ANDROGENS AND CORONARY ARTERY DISEASE Chapter 16 Carolyn A Allan, Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia Moulinath Banerjee, Cardiovascular Research Group, Manchester University, 43 Grafton Street, Manchester M13 9NT Fredrick C.W. Wu, Department of Endocrinology, Manchester Royal Infirmary, University of Manchester, Oxford Road, Manchester M13 9WL, United Kingdo Revised 24 JAN 2011	Index Contributors Search

1 INTRODUCTION

Coronary artery disease (CAD) is one of the leading causes of mortality in men and women, being 5 ^th in the rank order of disabilities in 1990. CAD is predicted to become the leading global cause of disease burden by 2020 [1] . The age-adjusted morbidity and mortality rates from CAD are 2.5- to 4.5-fold higher in men than in women, the sex-specific gap narrowing after the menopause [2] . The lifetime risk of CAD at the age of 50 years is 1 in 2 for men and 1 in 2.5 for women [3] . This male preponderance is remarkably consistent across 52 countries with hugely divergent rates of CAD mortality and lifestyles [4] . The universality of this disparity makes it likely that there is an intrinsic sexual dimorphism in susceptibility to CAD that may involve genetic, hormonal, lifestyle or ageing factors. Sex hormones can influence a multitude of factors implicated in the pathogenesis of atherosclerosis and coronary artery disease - the traditional view being that androgens are harmful and estrogens beneficial but the oestrogen hypothesis was seriously challenged in light of the results from the Women’s Health Initiative [5, 6] . Indeed, the sex-specific disparity in CAD may involve diverse mechanisms ranging from in utero sex hormone imprinting, gender-specific behaviour, distribution of visceral body fat to vascular and myocardial structural/functional adaptation to ageing, pressure overload and disease [7] . As the therapeutic indications for male androgen therapy widen to ‘non-classical’ indications [8] including male contraception, physiological ageing, chronic debilitating conditions and hormone replacement in postmenopausal women [9] , it becomes increasingly pertinent to consider whether natural or induced (e.g. androgen deprivation therapy for prostate malignancy) changes in levels of testosterone (T) or dehydroepiandrosterone (DHEA) will impact on the risks of coronary artery disease in men and women. This question is one of the major safety issues for androgen therapy. This chapter synthesises data from Medline publications on a variety of disciplines into a global assessment of the relationship between androgens and CAD in men. The role of androgens in CV diseases in women is dealt with in the Female Reproduction section.

2 Testosterone (T) and Coronary artery Disease

2.1 Observational Clinical Studies

It is important to emphasize the limitations of observational studies on the associations between serum levels of endogenous androgens and CAD. The CAD endpoints are extremely variable (mortality, morbidity such as myocardial infarction and angina, angiography, ultrasound, arterial calcification, post-mortem findings and unspecified ‘cardiac events’). Study groups are heterogeneous in terms of age, number of subjects and selection criteria. Most CAD patients will be on medications and have modified their lifestyle. In some studies, selection of poorly-matched controls may have introduced biases. The time interval from MI to blood sampling varied from 3 months to many years and was not always standardised for diurnal variation of hormone levels. Only a minority of studies adjusted for confounding factors such as smoking, blood pressure, obesity, diabetes, and dyslipidemia. Finally, chronic illnesses including CAD can lower serum levels of T .

2.1.1 Endogenous T and CAD

2.1.1.1 Cross-Sectional Studies

Of the cross-sectional studies [10-47] investigating the relationships between circulating testosterone (T) and CAD in men, 20 of 38 showed no association while the remainder found lower levels of T in cases compared to controls (Table 1a). Many of the studies did not have sufficient statistical power due to the small number of subjects investigated. Two of the largest studies [18, 26] found a negative relationship between T and ischaemic heart disease or abdominal aortic calcification. As with all cross-sectional studies, the directionality between cause and consequence and the significance of the observed negative relationship is unclear, e specially when CAD or generalized atherosclerosis can lower serum levels of T.

Table 1a Relationships between circulating testosterone levels and coronary artery disease in men

T - total testosterone, free T – unbound testosterone measured by equilibrium dialysis or analogue assay, free T index - unbound testosterone derived from total T and SHBG, bioT - bioavailable (non-SHBG bound) testosterone *adjusted for cardiovascular risk factors

CAD coronary artery disease CHD coronary heart disease IHD ischaemic heart disease

MI myocardial infarction Angio coronary angiography OR – odds ratio (95% confidence intervals)

Negative relationship indicates lower T levels in patients with CAD compared to controls and a null relationship indicates no difference between cases and controls.

2.1.1.2 Prospective Cohort or Case-Control Studies

Nine of ten [26, 48-56] non-cross sectional studies showed no significant relationship or predictive value between T and clinical or biochemical indicators of CAD (Table 1b). The six prospective cohort studies followed 1009 Californian men aged 40-79 over 12 years [49] , 2512 men aged 45-59 in South Wales (Caerphilly) for 5 years [52] , 890 Baltimore men aged 53.8  16 yr for up to 31 years [54] , 2084 men aged 56  12 yr from Framingham, Massachusetts for 10 yrs [55] , 1568 men from Tromso, Norway aged 59.6  10 yrs for 10 years [56] and 2 82 Dutch men for 6.5 years [26] . In the first five, there was no correlation between baseline T levels and subsequent development of fatal or non-fatal CAD, stroke or heart failure after adjusting for relevant confounders. In the last (Rotterdam) study, low T was associated with progression of abdominal aortic atherosclerosis detected by radiological calcification. In the 4 case-control studies, baseline T levels in cases of CAD and matched controls from the Multiple Risk Factors Interventional Trial [48] , Honolulu Heart Programme [50] , Baltimore Longitudinal Study of Ageing [51] and the Helsinki Heart Study [53] did not predict CAD events during observation periods of 6-8, 19-20, 9.5 and 5 years, respectively.

Table 1b Prospective cohort or nested case-control studies - the number of cases (first n) and controls (second n) and duration of follow

There have been a number of recent publications studying the association between serum testosterone levels and cardiovascular mortality in middle-aged and older men followed for 7 to 16.5 years [55-60] (Table 1c). Whilst some have reported an inverse correlation between testosterone and death due to cardiovascular disease [59] , others have failed to find an association [55-58] . In the Rancho-Bernardo cohort, after adjusting for health status, adiposity and other cardiovascular risk factors, men with testosterone levels in the lowest quartile (mean 7.1nmol/L) had a 40% increased risk of cardiovascular death when compared with men in the highest quartile (mean 15.1nmol/L) [59] . The association remained significant after accounting for the possibility of reverse causality by excluding deaths that occurred within the first five years of follow-up. When all cause mortality in this cohort was analysed as a function of deciles of total testosterone, no additional survival benefit was evident for men with a testosterone level above 10nmol/L. In the EPIC-Norfolk prospective population study [60] men with testosterone levels of 9.5nmol/L had an almost 50% increased risk of cardiovascular death, after adjusting for other risk factors, when compared to men with testosterone levels of 24.2nmol/L. Again the association remained evident after excluding deaths within the first two years of follow-up.

