcome measures; and 3) age is a critical interactive factor in hormone action, particularly in the timing of the initiation of HT relative to the onset of menopause. An additional and perhaps crucial element in accounting for the study disparities is our emergent understanding of estrogen’s interface with inflammation and with the adipose/metabolic system. There are direct effects of estrogen on the inflammatory process and on adipose tissue distribution and function, and the question is how does this contribute to the phenotypic shifts in dyslipidemia, diabetes, and cardiovascular and neurodegenerative diseases in women through their life span. Recent discoveries in these areas allow us to consider novel concepts regarding the varied roles of estrogen and the design of future hormone therapies.

Here, we review the basic pharmacology of estrogens and progestins and provide an overview of the inflammatory response. On this background, we examine the points raised above with a focus in three major areas: adipose/metabolic, cardiovascular, and central nervous systems.

	II. Pharmacology of Estrogens and Progestins

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
A. Menopausal hormone therapy (HT)
Publication of results from clinical trials, such as the WHI and the Heart and Estrogen/Progestin Replacement Study (HERS) (10), has prompted an extremely useful reexamination of the roles of ovarian hormones outside of the reproductive axis. An equally constructive result has been an increased awareness of lapses that can occur in the dialogue between clinical and basic scientists on estrogen and progestin actions.

In comparing clinical studies and animal models of HT, the focus most often is on subject characteristics and main outcome measurements and less frequently on the specifics of the intervention. Here, we address the principle that not all estrogens or progestins are equal and briefly review the consequences of differences in receptor activation, delivery modes, and timing of hormone treatment. The reader is also directed to reviews by Shoham and Kopernik (18, 19) that provide a wonderfully lucid and insightful perspective on the history, pharmacology, and bioactivity of HT formulations.

1. Are all estrogens or progestins equal in action?
Clearly, the answer is no. "Estrogen" and "progestin" are generic terms covering an array of endogenous hormones and synthetic compounds. Although useful as shorthand, these nonspecific terms obscure the importance of the unique actions of individual estrogens or progestins. As reviewed in Refs. 3, 20 , and 21 , binding of ligand to a steroid receptor induces distinct structural alterations in the receptor. Instead of functioning as simple switches for receptor activation, ligands have a more complex role in that different ligands induce unique conformations in a receptor. This is crucial to the ultimate cellular response because recruitment of appropriate coregulators to the ligand-receptor complex is conformation-dependent. The coregulator complement, in turn, confers cell specificity. The final conformation of the ligand-receptor-coregulator complex affects how its activity is modified (by phosphorylation, etc.) and ultimately determines target-gene promoter specificity or, in the case of extranuclear receptors, signaling targets (21, 22, 23, 24, 25, 26). This versatile system is made more so by at least two different forms of the estrogen receptor (ER and ERß) and the progesterone receptor (PR-A and PR-B). Within their ligand binding domains, the ER isoforms differ by more than 40% in amino acid sequence, thus providing further opportunity for different estrogens to induce distinct conformational changes in an ER-coregulator complex to elicit discrimination in function (25). The isoforms within a receptor type are differentially expressed and regulated across cell types and have both common and distinct target genes (27, 28, 29, 30, 31, 32, 33, 34).

The significance of ligand-dependent receptor conformation cannot be overstated in comparing the actions of different types of estrogens or progestins. Examples of the influence that structural differences in ligand have on outcome measures can be seen with selective ER modulators (SERMs), such as tamoxifen or raloxifene, which function as either ER agonists or antagonists depending on the cell type and coregulator complement (29, 35). SERMs, acting through ER isoforms, have been shown to regulate different gene subsets compared with the classic ovarian estrogen, 17ß-estradiol (E₂) (36). Receptor isoform-specific regulation of gene expression, which has been demonstrated for the PR isoforms as well as for ER isoforms, is being exploited for structure-based design for ligands with individualized tissue effects (34, 37, 38, 39, 40, 41). This work and other research, for example investigating specific coregulator recruitment (42, 43), use mechanism-based approaches that show great promise for producing therapeutic tools that are function-specific. These approaches also are being explored for extranuclear steroid receptors, for example in the design of ER ligands that are cytosolic signaling pathway-selective, with the goal of target-specific therapy (44). The area of extranuclear steroid receptors and rapid signaling, including the possibility of nonreceptor-mediated effects, is too complex and unique a topic to be succinctly summarized here, but we strongly recommend recent excellent and comprehensive reviews of the subject (45, 46).

Although SERMs and selective PR modulators represent well-known examples of compounds that induce unique receptor conformations and exhibit distinct properties in specific cell types (35), what about the estrogens and progestins commonly used in HT? By far the most frequently prescribed treatment in the United States for menopausal HT is oral CEE, with or without MPA (Table 1) (47). CEE, which is derived from the urine of pregnant horses, is comprised of at least 10 different estrogens, as well as some androgens and progestins (48, 49). In addition to sulfated forms of estrone (E₁) and E₂, the estrogen component of CEE includes several sulfated estrogens that are unique to the horse, the most prominent being equilin (49, 50). The critical question is what are the functional, tissue-specific effects of the CEE component estrogens on the function of ER isoforms in women? The few studies that directly addressed this question suggest a wide range of activities reflecting individual metabolic clearance rates as well as estrogen type-specific activity at the ER that can translate into cell type-specific responses (48, 50, 51). For example, a CEE component, ^8,9-dehydroestrone sulfate, has a tissue selectivity and activity profile distinct from that for E₂ with full agonist effects on several central nervous system endpoints and little or no efficacy in certain hepatic or vascular endpoints (48, 52). In general, the CEE components demonstrate the principle that binding affinity for the ER does not necessarily predict biological activity (48). Thus, the clinical response to CEE is a composite of the pharmacokinetic profiles and individual actions of the steroid components as well as the interactions of the components at ER isoforms that could either mitigate or magnify a response.

Drospirenone (DRSP) is a progestin with antialdosterone and antiandrogenic activities that is in active use in HT and contraceptive formulations in many countries outside the United States, and especially Europe (67). In clinical trials, DRSP is effective against menopausal symptoms and is able to provide endometrial protection and maintain amenorrhea in a majority of women. In the doses used in HT formulations, DRSP also has a modest blood pressure-lowering effect, which may prove to be beneficial in some patients. The role of the mineralocorticoid receptor in cardiovascular physiology currently is receiving increasing attention, and the presence of functional mineralocorticoid receptors regulating gene expression in vascular tissues has been reported recently (68), raising the possibility that some of the protective effects of antialdosterone compounds like spironolactone or eplerenone in CVDs (69, 70) may be exerted in nonrenal tissues, including the blood vessel (71, 72).

2. HT delivery route.
In postmenopausal literature, the term "HT" frequently is used as if it were a single entity. However, in addition to the profound effect that the specific estrogen or progestin formulation may have on outcome, a perhaps equally critical variable is how HT is delivered. In 2003, an estimated 80% of prescriptions dispensed in the United States for HT were for estrogen +/– progestin formulations delivered orally, and the majority of these contained CEE as the estrogen component (Table 1) (47). In Europe, micronized E₂ is one of the most common estrogens used orally, but transdermally administered E₂ is widely used as well (19, 73, 74, 75). For basic research in animal models, steroid hormones are usually given as E₂ and progesterone in sc or im depots and are rarely administered orally. This difference in drug delivery methods is a critically important factor when extrapolating knowledge gained from animal models to humans, for which oral HT is by far the most common delivery route.

Oral CEE and oral E₂ are similar in that a peak in plasma concentration is reached within the first 3 h, followed by a decline, and both oral formulations result in higher plasma E₁ levels than E₂, which is unlike the normal menstrual cycle ratio. The pharmacokinetics of oral CEE is complex due to the multiple estrogen components, with varying binding affinities for transport proteins and different metabolic clearance rates (49). For transdermal HT, the estrogen component is E₂. Transdermal application of E₂ avoids the exaggerated peaks and nadirs in plasma estrogen concentration that are a consequence of oral HT and results in a rate of conversion to E₁ that produces an E₂:E₁ ratio more like that found in a menstrual cycle.

It has long been recognized that estrogens taken orally have the potential to modulate liver function due to first-pass effects on hepatic ERs. Clinical studies reporting side-by-side or crossover comparisons provide specific and striking examples of effects induced by oral but not transdermal estrogen on liver production of proteins involved in inflammation, lipid profile, thrombosis, and metabolism (Table 2). A key to these differences is the high concentration of oral estrogen that is required to escape first-pass hepatic metabolism and achieve a therapeutic concentration in peripheral circulation. For E₂, oral administration requires doses that are 20- to 40-fold higher than doses used for transdermal therapy to achieve comparable systemic E₂ levels. For the examples shown in Table 2 comparing oral and transdermal E₂, the resulting circulating E₂ levels averaged 40–150 pg/ml for either treatment modality; the difference was in the dose delivered to the liver.

Recent studies have generated insight into mechanisms responsible for some of the hepatic actions of estrogen. For example, the cytokine signaling pathway used by GH to stimulate production of IGF-I by liver cells has a built-in brake system via a parallel induction of suppressor proteins from the SOCS (suppressor of cytokine signaling) family. In vitro studies now show that E₂, acting through nuclear ER, can suppress GH signaling by induction of SOCS-2, thus providing one possible target mechanism for elevated hepatic estrogen (78). It will be important to establish that a concentration-dependent estrogen suppression of GH signaling occurs in vivo in the liver and to determine whether this estrogen-stimulated mechanism is operative at other GH targets, e.g., affecting GH-stimulated lipolysis in adipose tissue.

Determining the impact of oral progestins on hepatic proteins is complex due to the multiple formulations and delivery routes used therapeutically and also to the experimental challenge of sorting out effects attributable to the androgenicity of the compounds. It has been shown, however, that the most commonly prescribed progestin, oral MPA, blunts the increase in high-density lipoprotein (HDL) induced by oral CEE in a dose-dependent manner, yet oral micronized progesterone has no effect on this endpoint (79, 80). For the more androgenic progestins, such as norethindrone or levonorgestrel, both of which can be taken either orally or transdermally, the few side-by-side trials in postmenopausal women suggest that the differences in outcomes such as thromboembolic events or effects on lipid profile are more related to chemical structure than to delivery route (81, 82, 83).

3. HT timing.
Two design aspects of the WHI and HERS that have been particularly controversial are the age of the participants and the duration of estrogen deficiency before HT was initiated. With a mean age of 63 yr (with nearly 70% between the ages of 60 and 79) and estrogen deficiency for more than a decade in the majority of the women, the WHI in effect becomes a study more of aging than of menopause (84, 85).

The physiological consequence of estrogen reintroduction depends on an interaction between the target system, the health of that system, and the duration of estrogen deprivation. For example in the cardiovascular system, premenopausal ovariectomy or premature ovarian failure has been associated with an increase in peripheral vascular resistance and blood pressure, impaired endothelial function, and increased risk of coronary heart disease that can be reversed by early replacement therapy with estrogen (86, 87, 88). That the timing of estrogen therapy (ET) initiation is critical was demonstrated in a monkey model of diet-induced atherosclerosis. After surgically induced menopause in these monkeys, if ET was initiated immediately there was a reduction in coronary artery atherosclerosis compared with placebo-treated ovariectomized monkeys, but delaying ET by an equivalent of about 6 human years completely eliminated the beneficial reduction in atherosclerosis (89). The effects of estrogen reintroduction after a disease process has begun will vary depending on the end organ target, its plasticity, and any consequences aging may have had in the interim. Evaluation of these system-dependent variables must be made before conclusions can be drawn regarding beneficial, neutral, or harmful effects of HT.

