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New Perspectives on Mars and Venus: Unravelling the Role of Androgens in Gender Differences in Cardiovascular Biology and Disease
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Original Article
New Perspectives on Mars and Venus: Unravelling the Role of Androgens in Gender Differences in Cardiovascular Biology and Disease Martin K.C. Ng a,b,∗ a
Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia b Heart Research Institute, Camperdown, NSW 2050, Australia
There are substantial gender differences in the pattern, severity and clinical outcomes of coronary heart disease independent of environmental risk factor exposure. As a consequence, there has been considerable interest in the potential role of sex hormones in atherogenesis, particularly the potential protective effects of oestrogen. However, the failure of the recent clinical randomised trials to show a cardioprotective effect for oestrogen coupled with a growing interest in androgen replacement therapy in elderly men has refocused interest on the role of androgens in cardiovascular biology and disease. Over the last decade, compelling evidence has emerged that sex differences in vascular biology are not only determined by gender-related differences in sex steroid levels but also by gender-specific tissue and cellular characteristics which mediate sex-specific responses to a variety of stimuli. In the vasculature, androgens often act in a gender-specific manner, with differential effects in male and female cells. This gender-dependent regulation may have important implications for understanding the basis of the gender gap in atherosclerosis and may eventually lead to the development of sex-specific treatments for cardiovascular disease. This review will summarise the current data for the role of androgens in gender differences in coronary heart disease and cardiovascular biology. (Heart, Lung and Circulation 2007;16:185–192) © 2007 Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia and New Zealand. Published by Elsevier Inc. All rights reserved. Keywords. Coronary heart disease; Vascular biology; Gender; Hormones and androgens
Introduction
T
here are substantial gender differences in patterns of health and illness throughout the world. Most strikingly, men in almost all societies exhibit a shorter life expectancy than women of the same age and socioeconomic background.1 In fact, men have a shorter life expectancy than women in 186 of 191 (97%) United Nation member states, with men living an average of 5.6 years shorter in those countries above a minimal level of economic development. Despite this pervasive difference, the gender gap in life expectancy and mortality remains incompletely understood and remains one of the most interesting and important questions in biology and medicine. This gender gap in life expectancy is driven largely by the steep male predisposition to the clinical manifestations of atherosclerosis, particularly coronary heart disease. With economic development and the associated Available online 19 April 2007 ∗
Correspondence address: Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia. Tel.: +61 2 95156111; fax: +61 2 95506262. E-mail addresses: [email protected], [email protected]
decline in communicable diseases globally, cardiovascular disease is now the leading cause of mortality worldwide and is expected to remain so well into the twenty-first century.2,3 Hence, sex differences in the onset, extent and severity of coronary heart disease are a major contributor to the gender gap in longevity. It is well known that coronary deaths in men consistently exceed those in agematched women by 2.5–4.5-fold up until the age of 75.4 This sex difference is remarkably consistent across countries with very different racial compositions, rates of heart disease and lifestyles.4 There is a higher prevalence of conventional cardiovascular risk factors in men but this does not entirely explain the gender gap. In fact, a significant gender difference in coronary disease remains even when the cardiovascular risks associated with systolic hypertension, smoking, hypercholesterolaemia, hyperglycaemia and obesity are controlled by multivariate logistic analysis.5 A less well appreciated aspect of sex differences in coronary heart disease is the observation that once women develop coronary disease, they lose their survival advantage. Indeed, this gender relationship appears to be reversed with respect to clinical outcomes following acute coronary syndromes (ACS): compared to men, women consistently have worse short- and long-term
© 2007 Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia and New Zealand. Published by Elsevier Inc. All rights reserved.
1443-9506/04/$30.00 doi:10.1016/j.hlc.2007.02.108
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outcomes following ACS, independent of co-morbidities and differences in management.6–8 For example, using a nationwide database containing hospitalisation data for acute myocardial infarction (AMI) for over 380,000 patients, Vaccarino et al. found that women under 75 years of age had a higher mortality compared to men, with a greater than two-fold increase in mortality for women less than 50 years of age.7 After adjustment for risk factors, co-morbidities and differences in management, female gender was still associated with a higher risk of mortality, consistent with a gender-dependent phenomenon. Similarly, in a study involving over 49,000 patients, women had lower three-year survival rates following AMI up to age 70 years: women had a 69% higher death rate in the age group 30–49 years and a 21% higher death rate in the age group 50–69 years.6 Such differences in clinical outcome post ACS raise the possibility that an intrinsic gender difference may exist not only with respect to atherogenesis but also with respect to cardiovascular adaptation/repair in response to critical ischaemia/infarction. Such observations have produced considerable interest in the potential role of sex hormones in cardiovascular biology and disease. Much of this attention has to date centred on the apparent atheroprotective effect of the female sex steroid, oestrogen. However, the failure of the recent clinical randomised trials to show a cardioprotective effect for oestrogen9,10 coupled with a growing interest in androgen replacement therapy in elderly men has refocused interest on the role of androgens in cardiovascular biology and disease. Over the last decade compelling evidence has emerged that that sex differences in vascular biology are not only determined by gender-related differences in sex steroid levels but also by gender specific tissue and cellular characteristics which mediate sex-specific responses to a variety of stimuli. Hence, androgens may act in a gender-specific manner, with differential effects in male and female cells. This gender-related regulation may have important implications for understanding the basis of the gender gap in atherosclerosis and may eventually lead to the development of sex-specific treatments for cardiovascular disease. This review will summarise the current data for the role of androgens in gender differences in coronary heart disease and cardiovascular biology. In particular, new data regarding gender specific mechanisms of androgen action in the vasculature and their implications of understanding gender differences in cardiovascular biology and disease will be discussed.
The Author’s Links to Professor John Uther and Westmead Hospital The author undertook his advanced training in cardiology at Westmead Hospital in 1998 and 1999 when Professor John Uther was Chairman of the Division of Medicine at Westmead Hospital. Whilst at Westmead he developed an interest in atherogenesis and began using molecular techniques to detect evidence of bacterial presence in human coronary artery plaques. Extending these interests, he subsequently completed a PhD at the Heart
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Research Institute, Sydney on the role of androgens in the pathogenesis of atherosclerosis under the supervision of Professor David Celermajer. On completion of his PhD, the author undertook a NHMRC CJ Martin Postdoctoral Fellowship at Stanford University in 2004 and 2005. At Stanford, he worked with Professor John Cooke and Assistant Professor William Fearon on the role of the nicotinic cholinergic pathway in microvascular endothelial cell biology/angiogenesis and on invasive assessment of microvascular physiology. The author is currently a Staff Specialist in Cardiology at Royal Prince Alfred Hospital and is a Research Group Leader at the Heart Research Institute where he continues work on androgens and cardiovascular biology.