Table 1c Relationships between circulating testosterone levels and cardiovascular mortality in men

Q=quartile; *Hazard Ratio 1.38 [95%CI: 1.12-1.69] for lowest compared to highest quartile; **Odds Ratio 0.53 for highest compared to lowest quartile

Possible explanations for the lack of a relationship between testosterone and cardiovascular mortality in other cohorts include younger age of the men studied [55, 57, 58] , a relatively small number of deaths [56-58] and higher testosterone levels in men in the lowest quartile of serum testosterone [55, 57] .

In summary, none of the studies, cross-sectional or longitudinal, have shown a positive relationship between endogenous T and CAD to suggest that high levels of this androgen may be a risk factor. On the contrary, CAD and cardiovascular death may be more common in men with low T. Although the cause and effect nature of the relationship remains to be determined, it is possible that testosterone is a biomarker for cardiovascular health.

2.1.1.3 Klinefelter Syndrome

Klinefelter syndrome, characterized by small testes, azoospermia, gynaecomastia and a variable degree of hypogonadism with a 47,XXY karyotype has a prevalence of 1 in 500 to 640 men [61] . It is estimated that more than 50% of cases are not diagnosed making Klinefelter syndrome potentially the largest single unrecognized (and thus untreated) cause of androgen deficiency in young men [62] . Other clinical manifestations also occur in many systems aside from infertility and androgen deficiency [63] with increased mortality due to a variety of causes [64, 65] resulting in a reduced life expectancy of 2.1 years in one study [64] . An increased IHD risk has been documented both before and after the diagnosis of Klinefelter syndrome [66] . However, while circulatory system mortality was increased by 30-40% [64, 65] , the excess mortality was due to deaths from pulmonary embolism and peripheral vascular disease and IHD mortality was actually reduced (HR 0.7 [0.50-0.90]) [64, 65] . The relative contributions of the chromosomal abnormality itself, direct cardiovascular effects of testosterone, indirect androgen effects including adiposity and diabetes mellitus, and lifestyle factors, such as smoking, are not known. These studies are also unable to account for the adequacy of testosterone replacement therapy.

2.2 Interventional Clinical Studies

The potential consequence of decreasing endogenous or increasing circulating levels of T on CAD is discussed under various clinical interventional scenarios.

2.2.1 Endogenous Androgen Deprivation

2.2.1.1 Castration

A frequently cited but misquoted study [67] compared the life expectancy of institutionalised mentally retarded castrated with intact white males; castrated males lived an average of 13.6 years longer than intact controls. However, the excess mortality in intact inmates was due to infections with no difference in cardiovascular disease mortality between the two groups. The authors correctly concluded that post-pubertal castration did decrease the frequency of deaths due to cardiovascular disease.

2.2.1.2 Cross Gender Hormone Therapy

Cross-gender anti-androgen treatment in 816 male-to-female orchidectomised transexuals aged 18-86 years [68] by administration of ethinylestradiol 100µg/day and cyproterone acetate 100mg/day for 7734 patient-years was not associated with any significant difference in arterial cardiovascular mortality or morbidity compared to the general male population despite a 20-fold increase in venous thromboembolic complications.

2.2.1.3 Androgen Deprivation Therapy in Prostate Cancer

Androgen deprivation therapy (ADT) is the mainstay of treatment for metastatic prostate cancer and is increasingly used in locally advanced disease. The resultant profound hypoandrogenism is associated with an adverse cardio-metabolic profile characterized by an accumulation of body fat [69] and a reduction in insulin sensitivity [70] . More recently it has been appreciated that the risk of cardiovascular disease [71-75] is also significantly increased, and this, in addition to diabetes, is the leading cause of non-cancer deaths in prostate cancer survivors.

An observational study of 73,196 men aged 66 years and older followed for 10 years found that GnRH agonist use was associated with a 16% increase in the risk of each of coronary heart disease and sudden cardiac death; the incident diabetes risk was increased by 44% [71] . The increase in risk was evident after only 4 months of ADT and remained elevated for the duration of treatment. In a further study of 5077 men stratified according to co-morbidities, established coronary artery disease, and the presence of cardiovascular risk factors, the excess risk was confined to those men with a history of myocardial infarction or heart failure. After 5 years these men had a 2-fold increase in all-cause mortality with no increase seen in men with either no co-morbidities or a single cardiovascular risk factor [76] .

2.2.2 Androgen Excess from Anabolic Steroid Abuse

It should be emphasised that pathological data from men abusing anabolic-androgenic steroids (AAS) in doses several orders of magnitude higher than those prescribed in clinical practice should not be extrapolated to the legitimate therapeutic use of approved testosterone preparations or indeed to androgen physiology.

AAS abuse, previously only prevalent in athletes and body builders [77, 78] , is said to have increased significantly in recent years especially amongst adolescents [79, 80] . Case reports of cardiovascular events in young male body builders abusing pharmacological doses of multiple anabolic agents include arrhythmia, hypertension, acute myocardial infarction, cardiomyopathy and stroke [81] . Postmortem examinations in 2 young body builders using AAS who died of acute myocardial infarction did not reveal any lesions in the coronary arteries [82] . It is not possible to draw firm scientific conclusions about the cardiac toxicity or atherogenicity of AAS from these sporadic case reports, when the baseline denominator information on prevalence and extent of exposure is shrouded in uncertainty and secrecy. Nevertheless, it has been conjectured that dose-dependent androgen– induced vasospasm, endothelial dysfunction, platelet aggregation, activation of the coagulation cascade, atherogenic lipid profiles (decreased HDL–cholesterol and increased LDL-cholesterol), and direct myocardial injury leading to abnormal left ventricular function (including diastolic dysfunction) and hypertrophy are relevant mechanisms precipitating sudden cardiac deaths in young power athletes and body builders [81, 83] .

2.2.3 Exogenous T Treatment in Ageing Men

A recent meta-analysis was unable to discern any effect of testosterone on rates of death, myocardial infarction, revascularization procedures or cardiac arrhythmias in men with low/low-normal testosterone levels treated with testosterone for at least 3 months [84] . However such risk estimates are imprecise because of the small numbers of subjects, short duration of treatment and limited number of events. None of the included studies was powered for actual cardiovascular events; additionally, several of the studies included men with significant co-morbidities.

The first report of a trial of testosterone therapy terminated early as a result of adverse cardiovascular events was the Testosterone in Older Men with Mobility Limitations (TOM) trial, designed to determine the effects of testosterone for 6 months on leg strength and physical function in older men [85] . Of the 209 men (mean age 74 years) randomized, 23 in the testosterone group compared to 5 in the placebo group experienced a cardiovascular-related adverse event. These men had a high rate of pre-existing cardiovascular disease (53%) and cardiovascular risk factors (hypertension 85%, diabetes 24%, hyperlipidaemia 63%, obesity 45%) at baseline and were treated with a high dose (100mg/day) of testosterone gel to achieve a serum testosterone of 17.4-34.7mmol/l (500-1000ng/dL). Men exposed to testosterone levels in the highest quartile were at greatest risk (hazard ratio of 2.4). The trial was not designed to assess cardiovascular risk and the authors urge caution in interpreting the results because of the small number of events and limitations with respect to ascertainment of the adverse events. Furthermore, a very similar trial in frail elderly (>65 yr) men [86] using a more conventional dose (50mg/day) of testosterone gel daily showed no increase in CV events in the T treated group during and after treatment. Nevertheless, the unexpected finding of the Basaria study should alert physicians and clinical investigators to the possibility of harm from testosterone treatment, particularly with high doses, in older (>65 yr) men with existing cardiovascular disease and/or risks factors.