In summary, the initial aim of HT was to alleviate the obvious signs of estrogen withdrawal such as vasomotor flushes and vaginal dryness. Over the years, these relatively simple aims have grown into a long and complex list of targets outside of the reproductive system for which a "one formulation/one size fits all" approach is not tenable. CEE/MPA or other HT formulations were not meant to be a panacea for aging, nor were they designed to be optimal for the physiological/endocrinological status of all women over the age of 50. When some risk to health for HT users is suggested, as in the WHI, all estrogens and progestins should not be denounced because each of the formulations has a unique action profile and pharmacokinetic properties. Additionally, oral HT has a wide range of effects on liver proteins, and the consequences of these changes and their impact on other organ systems must be a central consideration in the interpretation of clinical studies as well as in comparing human studies with animal model studies, for which the steroids rarely are administered orally. Other influential factors include a woman’s age and health status if HT is initiated after a lapse of several years. To paraphrase Stevenson and Whitehead (90), survival of the human species over 2 million years implies that ovarian hormones by themselves are not a health hazard; if harm is suspected, the judicious research inquiry should focus on the types and doses of hormone substitutes being used as well as their delivery routes and treatment protocols. And we would add that an additional focus should be to understand the underlying physiology for the many targets of E₂ and progesterone outside of the reproductive system and the adaptive roles they have in the aging population.

B. Estrogen/progestin therapy before menopause
The most common use of exogenous estrogens and progestins in premenopausal women is for contraception. The pharmacology of the wide-ranging choices for types of contraceptive drugs and delivery routes is outside the scope of this review, but a limited examination of common oral formulations and the recently available transdermal preparation is presented for comparison with the regimens and outcomes of postmenopausal HT.

1. Oral contraceptive (OC) hormones.
Preparations containing 17-ethinyl estradiol (EE) and a progestin (e.g., norethindrone, levonorgestrel, or norgestimate) are the most frequently prescribed oral hormonal contraceptives. Since U.S. approval 46 yr ago, the primary changes in OCs have been a decrease in EE content and the development of new generations of progestins in an effort to reduce side effects while maintaining LH/FSH inhibition to prevent ovulation. Current low-dose oral EE is no greater than 35 µg/d.

How does EE compare to E₂? At the nuclear ER, the two ligands have similar binding affinity (91, 92) and ability to induce rat uterine target genes (93), although this similarity in efficacy has not been established for all systems or for nonnuclear ERs. In fact, there are surprisingly few studies evaluating specific actions of EE outside of the reproductive system. Earlier studies suggested that EE had enhanced hepatic effects and systemic potency compared with that following oral E₂, but this is likely related to the increased bioavailability of EE due to its marked resistance to metabolism by hepatic 16-hydroxylation as well as reduced conversion to E₁, thus resulting, in effect, in a higher concentration of EE presented to hepatic cells (see Refs. 49, 93 , and 94 and references therein). Another crucial contribution to the bioavailability of EE is its minimal binding to SHBG, instead being transported by albumin (49). A 35-µg dose of oral EE in premenopausal women results in an average total serum concentration of about 50 pg/ml, a large fraction of which is free due to the low-affinity binding of EE to albumin (95). In comparison, at least 1000 µg of oral micronized E₂ would be required to achieve comparable average total serum levels. However, in contrast to EE, less than 5% of total E₂ in circulation is free due to SHBG binding (96).

Not unexpectedly, because of the first-pass hepatic phenomenon and increased bioavailability, oral EE affects liver proteins similarly to oral E₂ (Table 2). For example, OCs are associated with increased incidence of thromboembolic disease (81, 82, 97). One of the most common risk factors for venous thromboembolism is hereditary resistance to hepatic protein C, which when activated essentially down-regulates thrombin formation. OCs have been shown to induce resistance to activated protein C (APC) in the absence of a hereditary mutation, so-called acquired APC resistance, and this extends to oral postmenopausal HT as well (Table 2) (98, 99, 100, 101). Although oral estrogen by itself can lead to increased APC resistance, the progestin component, particularly third generation progestins such as norgestimate, can contribute to the thrombotic effects of oral estrogen (81, 82, 98, 99, 102). In addition to its role in antithrombin formation, APC has been shown to have antiinflammatory activity (103, 104), but whether this function is affected by OC- or oral HT-induced APC resistance has not been established.

Venous thrombosis is a complex process involving procoagulant, anticoagulant, and fibrinolytic elements, and oral estrogen has some beneficial effect on other components of the pathway despite its overall adverse effect on thrombosis. This becomes more apparent when first-pass liver effects are bypassed; studies in postmenopausal women suggest that transdermally administered E₂ has beneficial effects on several hemostatic markers for anticoagulant activity (105). It is significant that transdermal E₂ used for HT, unlike oral E₂, is not associated with risk for venous thrombosis and does not induce APC resistance (Table 2) (75, 100, 106). It remains to be determined whether the same applies to transdermal EE (see Section II.B.2) and to premenopausal women.

2. Transdermal contraceptive hormones.
A recent addition to the contraceptive armamentarium is a combined estrogen/progestin formulation given as a transdermal patch. Instead of containing E₂ as in the patch used for HT, the currently available contraceptive patch contains EE. The formulation results in average serum levels of 50–70 pg/ml with the same increased bioavailability as oral EE but without the peaks and troughs and, consequently, an overall higher 24-h level of serum EE compared with that found with oral EE as described above (95, 107). The progestin in the contraceptive patch is norelgestromin, the primary active metabolite of a common component of OCs (norgestimate).

Given the importance of overall health status and the indeterminate effects of aging in the response of organ systems to the modulating actions of estrogen, the premenopausal population represents an excellent opportunity for a direct comparison of oral vs. transdermal administration of the same estrogen/progestin formulation in younger women. This would be particularly useful for the examination of cardiovascular and central nervous system endpoints affected by the confounding variable of oral estrogen’s hepatic action. For example, a study of oral vs. transdermal E₂ rather than EE in premenopausal women to evaluate indicators of venous and arterial thromboses could be highly informative for comparison with existing data for oral vs. transdermal E₂ in postmenopausal women.

In summary, hormone treatment can be considered as a continuum from contraception through postmenopause. Although similarities are many, the differences may be informative. For example, unlike most regimens for HT, the standard protocol for contraceptive hormones includes a periodic interruption for a week without exogenous hormones, which may have consequences for ER isoform expression level and ratios. Additionally, some differences in formulation and dose are found between contraceptive hormones and HT, but the significance of these variances to targets outside the reproductive system has not been firmly established. Confounding such analysis are endogenous hormones. Unlike that in postmenopausal women, the ovarian contribution to circulating estrogen levels still can be significant in premenopausal women on contraceptive HT. However, the current trend toward an increase in body mass index (BMI) for both pre- and postmenopausal women may mute this difference in endogenous hormone levels because of conversion of adrenal androgens to estrogens by adipocyte aromatase. And perhaps the most critical difference between premenopausal and postmenopausal populations is that of overall health status and background into which HT is introduced, a consideration particularly relevant for subclinical disease progression when HT initiation is delayed.

	III. The Inflammatory Process

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
A. Inflammation in brief
Inflammation research has experienced remarkable progress over the last decade and has generated increased appreciation for the complexity of the inflammatory process. A detailed presentation of the area is well beyond the scope of this review, but an outline of some of what is known of the ground rules and the players in inflammation is necessary for considering how sex steroid hormones might affect this process.

As a simple working definition, inflammation is a well-coordinated response of the immune, endocrine, and metabolic systems to tissue damage. Although an acute inflammatory response, for instance to a skin injury or infection, is the type that most readily comes to mind, at the other end of the spectrum is chronic inflammation, which is thought to be at the base of disorders such as arteriosclerosis, osteoarthritis, inflammatory bowel diseases, and several neurodegenerative diseases. The highly regulated acute inflammatory response that results in damage control and tissue restoration normally includes antiinflammatory mechanisms that contribute to the termination of the response. However, if the initiating injury or perturbation is recurrent or nonresolving or if a genetic alteration interferes with the sequence, the result is chronic inflammation.

The primary signaling molecules in the inflammatory response are cytokines produced on site (by, e.g., macrophages, monocytes, lymphocytes, activated microglial cells) and peripherally (e.g., visceral adipose tissue). The proinflammatory cytokines (e.g., TNF, IL-1, IL-6, and interferon-) along with growth factors (e.g., macrophage colony-stimulating factor) and chemokines (e.g., IL-8) initiate a coordinated sequence including, for example, recruitment of appropriate leukocytes and immune cells, up-regulation of pattern-recognition receptors for innate immunity including scavenger receptors and Toll-like receptors, positive feedback for continued production of proinflammatory cytokines, and increased synthesis of prostaglandins and leukotrienes. In contrast to this group of mediators, as part of the turn-off mechanism, antiinflammatory cytokines (e.g., IL-4, IL-10) inhibit production or block actions of the proinflammatory cytokines.

The initiating signals that stimulate production of proinflammatory molecules can range from peroxidation products from excessive lipid oxidation to mechanical stress or a combination of cell-specific insults. Regardless of the particular perturbation, nuclear factor-B (NF-B)/Rel transcription factors play pivotal roles in transducing a signal that leads to production of cytokines. The NF-B system also acts as mediator of inflammatory cytokine action, as do the MAPK signaling pathways and c-Jun N-terminal kinase (JNK1) (reviewed in Refs. 108, 109, 110, 111). In addition to cytokines such as TNF-, JNK1 also is activated by free fatty acids, and the activity of this kinase is abnormally elevated in mouse models of obesity (112).

Another key player in the regulation of inflammatory events is nitric oxide (NO), a diffusible uncharged gas with a half-life in seconds and having multiple molecular targets, for example cytokine production, inhibition of immune cell proliferation, antimicrobial activity, cyclooxygenase-2 induction, and vascular permeability and vasodilation (see Refs. 113, 114, 115 and references therein). Depending on its concentration and the cell- and event-specific activity, NO can have either pro- or antiinflammatory effects. NO production is regulated by isoforms of NO synthase (NOS): inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). All three isoforms operate in the immune system and are expressed in multiple cell types. Regulation of iNOS is via transcription activated by cytokines and costimulatory molecules signaling through several pathways, including the NF-B system. Activation of eNOS and nNOS predominantly is via a change in activity of existing protein triggered by a wide array of stimuli, including vasoactive substances, neurotransmitters, shear stress, and cytokines.