Gender-dependent Mechanisms of Androgen Action in the Human Vasculature Evolving knowledge of the biology of androgen action has shed light on potential mechanisms for androgen actions on the vasculature. Androgens exert complex effects on a multiplicity of vascular biological processes, often in a sex-specific and age-dependent fashion.11 A critical feature of androgen action involves steroid binding to the ‘classic’ androgen receptor—a member of a superfamily of intranuclear transcription factors that mediate the genomic effects of steroid hormones. Activation of the androgen receptor by ligand binding triggers a cascade of events that leads to receptor dimerisation and subsequent dimer binding to specific DNA sequences on target genes, which ultimately leads to regulation of the transcriptional activity of androgen-sensitive genes.12 Tissue androgen sensitivity is determined in large part by the intensity of androgen receptor expression and also by a complex array of biological determinants including the regulation of androgen receptor co-regulators, functional androgen receptor polymorphisms, androgen metabolic enzymes and possibly also non-genomic mechanisms. In recent years, work by Celermajer and others has demonstrated that expression of the classic androgen receptor on key cells involved in atherogenesis is highly gender-dependent.13,14 Indeed, male human monocytederived macrophages express at least four-fold higher levels of androgen receptor mRNA than female donor cells15 whilst endothelial cells from male donors have two-fold higher androgen receptor protein than endothelial cells from female donors.14 Moreover, in rat aortic smooth muscle cells, AR protein levels are higher in male than in female animals.16 Using immunohistochemistry Death et al. demonstrated a strikingly gender-dependent expression of the androgen receptor in the human arterial wall, with arterial samples from male donors having five-fold more androgen receptor positive cells than those from female donors.14 These findings suggest that key players/tissues in atherogenesis are likely to possess sex-specific differences in androgen-sensitivity with implications that androgens may regulate key vascular biologic processes in a gender-specific manner. Whether the greater expression of androgen receptor by male cells is due to genetic or environmental factors
has been subject of a study by Sader et al. who measured androgen receptor expression levels in genetically female leucocytes from male bone marrow transplant recipients who had received bone marrow from female donors.17 When exposed to a male androgenic milieu, androgen receptor expression by genetically female leucocytes increased to levels similar to those for young healthy males—a finding which suggests that gender differences in androgen receptor levels are predominantly determined by sex differences in endogenous androgen levels rather than by genetic factors. By contrast to endogenous androgens, exogenous androgen administration consistently downregulated androgen receptor expression in males and females. In addition to their ‘classic’ genomic effects, there is increasing evidence that androgens exert rapid-onset non-genomic effects that are thought to be the result of androgen binding to a (as yet uncharacterised) membrane receptor.18 This activates second messengers such as calcium and protein kinases, which produce rapid responses such as smooth muscle relaxation. For example, the vasodilatory effects of supraphysiological doses of testosterone have been attributed to a non-genomic effect.
Gender-dependent Differences in Androgen Action at the Genomic Level Monocyte-derived macrophages (MDMs) play a key role in both the early (via foam cell formation) and late (via inflammatory and other mediators) stages of atherosclerosis. As male-donor MDMs express much higher levels of the androgen receptor than female-donor cells, we hypothesised that the genomic effects of androgen exposure on MDMs would be gender-dependent.11 We utilised cDNA array analysis to systematically investigate the effects of androgen exposure on gene expression in maleand female-donor human MDMs. In male-donor human MDMs, androgen exposure upregulated the expression of 27 genes with diverse atherosclerosis-related functions, including lipoprotein metabolism, adhesion, inflammation, coagulation and angiogenesis.11 Strikingly, by contrast, no genes were affected in female-donor MDMs. These array findings were confirmed by realtime RT-PCR findings, as well as by the demonstration of functional effects of androgen exposure on macrophage lipoprotein metabolism. These findings suggest that gender differences in androgen receptor content, by regulating genomic effects of androgen exposure, may be a key mediator of gender differences in vascular biology.
Androgens and Coronary Heart Disease—Clinical Studies The universality of the male excess risk for coronary heart disease suggests that there is an intrinsic sexual dimorphism in the pathogenesis of atherosclerosis. A popular alternate explanation to the oestrogen protection hypothesis to explain gender differences in coronary heart disease is that androgens are proatherogenic. To date, no
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clinical trials of testosterone therapy with cardiovascular outcomes in men have been reported. However, epidemiological studies have not demonstrated a consistent relationship between endogenous androgen levels and cardiovascular event rates in men or women.19,20 Whilst the majority of cross-sectional studies have reported either a neutral or inverse correlation between serum testosterone and coronary heart disease, all prospective studies have shown no correlation between testosterone and coronary heart disease in either men or women.20 A caveat of these epidemiological studies is that intra-gender differences in circulating androgens are small and may not clearly correlate with cardiovascular risk, whilst the large differences in androgenic milieu between normogonadal men, hypogonadol men and women may contribute to the sex differences in coronary heart disease. Indeed, men with hypopituitarism and untreated gonadotrophin deficiency do have increased cardiovascular mortality and increased overall mortality in comparison with treated men.21,22 In a recent prospective study of elderly men, many of whom have low circulating androgen levels, serum free testosterone levels were inversely related to progression of carotid atherosclerosis as assessed by intima-media thickness (IMT), a finding consistent with recent cross-sectional studies.23,24 These findings suggest that large changes in androgen levels indeed may affect atherogenesis. They also challenge the notion of androgenic predisposition to atherosclerosis and suggest instead, that very low androgen levels in men may, in fact, be deleterious.