2.2.4 Exogenous T Treatment in Men with CAD and Heart Failure

Uncontrolled studies from the 1940s, largely of historical interests only, suggested that T may improve symptomatic CAD in men. More recent data are considered below.

Bolus intravenous or intracoronary injections of pharmacological doses of T acutely improved myocardial ischaemia or induced coronary artery dilatation in a small number of men with CAD [87-89] . Whether this acute pharmacological action of T can be translated into a therapeutic effect remains to be determined.

In a randomised placebo-controlled double-blind study, T cypionate 200mg i.m. weekly for 8 weeks decreased ST segment depression during exercise testing in 25 men with positive tests [90] . In a placebo-controlled crossover (but not double-blinded) study in 62 elderly men with CAD, oral T undecanoate for 4 weeks improved subjective symptom scores and resting ECG [91] . Transdermal testosterone 5mg daily for 12 weeks increased the time to 1-mm ST segment depression in men with symptomatic CAD [92] and oral testosterone selectively improved myocardial perfusion to areas supplied by unobstructed coronary arteries in men with coronary heart disease and baseline testosterone levels of 9nmol/L [93] .

These preliminary data suggest ECG changes can improve after (maximum of 12 weeks) short-term T supplement in CAD patients with low T levels. A small study of 13 men with testosterone levels <12nmol/L and angina found that the benefits of intramuscular testosterone on myocardial ischaemia (as determined by ST segment depression) were maintained at 12-months [94] . It is unclear if the effects of T are based on specific cardiac actions or non-specific improvements in skeletal muscle performance during exercise testing or a central action in the brain. Whether there are real symptomatic or functional benefits or decreased cardiovascular mortality from T treatment in men with ischaemic heart disease remains uncertain.

Currently, heart failure is a labeled contra-indication for testosterone treatment. However, there are recent limited data exploring the use of testosterone in patients with cardiac failure who frequently have low circulating testosterone levels. Cardiac output was acutely increased after two doses of buccal testosterone in men with heart failure [95] , and 12 weeks of intramuscular therapy improved peak oxygen consumption in elderly men with stable chronic heart failure [96] . Over 12-months of follow-up, transdermal testosterone improved functional capacity in men with moderately severe heart failure when compared to placebo [97] . These preliminary findings require validation in rigorously controlled clinical trials and do not alter the current established indications and contra-indications for testosterone therapy.

In summary, whilst some examples of endogenous androgen deprivation (castration, Klinefelter Syndrome) have not been associated with increased cardiovascular risks, ADT for advanced prostate cancer does appear to increase cardiovascular morbidity and mortality. Although some preliminary information hints at possible short-term favourable effects of exogenous testosterone on myocardial ischaemia, prospective controlled data on cardiovascular disease endpoints (myocardial infarction, angina, mortality) from large-scale interventional studies using physiological doses of androgens are currently lacking. A single study has suggested excess cardiovascular risk in frail older men with prevalent baseline cardiovascular risk factors treated with a high dose of testosterone [85] . This serves as a warning signal to remind physicians of the possibility of (serious) harm from testosterone treatment in some men. The balance of potential risk versus possible benefit and the current lack of definitive safety data should be carefully discussed with each individual patient before starting treatment.

3. Dehydroepiandrosterone (DHEA) and Coronay artery disease (CAD)

DHEA and its sulphate , DHEAS, are weak but highly abundant adrenal androgens which show a progressive age-related decline from the third decade onwards [98, 99] There is a body of opinion suggesting that DHEA supplementation may be beneficial to the elderly in a variety of physiological functions including the prevention of cardiovascular disease [100, 101] . It has been implied that, against an androgenic milieu in men, DHEA acts as a pro-hormone for conversion to metabolites with predominantly oestrogenic effects and potentially anti -atherogenic actions [102, 103] .

3.1 Observational Clinical Studies

3.1.1. DHEA and CAD

Many observational studies in men have attempted to demonstrate a correlation between serum DHEAS levels with different CAD endpoints including the extent of atherosclerosis assessed by autopsy, coronary angiography, carotid vessel thickness/pulse wave, aortic calcification and clinical disease states including angina, myocardial infarction and mortality ( Table 2 ). These have shown either an inverse [35, 38, 40, 104-109] (mostly cross-sectional), null [26, 38, 42, 106, 109-116] or positive [29, 53] relationship between DHEAS levels and CAD ( Table 2 ).

Table 2 Relationships between circulating DHEA and DHEAS levels and coronary artery disease in men

Of the nested case control or prospective cohort studies, all but four [53, 104, 106, 108] showed no association between DHEAS levels and incident CAD (Table 2). In the Helsinki Heart Study of middle-aged dyslipidaemic men, higher DHEAS levels were associated with an increased risk of CAD [53] . In the Honolulu Heart Study of 6000 men of Japanese descent followed for 18 years [106] , low DHEA was associated with fatal but not non-fatal CAD. In the Rancho Bernardo cohort study, a preliminary report of 242 men also showed a negative relationship between DHEAS and CAD mortality [104] . However, in the full analysis of the same study on 942 men over 19 years [109] , there was only a modest negative relationship between DHEAS and those that survived their cardiac events, but none with CAD mortality. DHEAS levels appear to be associated with increased mortality from all causes of death in men over the age of 50 [106, 109, 111] , giving rise to the notion that this is a non-specific marker of poor health and lack of adaptive capacity to acute illnesses or a secondary phenomenon consequent upon various diseases of ageing such as malignancies and heart failure [98, 115, 117] . Yet among 963 men and 1161 women of age 65-76 years and followed up for 7.4 years, there was no association of serum DHEA-S with age adjusted all cause and CVD-mortalities [112] .

3.1.2 Interventional Studies with DHEAS

In a mixed population of Addison’s disease (n=40), DHEA supplementation for 12 weeks normalized DHEAS and androstenedione. Apart from minor reductions of LDL-cholesterol, there were no other metabolic or vascular changes seen at the end of the study period [118] . Administration of DHEA (25 mg od for 4 weeks) to 12 eugonadal, hypercholesterolaemic men (mean age 54 years) serum PAI-1 concentration when compared to a matched group treated with placebo [119] . The only RCT which has investigated the effects of 50 mg of DHEA administration over 1 year in both men and women (n=280, age range: 60-79 years) found that there were no significant benefits in men. Women above the age of 70 years, benefitted by increased bone density but aortic pulse wave velocity (aPWV) remained unchanged in either gender [120] .