B. Estrogen and the inflammatory process
Experimental evidence for the role of estrogen in inflammation within the adipose/metabolic, cardiovascular, and neural systems will be reviewed in subsequent sections, but it is useful here to consider recent studies that establish the mechanistic potential for estrogen to affect the inflammatory process. ER and, in some cases, ERß are present in front line immune and cytokine-producing cells, such as macrophages and microglia, and E₂-activated ER has been shown in vitro to affect release of proinflammatory cytokines from these cells and to interfere with the action of cytokines (see Refs. 116, 117, 118, 119, 120 and references therein). These critical E₂ actions can be explained, at least in part, by the ability of ER to function as a transcriptional repressor by inhibiting the activity of NF-B through a protein-protein interaction of agonist-bound ER with a subunit of activated NF-B (121, 122, 123). The extent of the inhibitory action of E₂ on NF-B function may be context- and target gene-selective, thus opening intriguing possibilities for mechanism-based design of SERMs (30, 123, 124).

E₂ also is implicated in the activation of eNOS and nNOS as well as the regulation of expression of all three NOS isoforms in the immune, cardiovascular, and central nervous systems (see Refs. 37, 115 , and 125, 126, 127 and references therein). Both nuclear and extranuclear pathways are used by E₂ to affect the NOS isoforms. For example, the rapid release of NO in endothelial cells that results in vasodilation can be accomplished by E₂ activation of membrane ER and subsequent signaling through a phosphatidylinositol 3-kinase/Akt signaling pathway leading to eNOS activation (reviewed in Refs. 45 and 46). Progesterone has been shown to potentiate E₂ effects on the generation of NO in human endothelial cells in vitro, whereas MPA impairs E₂ signaling in this model (61).

In summary, the widespread presence and participation of ERs in multiple cell types of the immune system and the inflammatory response is remarkable and represents just one facet of the pleiotropic action of estrogens. Although much work remains to be done in defining the complex interaction of estrogens with the inflammatory process, the studies to date establish E₂ as a significant player, as will be explored in the following.

	IV. Adipose/Metabolic System and Estrogens

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
A. Visceral adipose tissue
Visceral fat produces an array of hormones involved in energy homeostasis, the fibrinolytic system, the immune system, vascular homeostasis, and the inflammatory response. This multifunctional endocrine/paracrine organ has been the focus of many excellent reviews (for example, Refs. 128, 129, 130, 131), and only a few highlights will be considered here.

Recent interest has been sparked, in part, by the observation that increased visceral adiposity is closely associated with insulin resistance, hypertension, dyslipidemia, and CVD. Adipose-derived hormones (adipokines) involved in these disorders have been examined in clinical, animal knockout, and in vitro studies, and prominent on any list have been the inflammatory cytokines and related proteins (Fig. 1) (131, 132, 133). For many of the adipokines shown in Fig. 1, there is a direct relationship between visceral adipose mass and the amount of product. An important exception is adiponectin, which has an inverse relationship to fat mass; in addition, an inverse association between plasma adiponectin levels and both insulin resistance and chronic inflammation has been reported (131, 134).

View larger version (36K): [in this window] [in a new window]   FIG. 1. Overview of some of the endocrine and paracrine products of visceral adipose tissue. The products are grouped into broad functional categories with examples shown for each category. Many of the secretory products (adipokines) have targets within visceral fat as well as in the periphery. The enzyme 11ß-hydroxysteroid dehydrogenase-1 expressed in adipocytes is responsible for the conversion of circulating cortisone (inactive) to cortisol (active); adipose aromatase converts circulating androgens to estrogens. Receptors for cortisol and for E2 are present in adipocytes. PAI-1, Plasminogen activator inhibitor-1; FFA, free fatty acids; VEGF, vascular endothelial growth factor.

Rodent models have revealed that the selective surgical resection of visceral fat pads led to marked improvements in insulin resistance, which was not seen with the removal of equivalent amounts of sc adipose tissue (142), supporting the concept that sc adipose tissue is a less important determinant of insulin action and lipid metabolism than visceral adipose tissue. Consistent with the functional distinctiveness of adipose depots is the recent identification of visfatin, a novel adipokine preferentially produced by human abdominal visceral adipose that facilitates adipogenesis but intriguingly also acts as an insulin-mimetic (143, 144, 145). Although much work remains in establishing its physiological roles, visfatin offers new prospects in obesity research.

The regulation of visceral adipose activity is complex, which is not surprising given its multifunctional and integrative characteristics. In addition to receptors for multiple cytokines and metabolic hormones, visceral adipocytes express most members of the nuclear hormone receptor family, notably peroxisome proliferator-activated receptor- (PPAR), liver X receptor (LXR), thyroid hormone receptor, glucocorticoid receptor, AR, PR, and ER. An example of visceral adipose as a complex, functionally integrated unit comes from studies of glucocorticoid receptor and its ligand cortisol, which is produced locally from inactive cortisone by 11ß-hydroxysteroid dehydrogenase-1 (11ßHSD-1) (Fig. 1). This enzyme, which is highly expressed in human adipocytes, results in increased cortisol concentration within adipose without significantly affecting systemic cortisol levels (reviewed in Refs. 128, 146 , and 147). In addition to its effect on adipocyte lipolysis and sensitivity to insulin, cortisol promotes differentiation of human preadipocytes into mature adipocytes (148). Although questions remain about the regulation and dysregulation of 11ßHSD-1 and its role as cause or effect in visceral obesity (146, 149, 150), this cortisol-generating system within adipose is significant for its local integration of energy homeostasis under normal conditions and for its potential as a target for pharmaceutical manipulation.

B. Influence of estrogen on adipose tissue
1. Estrogen and body fat distribution.
It is now clear that sex steroid hormones are major determinants of body fat distribution and that reproductive hormones in general may be considered adiposity signals. There is a gender dimorphism in body fat distribution, because women generally accumulate sc fat in the gluteal and femoral regions, whereas men develop an android pattern of body fat distribution with weight gain. Women generally have a higher percentage of total body adiposity, whereas BMI-matched men have about twice as much visceral adipose tissue as premenopausal women (151). The lower amount of visceral adipose tissue in women is thought to contribute to the lower prevalence of dyslipidemia, hypertension, diabetes, and CVD in premenopausal women compared with men and postmenopausal women.

The influence of hormones on body fat distribution appears to be related both to adipose tissue-specific expression of steroid receptors and to local tissue steroid hormone metabolism. ER and ERß are expressed in both sc and visceral fat tissues; however, ERß appears to be preferentially expressed in sc adipose (152). Steroidogenic enzymes that metabolize sex steroid hormones, e.g., 17-hydroxylase and aromatase, are differentially expressed in sc and visceral adipose depots and regulate local steroid conversion (153). Adipose aromatase itself can be regulated, for example, by locally produced cytokines and prostaglandins (154). In human adipose stromal cells, cortisol produced locally from cortisone is a potent stimulator of the conversion of androgens to estrogens through an action on aromatase (155).

Lipoprotein lipase (LPL), a triglyceride hydrolase that directs the deposition of triglyceride into adipocytes cells, is thought to play an important role in regional body fat distribution. Tchernof et al. (156) recently showed regional differences in adipocyte metabolism with menopausal status, independent of age, body fat mass, and visceral adipose tissue accumulation. The authors found that omental adipocytes from postmenopausal women were larger and had higher LPL activity compared with premenopausal women, but they found no menopause-related differences in sc adipocytes, reflecting a shift toward visceral fat storage. A role for estrogen in LPL regulation is supported by studies in aromatase knockout mice, which have a marked increase in visceral adiposity associated with an increase in LPL expression and a metabolic syndrome phenotype. E₂ treatment resulted in a marked decrease in visceral fat mass and a profound inhibition of LPL expression in the aromatase knockout (reviewed in Ref. 157).

2. Estrogen deficiency.
Menopausal estrogen deficiency is associated with significant changes in metabolic parameters that appear to be partially related to the shifts in body fat distribution with menopause (158). Premenopausal women have a less "atherogenic" lipid profile than men due to higher HDL, higher levels of large antiatherogenic HDL₂ subspecies, and lower triglyceride levels, all of which are closely associated with lower central fat accumulation. Menopause, either natural or surgical, is coupled with rapid adverse changes in lipid metabolism, because reduced HDL and HDL₂ and increased triglyceride and low-density lipoprotein (LDL) are seen within 3 months of amenorrhea (159, 160). The rapidity of the lipid changes implies that there may be both direct effects of sex steroid hormones and indirect effects of visceral fat accumulation on lipid metabolism.

Cross-sectional (156, 161) and longitudinal studies (162, 163) have shown that the menopausal transition is associated with an increase in visceral adiposity, and this effect of menopause is independent of the effect of age and total body adiposity or BMI (164, 165, 166). Establishing a mechanistic relationship in humans between the absence of E₂ and an increase in visceral adipose has been elusive, due in part to the complexities contributed by a paradoxical increase in serum E₂ associated with increases in adiposity in older menopausal women (see Ref. 167 and references therein). An additional factor in attempting to sort out the cause/effect role of estrogen and adiposity is the negative correlation of serum SHBG levels with BMI in peri- and postmenopausal women, resulting in a greater fraction of bioactive E₂ in circulation in obese individuals (168, 169). Another consideration is the source of postmenopausal E₂, which primarily is from extragonadal conversion of testosterone by aromatase. Consistent with the positive correlation of serum E₂ and BMI in postmenopausal women is the observation that adipose aromatase activity increases with age (170). So influential is the regulation by aromatase that the argument has been made that, in postmenopausal women, E₂ functions not as a circulating hormone but as a paracrine factor at its sites of production (e.g., adipose, brain, breast, and bone) (157). Clearly, the availability of testosterone and its derivation from adrenal and ovarian androstenedione and dehydroepiandrosterone must be major considerations in attempting to decipher the complex relationship of E₂, body weight, and metabolism in menopausal women.

3. Exogenous estrogen.
Contrary to popular belief, neither postmenopausal ET nor OCs have been shown to cause weight gain (171, 172), and OCs do not appear to have an effect on body fat distribution (173). However, oral ET has been shown to have a modest effect in reducing the postmenopausal weight gain (174). Mattiasson et al. (175) showed a selective reduction in visceral fat in early postmenopausal women treated with oral E₂ plus cyclic MPA that was not seen in the placebo-treated women. The HERS and WHI studies showed that combination HT (oral CEE + MPA) led to improvements in BMI and waist circumference in women with (372) and without (176) coronary heart disease.

Rodents rapidly become obese after ovariectomy, and E₂ administration is known to prevent the increase in adiposity (177). Recent data from D’Eon et al. (178) showed that E₂ treatment in ovariectomized mice promoted a reduction in adipose tissue mass and adipocyte size compared with pair-fed ovariectomized controls. The reductions in adiposity were seen in the intraabdominal fat but not the sc fat depots. The authors found increased levels of adipocyte lipolysis and down-regulation of associated genes such as LPL and LXR- in intraabdominal fat from the E₂-treated mice. Conversely, in muscle they found that E₂ led to up-regulation of LPL and muscle-specific PPAR, as well as several of its downstream targets, suggesting that E₂ promotes the use of lipid as a fuel by promoting fat oxidation in the muscle and enhancing adipocyte lipolysis (178).

If ET is associated with decreased visceral adipose, the critical question is whether there are beneficial metabolic consequences in humans. When insulin sensitivity is used as an endpoint, results are conflicting with reports of either increases, decreases, or no change depending on the age of the women, treatment route, and methodology for assessing insulin resistance (175, 176, 179). Similar inconsistencies are found with other endpoints and underscore the complexity of interactions between the paracrine and the endocrine functions of adipose, their integration with liver metabolic activity and inflammation, and the multiple estrogen targets within these tissues. Studies to disentangle this complex area are beginning to produce some answers, but much work remains in understanding the roles for estrogen in these relationships.