Gender-dependent Relationships between Androgens and Cardiovascular Risk Factors Observational studies show that the relationship between serum testosterone and cardiovascular risk factors are in opposing directions for men and women, consistent with the observation that androgens exert gender-dependent vascular biologic effects. In most studies, a positive correlation between plasma testosterone and plasma high-density lipoprotein cholesterol (HDL-C) concentrations was reported in adult men.25 On the other hand, adult male plasma testosterone levels have been found to inversely correlate with levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and the thrombogenic factor fibrinogen,26 potentially atheroprotective effects. However, these correlations between endogenous androgens and coronary risk factors in men are confounded by even stronger inverse relationships that exist between serum testosterone levels and risk factors for the metabolic syndrome, including body mass index, amount of visceral fat, waist-hip ratio and serum levels of insulin, leptin and free fatty acids.20,27 These findings indicate that low serum testosterone in men may be a component of a metabolic syndrome, characterised by obesity, insulin resistance, hypertension, hypertriglyceridaemia, low HDL-C, and a procoagulatory and antifibrinolytic state. In contrast to men, women suffering from polycystic ovary syndrome (PCOS), the most common cause of
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hyperandrogenism in women, have elevated testosterone levels, ‘atherogenic’ lipoprotein profiles (e.g. low levels of HDL-C and increased ratios of LDL to HDL-C28 ), elevated fibrinogen and PAI-1 and an increased risk for myocardial infarction.29 Furthermore, female hyperandrogenism, rather than hypoandrogenism, is associated with high body mass index, elevated waist-hip ratio and high levels of leptin and insulin and low levels of HDLC.30 In total, these data suggest that androgens exert gender-dependent effects on a myriad of cardiovascular risk factors, with implications for gender differences in cardiovascular disease.
Animal Studies—Androgens and Atherosclerosis Animal studies evaluating the role of androgens in atherogenesis have yielded differing results, in a variety of different animal models. Testosterone has been found to exacerbate atherogenesis in male chicks31 and female cynomolgus monkeys,32 whilst experiments in rabbits and mice have yielded conflicting results with proatherogenic, neutral or anti-atherogenic effects reported depending on the specific animal model and hormone intervention involved.13,33,34 Nevertheless, sex-specific effects of androgens in atherogenesis have also been reported. In a study by Bruck et al.,35 testosterone was found to have an inhibitory effect on aortic arch intimal thickening in male but not female rabbits. By contrast, in a study using apoEdeficient mice, von Dehn et al. reported a proatherogenic effect of testosterone in males but an anti-atherogenic effect in females.33 Administration of a gonadotropinreleasing hormone (GnRH) antagonist Cetrorelix in this study produced a moderate anti-atherogenic effect in both sexes. The androgen effect in any in vivo model may depend on variations in the balance between male and female hormones in each animal species as well as the extent of aromatisation of testosterone to oestrogen metabolites. The diverse results from the animal studies also reflect the fact that many different mechanistic events in atherogenesis are potentially influenced by androgen exposure depending on the animal model, as well as gender-specific responses to androgens. Findings from animal studies evaluating gender differences in atherogenesis must be interpreted with some caution, for (with the exception of the non-human primate) the human gender difference in atherosclerosis is not found in most conventional animal models.32,36,37
Gender-specific Effects of Androgens in Vascular Biology Effects of Androgens on Macrophage Foam Cell Formation A prominent early event in atherogenesis is the adherence of monocytes to endothelial cells and their subsequent transmigration into the subendothelial space to differentiate into macrophages.38 The macrophages then accumulate very large amounts of cholesteryl esters (CE), giving them a foamy appearance (hence ‘foam’ cell). In recent in vitro studies, we reported that exposure to
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either dihydrotestosterone or the adrenal androgen dehydroepiandrosterone produced dose-dependent, androgen receptor-mediated increases in CE accumulation in human male-donor monocyte-derived macrophages.15,39 By contrast, androgen exposure had no effect on foam cell formation in female-donor monocyte-derived macrophages, a finding in keeping with gender differences in androgen receptor expression in macrophages. In subsequent studies using cDNA arrays, androgen exposure was found to exert widespread effects on the expression of genes involved in lipoprotein metabolism in male but not female monocyte-derived macrophages.11 In male macrophages, androgen effects included upregulation of genes involved in: (1) lysosomal lipoprotein processing (lysosomal acid lipase) and; (2) post lysosomal processes including intracytoplasmic cholesterol esterification (acyl coA:cholesterol acyl transferase (ACAT) and cholesteryl ester hydrolysis (hormone-sensitive lipase). The functional significance of these findings were validated by lipoprotein metabolism studies demonstrating androgen-dependent upregulation of lysosomal acid lipase activity in male-donor monocyte-derived macrophages.11 These findings suggest that androgens may be involved in gender-dependent regulation of atherogenic processes within the vessel wall. Whilst macrophage CE accumulation (i.e. foam cell formation) has been historically viewed as a key proatherogenic event, recent data suggests that macrophage CE accumulation in atherosclerotic lesions functions as a key defensive mechanism to protect the macrophage against the cytotoxic effects of excess intracellular free cholesterol as a result of phagocytosis of modified lipoproteins in the subintimal space.40 Attempts at inhibiting atherosclerosis by inhibition of macrophage cholesteryl esterification by gene knockdown of ACAT in mice or by pharmacological inhibition of ACAT in man have both failed and have resulted in paradoxical increases in atherosclerotic lesion size, probably as a consequence of increased macrophage death due to toxicity from unesterified cholesterol.40,41 In the light of these recent data, the androgenic promotion of macrophage CE accumulation in males may be viewed a ‘plaque stabilising’ event rather than as a proatherogenic one.