Taken together, data from observational and interventional studies in men and women do not support the hypothesis that DHEAS ‘deficiency’ is a risk factor for CAD fatalities or that DHEA may confer an anti-atherogenic action in men or women. The interventional studies, despite demonstrating attainment of physiological androgen levels in women, showed there were no discernible effects on vascular function in either short or long term in either gender. DHEA may be a non-specific marker for ill health in general.

4. Androgens and arterial disease - Animal studies

The influence of androgens on the development and progression of experimentally-induced atherosclerosis has been investigated in animal models of diet-induced atherosclerosis and atherosclerosis-susceptible transgenic mouse models (Table 3).

Table 3 Relationship between androgens and atherosclerosis in animals fed on atherogenic cholesterol-enriched diets

Ovx ovariectomised Odx orchidectomised T testosterone E2 oestradiol DHEA dehydroepiandroterone

§ apoE-/- mice: apoE deficient knockout mice  LDLR-/-mice: LDL-receptor deficient knocknout mice ¶ Cetrorelix – GnRH antagonist

The effects of androgens on atherosclerosis indicated are lipid- independent unless otherwise stated.

* T reversed atherosclerosis-related impairment of endothelium-dependent vasodilatation response i.e. functional benefit

4.1 Animal Studies with T

4.1.1 Accelerated Atherosclerosis

Larsen et al investigated the effects of I.M. T enanthate in castrated male rabbits fed on cholesterol-rich diet. There was no difference in the cholesterol content of abdominal aorta lesion after 17 weeks [121] . Bruck et al demonstrated sex-specific effects of T and oestradiol in castrated male and female rabbits fed an atherogenic diet. After 12 weeks, aortic arch intimal thickness was reduced by I.M. oestradiol valerate 1mg/kg/week in females but not males, and T enanthate 25mg/kg/week in males but not females, and by combined oestradiol and T administration in both sexes. Interestingly, T treatment in female rabbits increased plaque sizes but oestradiol had no effect in male rabbits. These sex-specific anti-atherogenic effects of T and oestradiol were independent of changes in plasma lipoproteins [122] . Alexandersen et al showed that castration per se in male rabbits resulted in a doubling of aortic atherosclerosis compared to sham-operated controls. This was able to be reversed by oral T undecanoate 80mg daily or DHEA 500mg daily via lipid-dependent mechanisms. In addition, I.M. T enanthate 25mg twice weekly, which raised circulating T levels by 10-fold, decreased aortic atherosclerosis [123] . This suggests that androgens in pharmacological doses may exert effects on the vasculature that protects against atherosclerosis.

Treatment of male chicks with T resulted in a dose-dependent increase in aortic atherosclerosis [124] . Similarly, T treatment in female ovariectomised cynomolgus monkeys fed an atherogenic diet for 24 months increased the extent of coronary atherosclerosis by two-fold compared to untreated intact and ovariectomised controls [125] . These effects were independent of changes in lipid levels. Surprisingly, the acetylcholine-induced atherosclerosis-related coronary artery vasoconstriction was reversed by T treatment suggesting an improvement in endothelial reactivity [125] . Vascular tone was increased in the branches of middle cerebral artery in orchiectomised rats after treatment with T when compared to those without such treatment [126] . Thus, in contrast to the neutral or beneficial effects of androgens observed in male animals, experimentally induced hyperandrogenism may enhance atherosclerosis in female animals. In view of the major sexually-dimorphic action of T, data from experimentally-induced atherosclerosis in female animals should clearly not be extrapolated to males.

4.1.2 Transgenic Mouse Models

Two studies investigating the effect of castration and exogenous T on atherosclerosis of apoE-deficient mice yielded conflicting results. In the study by Elhage et al., castration at the age of 4 weeks had no effect on atherosclerosis in either male or female mice. In both sexes, application of subcutaneous T pellets for 8 weeks significantly decreased serum levels of cholesterol and inhibited the development of fatty streak lesions in the sinus aortae by about 30% [127] . In the study by von Dehn et al, chemical castration by a GnRH antagonist to suppress endogenous T led to a decrease in atherosclerosis in both the sinus aortae and the ascending aorta despite increased cholesterol in male and decreased HDL-C in female mice. Raising T levels by silastic T implants in male mice led to small but significant increases of cholesterol levels and increased atherosclerotic lesions. Increasing serum T using silastic implants in female mice induced fewer atherosclerotic lesions and no changes in lipids [128] . The discrepancy between the two studies may have resulted from the higher dosages of T in the second study. Another study performed in LDL-receptor knock-out mice also found an anti-atherogenic effect of T which was blunted by the parallel use of an aromatase inhibitor [129] . The anti-atherogenic effect was therefore ascribed to oestradiol rather than T.

4.2 Animal Studies with DHEA

In contrast to the conflicting data on the effects of exogenous T on diet-induced atherosclerosis, DHEA administration to rabbits seems to consistently decrease atherosclerosis. Thus all 4 studies (Table 3) [123, 130-132] in intact or castrated male and female rabbits treated by DHEA for between 5-30 weeks showed a significant reduction in the extent of aortic or cardiac transplant atherosclerotic lesions independently of changes in lipids. DHEA may therefore be considered favourable under these rather artificial experimental conditions. Although oestrogens were not measured in these studies, it is probable that DHEA administered in pharmacological doses to animals (rabbits) with little endogenous adrenal androgen production, would be converted to oestrogenic metabolites with potent actions on the vascular endothelium. This is supported by the findings of Hayashi et al [132] who demonstrated that the anti-atherogenic effects of DHEA in ovariectomised female rabbits can be partially (50%) blocked by the aromatase inhibitor fadrozole. Together with the fact that no specific receptors for DHEA have yet been identified, it is plausible that this steroid primarily acts as a pro-hormone for more potent metabolites. One should be circumspect in extrapolating the apparent beneficial actions of DHEA suggested by these animal studies to man since exposure to similarly high pharmacological doses has not been investigated.

5. Effects of androgens on cardiovascular risk factors

The net effect of androgens on cardiovascular risk is difficult to assess for at least six reasons. First, the risk factors for cardiovascular disease are numerous and ever increasing. T can influence several risk factors simultaneously, some of which at first sight appear beneficial, (e.g. lowering lipoprotein(a) (Lp(a)), insulin, fibrinogen, and plasminogen activator type 1 (PAI-1)), while others are considered adverse (e.g. suppressing HDL-C). Second, endogenous T appears to have opposite effects on cardiovascular risk factors to that of exogenously administered T. Third, the associations between serum concentrations of endogenous T and cardiovascular risk factors are confounded by the complex interactions between endogenous androgens, body fat distribution, and insulin sensitivity. Fourth, a causal relationship between some of the aforementioned changes in risk factors and atherosclerosis has not been proven. Of particular importance is the example where exogenous T-induced suppression of HDL-C may not necessarily be accompanied by changes in cardiovascular risk. Fifth, T can exert its metabolic effects directly or via its metabolites E2 and dihydrotestosterone. The effects of T and E2, in particular, can be either be additive (e.g. on Lp(a)) or counter-regulatory (for example on HDL-C). Sixth, polymorphisms in the genes encoding the androgen receptor, sex hormone binding globulin (SHBG) and 5reductase regulate genomic effects and bioavailability of T and dihydrotestosterone respectively. Thus, at any given serum concentration of T, the metabolic effects at individual target tissue sites are pleiotrophic and complex.