C. The influence of estrogen on markers of inflammation
Acute phase proteins serve as examples of the interface between adipose tissue and the inflammatory response. Markers of subclinical inflammation, such as C-reactive protein (CRP), IL-6, and serum amyloid A (SAA), have been linked to insulin resistance, diabetes mellitus 2, and CVD. In vivo and association studies have shown a strong link between obesity and increased inflammatory proteins, and obesity is associated with increased infiltration of adipose tissue by macrophages (180, 181).

CRP is a marker of the presence of subclinical inflammation that has been proposed to be an independent risk factor for cardiac events (182) and is a hallmark of the inflammatory processes that convert "fatty streak" plaques into complex atherosclerotic lesions. Although CRP is synthesized mainly in the liver, several extrahepatic sources of CRP have been reported, for example, adipocytes, macrophages, coronary artery smooth muscle cells, and neurons (131, 183). Circulating CRP levels are positively associated with total body fat and visceral adipose, and weight loss by hypocaloric diet or surgical intervention has been shown to reduce CRP levels (reviewed in Ref. 131). Oral estrogen significantly increases CRP levels through the first-pass hepatic effect, and some have hypothesized that increased CRP may have played a role in the higher rates of cardiovascular events and stroke observed in the HERS (10) and WHI (11) trials. Transdermal E₂, however, has no effect on systemic CRP levels (Table 2). It would be important to determine whether E₂ can affect local production of CRP within adipose or other target sites.

IL-6 is a proinflammatory cytokine produced at many sites, for example monocytes/macrophages, microglia, bone, and adipose, and has multiple functions in the inflammatory cascade, including increasing CRP and SAA production. Elevated IL-6 levels have been associated with increased BMI and risk of cardiovascular death (184). IL-6 has been shown to be preferentially secreted by visceral adipocytes, and removal of sc fat by liposuction did not reduce IL-6 levels (141). In addition to the positive association with BMI, circulating levels of IL-6 correlate positively with age (116, 186). Whether menopausal ET can reverse elevated serum IL-6 levels has been the subject of many studies with conflicting results, depending on treatment formulations and the age and BMI status of the women (116, 186, 187, 188, 189). For example, data from the Postmenopausal Estrogen Progestin Intervention (PEPI) trial and other randomized clinical trials have shown no significant effect of oral ET (CEE), with or without progestin, on IL-6 levels (188), but several observational studies have shown variable effects (no change, reduction, or elevation) on IL-6 levels in postmenopausal women using oral ET (reviewed in Ref. 190). In contrast, in vitro studies establish that E₂ can decrease IL-6 production and interfere with its actions in several cell types secondary to a cell-type specific disruption of NF-B signaling (116, 123, 191, 192). That E₂ modulates IL-6 activity primarily at the local tissue level is an important consideration and requires further study.

SAA is a marker of systemic inflammation that predicts future cardiovascular events and responds simultaneously with CRP to inflammatory stimuli. The SAA family is a group of differentially expressed apolipoproteins that are synthesized primarily in the liver and associate with HDL particles (187). The association of elevated SAA with HDL has been hypothesized to promote a proatherogenic phenotype by impairing reverse cholesterol transport (193). During inflammation, SAA-associated HDL particles become depleted of ApoA-1 (the apolipoprotein of the more antiatherogenic HDL subspecies), have a lower affinity for hepatocytes than HDL, and induce activity of phospholipases that lower HDL levels (187). Like CRP and IL-6, SAA is positively associated with BMI and waist circumference and has been found to be expressed in adipose tissue (194). Both IL-6 and SAA are up-regulated in adipocytes in the insulin-resistant state (see Ref. 129 and references therein). Abbas et al. (195) recently compared the effects of 8 wk of oral estrogens (CEE) or transdermal E₂ to placebo in postmenopausal women. They found that oral estrogen significantly increased total SAA and HDL-associated SAA levels (proatherogenic phenotype), whereas transdermal E₂ resulted in a significant reduction in SAA levels compared with placebo. Thus, presumed beneficial effects of oral estrogens resulting from an increase in HDL particles may be obviated by the parallel increase in SAA and interference with HDL function. The observation that transdermal E₂ is associated with a decrease in SAA reinforces the need to establish the action of E₂ on the production of acute phase proteins at nonhepatic sites.

	V. Cardiovascular System and HT

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
In postmenopausal women, the effect of HT on cardiovascular events has become an intensely controversial issue. Several major recent clinical trials, especially the WHI study, have caused basic and clinical scientists to reconsider the fundamental biology and physiological complexities that impact upon HT in postmenopausal women (3). The lack of an effect of HT on cardiovascular endpoints in WHI and several other recent prospective clinical trials has caused confusion and controversy because the outcomes of these trials seem in opposition to a large body of observational data that demonstrate a protective cardiovascular effect of hormone replacement in postmenopausal years. However, recent clinical trials like the WHI studied cardiovascular endpoints after de novo initiation of HT to a mostly older postmenopausal population without a prior history of hormone use, which contrasts sharply with most observational cohorts studied in which the initiation of HT occurred in younger women who usually were enrolled in these trials for treatment of active perimenopausal symptoms.

The strengths and weaknesses of the WHI trial have been reviewed recently (3). Consensus now has emerged that it is important to acknowledge the strengths and limitations of WHI, but also to move past the study by emphasizing the enormous need that exists to strengthen interactions between preclinical and clinical research scientists and to improve our understanding of menopause and of the biology of age and gender differences in the cardiovascular system (3, 196, 197). The biology of the perimenopause remains poorly understood at present, and enormous gaps remain in our knowledge of the gender differences of relevance to cardiovascular function that may occur during this transition (197). It remains unclear, for example, whether or not there is a critical window for hormonal treatment that will confer cardioprotection, whether the duration of any such therapeutic advantage conferred by HT is limited, and what postmenopausal time course underlies the loss of efficacy and/or emergence of adverse effects seen with administration of HT to older women. The cardiovascular implications of discontinuous vs. continuous administration of HT also are unclear, as are the hormonal formulations and selective receptor modulators that may optimize cardiovascular functions and protection (197, 198). To address these issues, it will be necessary to understand the mechanisms of sex steroid hormone effects on cardiovascular tissues in both normal physiology and disease.

A. Sex steroid hormone receptor action and cardiovascular physiology
Steroid hormones all activate members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. The biology of these receptors has been reviewed in Section II.A and will not be discussed in detail here. The general presence of functional sex steroid hormone receptors (SSHR) in the cardiovascular system is well established, their expression in both heart and blood vessels having been recognized for several decades (reviewed in Refs. 199, 200, 201, 202). E₂, progesterone, testosterone, and dihydrotestosterone all bind specifically to atrial and ventricular myocardial fibers and to vascular endothelial and smooth muscle cells of human and nonhuman primates (203, 204, 205, 206, 207, 208, 209). In a classic study, specific saturable receptors for estrogens, androgens, and glucocorticoids were demonstrated in canine coronary arteries, and PRs, absent at baseline, were induced by treatment with physiological E₂ concentrations (205). PRs also are expressed early in life, in human term fetoplacental vessels (210). ER and ERß expression in vascular smooth muscle and endothelial cells is regulated by injury and by E₂ (see Refs. 199, 200 , and 202 and references therein)_. Aromatase, the enzyme that produces estrogens from androgens, is expressed in primate coronary arteries (211) and human venous and arterial tissues, including vascular smooth muscle cells and, in some but not all studies, endothelial cells (212, 213, 214, 215, 216). Vascular aromatase mediates normal vasomotion in males, and normal young males given aromatase inhibitors develop endothelial dysfunction that is reversible upon cessation of the drug (202, 217, 218, 219). Surprisingly, the effects of aromatase inhibition on vascular function in females have not yet been studied, despite the increasing use of these compounds in the therapy of breast cancer. Furthermore, the precise distribution and regulation of expression of SSHR and aromatase in heart and vascular tissues is not known, nor is it clear yet whether vascular beds differ, whether there are gender differences in the levels of expression of these proteins, and whether their level of expression is modified by the presence of atherosclerosis.

The hypothalamic-pituitary-gonadal axis undergoes perinatal maturation. Mammalian hypothalamic-pituitary-gonadal axis function begins in utero, when testosterone induces male reproductive organ development, whereas ovarian endocrine activity begins after birth (220). However, the cardiovascular developmental effects of sex steroid hormones and the time course of expression of SSHR in the development of cardiovascular tissues have not been studied. An important hypothesis in maternal-fetal medicine, the Barker or fetal programming hypothesis arose from recognition of a statistical correlation between conditions prevailing at the time of birth and morbidity and mortality due to chronic diseases late in life, such as the strong and inverse relationship observed between coronary heart disease mortality and perinatal weight (221). However, little data examining this hypothesis at the level of cardiovascular endocrinology and physiology exist at present. One recent epidemiological report suggested that low birth weight is only associated with increased carotid intimal medial thickening in young adulthood in subjects who have experienced both severe intrauterine growth retardation and exaggerated postnatal growth (222).

Another interesting perinatal issue is the recently described presence of fetal cells in myocardial tissues of the mother (223). The potential biological effects of fetal microchimerism on adult physiology have been suggested, but the cardiovascular role of these cells in gender differences for vascular or myocardial disorders also has not been explored.

Sex steroid hormones are critical determinants of cardiovascular gender differences. The majority of research has focused on the effects of estrogens and ER on cardiovascular physiology and disease, whereas progesterone and testosterone, their receptors, and the enzyme Cyp19 or aromatase all have received far less attention (202). Although SSHR interactions with their respective hormones, transcriptional coregulatory proteins, and specific DNA response elements have been studied extensively in reproductive target organs, these interactions have not yet been explored sufficiently in cardiovascular physiology. One common mechanism regulating hormone action that is likely to be of major importance for cardiovascular physiology involves coactivator and corepressor proteins that interact directly with the SSHR. The regulation of these coregulator proteins, in turn, through posttranslational modifications allows cells to alter the genes regulated by SSHR and the timing of transcriptional events (3, 21, 43). Coregulator biology also is important to the development of novel cardiovascular selective SSHR modulators (43, 198). Examples of cardiovascular coregulator specificity include the relatively specific myocardial AR coactivator, FHL2 (224) and the vascular role of the coactivator protein steroid receptor coactivator 3 (SRC-3; also known as AIB1, pCIP, ACTR, and TRAM-1) in mediating estrogen inhibition of vascular injury (225).