Effects of Androgens on Cellular and Systemic Inflammation Local cellular and systemic inflammation are key biologic processes which promote atherogenic risk.42 Elevated C-reactive protein (CRP) levels, a systemic marker of inflammation, have been shown to predict adverse cardiovascular outcomes in men and women.43,44 At a cellular level, the binding and recruitment of circulating monocytes to the vascular endothelium, key processes in early atherogenesis, are facilitated by a family of cellular mediators of inflammation or cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Elevated concentrations of soluble ICAM-1 predict the risk of myocardial infarction in men.45 Furthermore, high cir-
culating levels of VCAM-1 have been associated with increased carotid intimal-medial thickness (a marker of subclinical atherosclerosis),46 with the extent of peripheral vascular disease as evaluated by angiography47 and with future risk of cardiovascular death in patients with angiographically documented coronary artery disease.48 In in vitro experiments using male-donor human umbilical vein endothelial cells (HUVECs), McCrohon et al. found that dihydrotestosterone increased monocyteendothelial adhesion via androgen receptor-dependent upregulation of VCAM-1.49 By contrast, dihyrotestosterone had no effect on VCAM-1 expression in femaledonor HUVECs,50 whilst testosterone attenuated TNF∝induced VCAM-1 expression by aromatase-dependent conversion to oestradiol. In subsequent mechanistic studies by Death et al., androgen effects on endothelial cell VCAM-1 expression found to be due to gender-specific activation of the key inflammatory transcription factor, nuclear factor-B.14 Consistent with these findings in endothelial cells, we found by cDNA array analysis that androgen exposure in macrophages produced a sexspecific upregulation of genes implicated in promoting the adherence of monocytes to vascular endothelium (including CD40, leukotriene B4 receptor and cadherin 19). CD40 ligation in human macrophages has been shown to trigger the expression of adhesion molecules including lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1.51 Leukotriene B4, a potent chemoattractant and proinflammatory mediator, has also been implicated in monocyte recruitment, activation and adhesion. These results suggest that androgens exert strongly gender-dependent effects on adhesion and cellular inflammation. As androgens exert gender-specific effects on cellular adhesion and inflammation, we studied the effects of androgen replacement therapy on serum inflammatory markers, including high sensitivity C-reactive protein (CRP), soluble VCAM-1 (sVCAM-1) and soluble ICAM-1 (sICAM-1), in healthy older men with partial androgen deficiency (serum testosterone levels <15 nmol/L) in two randomised double-blind placebo-controlled trials.52 One trial evaluated the effects of a transdermal gel of the non-aromatisable androgen dihydrotestosterone whilst a second trial evaluated the effects of subcutaneous recombinant human chorionic gonadotropin (rhCG), a placental hormone which is a close analog of pituitary luteinising hormone (LH) and acts upon the same Leydig cell receptor to increase testosterone production and thereby also estradiol formation, via aromatisation of testosterone. Therefore treatment with rhCG, unlike dihydrotestosterone, produces increases in both serum testosterone and oestradiol levels. We found that androgen replacement in older men to eugonadal levels, with either dihyrotestosterone or rHCG, for three months had no significant effects on the serum levels of hsCRP, sICAM-1 or sVCAM-1. Our findings are in contrast to those for oestrogen hormone replacement therapy in postmenopausal women, which produces a sustained increase in hsCRP levels.53,54 Oestrogen therapy also reduces circulating levels of cell adhesion molecules in postmenopausal women such as
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sICAM-1, sVCAM-1 and E-Selectin in.55,56 In view of the absence of an effect on inflammatory markers with DHT, a pure androgen, results from our rHCG study suggest that oestrogens may not have a significant effect on circulating ICAM-1, VCAM-1 or CRP in older men, unlike their effects in postmenopausal women. Our observations suggest that a gender difference may exist with regard to the effects of oestrogens on serum inflammatory markers.
Effects of Androgens on Platelet Function Platelet aggregation is a prerequisite for thrombosis, leading to acute coronary syndromes. Ajayi et al. reported that testosterone administration in eugonadal men is associated with increased ex vivo platelet aggregation in response to the thromboxane analogue, I-BOP.57 Furthermore, Higashiura et al. reported that dihydrotestosterone upregulated thromboxane A2 receptor density on rat aortic smooth muscle cells in a genderdependent fashion—with male cells having a two-fold greater response in comparison to female cells.16 The authors also reported that male-derived rat aortic smooth muscle cells expressed significantly higher levels of androgen receptor protein than female-derived cells—thereby implicating the androgen receptor in mediating genderdependent platelet responses.
Androgens and Angiogenesis Angiogenesis is a fundamental process by which new blood vessels are formed.58 It is a prerequisite for development and plays a critical role in postnatal physiological processes such as wound healing and in tissue neovascularisation in response to ischaemia. There is considerable evidence for a direct effect of oestrogens in the modulation of angiogenesis.59 Such evidence includes the recurrent hormone-regulated neovascularisation of the female reproductive tract, impaired angiogenesis in oestrogen receptor knockout mice, and a positive correlation between oestrogen receptor expression and the degree of angiogenic activity and breast tumour invasiveness. In vitro, oestrogen has been demonstrated to promote key cellular activities necessary for angiogenesis including endothelial proliferation and migration. Whilst far less studied than oestrogen, multiple lines of evidence point to a role for androgens in the modulation of angiogenesis. It is well known that androgens promote tumour neovascularisation and tumour growth via androgen receptor activation in the context of prostate cancer. However, to date, the role of androgens in angiogenesis outside this neoplastic context has been largely ignored. Nevertheless, there is some evidence for a physiologic role of androgens in angiogenesis in a variety of tissues. We have shown that androgens upregulate expression of vascular endothelial growth factor (VEGF) 165 receptors 1 and 2 in male but not female monocytederived macrophages.11 In rats, testosterone stimulates pulmonary vascular endothelial cell proliferation in malebut not female-donor cells.60 In avian higher vocal centres, testosterone strongly stimulates endothelial cell proliferation and angiogenesis, an effect associated with
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upregulation of VEGF and VEGF receptor-2.61 Consistent with this, dihydrotestosterone upregulates the expression of VEGF mRNA and VEGF biological activity in adult rat prostates62 and testosterone increases VEGF expression in an AR-dependent fashion in an immortalised cell line S115.63 In total, these data suggest that a pathway for androgen receptor-mediated VEGF-dependent angiogenesis exists in a variety of tissues, including vascular endothelial cells.
Conclusion There is an emerging body of evidence that suggests that gender-specific effects of androgens in the vasculature may contribute to the striking gender differences in the pathophysiology, prevalence and severity of atherosclerosis. A critical feature of androgen action is the function of the ‘classic’ androgen receptor as a nuclear transcription factor that regulates the transcriptional activity of androgen-sensitive genes. Recent studies show that gender differences exist in androgen receptor expression in key cells and tissues in the human vasculature with consistently higher levels in men than women. As a consequence there appear to be gender differences in genomic responses to androgen exposure in male compared to female cells. Indeed, androgens exert complex effects on a multiplicity of vascular biological processes, often in a sex-specific fashion. Some androgen effects appear to be proatherogenic whilst others potentially atheroprotective, making a global assessment of androgen effects complex and difficult. Current evidence suggests that the long held assumption that the gender gap in atherogenesis is attributable to favourable vascular effects of oestrogens in contrast to the assumed unfavourable effects of androgens is, at best, an inaccurate oversimplification. In fact, some emerging data challenge this notion and suggest that androgens may instead be cardioprotective in men but not in women. There is a need to better address the possibility of differential gender effects in future research in the field of sex hormones and atherogenesis. Given the inherently genomic nature of androgen and oestrogen action, further functional genomic and proteomic characterisation of sex steroid actions on a variety of male and female vascular cells and tissues will substantially enhance our understanding of the role of sex steroids in atherogenesis. Such observations may eventually lead to the novel concept of ‘gender specific’ treatments for cardiovascular disease.