5.1 Associations of Endogenous T with Cardiovascular Risk Factors

Cross-sectional population studies ha ve found statistically significant correlations between plasma levels of T and various risk factors. A study of middle-aged men classified by total testosterone levels (10 vs. 20nM) found that systolic blood pressure, fasting glucose and total and LDL cholesterol levels were inversely related to testosterone, but after adjusting for measures of adiposity and insulin resistance, only insulin levels and triglycerides remained significantly correlated with testosterone [133] . Negative correlations between testosterone and hypertension [134] , fasting plasma glucose [135] , hyperinsulinaemia [133] and visceral adiposity [136] have been documented, but the association with HDL-cholesterol was uncertain [137, 138] . Overall the observational evidence suggests a neutral or beneficial effect of endogenous testosterone on cardiovascular risk factors in middle-aged and older men [139] .

It is increasingly appreciated that low serum T in men is associated with components of the metabolic syndrome. The latter is characterized by obesity, insulin resistance/glucose intolerance or overt type 2 diabetes mellitus, arterial hypertension, hypertriglyceridemia, low HDL-C, a pro-coagulatory and anti-fibrinolytic state. Indeed, m en with type 2 diabetes frequently have lower total testosterone levels than non-diabetic men [135, 140, 141] . The level of SHBG is usually low [142] presumably as a result of hyperinsulinemia [143]. However, both total and free testosterone levels, remain lower in diabetic men after controlling for body mass index [135, 141, 142] and are negatively correlated with insulin resistance [141] . Low LH levels suggest hypogonadotropic hypogonadism as a mechanism [140].

The relationship between androgens, body fat distribution, and insulin sensitivity, of which the latter two are also involved in the regulation of HDL and triglyceride metabolism, is complex [144, 145] . It is not clear whether androgens regulate adipose tissue and insulin sensitivity or whether, vice versa adipocytes and insulin regulate T levels; probably a bi-directional relationship exists. Morbidly obese and insulin resistant men frequently have low serum levels of T which increase upon weight loss [146, 147] . E2 levels show the opposite changes to T with obesity and weight loss. It has therefore been suggested that obesity may cause hypotestosteronemia by increased aromatisation of T to E2 in the adipose tissue but direct evidence for this is lacking. Supporting a role of insulin in the determination of T levels in men, hyperinsulinemic-euglycemic clamp studies in young healthy men documented that increasing insulin resistance was associated with a decrease in Leydig cell T secretion [148] . In support of the notion that testosterone is the dominant driving factor in the bi-directional relationship however, quantitative CT analysis of hypogonadal men (mean age 52 years) has shown that they have a greater subcutaneous fat area and a trend towards an increased visceral fat area when compared to age-matched eugonadal men [149] .

Further evidence for the dominant role of testosterone is discussed below in the section entitled: Effects of exogenous T and DHEA on cardiovascular risk factors.

5.2 Role of the Androgen Receptor

As androgen action is inversely proportional to the number of CAG repeats in exon 1 of the androgen receptor gene [150, 151] , it has been suggested that there may be an association between CAG repeat length and cardiovascular disease. Shorter CAG repeat length has been associated with lower levels of HDL-cholesterol [152, 153] and reduced flow-mediated vasodilatation [152] but also with lower body fat and plasma insulin in healthy men [154] and with reduced body fat in men with Type 2 Diabetes [155] or CAD [156] . A shorter CAG repeat length was correlated with more severe CAD in men aged 36-86 years of age [156] but not with CAD or MI in middle-aged men [153] . However the above preliminary results have not been replicated in larger population studies. AR CAG repeat numbers did not show any cross-sectional associations with serum lipid levels in over 3000 European men [157] nor predicted incident heart disease (or changes in HDL-cholesterol, LDL-cholesterol or BMI) in American men followed for 15 years [158] .

5.3 Effects of Exogenous T and DHEA on Cardiovascular Risk Factors

The effects of exogenous androgen on cardiovascular risk factors varies with the dose, route, duration and type of treatment, as well as the age, gender and conditions of the recipients (for review see [159] ). The most consistent findings were decreases in plasma levels of HDL cholesterol, lipoprotein(a) (Lp(a)) and fibrinogen, which are accompanied by much less prominent declines of LDL cholesterol and triglycerides .

5.3.1 Visceral Fat Mass

Placebo controlled trials of testosterone replacement therapy in ageing men have shown decreases in total fat mass ranging from 1- 4.5 kg in studies of 3 – 36 months duration [160] . Data on the effects of exogenous testosterone specifically on visceral fat are limited. Intra-abdominal visceral fat loss was seen (on MRI) in non-obese ageing men treated for 12 months with transdermal testosterone [161] and 6 months with intramuscular testosterone [162] . Similar results have been reported in obese men [163, 164] .

These data indicate that in men, the dominant action in the bi-directional relationship is that T reduces fat mass especially in the abdomen and improves insulin action. In agreement, androgens activate the expression of adrenergic receptors, adenylate cyclase, protein kinase A and hormone sensitive lipase in adipocytes [165] . As a result, T stimulates lipolysis and thereby reduces fat storage in adipocytes.

5.3.2 Lipids and Lipoproteins

5.3.2.1 HDL-Cholesterol

In the majority of studies, substitution of T in hypogonadal men and men with low/low-normal testosterone levels decreased HDL-C levels [84, 159] . In normal young men, supraphysiological doses of T or androgen-like anabolic steroids decreased HDL-C by about 20% or more. Conversely, surgical castration or medical castration by GnRH analogues increased HDL-C [159] . In hypogonadal patients or in elderly men, substitution of testosterone led to minor or no decrease in HDL-C. Meta-analyses have suggested that suppression of HDL-C is directly correlated with the dose of T but inversely related to the age and duration of treatment [166-168] . Transdermal application of testosterone or dihydrotestosterone exerted less effect on HDL-C than oral and intramuscular administration.

Since low HDL-C is an important CAD risk factor and HDL exerts several potentially anti-atherogenic actions, lowering of HDL-C by T treatment may potentially increase cardiovascular risks. However, the epidemiological association of low HDL-C with CAD has not been proven to represent a causal relationship. Instead, low HDL-C often coincides with other components of the metabolic syndrome, and may therefore merely be a surrogate marker for other co-existing pro-atherogenic condition(s). Moreover, in transgenic animal models, only increases of HDL-C induced by apoA-I overproduction but not by inhibition of HDL catabolism were consistently found to prevent atherosclerosis [169] . Therefore, the mechanism of HDL modification or turnover rather than levels of HDL-C per se, appear to determine the (anti)-atherogenicity of HDL modification (Figure 1). Two genes involved in the catabolism of HDL are up-regulated by T, namely scavenger receptor B1 (SR-B1) and hepatic lipase (HL). SR-B1 mediates the selective uptake of HDL lipids into hepatocytes and steroidogenic cells including Sertoli and Leydig cells of the testes as well as cholesterol efflux from peripheral cells including macrophages. T up-regulates SR-B1 in the human hepatocyte cell line HepG2 and in macrophages and thereby stimulates hepatic selective cholesterol uptake and peripheral cholesterol efflux, respectively [170] . HL hydrolyses phospholipids on the surface of HDL thereby facilitating the selective uptake of HDL lipids by SR-B1. The activity of HL in post-heparin plasma is increased after administration of exogenous T [171] and slightly decreased by suppression of T after GnRH antagonist treatment [172] . Increasing both SR-B1 and HL activities are consistent with the HDL lowering effect of T. Interestingly, in transgenic mice, overexpression of SR-BI or HL caused a dramatic fall in HDL-C but inhibited rather than enhanced atherosclerosis [169] . This again demonstrates the fallacy of extrapolating the HDL-C lowering effect of T to increased cardiovascular risk.