Yuan et al. (225) recently studied the role of SRC-3 in estrogen-mediated vascular protection after vascular injury. These authors created mice harboring a knock-in of the LacZ reporter into the SRC-3 gene. Heterozygous mice were phenotypically normal and provided a sensitive marker to characterize SRC-3 expression within the cardiovascular system, where SRC-3 expression was noted in vascular smooth muscle cells and endothelial cells but not in myocardial cells. A carotid ligation injury model was used to examine the extent of neointima formation in mice homozygous for SRC-3 disruption (SRC-3^–/– mice). Vascular injury was more exuberant in SRC-3^–/– mice than in wild-type mice, and this difference was diminished after ovariectomy. E₂ treatment after ovariectomy in wild-type mice inhibited the neointimal response, as has been reported previously (226, 227), but the ability of E₂ to inhibit the neointimal response and cellular proliferation in SRC-3^–/– mice was attenuated. Thus, the loss of SRC-3 function interfered with the protective effects of estrogen in this vascular injury model, suggesting the potential importance of this coactivator in vascular biology. SRC-3 has been shown recently to integrate genomic responses to multiple cellular signaling pathways (228), suggesting that it may be an especially critical coactivator deserving of further study in cardiovascular cells and tissues (Fig. 2).

View larger version (75K): [in this window] [in a new window]   FIG. 2. Overview of the vascular and blood cell types that are influenced by estrogens. The effects of estrogens on the blood vessel wall depend upon the extent and complexity of atherosclerotic disease present at the time therapy is initiated. As atherosclerosis evolves, the early vascular protective mechanisms of estrogen ("early HT"; e.g., increased NO and cyclooxygenase-2 and decreased TNF-, cell adhesions molecules, LDL oxidation/binding, platelet activation, and vascular smooth muscle cell proliferation) recede and are replaced by estrogen responses in the setting of underlying advanced and unstable lesions that may be deleterious ("delayed HT"). The pleiotropic effects of HT on the vascular endothelium, vascular smooth muscle cells, and inflammatory cells thus differ, depending on the stage of atherosclerosis in the underlying blood vessel (the Timing Hypothesis, Ref. 202 ). The relative roles of steroid hormone receptors, other than ERs, and of the coregulatory molecules involved in nuclear receptor action in the vasculature remain less well understood.

Another type of nuclear hormone receptor that has not yet been adequately explored for regulation by sex steroid hormones and as a contributor to the gender differences in CVDs is the LXRs that regulate cholesterol homeostasis. Like the SSHR, LXRs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, and there is evidence that LXR expression can be affected by E₂ (232, 233, 234) (see below). The LXRs regulate genes whose protein products control cholesterol homeostasis, such as proteins in the reverse cholesterol transport pathway involved in cholesterol removal from peripheral tissues, as well as proteins involved in the breakdown and excretion of cholesterol from the body, which can directly influence the rate and progression of atherosclerosis (232). LXR- and LXR-ß, the two known LXRs, are highly expressed in liver, adipose tissue, and macrophages, and LXR-ß is expressed as well in many other tissues. Lundholm et al. (234) have recently used a gene profiling approach to examine estrogen-regulated genes in mouse adipose tissue. In these studies, E₂ decreased mRNA expression of LXR, as well as several known LXR target genes involved in cholesterol homeostasis, including sterol regulatory element-binding protein 1c, apolipoprotein E, and the ATP-binding cassette A1. That E₂ can decrease expression of LXR and several of its target genes in adipose tissue suggests that further study of LXR regulation by sex steroid hormones may be fruitful. This sort of crosstalk between SSHR and other nuclear hormone receptors important to cardiovascular physiology and disease deserves greater attention (Fig. 2). In summary, better understanding of the emerging regulatory mechanisms for SSHR is central to advancing our understanding of sex steroid hormones in cardiovascular physiology and disease (202).

B. Vascular effects of sex steroid hormones, atherosclerosis, and ischemic cardiovascular diseases
The importance of the timing of HT to the effects on vascular function has been reviewed recently (202). The largest body of scientific evidence in the field, the seminal primate studies of Clarkson and colleagues (89, 235), supports the conclusion that HT can be beneficial when initiated in the early stages of atherogenesis but that its effects are lost in the setting of more advanced atherosclerosis (202) (Fig. 2). Sex steroid hormone therapies will require better attention to timing, as well as dose and formulation of hormone, like other hormonal replacement therapies for endocrine organs that are losing or have lost their capacity to synthesize hormone (such as in thyroid disease). The effects of endogenous and exogenous hormones on cardiovascular SSHR have been discussed at length (201, 202). Here, we highlight several newer areas of hormone action in cardiovascular tissues that have not been discussed as thoroughly.

Two important systems regulated by SSHR that impact on cardiovascular physiology and disease are located principally outside of the cardiovascular tissues: those controlling lipoprotein metabolism and hemostasis-thrombosis. The role of SSHR in regulating lipoprotein synthesis and circulating lipid levels is well studied, especially for estrogens and progesterone, and has been reviewed extensively (201, 236, 237, 238). The liver expresses ER but not ERß and also expresses AR. Hormonal stimulation of hepatic receptors is largely responsible for the decreases in LDL and total cholesterol, the modest rise in HDL, and the increase in triglycerides that characterize oral HT.

Oral HT regimens and SERMs studied to date all lead to an increase in venous thromboembolic events, which may be relevant to the increased incidence of stroke seen in older women started de novo on HT in the WHI. Surprisingly, however, too little is known about the effects of sex steroid hormones on coagulation, fibrinolysis, and arterial thrombosis, and this area is ripe for increased research attention (196, 237, 239). Oral HT and OCs alter coagulation-fibrinolytic pathway protein expression, increasing levels of factor VIIc and VIIa and platelet fibrinogen binding, while decreasing circulating fibrinogen and the tissue plasminogen activator inhibitor, plasminogen activator inhibitor-1 (reviewed in Ref. 239). Simple correlations are not likely to be informative (for example, oral estrogen decreases fibrinogen levels, but increases thrombosis). Genetic variants of coagulation and fibrinolytic mediators also need greater exploration (196), because early evidence supports the hypothesis that specific variants in several of these proteins predisposes to arterial thrombosis (239). A leading candidate for increased venous thrombosis seen with ET is the common variant factor V Leiden (FVL), which may predispose to thrombosis by causing functional resistance to the fibrinolytic protein C, as a recent meta-analysis suggests (240). Oral but not transdermal estrogen has been shown to induce resistance to APC in the absence of FVL, but the risk of thrombotic events increases severalfold further in women with FVL, supporting the importance of further studies of FVL in peri- and postmenopausal women (75, 98, 106, 239, 241).

Genotypic-phenotypic studies are just beginning to move past identification of simple associations to the current preferred standards of: 1) confirmation of associations in two or more independent DNA datasets; and 2) in vitro and in vivo studies of the physiological consequences of different variants identified. There also are few studies of hormonal effects on thrombosis with in vivo models or at the menopausal transition. Studies of carotid injury in male ER knockout mice and their wild-type littermates have been technically limited by frequent early thrombosis at the site of injury and the failure of the ER knockout male mice to survive the procedure (M. E. Mendelsohn, unpublished results). Megakaryocytes contain ERß and AR mRNA, but not ER or PR (242). Platelet aggregation and secretion change with sexual maturity differently in females and males (243). Platelets of female mice are more reactive than those of males (244), but studies of platelet function in gonadectomized animals or in mice harboring disruptions of various SSHR are lacking.

C. Sex steroid hormones and the myocardium in health and disease
The effects of hormones on SSHR receptors in the heart also have been discussed at length (199, 201, 202). Several newer areas of hormone action in the heart have not been discussed as thoroughly and are now highlighted. E₂, progesterone, and testosterone all bind specifically to animal and human atrial and ventricular myocardial tissue (201, 202, 203, 206, 245). Although recent controversy has arisen as to whether murine hearts express ERß (246), both ER and ERß have been identified in animal and human myocardial cells (247, 248, 249, 250, 251, 252). Local production of estrogen occurs in murine cardiac myocytes (250). Myocardial cells and tissue of lower animals and human and nonhuman primates also express ARs, which respond to agonist stimulation with a hypertrophic response (245, 253, 254, 255). In diseased hearts, AR expression is increased, and testosterone can alter expression of cardiac-specific genes in diseased hearts, suggesting that heart-specific steroid metabolism influences cardiac hypertrophy (255).

The electrical (conduction) system of the heart also is influenced by sex steroid hormones. There are clear gender differences in cardiac electrophysiological function, the subject of several excellent recent reviews (256, 257, 258). Women normally have higher resting heart rates, due in part to intrinsic differences in the sinus node pacemaker (257). This difference is apparent at birth, when newborn boys display lower baseline heart rates than girls (259). Evidence also supports the fact that women have differences in autonomic nervous system activity and in myocardial repolarization (the electrocardiographic QT interval) that are hormonally influenced (257, 258, 259). The QT interval is consistently found to be longer in women than in men, and women are more susceptible to drug-induced ventricular tachycardia due to this QT prolongation. At puberty, QT shortening is noted in boys, suggesting that androgens may be responsible for this gender difference. Heart rate is lower during menses than during the follicular or luteal phases of the menstrual cycle, and spontaneous episodes of tachycardia appear to be correlated directly with progesterone levels and inversely with E₂ levels, although the mechanism is unknown. Several atrial and ventricular arrhythmias are more common in women, including especially supraventricular atrial tachycardias and ventricular tachycardias due to prolonged myocardial repolarization. Ion channel effects of sex steroid hormones likely contribute to gender differences in arrhythmias. Myocardial L-type calcium channel density is down-regulated via an estrogen-ER mechanism. E₂ also decreases potassium channel currents and prolongs repolarization in animal models, whereas androgen shortens repolarization in mice (260), supporting roles for both sex steroid hormones in the gender differences. More studies are needed of SSHR expression in cardiac atrial and ventricular tissues and the effects of different HT preparations on ion channel function and cardiac electrophysiology.

There are clear gender differences in myocardial hypertrophy and heart failure that have been noted mainly in animal and in vitro models (reviewed in Refs. 261 and 262). SSHR-mediated changes in the levels and regulation of myocardial calcium-contractility coupling proteins in the heart are likely involved in the effects of sex steroid hormones on myocardial hypertrophy and heart failure. Testosterone induces expression of both AR and the Na/Ca exchanger in isolated rat ventricular myocytes (263). Cardiac myocytes from male rats have an enhanced response to ß-adrenergic stimulation with a greater influx of calcium than females (264). Disruption in mice of the gene for the FK506 immunosuppressant binding protein FKBP12.6, which interacts with cardiac ryanodine receptors important to regulation of intracellular calcium, results in hypertension and cardiac hypertrophy in male but not female mice (265). Treatment of female FKBP12.6-null mice with the mixed ER agonist-antagonist tamoxifen induces cardiac hypertrophy, suggesting that estrogen has vascular and/or cardiac effects that oppose loss of the FKBP12.6 gene (265). These and other notable studies underscore the potential for sex steroids to profoundly affect myocardial function, the gender difference in that action, and the need for continuing mechanistic studies that can form the basis for therapeutic exploitation.

	VI. Central Nervous System and Sex Steroids

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
This component of the review will focus on our understanding of the role of estrogens on the central nervous system. The first portion will emphasize studies that were performed using in vivo and in vitro models. The second portion will focus on studies performed in humans, to illustrate instances in which these animal models provide insights that will help to improve the design of clinical and epidemiological studies in the future. Over the past 20 yr, we have come to understand that the presence of ovarian steroids is essential to the normal development and function of the brain, spinal cord, and peripheral nervous system (266, 267, 268). In addition, a significant, but not universal literature suggests that ovarian steroids are involved in the prevention of neurodegeneration brought about by disease or injury.