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6. Kostis JB, Wilson AC, O’Dowd K, Gregory P, Chelton S, Cosgrove NM, Chirala A, Cui T. Sex differences in the management and long-term outcome of acute myocardial infarction. A statewide study. Circulation 1994;90: 1715–30. 7. Vaccarino V, Parsons L, Every NR, Barron HV, Krumholz HM. Sex-based differences in early mortality after myocardial infarction. National Registry of Myocardial Infarction 2 Participants. N Engl J Med 1999;341:217–25. 8. Vaccarino V, Krumholz HM, Yarzebski J, Gore JM, Goldberg RJ. Sex differences in 2-year mortality after hospital discharge for myocardial infarction. Ann Intern Med 2001;134:173–81. 9. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA 1998;280:605–13. 10. Manson JE, Hsia J, Johnson KC, Rossouw JE, Assaf AR, Lasser NL, Trevisan M, Black HR, Heckbert SR, Detrano R, Strickland OL, Wong ND, Crouse JR, Stein E, Cushman M. Estrogen plus progestin and the risk of coronary heart disease. N Engl J Med 2003;346:523–34. 11. Ng MK, Quinn CM, McCrohon JA, Nakhla S, Jessup W, Handelsman DJ, Celermajer DS, Death AK. Androgens upregulate atherosclerosis-related genes in macrophages from males but not females: molecular insights into gender differences in atherosclerosis. J Am Coll Cardiol 2003;42:1306–13. 12. Prins GS. Molecular biology of the androgen receptor. Mayo Clin Proc 2000;75:S32–5. 13. Ng MK, Jessup W, Celermajer DS. Sex-related differences in the regulation of macrophage cholesterol metabolism. Curr Opin Lipidol 2001;12:505–10. 14. Death AK, McGrath KC, Sader MA, Nakhla S, Jessup W, Handelsman DJ, Celermajer DS. Dihydrotestosterone promotes vascular cell adhesion molecule-1 expression in male human endothelial cells via a nuclear factor-kappaB-dependent pathway. Endocrinology 2004;145:1889–97. 15. McCrohon JA, Death AK, Nakhla S, Jessup W, Handelsman DJ, Stanley KK, Celermajer DS. Androgen receptor expression is greater in macrophages from male than from female donors. A sex difference with implications for atherogenesis. Circulation 2000;101:224–6. 16. Higashiura K, Mathur RS, Halushka PV. Gender-related differences in androgen regulation of thromboxane A2 receptors in rat aortic smooth muscle cells. J Cardiovasc Pharmacol 1997;29:311–5. 17. Sader MA, McGrath KC, Hill MD, Bradstock KF, Jimenez M, Handelsman DJ, Celermajer DS, Death AK. Androgen receptor gene expression in leucocytes is hormonally regulated: implications for gender differences in disease pathogenesis. Clin Endocrinol (Oxf) 2005;62:56–63. 18. Losel R, Wehling M. Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol 2003;4:46–55. 19. Alexandersen P, Haarbo J, Christiansen C. The relationship of natural androgens to coronary heart disease in males: a review. Atherosclerosis 1996;125:1–13. 20. Wu FC, von Eckardstein A. Androgens and coronary artery disease. Endocr Rev 2003;24:183–217. 21. Rosen T, Bengtsson BA. Premature mortality due to cardiovascular disease in hypopituitarism. Lancet 1990;336:285–8. 22. Tomlinson JW, Holden N, Hills RK, Wheatley K, Clayton RN, Bates AS, Sheppard MC, Stewart PM. Association between premature mortality and hypopituitarism. West Midlands Prospective Hypopituitary Study Group. Lancet 2001;357:425–31.
23. Muller M, van den Beld AW, Bots ML, Grobbee DE, Lamberts SW, van der Schouw YT. Endogenous sex hormones and progression of carotid atherosclerosis in elderly men. Circulation 2004;109:2074–9. 24. Makinen J, Jarvisalo MJ, Pollanen P, Perheentupa A, Irjala K, Koskenvuo M, Makinen J, Huhtaniemi I, Raitakari OT. Increased carotid atherosclerosis in andropausal middleaged men. J Am Coll Cardiol 2005;45:1603–8. 25. Khaw KT, Barrett-Connor E. Endogenous sex hormones, high density lipoprotein cholesterol, and other lipoprotein fractions in men. Arterioscler Thromb 1991;11: 489–94. 26. Barrett-Connor E. Testosterone and risk factors for cardiovascular disease in men. Diabetes Metab 1995;21:156–61. 27. Simon D, Charles MA, Nahoul K, Orssaud G, Kremski J, Hully V, Joubert E, Papoz L, Eschwege E. Association between plasma total testosterone and cardiovascular risk factors in healthy adult men: The Telecom Study. J Clin Endocrinol Metab 1997;82:682–5. 28. Conway GS, Agrawal R, Betteridge DJ, Jacobs HS. Risk factors for coronary artery disease in lean and obese women with polycystic ovary syndrome. Clin Endocrinol 1992;37:119–25. 29. Talbott E, Guzick D, Clerici A, Berga S, Detre K, Weimer K, Kuller L. Coronary heart disease risk factors in women with polycystic ovary syndrome. Arterioscler Thromb Vasc Biol 1995;15:821–6. 30. Hergenc G, Schulte H, Assmann G, von Eckardstein A. Associations of obesity markers, insulin, and sex hormones with HDL-cholesterol levels in Turkish and German individuals. Atherosclerosis 1999;145:147–56. 31. Toda T, Toda Y, Cho BH, Kummerow FA. Ultrastructural changes in the comb and aorta of chicks fed excess testosterone. Atherosclerosis 1984;51:47–57. 32. Adams MR. Effects of androgens on coronary artery atherosclerosis and atherosclerosis-related impairment of vascular responsiveness. Arterioscler Thromb Vasc Biol 1995;15:562–70. 33. von Dehn G, von Dehn O, Volker W, Langer C, Weinbauer GF, Behre HM, Nieschlag E, Assmann G, von Eckardstein A. Atherosclerosis in apolipoprotein E-deficient mice is decreased by the suppression of endogenous sex hormones. Horm Metab Res 2001;33:110–4. 34. Nathan L, Shi W, Dinh H, Mukherjee TK, Wang X, Lusis AJ, Chaudhuri G. Testosterone inhibits early atherogenesis by conversion to estradiol: critical role of aromatase. Proc Natl Acad Sci USA 2001;98:3589–93. 35. Bruck B, Brehme U, Gugel N, Hanke S, Finking G, Lutz C, Benda N, Schmahl FW, Haasis R, Hanke H. Gender-specific differences in the effects of testosterone and estrogen on the development of atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol 1997;17:2192–9. 36. Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerosis lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H. Atherosclerosis 1987;64:215–21. 37. Hanke H, Hanke S, Finking G, Muhic-Lohrer A, Muck AO, Schmahl FW, Haasis R, Hombach V. Different effects of estrogen and progesterone on experimental atherosclerosis in female versus male rabbits. Quantitation of cellular proliferation by bromodeoxyuridine. Circulation 1996;94:175–81. 38. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801–9. 39. Ng MK, Nakhla S, Baoutina A, Jessup W, Handelsman DJ, Celermajer DS. Dehydroepiandrosterone, an adrenal androgen, increases human foam cell formation: a potentially proatherogenic effect. J Am Coll Cardiol 2003;42:1967–74.