Figure 1 Pathways of HDL metabolism and regulation by testosterone and oestradiol
Mature HDL ₃ and HDL ₂ are generated from lipid-free apoA-I or lipid-poor pre β-HDL as the precursors. These precursors are produced as nascent HDL by the liver or intestine or are released from lipolyzed VLDL and chlyomicrons, or by interconversion of HDL ₃ and HDL ₂ . ABCA1-mediated lipid efflux from cells is important for initial lipidation; LCAT-mediated esterification of cholesterol generates spherical particles which continue to grow upon ongoing cholesterol esterification, and PLTP-mediated particle fusion and surface remnant transfer. These mature HDL particles also continue to accept cellular cholesterol by processes which are facilitated by the scavenger receptor BI (SR-BI) and LCAT. Larger HDL ₂ are converted into smaller HDL ₃ upon CETP-mediated export of cholesteryl esters from HDL onto apoB-containing lipoproteins, SR-B1-mediated selective uptake of cholesteryl esters into liver and steroidogenic organs, and HL- and EL-mediated hydrolysis of phospholipids. HDL lipids are catabolized either separately from HDL proteins, i.e. by selective uptake or via CETP-transfer, or together with HDL proteins, ie. via uptake through as yet unknown HDL receptors or apoE receptors. Both the conversion of HDL ₂ into HDL ₃ and the PLTP-mediated conversion of HDL ₃ into HDL ₂ liberate lipid-free or poorly lipidated apoA-I, which is either re-used for the formation of mature HDL or is filtrated into the kidney. Grey arrows represent lipid transfer processes, black arrows represent protein transfer processes. The hepatic expression and activity of both HL and SR-B1 was shown to be up-regulated by testosterone and down-regulated by oestradiol. In addition oestradiol up-regulates the hepatic expression and secretion of apoA-I. These actions of testosterone and oestradiol are in good agreement with their lowering and increasing effect on HDL cholesterol, respectively. In addition both testosterone and oestradiol stimulate SR-BI expression in macrophages and thereby cholesterol efflux from these cells onto lipidated HDL.

Any effects of exogenous DHEA on cardiovascular risk factors appear to be marginal. In men aged 60 – 84 yr, DHEA 100mg daily for 3 months decreased total and HDL-cholesterol [173] but this was not confirmed in a larger study [174] .

5.3.2.2 Lipoprotein(a)

Results of many case-control studies and most prospective population studies demonstrate that lipoprotein(a) (Lp(a)) levels higher than 30 mg/dl are an independent risk factor for coronary, cerebrovascular, and peripheral atherosclerotic vessel diseases , especially if it coincides with other cardiovascular risk factors [175] . Although Lp(a) levels are predominantly determined genetically, administration of T to men consistently decreased serum levels of Lp(a) significantly by 25% to 59% [159, 176] . Conversely Lp(a) levels were increased by 40% to 60% in men in whom endogenous T was suppressed by treatment with the GnRH analogues [159, 177, 178] . The Lp(a) lowering effect of T is independent of E2, which also reduces Lp(a) levels. It is not known how T regulates Lp(a). It is also not known whether the decrease in circulating Lp(a) induced by T will reduce cardiovascular risk but it illustrates the complex multi-faceted actions of T on lipid metabolism, the interpretation of which is prone to over-simplification.

5.3.2.3 Lipid Constituents

Unregulated uptake of oxidatively modified lipoproteins via type A scavenger receptors leads to the intracellular accumulation of cholesteryl esters in macrophages and to foam cell formation [169] . T increases the oxidation of LDL by placental macrophages in vitro [170] . DHT dose-dependently stimulates the uptake of acetylated LDL by scavenger receptor type A and, hence, the intracellular cholesteryl ester accumulation in macrophages. In addition to the higher expression of the androgen receptor in male donors, this effect was only seen in macrophages of male but not female donors. The stimulatory effect of DHT was blocked by the androgen receptor antagonist hydroxyflutamide [179] .

Non-hepatic and non-steroidogenic cells cannot metabolize cholesterol and, therefore, can only dispose of excess cholesterol by secretion. Cholesterol efflux from cells is hence central to the regulation of the cellular cholesterol homeostasis. Non-specific and passive (i.e. aqueous diffusion) as well as specific and active processes (i.e. receptor-mediated) are involved. To date, two plasma membrane proteins are known to facilitate cholesterol efflux. Interaction of the scavenger receptor B1 with mature lipid-containing HDL is thought to facilitate cholesterol efflux by re-organizing the distribution of cholesterol within bilayer plasma membrane (Figure 1) . The ATP binding cassette transporter A1 mediates phospholipid and cholesterol efflux to extracellular lipid-free apolipoproteins by translocating these lipids from intracellular compartments to the plasma membrane and/or by forming a pore within the plasma membrane, through which the lipids are secreted [93] . We have found that T up-regulates the expression of the scavenger receptor B1 in human monocyte-derived macrophages thereby stimulating HDL-induced cholesterol efflux. No effect of T was seen on the expression of the ATP binding cassette transporter A1 [180-182] .

In a cross-sectional study of 65 men (mean age: 60 8 years), serum testosterone had strong inverse correlation with anti-oxidised LDL-cholesterol antibody (r=-0.346, p=0.0047). The relationship persisted after adjusting for other common CVD risk factors. This would be suggestive of either the direct role of testosterone on oxidised LDL-cholesterol metabolism or immune response to oxidised LDL-cholesterol [183] . Testosterone replacement in hypogonadal men with CAD resulted in a lower HDL-cholesterol and Apolipoprotein A1 when compared to placebo [184] .