One of the great paradoxes today is that the vast majority of in vivo and in vitro studies performed using laboratory animals clearly demonstrates that E₂ is critical in the development, maintenance, and recovery of normal neural structures and function; E₂ is important in maintaining normal neuronal morphometry, synapse formation and function, and morphological and functional relationships among neurons, astrocytes, and microglia (267, 269, 270). Furthermore, these basic science studies show that E₂ is a potent neuroprotective factor that attenuates neuronal cell death and proliferation of new neurons and helps to maintain normal neural function in the face of injury or disease. The relationship between trophic actions of E₂ and its ability to protect against neurodegeneration is not clear. Moreover, the apparent dichotomy between the beneficial actions of E₂ on the brains of experimental animals and the WHI report that hormone treatment increases the risk of probable dementia in humans (271, 272) or risk of cerebrovascular stroke (273) is a great conundrum. A review of the basic science literature may offer insights into the complexities of hormone actions and provide a rationale for the design of future clinical trials. The majority of the studies in animal models have used sc E₂, and not CEE or SERMs. However, a growing body of experimental studies have begun to use these compounds (6, 274, 275, 276, 277, 278). Many of these recent studies continue to support the concept that estrogens are efficacious neuroprotective agents, although a few studies report contradictory results in terms of whether these compounds mimic the actions of E₂ (276, 277, 278, 279, 280, 281, 282, 283). For example, Rossberg et al. (282) and Ciriza et al. (275) found that raloxifene protected the brain against kainic acid or stroke injury in rats; however, Lacreuse et al. (276) found that it does not improve aspects of spatial working memory in rhesus monkeys. Also, studies performed in vitro are inconsistent in terms of demonstrating that SERMs act as estrogens in protecting against cell death (277, 278). Interestingly, a recent study conducted in older women showed that raloxifene protected against the risk of mild cognitive impairment and developing AD but that it was dose related (see Section VI.G) (283).

A. Comparisons between basic science studies and the WHI
Why should animal studies and human studies draw such opposing conclusions? It is critical to consider several differences between the design of most of the animal studies, differences in physiology of the animal models and humans, and the design of the WHI.

First, most studies performed in animals induce a stroke-like environment and then examine the extent of injury after the experimental manipulation. In contrast, the WHI assessed the risk of a stroke in the future if women are exposed to HT.

Second, the vast majority of experiments performed in animals initiate hormone treatment before or immediately after they are ovariectomized. Ovariectomy of younger animals is used to mimic the postmenopausal state, because all ovarian hormones are removed. However, this model may have inherent problems as a model of the menopause, because the effects of aging are not considered, the abruptness of the removal of steroids is unlike the normal gradual sequence of events that accompany the menopausal transition, and the changes in gonadotropin levels that accompany ovariectomy are not identical to those that accompany the menopause. Other animal models of the menopause are being developed; however, they are not without their own challenges, and they have not been thoroughly characterized (284, 285).

Finally, extrapolations from animal models to humans always must be done with the full appreciation that there are inherent species differences. In vitro models, while simplifying the cellular environment, by necessity, do not mimic the complexity of neural interactions but do provide an excellent starting point for investigating that complexity.

B. Studies performed using animal models
During fetal development and the early postnatal period, E₂ is a potent trophic factor that influences brain development and differentiation by modulating neurogenesis (286), cell death (287), cell migration (288, 289), neuronal somatic and dendritic growth (289), synapse formation and elimination (290), and glial differentiation and neuronal morphology (291). Several investigators have found that addition of E₂ in vitro to organotypic cultures or dispersed cells derived from embryonic or neonatal rodent hippocampus, cerebral cortex, or hypothalamus stimulates neurite outgrowth, arborization of their branches, and synaptogenesis (for reviews, see Refs. 292, 293, 294). More recently, we recognized that these neurotrophic actions extend into adulthood. The elegant work of Woolley and McEwen and their colleagues (reviewed in Ref. 295) over the past 15 yr clearly shows that removal of endogenous ovarian hormones results in a reduced number and density of dendritic spines and axospinous synapses in the CA1 region of the hippocampus. This decrease can be either prevented or reversed by treatment with E₂ and requires activation of N-methyl-D-aspartate (NMDA) receptors. The electrophysiological consequences of the structural changes appear to be greater sensitivity to NMDA receptor-mediated input that is directly correlated with the density of dendritic spines. A recent study by Hao et al. (296) showed that the E₂ treatment has similar morphological and functional effects related to increased dendritic spine density and NMDA receptor-mediated activity in the prefrontal cortex of nonhuman primates (see Section VI.F).

Several laboratories have shown that E₂ not only acts during development and adulthood to establish and then maintain normal brain structure and function, but exerts protective actions when the neuron is vulnerable to cell death when faced with injury or disease. Thus, E₂ exerts neuroprotective actions in adult animals and protects against a wide variety of injuries, such as cerebral contusion (297), hypoxia (298), administration of excitatory amino acids (299), and drug-induced toxicity (300). Studies using animal models of stroke have been developed and used extensively as a model of neural injury. Results from these studies provide particularly strong evidence that E₂ is a neuroprotective factor that attenuates the degree of ischemic brain injury (301, 302, 303, 304, 305, 306). The studies in animal models clearly establish that females uniformly suffer less stroke injury than males. For example, intact female rats sustain over 50% less injury than intact males and ovariectomized female rats after ischemia induced by transient middle cerebral artery occlusion (MCAO) (305, 307). Furthermore, gonadectomized animals given ET (301, 302, 303, 308, 309) have smaller infarcts than E₂-depleted controls. Estrogens appear to protect against ß-amyloid-induced cell death (300, 310, 311) and to enhance memory (312) in animal models and in vitro culture models. Together, these studies are particularly perplexing because, on first glance, they lead to opposite conclusions compared with the results of the WHI: both CEE (Premarin) and the combination of CEE with MPA (Prempro) were found to exacerbate the risk of stroke and dementia.

In vitro studies complement and confirm the results of in vivo studies and clearly establish that estrogens exert protective effects against a variety of neurotoxic insults. Studies have induced injury through conditions that mimic AD toxicity (300, 310, 313), hypoxia and oxidative stress (314, 315, 316, 317), excitotoxicity (317, 318, 319), and physical injury. Thus, studies have examined whether E₂ can salvage cells from death induced by inhibition of mitochondrial function, suppression of glucose metabolism, alteration of NO production, or administration of substances such as ß-amyloid peptide, excitatory amino acids, free radicals, and gp120. Although the modes of injury are variable, they may share similar mechanisms of toxicity and face final common pathways in the induction of cell death. It remains to be determined whether E₂ protects against cell death through parallel or divergent pathways in the different modes of neuronal injury.

C. Mechanisms of estrogen action in the central nervous system
What are the mechanisms by which E₂ exerts its effects? What determines which mechanism is used? Are similar mechanisms used when estrogen-like compounds are used? Answers to these questions are only beginning to emerge.

It appears that when low physiological concentrations of E₂ exert trophic or protective actions, ERs are critical and they appear to act either as transcription factors that induce or repress the expression of genes that favor neuronal differentiation and survival (300, 320, 321, 322) or in conjunction with membrane-associated second messenger systems (292, 318, 323, 324, 325, 326, 327, 328). In these cases, trophic effects and neuroprotective actions require hours to days of exposure, and the exposure must precede the injury by at least 12–24 h (301, 316, 329). The absence of the receptor (330) or the presence of receptor antagonists (316, 325) blocks the trophic and protective actions of the hormone. However, it is also clear that receptors are not the only element that is critical, but that they may interact with other growth factors (331) to activate nongenomic actions. Thus, using cultured hippocampal neurons, Murphy and colleagues (327, 332, 333, 334) showed that E₂ induced a coordinated multistep sequence of cellular events: activation of NMDA receptors, decreased -aminobutyric acid neurotransmission, increase in cAMP binding protein and phosphorylated cAMP-response element binding protein, and a decrease in brain-derived neurotrophic factor. All of these signals were required to mediate the influence of E₂ on synapse formation and turnover. Several other E₂-induced changes may contribute to changes in excitability and synaptogenesis (335, 336). However, their roles in this experimental paradigm have not been tested.

When higher, pharmacological concentrations are used, E₂ appears to act in a receptor-independent manner. When these concentrations are used, treatment before injury is not necessary; in fact, E₂ protects against stroke-like injury when it is administered as late as 3 h after MCAO (308). Pharmacological levels of E₂ can rapidly and reversibly decrease NMDA induced currents (319), suggesting that it may reduce excitatory cell death caused by neurodegenerative injury. Furthermore, estrogens can influence members of the NOS family to induce vasodilatory actions on cerebral blood vessels (337) and thus improve blood flow to compromised brain regions. Estrogens can also act as potent antioxidants and inhibit lipid peroxidation (304, 313, 338, 339, 340) through actions that have been shown to occur via the C3 hydroxyl group located on the phenolic A ring of the steroids (341). These studies demonstrate that this antioxidant mechanism requires supraphysiological levels of estrogens, findings that may be key in the development of therapeutic approaches aimed at achieving neuroprotection against injury induced by oxidative stress.

More recently, we have begun to appreciate the importance of inflammation in neurodegeneration (342, 343, 344) and the role of E₂ acting as an antiinflammatory factor (117, 118, 119, 125). The inflammatory response associated with stroke is complex, but significant experimental evidence shows that estrogens may directly or indirectly regulate three components of the inflammatory response: 1) microglial activation; 2) activation of the enzyme, iNOS; and 3) the activation of cytokines/chemokines (345). These components of inflammation may interact with each other and are not mutually exclusive. Microglia are the resident immune cells of the central nervous system (Fig. 3). They become activated in response to injury and initiate a pattern of events marked by proliferation, migration to the site of injury, and changes in both morphology and cell surface markers (346). Recent work establishes that microglia play a critical role in response to injury (347, 348). Both in vivo and in vitro studies have shown that E₂ suppresses microglial activation and that this response is regulated by ERs (117, 119, 125, 349). The region surrounding the ischemic core (penumbra) is vulnerable but responsive to protective factors. Experimental results suggest that microglial activation in the ischemic penumbra is a critical component of the inflammatory response associated with cerebral ischemic injury. Infiltrating monocytes and macrophages from the periphery also migrate to the penumbra. Microglial activation can be either deleterious or beneficial, depending on the microenvironment and time course of the stroke injury (343, 350, 351). Currently, we believe that E₂ may suppress deleterious microglial activation pathways, leading to decreased cell death in the ischemic penumbra.

View larger version (80K): [in this window] [in a new window]   FIG. 3. Overview of cell types and examples of neurotransmitters and neuromodulators that are influenced by estrogens. The ability of estrogens to exert trophic and protective actions depends upon their ability to alter the birth and death of neurons, synaptogenesis, and neuritogenesis. Their effects on microglia, through modulation of the number of microglia and synthesis and secretion of pro- and antiinflammatory cytokines, result in altering the inflammatory state of the brain. Finally, estrogen effects on astrocytes influence astrocyte morphometry and function as well as neuron-neuron, neuron-astrocyte, and astrocyte-astrocyte interactions. VIP, Vasoactive intestinal peptide; BDNF, brain-derived neurotropic factor; PGE2, prostaglandin E2; bFGF, basic fibroblast growth factor; IFN, interferon-; MMP-9, matrix metalloproteinase-9.