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40. Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese RVJ. Increased atherosclerosis in LDL receptornull mice lacking ACAT1 in macrophages. J Clin Invest 2001: 163–71. 41. Nissen SE, Tuzcu EM, Brewer HB, Sipahi I, Nicholls SJ, Ganz P, Schoenhagen P, Waters DD, Pepine CJ, Crowe TD, Davidson MH, Deanfield JE, Wisniewski LM, Hanyok JJ, Kassalow LM. Effect of ACAT inhibition on the progression of coronary atherosclerosis. N Engl J Med 2006;354:1253–63. 42. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–26. 43. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 1997;336: 973–9. 44. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836–43. 45. Ridker PM, Hennekens CH, Roitman-Johnson B, Stampfer MJ, Allen J. Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancet 1998;351: 88–92. 46. Rohde LE, Lee RT, Rivero J, Jamacochian M, Arroyo LH, Briggs W, Rifai N, Libby P, Creager MA, Ridker PM. Circulating cell adhesion molecules are correlated with ultrasound-based assessment of carotid atherosclerosis. Arterioscler Thromb Vasc Biol 1998;18:1765–70. 47. Peter K, Nawroth P, Conradt C, Nordt T, Weiss T, Boehme M, Wunsch A, Allenberg J, Kubler W, Bode C. Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin and thrombomodulin. Arterioscler Thromb Vasc Biol 1997;17:505–12. 48. Blankenberg S, Rupprecht HJ, Bickel C, Peetz D, Hafner G, Tiret L, Meyer J. Circulating cell adhesion molecules and death in patients with coronary artery disease. Circulation 2001;104:1336–42. 49. McCrohon JA, Jessup W, Handelsman DJ, Celermajer DS. Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1. Circulation 1999;99(17):2317–22. 50. Mukherjee TK, Dinh H, Chaudhuri G, Nathan L. Testosterone attenuates expression of vascular cell adhesion molecule1 by conversion to estradiol by aromatase in endothelial cells: implications in atherosclerosis. Proc Natl Acad Sci USA 2002;99:4055–60. ¨ 51. Schonbeck U, Libby P. CD40 signaling and plaque instability. Circ Res 2001;89:1092–103. 52. Ng MK, Liu PY, Williams AJ, Nakhla S, Ly LP, Handelsman DJ, Celermamajer DS. Prospective study of effect of androgens on serum inflammatory markers in men. Arterioscler Thromb Vasc Biol 2002;22:1136–41. 53. Cushman M, Legault C, Barrett-Connor E, Stefanick ML, Kessler C, Judd HL, Sakkinen PA, Tracy RP. Effect of postmenopausal hormones on inflammation-sensitive proteins. The Postmenopausal Estrogen/Progestin Interventions (PEPI) Study. Circulation 1999;100:717–22. 54. Ridker PM, Hennekens CH, Rifai N, Buring JE, Manson JE. Hormone replacement therapy and increased plasma concentration of C-reactive protein. Circulation 1999;100:713–6. 55. Caulin-Glaser T, Farrell WJ, Pfau SE, Zaret B, Bunger K, Setaro JF, Brennan JJ, Bender JR, Cleman MW, Cabin HS, Remetz MS. Modulation of circulating cellular adhesion molecules in
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postmenopausal women with coronary artery disease. J Am Coll Cardiol 1998;31:1555–60. van Baal WM, Kenemans P, van der Mooren MJ, Kessel H, Emeis JJ, Stehouwer CDA. Increased C-reactive protein levels during short-term hormone replacement therapy in healthy postmenopausal women. Thromb Haemost 1999;81:925–8. Ajayi AA, Mathur R, Halushka PV. Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses. Circulation 1995;91:2742–7. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931–4. Losordo DW, Isner JM. Estrogen and angiogenesis: a review. Arterioscler Thromb Vasc Biol 2001;21:6–12.
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60. Liu J, Wu S, Wei H, Zhou K, Ruan Y, Lai W. Effects of sex hormones and their balance on the proliferation of rat vascular endothelial cells. Horm Res 2002;58:16–20. 61. Louissaint AJ, Rao S, Leventhal C, Goldman SA. Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain. Neuron 2002;34:945–60. 62. Sordello S, Bertrand N, Plouet J. Vascular endothelial growth factor is up-regulated in vitro and in vivo by androgens. Biochem Biophys Res Commun 1998;251:287–90. 63. Ruohola JK, Valve EM, Karkkainen MJ, Joukov V, Alitalo K, Harkonen PL. Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells. Mol Cell Endocrinol 1999;149:29–40.