5.3.2.4 Macrophage Functions

Circulating monocytes migrate into the vascular wall and differentiate into macrophages. They bind to modified lipoproteins which have permeated the vascular endothelium. The uptake of modified (e.g. by oxidation) lipoproteins by macrophages leads to the formation of large foam cells. These, together with T-lymphocytes, release inflammatory mediators which stimulate the proliferation and migration of smooth muscle cells. Human monocyte-derived macrophages express the androgen receptor in a gender-specific manner. Macrophages from men exhibit a fourfold higher expression of the androgen receptor than macrophages from women [185] . There is also evidence that T regulates macrophage function by non-genomic effects via a G-protein-coupled, agonist-sequestrable plasma membrane receptor which initiates calcium- and 1,4,5-triose-phosphate-signaling [186]

5.3.3 Insulin Resistance

Young, lean men did not demonstrate any change in insulin sensitivity across a wide range of serum testosterone levels in a dose-response study despite a dose-related reduction in fat mass [187] . Centrally obese middle-aged men receiving testosterone showed an improvement in insulin sensitivity (by hyperinsulinaemic / euglycaemic clamp studies) and a lowering of serum insulin levels [163] . In ageing men hCG administered for 3 months did not affect insulin sensitivity (as measured by euglycaemic clamp) [188] . It is unclear whether changes in serum testosterone regulate insulin sensitivity independent of their effect on fat mass (specifically visceral fat). Comparison of data sets is difficult as those middle-aged men showing improved insulin sensitivity had higher fat mass and greater waist circumference at baseline [163] than the ageing men treated with hCG [188] . Whilst anabolic steroids (oxandrolone) demonstrate a significant reduction in abdominal fat [164] they have been associated with insulin resistance, possibly mediated through a direct hepatotoxic mechanism.

5.3.4 The Haemostatic System

In agreement with an important role of thrombus formation in the pathogenesis of acute coronary events and stroke, prospective studies have identified various haemostatic variables as cardiovascular risk factors [189] among them fibrinogen and the fibrinolysis inhibitor PAI-1 or tissue plasminogen activator antigen. Administration of supraphysiological dosages of T to 32 healthy men in a trial of male contraception, led to a sustained decrease of fibrinogen by 15 to 20% over 52 weeks of treatment [190] . In this study the doubling of serum T levels initially also led to significant decreases of PAI-1, protein S, and protein C as well as to increases of antithrombin and beta-thromboglobulin. Likewise PAI-1 was decreased in men who received the anabolic androgen stanozolol. However, 12-months of a more ‘physiological’ transdermal testosterone did not alter PAI-1 or fibrinogen levels in men with chronic stable angina [191] .

Suppression of T in patients with benign prostate hypertrophy by GnRH analogues exerted no significant effects on plasma fibrinogen levels [192] . In agreement with the lowering effects of T on PAI-1, T inhibited the secretion of PAI-1 from bovine aortic endothelial cells in vitro. Taken together the current data indicate that T may lower fibrinogen and PAI-1, although the magnitude of effect is likely to vary according to the type and route of the administered androgen. However, these anti-coagulatory and pro-fibrinolytic effects may be opposed by pro-aggregatory effects on platelets since high dosages of androgens were found to decrease cyclooxygenase activity and thereby increase platelet aggregability [193] .

Testosterone increased platelet TX-A ₂ receptor density in rats [194] . Surgical or medical castration in prostate cancer patients showed reduced platelet TX-A ₂ receptor density but not affinity. This is associated with reduced platelet aggregation but not sensitivity to thromboxane mimetic [195] . Serum from hypogonadal men have been found to have pro-coagulant properties due to low tissue factor pathway inhibitor thereby promoting tissue factor induced coagulation [196] .

6. Effects of androgens on VASCULAR function

Blood vessels are specialised tissues lined by a single layered endothelium, have a middle muscular layer comprising of vascular smooth muscle cells (VSMC) and a fibrous coat - adventitia. The cellular components are held together by connective tissue framework and extracellular matrix. T can induce both vasodilation and vasoconstriction via endothelium-dependent or endothelium-independent mechanisms, and by genomic or non-genomic modes of actions. The diversity of these actions and mechanisms appears to be related to differences in species, gender, concomitant disease and, most importantly, dosage of T used.

6.1 Effect of Androgens on the Endothelium

Brachial artery flow-mediated dilation (bFMD) is commonly used to evaluate endothelial function [197] . Among normal healthy men, circulating total and free T but not DHEA-S are positively associated with bFMD, independently of age, adiposity, systolic blood pressure, total and HDL-cholesterol, fasting glucose, smoking status [198] . bFMD was reduced in hypogonadal compared to eugonadal patients with end stage renal disease; bFMD was inversely related to serum T levels [199] .

Testosterone replacement in hypogonadal men reduces plasma asymmetric dimethyl arginine concentration [ADMA], a known inhibitor of endothelial NO synthase [200] and increases circulating endothelial progenitor cells [201] ; both these changes are associated with improved endothelial function. Short term infusion of T at both low and high doses increased bFMD eugonadal men with CAD [202] . In contrast, bFMD worsened after T replacement in hypogonadal men after 3 months and 6 months of therapy [203, 204] . This may be related to non-physiological T pharmacokinetics produced by the T treatment which cannot replicate the physiological diurnal variation of T in eugonadal men [205] .

6.2 Effect of Androgens on Vascular Smooth Muscle Cells

T can also modulate vasoreactivity directly by endothelium- independent actions on VSMC. Rats treated with testosterone had increased Thromboxane-A ₂ (TX-A ₂ ) receptor density in aortic smooth muscle cells with consequent increase in response to TX-A ₂ mimetics [206-208] . This modulation of TX-A ₂ receptor density in VSMC is mediated by DHT (non-aromatisable to oestradiol) rather than T [209] and is more prominent in aortic VSMCs from males than female rats [210] .

The mechanism of vasorelaxant effect of T is mediated by ATP-sensitive potassium channels [211] , potassium induced EDHF [212] and stimulation of calcium activated potassium channels [213] . Other possible mechanisms for the vasodilatory properties of T is by inhibiting extra-cellular calcium entry into VSMCs either via L-type Calcium channels [214-218] or mediated by PGF _2α [219-221] . These effects of T on VSMC are believed to be mediated by membrane-associated mechanisms independent of the AR [222] .

T has also been shown to directly influence vasoconstriction via regulating Neuropeptide Y1 receptor density [223] , while oestradiol has a vasodilatory property by increasing PGI ₂ production [224] and decreasing cytosolic calcium [225] . These could be potential mechanisms responsible for gender differences in vascular function.

Abuse of anabolic steroids among bodybuilders spares endothelial function but affects VSMC function. This has been demonstrated by the change in aortic augmentation index (AIx) by salbutamol and GTN administration respectively [226] .

Low plasma free T inversely correlated cross-sectionally with carotid artery intima media thickness (cIMT) [199, 227-232] as well as progression of cIMT longitudinally [233] . T also correlated inversely with thoracic aortic IMT (taIMT) measured by trans-oesophageal echocardiogram, even after adjusting for other cardiovascular risk factors [234] . In men, therefore, a low endogenous T may be associated with VSMC proliferation and increased large to medium vessel IMT.

6.3 Effect of Androgens on the Vascular Framework

VSMCs are known to produce proteoglycans (PG) and glycoseaminoglycans (GAG), which are important constituents of the extra-cellular matrix. Sex steroids have no effects ex-vivo on GAG [235] or PG [236] synthesis by aortic VSMCs. In aortic VSMCs, T leads to preservation of collagen deposition, reduction of elastin/collagen ratio, reduction of fibrillin-1 deposition and increased Matrix metalloproteinase-3 expression [237] . These changes are potential mechanisms to explain the increased aortic stiffness observed in males compared to females. This gender difference in aortic stiffness might be contributory towards the difference in the cardiovascular risk attributed to the male gender.