Cytokines are secreted proteins that communicate the immune status of the organism between tissues and organs. They appear to play a critical role in the pathophysiology of human cerebral ischemia: there is a positive correlation between high levels of proinflammatory cytokine in serum and greater stroke severity. Conversely, increased levels of antiinflammatory cytokines correlate with diminished stroke severity and an improved outcome (345, 360, 361, 362). Cytokines in the brain perform pleiotropic functions in inflammation and are synthesized primarily by microglia and astrocytes, but also can be produced by neurons (363) (Fig. 3). In several different brain injury paradigms, sc E₂ generally suppresses proinflammatory cytokines and enhances the production of antiinflammatory cytokines (364, 365). Both TNF (366, 367, 368) and IL-6 (360) display critical roles as part of the proinflammatory response, but also display pleiotropic roles as mediators of neuronal survival.

It is important to remember that even in animal models, estrogens do not always exert beneficial effects (297, 369, 370, 371). Several points should be emphasized. First, although a large body of literature suggests that estrogens are potent neuroprotective factors under numerous experimental and clinical circumstances in vivo and in many in vitro experimental conditions, in other experimental and disease/injury paradigms, estrogens either fail to protect or even exacerbate neural injury. Some authors have shown that E₂ fails to attenuate cell death in some animal models (297, 370, 371). It appears that when the degree of injury is too severe, as may be the case in the hippocampus after prolonged global ischemia (370) or when a disease state is already in progress (369), estrogens cannot overcome this degree of injury.

D. Evaluation of the Women’s Health Initiative Memory Study (WHIMS): design and findings
The starting point for any discussion of the clinical effects of HT on cognitive function is WHIMS because it is the only randomized clinical trial to date to examine the effects of HT on dementia risk (272, 373). WHIMS was an ancillary study to the WHI. The WHI enrolled over 161,000 women across an observational study and included three partially overlapping randomized trials: dietary modification, calcium/vitamin D, and postmenopausal HT. There were over 27,000 women enrolled in the HT arm, and of these 29% were also enrolled in the dietary modification trial, 59% were enrolled in the calcium/vitamin D trial, and 18% were in all three trials (374). WHIMS volunteers were drawn from the HT study and met additional screening criteria relevant to the study of HT and primary prevention of dementia, including absence of dementia at screening and age 65 and older. Inclusion of only older women in WHIMS was due to the practical need for a sufficient number of incident dementia cases over the planned 8.5-yr study.

As in the main WHI trial, treatment assignment in WHIMS was carried out in two separate arms. Older hysterectomized women were randomized to receive CEE or placebo, and older naturally menopausal women were randomized to receive CEE/MPA or placebo. Dementia evaluations took place in four phases. In phase I, the modified Mini-Mental State Examination (3MSE) was given to screen for probable dementia and mild cognitive impairment (MCI)—the preclinical phase of dementia—and to assess global cognitive function. Patients who scored below designated 3MSE cut points underwent additional clinical evaluation in phase II, including a neuropsychological evaluation and interviews with the patient and an informant. Phase III involved a clinical dementia evaluation that yielded a determination of no dementia, MCI, or probable dementia. Phase IV included laboratory and imaging studies to aid in diagnosis of specific dementia subtypes (e.g., AD, vascular dementia). Central adjudication by three experts was carried out to reach one consensus on diagnosis (375).

Results from the two HT arms combined indicated that the risk for all-cause dementia was significantly increased with HT compared with placebo (373). In the combined estrogen plus progestin arm, dementia risk was doubled with HT (40 of 2229 vs. 21 of 2303) (272). In the estrogen-alone arm, dementia risk was not significantly increased with active treatment (373). The risk for MCI did not increase significantly in the two arms combined or in either arm individually (272, 373). A direct statistical comparison between the CEE/MPA and CEE alone arms found no difference in the effect of active treatment on risk of dementia (373).

E. WHIMS validity: internal and external
The internal validity of WHIMS was high, with no evident biases or confounding factors. Perhaps the most important questions remaining after WHIMS pertain to external validity: Do the results generalize to all women and HT regimens that were not directly tested in WHIMS? At the forefront of this issue is the question of whether these results apply to women for whom HT remains an indication, that is, younger women with menopausal symptoms. WHIMS addressed a critically important question: Can older women initiate HT after age 65 to reduce risk of dementia. Whether this interpretation of the data can be generalized to younger women who initiate earlier in the menopausal transition for the treatment of menopausal symptoms is unknown. This question is not likely to be addressed by a randomized clinical trial because such a study would be prohibitively expensive because it would require early initiation (around age 50) and a long-term follow-up until women were at an age of increased risk for dementia (until age 65 and older). However, the generalizability of the WHIMS findings to young women could be addressed in follow-up analyses because WHIMS dementia evaluations continued even after treatment was discontinued. Therefore, it will be possible to examine whether the risk of dementia remains once treatment is stopped. If the risk is no longer present after discontinuation, then this would suggest that early initiators are not at long-term risk for developing dementia. In light of the very low incidence of AD among women in their early 50s, concerns about both immediate and long-term risk could be appreciably alleviated.

F. Differences with previous clinical and observational studies: age of initiation of treatment
Other insights into the external validity of WHI can be found in observational studies of HT and dementia, randomized clinical trials of HT and cognitive test performance, and basic science studies. The observational studies that gave rise to the hypothesis that HT might reduce the risk of AD involved women with typical patterns of HT use early in the menopause. These observational studies suggested that HT might decrease the risk for AD by 39–50% (376, 377, 378). This contrasts with the 75% increase observed in WHI (373). What factors might explain such a dramatic inconsistency in findings? One possibility is that WHIMS controlled for confounders that were ubiquitous in prior nonrandomized studies and that when those were controlled, the "true" effect became evident. Confounding differences in education levels between hormone users and nonusers have been noted as important biases, and low education (<8 yr) is a risk factor for dementia, particularly in women (379). To date, there have been three prospective studies of HT and incident dementia cases, and of these, two reported no significant differences between hormone users and nonusers in education (376, 378), and the third reported modest but significant differences (380). In each, however, HT users were younger than nonusers.

Recent data suggest that age of initiation of therapy may explain this magnitude of difference in dementia risk. For example, data from Cache County, Utah, revealed an increased risk for AD in older women who initiated HT after age 64 in contrast to a decreased risk for dementia in older women who initiated HT before age 64. This is similar to reports from observational studies (378). This pattern of findings led to the "critical period hypothesis," which states that HT confers optimal neuroprotection when initiated close in time to the menopausal transition (381). Additional evidence consistent with the critical period hypothesis was found in a recent case-control study of women participating in the MIRAGE (Multi-Institutional Research in Alzheimer Genetic Epidemiology) study, which found a reduced risk for AD only in the youngest age group (aged 50–63) (382). One interpretation of these studies is that early initiation protects against AD; those in the youngest age group necessarily initiated HT at a younger age, but those in the older age groups could have initiated HT at any age.

Other insights into the critical period hypothesis come from studies examining performance on neuropsychological tests. For example, women in the Melbourne Midlife Women’s Health Study who initiated HT before the final menstrual period showed better verbal memory compared with women who initiated HT sometime after menopause (383). These findings parallel seminal findings from small randomized clinical trials that estrogen withdrawal, either through surgery or pharmacological suppression, is associated with decreased verbal memory, and add-back estrogen can reverse those deficits (384, 385, 386). Two recent randomized clinical trials reported enhanced verbal memory in women aged 50–65 randomized to receive HT (185, 387). Other trials showed neutral or detrimental effects of estrogen alone (388) or combined HT on verbal memory in women with an average age in the early 70s to 80s at randomization (389, 390, 391). There is some evidence from recent randomized clinical trials that HT enhances other cognitive measures, even when initiated later in life. For example, the largest clinical trial of HT and cognitive function, the Women’s Health Initiative Study of Cognitive Aging (WHISCA), recently published findings from a sample of 1416 women (mean age, 74) participating in the CEE/MPA arm of WHIMS (391). Results revealed a significant detrimental effect of HT on verbal memory and a significant beneficial effect on figural memory as measured by the Benton Visual Retention Test. Overall, clinical trials provide some support for the critical period hypothesis as applied to estrogen effects on tests of verbal memory but suggest that the critical period hypothesis does not apply to all cognitive domains.

Secondary analyses from large-randomized trials in elderly women suggest that the effects of HT on global cognitive function may depend on baseline cognitive function. A beneficial effect of E₂ alone on the MMSE was observed in the Women’s Estrogen for Stroke Trial (WEST) in a subset of women who had normal MMSE scores at baseline (388). WEST, a 3-yr trial of E₂ alone involving 664 women (mean age, 70) with a history of stroke, showed no detrimental effect of treatment on any cognitive function. Among women with a normal baseline MMSE score, those randomized to receive E₂ had a significantly lower rate of decline compared with the placebo group. The results are similar to findings from WHIMS showing that baseline 3MSE score significantly modified the effect of treatment on cognitive performance over time. In WHIMS, the detrimental effect of CEE on global cognitive function was evident among the 10% of women showing cognitive impairment at baseline. In contrast, for the 90% of women in the CEE alone arm who showed no cognitive impairment at baseline, CEE had no negative effect on global cognitive function (373). A similar effect was observed in the CEE/MPA arm, where women showing cognitive impairment at baseline showed significant declines in global cognitive function with treatment, whereas those with normal cognitive function at baseline showed no significant declines with treatment (272). These studies suggest that HT may be especially detrimental for women with impaired cognitive function and counter the earlier view that HT may be effective in the treatment of existing cognitive impairment. This set of findings is also consistent with the general view that estrogen appears better able to maintain healthy tissue than to repair damaged tissue (85).

This general view has been discussed with respect to HT and cardioprotection (see Sections II.A.3 and V.B; also reviewed in Refs. 85 and 202). The CEE alone arm of the WHI showed a 50% reduction in adverse coronary events in women ages 50–59 who were assigned to active treatment (12). Longitudinal studies report an increased risk for AD among those with cardiovascular risk factors, including hypercholesterolemia and hypertension, and a decreased risk among statin users (392, 393). The parallel between potential early benefit of HT on the cardiovascular system and brain raise interesting questions about possible common mechanisms of benefit and risk. The Kronos Early Estrogen Prevention Study, a 5-yr randomized, placebo-controlled trial, is planned to examine the effects of transdermal and oral HT on cardiovascular outcomes in women in early menopause (menses absent for at least 6 and not more than 36 months; E₂ and FSH levels in postmenopausal range) (394). Cognitive outcomes will be evaluated in an ancillary study with verbal memory as the primary outcome. This will be the first head-to-head trial comparing different forms and delivery methods of estrogen on cognition in a sample of early menopausal women.

Two of the primary mechanisms by which HT is thought to exert neuroprotection appear to be age-dependent, namely, effects on the hippocampus and interactions with the cholinergic system. Early studies performed using rats showed that estrogens exert direct effects on the morphology and connectivity of the hippocampus, a brain area critical to memory functioning. The density of dendritic spines in the hippocampus fluctuates with circulating E₂ over the estrous cycle and increases with ET in female rats. The mechanisms by which E₂ induces increases in hippocampal spine density are age-dependent, with younger but not older female rats showing the increase (395). E₂ interacts with the cholinergic system to reverse memory deficits in younger rats (396), but not in older rats (397). These studies suggest two mechanisms by which ET might confer protection to the hippocampus and memory in younger, but not older animals.