Original Article
New Perspectives on Mars and Venus: Unravelling the Role of Androgens in Gender Differences in Cardiovascular Biology and Disease Martin K.C. Ng a,b,∗ a
Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia b Heart Research Institute, Camperdown, NSW 2050, Australia
There are substantial gender differences in the pattern, severity and clinical outcomes of coronary heart disease independent of environmental risk factor exposure. As a consequence, there has been considerable interest in the potential role of sex hormones in atherogenesis, particularly the potential protective effects of oestrogen. However, the failure of the recent clinical randomised trials to show a cardioprotective effect for oestrogen coupled with a growing interest in androgen replacement therapy in elderly men has refocused interest on the role of androgens in cardiovascular biology and disease. Over the last decade, compelling evidence has emerged that sex differences in vascular biology are not only determined by gender-related differences in sex steroid levels but also by gender-specific tissue and cellular characteristics which mediate sex-specific responses to a variety of stimuli. In the vasculature, androgens often act in a gender-specific manner, with differential effects in male and female cells. This gender-dependent regulation may have important implications for understanding the basis of the gender gap in atherosclerosis and may eventually lead to the development of sex-specific treatments for cardiovascular disease. This review will summarise the current data for the role of androgens in gender differences in coronary heart disease and cardiovascular biology. (Heart, Lung and Circulation 2007;16:185–192) © 2007 Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia and New Zealand. Published by Elsevier Inc. All rights reserved. Keywords. Coronary heart disease; Vascular biology; Gender; Hormones and androgens
Introduction
T
here are substantial gender differences in patterns of health and illness throughout the world. Most strikingly, men in almost all societies exhibit a shorter life expectancy than women of the same age and socioeconomic background.1 In fact, men have a shorter life expectancy than women in 186 of 191 (97%) United Nation member states, with men living an average of 5.6 years shorter in those countries above a minimal level of economic development. Despite this pervasive difference, the gender gap in life expectancy and mortality remains incompletely understood and remains one of the most interesting and important questions in biology and medicine. This gender gap in life expectancy is driven largely by the steep male predisposition to the clinical manifestations of atherosclerosis, particularly coronary heart disease. With economic development and the associated Available online 19 April 2007 ∗
Correspondence address: Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia. Tel.: +61 2 95156111; fax: +61 2 95506262. E-mail addresses: [email protected], [email protected]
decline in communicable diseases globally, cardiovascular disease is now the leading cause of mortality worldwide and is expected to remain so well into the twenty-first century.2,3 Hence, sex differences in the onset, extent and severity of coronary heart disease are a major contributor to the gender gap in longevity. It is well known that coronary deaths in men consistently exceed those in agematched women by 2.5–4.5-fold up until the age of 75.4 This sex difference is remarkably consistent across countries with very different racial compositions, rates of heart disease and lifestyles.4 There is a higher prevalence of conventional cardiovascular risk factors in men but this does not entirely explain the gender gap. In fact, a significant gender difference in coronary disease remains even when the cardiovascular risks associated with systolic hypertension, smoking, hypercholesterolaemia, hyperglycaemia and obesity are controlled by multivariate logistic analysis.5 A less well appreciated aspect of sex differences in coronary heart disease is the observation that once women develop coronary disease, they lose their survival advantage. Indeed, this gender relationship appears to be reversed with respect to clinical outcomes following acute coronary syndromes (ACS): compared to men, women consistently have worse short- and long-term
© 2007 Australasian Society of Cardiac and Thoracic Surgeons and the Cardiac Society of Australia and New Zealand. Published by Elsevier Inc. All rights reserved.
1443-9506/04/$30.00 doi:10.1016/j.hlc.2007.02.108
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outcomes following ACS, independent of co-morbidities and differences in management.6–8 For example, using a nationwide database containing hospitalisation data for acute myocardial infarction (AMI) for over 380,000 patients, Vaccarino et al. found that women under 75 years of age had a higher mortality compared to men, with a greater than two-fold increase in mortality for women less than 50 years of age.7 After adjustment for risk factors, co-morbidities and differences in management, female gender was still associated with a higher risk of mortality, consistent with a gender-dependent phenomenon. Similarly, in a study involving over 49,000 patients, women had lower three-year survival rates following AMI up to age 70 years: women had a 69% higher death rate in the age group 30–49 years and a 21% higher death rate in the age group 50–69 years.6 Such differences in clinical outcome post ACS raise the possibility that an intrinsic gender difference may exist not only with respect to atherogenesis but also with respect to cardiovascular adaptation/repair in response to critical ischaemia/infarction. Such observations have produced considerable interest in the potential role of sex hormones in cardiovascular biology and disease. Much of this attention has to date centred on the apparent atheroprotective effect of the female sex steroid, oestrogen. However, the failure of the recent clinical randomised trials to show a cardioprotective effect for oestrogen9,10 coupled with a growing interest in androgen replacement therapy in elderly men has refocused interest on the role of androgens in cardiovascular biology and disease. Over the last decade compelling evidence has emerged that that sex differences in vascular biology are not only determined by gender-related differences in sex steroid levels but also by gender specific tissue and cellular characteristics which mediate sex-specific responses to a variety of stimuli. Hence, androgens may act in a gender-specific manner, with differential effects in male and female cells. This gender-related regulation may have important implications for understanding the basis of the gender gap in atherosclerosis and may eventually lead to the development of sex-specific treatments for cardiovascular disease. This review will summarise the current data for the role of androgens in gender differences in coronary heart disease and cardiovascular biology. In particular, new data regarding gender specific mechanisms of androgen action in the vasculature and their implications of understanding gender differences in cardiovascular biology and disease will be discussed.
The Author’s Links to Professor John Uther and Westmead Hospital The author undertook his advanced training in cardiology at Westmead Hospital in 1998 and 1999 when Professor John Uther was Chairman of the Division of Medicine at Westmead Hospital. Whilst at Westmead he developed an interest in atherogenesis and began using molecular techniques to detect evidence of bacterial presence in human coronary artery plaques. Extending these interests, he subsequently completed a PhD at the Heart
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Research Institute, Sydney on the role of androgens in the pathogenesis of atherosclerosis under the supervision of Professor David Celermajer. On completion of his PhD, the author undertook a NHMRC CJ Martin Postdoctoral Fellowship at Stanford University in 2004 and 2005. At Stanford, he worked with Professor John Cooke and Assistant Professor William Fearon on the role of the nicotinic cholinergic pathway in microvascular endothelial cell biology/angiogenesis and on invasive assessment of microvascular physiology. The author is currently a Staff Specialist in Cardiology at Royal Prince Alfred Hospital and is a Research Group Leader at the Heart Research Institute where he continues work on androgens and cardiovascular biology.