7. Oestrogen and CAD in Men

7.1 Endogenous Oestrogen

Persuasive evidence indicates that an increasing number of physiological actions of testosterone in men are mediated by the oestrogen receptors (ERs) following conversion to oestradiol by site-specific aromatases in target tissues [238] . The existence of two nuclear ER subtypes  and  as well as a membrane ER encoded by the same transcript as the  nuclear receptor attest to the potential for many different biological estrogen effects. ER, ER and aromatase are detectable in coronary arteries of monkey and man [239-241] . The extra-glandular production of estrogens (with circulating androgens as the immediate precursor substrate) may therefore play a role in male cardiovascular physiology and pathophysiology.

The importance of locally-produced oestrogens from aromatisation of testosterone in males for cardiovascular health is highlighted by human and transgenic mouse models of aromatase deficiency and oestrogen resistance. In two men with undetectable circulating oestradiol and oestrone and high testosterone due to P450 aromatase deficiency [242, 243] , dyslipidaemia with elevated total and LDL-cholesterol and triglyceride and decreased HDL-cholesterol was associated with insulin resistance (in the first patient only). These metabolic abnormalities were correctable by low dose oral or transdermal oestrogen replacement. Insulin resistance, acanthosis nigricans, low HDL-cholesterol, and impaired glucose tolerance were apparent in a 28 year old male with a null mutation in ER  gene causing oestrogen resistance [244] , with calcium deposition in a coronary artery indicating the presence of premature atherosclerosis [245] . Flow-mediated brachial artery endothelial dependent NO-activated vasodilatation (membrane ER-mediated) in response to hyperaemia was absent consistent with marked endothelial dysfunction [246] .

These clinical findings suggest that endogenous oestrogens play an important role in maintaining normal carbohydrate and lipid metabolism and endothelial function in men. They are compatible with data from transgenic knockout models confirming that ER is important in preventing adipocyte hypertrophy, obesity, insulin resistance and hypercholesterolaemia [247-249] and maintaining basal NO release from vascular endothelium [250] in male animals. ER in vascular smooth muscle may also regulate vascular reactivity to oestradiol [251] . The favourable effects of oestrogens on HDL-cholesterol are also in accord with findings in clinical of aromatase inhibitors in normal men (See section on Effects of exogenous testosterone on cardiovascular risk factors – HDL). In addition, many in vitro studies have demonstrated the direct actions of oestradiol in vasodilatation, inhibition of vascular smooth muscle cell proliferation/migration, inhibition of cytokine activation and expression of cell adhesion molecules in the vascular inflammatory response, and inhibition of platelet aggregation/adhesion (see Section 6 and for review see [252-255] ).

Despite this, reports of the associations between endogenous oestrogen levels and cardiovascular disease in men are conflicting. Epidemiological studies have linked higher endogenous serum oestradiol levels with coronary heart disease (in men in the Framingham cohort) [30, 256] , and increased carotid artery intima-media thickness [257] and peripheral arterial disease (with men in the Mr OS cohort) [258] , and yet low oestradiol levels predicted all cause mortality (independent of testosterone) in the Mr OS cohort [259] and increased cardiovascular disease in the subgroup of older men in the Framingham cohort [55] . There was no association with cardiovascular morbidity or mortality in the MrFIT [48] , Rancho-Bernardo [49] , Honolulu Heart Program [50] , BLSA [51] , Caerphilly [52] , MMAS [40] or Tromso [56] cohorts. Interestingly, in a study of men with heart failure, both the highest and lowest quartiles of oestradiol predicted increased mortality [260] . The relationships between oestradiol and the cardiovascular system in men are complex, and despite accounting for potential confounders such as lipids, glucose and insulin, the observational nature of these studies has inherent limitations in determining the cause and effect nature of the relationship. Specifically the effects of oestrogen on SHBG, and the contribution of serum testosterone and body fat, particularly visceral fat, are not well understood [261] . Furthermore, inaccuracies of measurement by immunoassay of low levels of oestradiol in men [262] in the vast majority of the above studies probably contributed to some of the conflicting findings.

7.2 Exogenous Oestrogen

The therapeutic role of e xogenous oestrogens has been studied in cardiovascular disease and prostate cancer. Huggins and Hodges first described the use of synthetic oestrogens to achieve androgen deprivation in 1941 [263, 264] but subsequently the use of oral diethylstilboestrol was found to result in an increased number of cardiovascular deaths, primarily due to myocardial infraction [265] . Although it was hypothesized that the toxicity of the oral oestrogens was due to their hepatic metabolism, and subsequent induction of a hypercoagulable state, the use of the parenterally administered compound, polyestradiol phosphate (PEP), also led to an increase in cardiovascular deaths in men with non-metastatic disease [266] and non-fatal cardiac events in men with metastatic disease [267] . High doses of oral conjugated equine oestrogens (2.5mg) resulted in excess deaths when administered to men post myocardial infarction in the “Coronary Drug Project” in the 1960’s [268] .

8. Conclusions and therapeutic implications

Current evidence indicates that the sex difference in CAD cannot be explained on the basis of ambient T exposure. Androgens can exert both beneficial and deleterious actions on myriad factors implicated in the pathogenic mechanisms of atherosclerosis and CAD. At present, it is not possible to determine the net effect of T on CAD. What are the clinical implications of this ongoing uncertainty? It is important to differentiate between 1) the concern for the possibility of cardiovascular side effects in androgen treatment of endocrine and non-endocrine conditions and 2) whether T may be used for the prevention or even treatment of CAD.

Efforts to exploit the therapeutic benefits of T for men in the treatment of osteoporosis, sarcop aenia, chronic debilitating disease and age-related hypoandrogenism in the ageing male population should take heed of recent concerns regarding the possibility of increased cardiovascular event risks.

Some clinicians argue that androgen replacement in the elderly male may have the potential to prevent CAD. It is hazardous to extrapolate from cross-sectional observational data examining cardiovascular risk factors, or in vitro data studying isolated mechanisms with pharmacological doses of androgens, to assume that manipulation of the sex steroid milieu will result in clinical benefits in a complex multifactorial condition such as CAD. The lessons from estrogen HRT in postmenopausal women are especially salutary. Despite the overwhelmingly positive but indirect evidence on risk factors and disease incidence, randomised controlled interventional studies recently have not confirmed estrogens to be effective in primary or secondary prevention of CAD in women [269-271] . Analogously, if HRT for ageing men were ever to become an acceptable therapeutic entity, the requirement for randomised controlled clinical interventional trials to assess the effects of androgens on CAD disease (and other) endpoints is obvious. In the absence of such information on T and DHEA currently, priority must be given to established modes of intervention, which have been proven to be effective in prevention or treatment of CAD (e.g. weight reduction, smoking cessation, exercise, aspirin, statins/fibrates, anti-hypertensives, and vasodilators). For men with established hypoandrogenism, however, there are no substantive data to suggest that physiological testosterone therapy is associated with increased cardiovascular risk. Finally, it is now recognized that ADT in the treatment of metastatic prostate cancer increases cardiovascular risk. These men should have their cardiovascular risk factors identified and treated accordingly.