Important recent studies in aged female rhesus monkeys addressed the question of the impact of E₂ on the neocortex. Two experimental design characteristics for these studies are noteworthy: first, the monkeys were comparable in age to 55- to 65-yr-old women when they were made surgically menopausal; and second, HT was low-dose E₂ administered im in a cyclical pattern over 2–3 yr. Compared with vehicle control, E₂ treatment substantially reversed the marked age-related impairment in the delayed response test of spatial working memory, a behavioral assessment that critically requires the prefrontal cortex (398). Investigation of possible neurobiological substrates showed that in E₂-treated monkeys there was a profound effect on spine morphology and an increase in spine density of pyramidal neurons in an area of the prefrontal cortex that mediates corticocortical integration responsible for cognitive processes (296). These results are highly significant in considering E₂-induced plasticity and potential for reversal of age-related decline in cognition. The characteristics of E₂ dose, treatment regimen, and time of initiation in this primate study have implications for clinical trial design.

G. Potential of SERMs as HT: efficacy on central nervous system parameters
There is accumulating evidence that some SERM compounds may protect against age-associated declines in cognition, even when initiated later in life. This evidence provides justification for continued investigation of SERMs as potential cognitive enhancers. For example, the impact of raloxifene on cognitive function was evaluated in a sample of almost 7500 postmenopausal women with osteoporosis (mean age, 66 yr; range, 31–80) enrolled in the Multiple Outcomes of Raloxifene Evaluation (MORE) (399). Women randomized to raloxifene showed a trend toward protection against significant declines in verbal memory and psychomotor speed over a 3-yr interval compared with women randomized to placebo. This effect reached significance in women age 70 and older, those who were at the highest risk for cognitive impairment. A subsequent report from MORE indicated that the risk of mild cognitive impairment was significantly lower among women randomized to receive 120 but not 60 mg/d of raloxifene compared with those randomized to receive placebo (283). This underscores the importance of further efforts to develop SERMs as treatments in the primary prevention of mild cognitive impairment and, consequently, primary prevention of dementia. The inclusion of cognitive outcomes in clinical trials of SERMs under development, including bazedoxifene, lasofoxifene, droloxifene, and idoxifene, would help to advance our understanding of potential cognitive benefits of these agents.

Because SERMs can act as cell type-specific ER antagonists as well as agonists, another priority for research on women’s cognitive health is to understand the possible detrimental cognitive effects of SERMs and other pharmacological tools such as aromatase inhibitors used in the treatment, for example, of breast cancer. Tamoxifen’s antiestrogenic actions in breast tissue underlie its efficacy in preventing recurrence of breast cancer. Effects on cognition are less well understood, and clinical studies performed to date have provided conflicting data. In a study of breast cancer survivors, standard-term and long-term tamoxifen users were more than twice as likely to report having seen a physician for memory problems after diagnosis than nonusers (400). Moreover, women in that study who had taken tamoxifen for at least 5 yr performed worse on cognitive tests than those who had taken tamoxifen for shorter periods, suggesting that tamoxifen may have detrimental longer-term effects on the brain. Another study of breast cancer survivors found that women who received both chemotherapy and tamoxifen performed worse on certain cognitive tests (i.e., visual memory, verbal learning, and visuospatial tests) compared with women who received chemotherapy alone (401). In contrast, tamoxifen use was associated with decreased risk of AD and enhanced decision-making skills in nursing home residents who had taken tamoxifen compared with those who had not (402). Thus, findings are inconsistent with respect to the effects of tamoxifen on cognition. The ongoing trial, Cognition in the Study of Tamoxifen and Raloxifene, should provide insights into this important question.

Although aromatase inhibitors have become more important in standard adjuvant therapy for early hormone receptor-positive breast cancer, little is known about their cognitive effects. Preliminary insights into the effects of aromatase inhibitors and tamoxifen on cognitive function were drawn from pilot data from a small study of breast cancer patients receiving anastrozole, tamoxifen, or combined treatment compared with community-dwelling healthy controls. Although treatment assignment remained blinded at the time of this analysis, patients performed significantly worse on tests of verbal memory and psychomotor speed compared with controls (403). In light of the recent U.S. Food and Drug Administration approval for the use of another aromatase inhibitor, letrozole, in the postsurgery treatment of postmenopausal women with hormone receptor-positive early breast cancer, cognitive studies of aromatase inhibitors take on greater importance.

H. Effect of progestins on cognition
Arguably one of the highest priorities in clinical research on HT and cognition is the need for studies examining the combined effects of estrogens plus various progestins and administered in different regimens (i.e., continuous vs. cyclic) on cognitive function. The pattern of results from WHIMS—a significant impact of CEE/MPA but not CEE alone on dementia risk—might seem to suggest that the progestin used in this study is responsible for the effect on dementia risk (272, 373). However, statistically significant differences in subject characteristics between the two studies at initial screening make cross-study comparisons difficult; compared with women in the CEE/MPA arm, those in the CEE alone arm were more likely to have used HT in the past, were more ethnically diverse, were more likely to have had a previous stroke or CHD, had lower educational attainment, and had lower 3MSE scores (373). Because of these cross-arm differences in factors that could influence dementia risk, WHIMS does not provide a clear answer with respect to how progestins may modulate the effects of estrogens on dementia.

To date, only two randomized, placebo-controlled studies have directly addressed the effects of estrogen plus progestin vs. estrogen alone on cognition test performance, and these studies are based on the same cohort of 49 early menopausal women (404, 405). The studies included a direct comparison of estrogen alone, estrogen plus progestin (estradiol valerate plus dinogest), and placebo. An advantage for estrogen alone over the other treatment conditions was observed in associative verbal memory, whereas an advantage for estrogen plus progestin was observed in numerical memory (404). This pattern is similar to the mixed pattern of effect observed in WHISCA in elderly women, i.e., improved figural memory and decreased verbal memory with CEE/MPA (391). The scarcity of studies on combined estrogens and progestin in younger women is particularly troublesome given that new prescriptions for symptomatic naturally menopausal HT users will include a progestin with estrogen to protect against uterine cancer. Thus, there is a pressing need for clinical trials aimed at comparing the effect of different progestins and different progestin regimens on cognitive outcomes in early menopausal women.

	VII. Summary/Conclusions

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 
The goal of this review was to discuss the dichotomy between the results of many clinical and basic studies and the results of the WHI. We present a summary of current knowledge of the action of E₂ on three major physiological systems: the adipose/metabolic, the cardiovascular, and the central nervous systems. We highlight that 1) different estrogens (and progestins) can have disparate actions within each physiological system; 2) routes of delivery, concentrations, and sequence of HT administration determine differential actions at various targets; and 3) the age, genetic background, health status, and previous hormone treatment environment determine the actions of HT (Fig. 4). This perspective reveals the incredible challenge that faces physicians and researchers when a single treatment may be desired by the public.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Composite overview of some of the pleiotropic actions of E2 on the adipose/metabolic, cardiovascular, and central nervous systems. Among its effects is a modulation of inflammatory processes within these three systems, thus placing E2 in a potentially integrative role. Whether E2 is stimulatory or inhibitory is a function of cell specificity, including ER type and expression level and coregulator complement, as well as E2 concentration and timing.

In that milieu, the WHI results were surprising, as was their broad interpretation. However, when the data are evaluated within the narrow context of the study design, insights emerge that encourage us to reassess fundamental assumptions and to consider how future clinical trials could be better designed. Our knowledge of the complexities of hormone action, particularly related to different types of estrogen, would predict that mechanism-based hormone therapies might lead to different, more individualized treatments during the menopausal and postmenopausal years. Thus, therapy design would recognize that estrogen treatment outcomes can be radically altered by the subject’s age and subclinical disease progression as well as her genetic or hormonal background, such as ER mutations or duration without E₂. For the future, clinical trials that balance empirical-based information and mechanism-based knowledge will allow better, more appropriate designs.

Finally, the results of the WHI and other recent prospective clinical trials emphasize the enormous need that exists to strengthen interactions between preclinical and clinical research scientists and to improve our understanding of menopause and of the biology of gender differences. In underscoring the complex actions of sex steroids and gaps in our knowledge regarding cardiovascular and neural function during the menopausal transition, postmenopause, and aging, the trial results provide a road map for future studies. We suggest that one of the most critical areas for focus is the interface of estrogens with the inflammatory response and the adipose/metabolic system and the consequences of this interface to cardiovascular and neural health in women through their lifespan. The need for basic studies that address these gaps and the role for HT is clear, as is the requirement for more clinical studies that build on this mechanism-based knowledge.

	Acknowledgments

 
We thank Dr. Dennis Waring for his critical review of the manuscript and constructive suggestions.

	Footnotes

 
This work was supported in part by National Institutes of Health (NIH)-National Institute of Child Health and Human Development Grant HD12137 (to J.L.T.); NIH-National Institute of Diabetes and Digestive and Kidney Diseases Grant DK66606 (to J.L.T.); NIH Grant K23RR016067 and the American Heart Association Grant 0465309Z (to M.C.C.); NIH-National Heart, Lung, and Blood Institute Grants HL50569 and HL63494 (to M.E.M.); NIH-National Institute on Aging Grants AG02224 and AG17164 (to P.M.W.); and the Ellison Medical Foundation (to P.M.W.).

First Published Online June 9, 2006

Abbreviations: AD, Alzheimer’s disease; APC, activated protein C; AR, androgen receptor; BMI, body mass index; CEE, conjugated equine estrogens; CRP, C-reactive protein; CVD, cardiovascular disease; DRSP, drospirenone; E₁, estrone; E₂, 17ß-estradiol; EE, 17-ethinyl estradiol; eNOS, endothelial NOS; ER, estrogen receptor; ET, estrogen therapy; FVL, factor V Leiden; HDL, high-density lipoprotein; HERS, Heart and Estrogen/Progestin Replacment Study; HT, hormone therapy; iNOS, inducible NOS; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LXR, liver X receptor; MCAO, middle cerebral artery occlusion; MCI, mild cognitive impairment; MMSE, Mini-Mental State Examination; MORE, Multiple Outcomes of Raloxifene Evaluation; MPA, medroxyprogesterone acetate; 3MSE, modified Mini-Mental State Examination; NF-B, nuclear factor-B; NMDA, N-methyl-D-aspartate; nNOS, neuronal NOS; NO, nitric oxide; NOS, NO synthase; OC, oral contraceptive; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; SAA, serum amyloid A; SERM, selective ER modulator; SRC, steroid receptor coactivator; SSHR, sex steroid hormone receptors; WHI, Women’s Health Initiative; WHIMS, WHI Memory Study; WHISCA, WHI Study of Cognitive Aging.

	References

Top Abstract I. Introduction II. Pharmacology of Estrogens... III. The Inflammatory Process IV. Adipose/Metabolic System and... V. Cardiovascular System and... VI. Central Nervous System... VII. Summary/Conclusions References

 

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