Gender-dependent Mechanisms of Androgen Action in the Human Vasculature Evolving knowledge of the biology of androgen action has shed light on potential mechanisms for androgen actions on the vasculature. Androgens exert complex effects on a multiplicity of vascular biological processes, often in a sex-specific and age-dependent fashion.11 A critical feature of androgen action involves steroid binding to the ‘classic’ androgen receptor—a member of a superfamily of intranuclear transcription factors that mediate the genomic effects of steroid hormones. Activation of the androgen receptor by ligand binding triggers a cascade of events that leads to receptor dimerisation and subsequent dimer binding to specific DNA sequences on target genes, which ultimately leads to regulation of the transcriptional activity of androgen-sensitive genes.12 Tissue androgen sensitivity is determined in large part by the intensity of androgen receptor expression and also by a complex array of biological determinants including the regulation of androgen receptor co-regulators, functional androgen receptor polymorphisms, androgen metabolic enzymes and possibly also non-genomic mechanisms. In recent years, work by Celermajer and others has demonstrated that expression of the classic androgen receptor on key cells involved in atherogenesis is highly gender-dependent.13,14 Indeed, male human monocytederived macrophages express at least four-fold higher levels of androgen receptor mRNA than female donor cells15 whilst endothelial cells from male donors have two-fold higher androgen receptor protein than endothelial cells from female donors.14 Moreover, in rat aortic smooth muscle cells, AR protein levels are higher in male than in female animals.16 Using immunohistochemistry Death et al. demonstrated a strikingly gender-dependent expression of the androgen receptor in the human arterial wall, with arterial samples from male donors having five-fold more androgen receptor positive cells than those from female donors.14 These findings suggest that key players/tissues in atherogenesis are likely to possess sex-specific differences in androgen-sensitivity with implications that androgens may regulate key vascular biologic processes in a gender-specific manner. Whether the greater expression of androgen receptor by male cells is due to genetic or environmental factors
has been subject of a study by Sader et al. who measured androgen receptor expression levels in genetically female leucocytes from male bone marrow transplant recipients who had received bone marrow from female donors.17 When exposed to a male androgenic milieu, androgen receptor expression by genetically female leucocytes increased to levels similar to those for young healthy males—a finding which suggests that gender differences in androgen receptor levels are predominantly determined by sex differences in endogenous androgen levels rather than by genetic factors. By contrast to endogenous androgens, exogenous androgen administration consistently downregulated androgen receptor expression in males and females. In addition to their ‘classic’ genomic effects, there is increasing evidence that androgens exert rapid-onset non-genomic effects that are thought to be the result of androgen binding to a (as yet uncharacterised) membrane receptor.18 This activates second messengers such as calcium and protein kinases, which produce rapid responses such as smooth muscle relaxation. For example, the vasodilatory effects of supraphysiological doses of testosterone have been attributed to a non-genomic effect.
Gender-dependent Differences in Androgen Action at the Genomic Level Monocyte-derived macrophages (MDMs) play a key role in both the early (via foam cell formation) and late (via inflammatory and other mediators) stages of atherosclerosis. As male-donor MDMs express much higher levels of the androgen receptor than female-donor cells, we hypothesised that the genomic effects of androgen exposure on MDMs would be gender-dependent.11 We utilised cDNA array analysis to systematically investigate the effects of androgen exposure on gene expression in maleand female-donor human MDMs. In male-donor human MDMs, androgen exposure upregulated the expression of 27 genes with diverse atherosclerosis-related functions, including lipoprotein metabolism, adhesion, inflammation, coagulation and angiogenesis.11 Strikingly, by contrast, no genes were affected in female-donor MDMs. These array findings were confirmed by realtime RT-PCR findings, as well as by the demonstration of functional effects of androgen exposure on macrophage lipoprotein metabolism. These findings suggest that gender differences in androgen receptor content, by regulating genomic effects of androgen exposure, may be a key mediator of gender differences in vascular biology.
Androgens and Coronary Heart Disease—Clinical Studies The universality of the male excess risk for coronary heart disease suggests that there is an intrinsic sexual dimorphism in the pathogenesis of atherosclerosis. A popular alternate explanation to the oestrogen protection hypothesis to explain gender differences in coronary heart disease is that androgens are proatherogenic. To date, no
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clinical trials of testosterone therapy with cardiovascular outcomes in men have been reported. However, epidemiological studies have not demonstrated a consistent relationship between endogenous androgen levels and cardiovascular event rates in men or women.19,20 Whilst the majority of cross-sectional studies have reported either a neutral or inverse correlation between serum testosterone and coronary heart disease, all prospective studies have shown no correlation between testosterone and coronary heart disease in either men or women.20 A caveat of these epidemiological studies is that intra-gender differences in circulating androgens are small and may not clearly correlate with cardiovascular risk, whilst the large differences in androgenic milieu between normogonadal men, hypogonadol men and women may contribute to the sex differences in coronary heart disease. Indeed, men with hypopituitarism and untreated gonadotrophin deficiency do have increased cardiovascular mortality and increased overall mortality in comparison with treated men.21,22 In a recent prospective study of elderly men, many of whom have low circulating androgen levels, serum free testosterone levels were inversely related to progression of carotid atherosclerosis as assessed by intima-media thickness (IMT), a finding consistent with recent cross-sectional studies.23,24 These findings suggest that large changes in androgen levels indeed may affect atherogenesis. They also challenge the notion of androgenic predisposition to atherosclerosis and suggest instead, that very low androgen levels in men may, in fact, be deleterious.
Gender-dependent Relationships between Androgens and Cardiovascular Risk Factors Observational studies show that the relationship between serum testosterone and cardiovascular risk factors are in opposing directions for men and women, consistent with the observation that androgens exert gender-dependent vascular biologic effects. In most studies, a positive correlation between plasma testosterone and plasma high-density lipoprotein cholesterol (HDL-C) concentrations was reported in adult men.25 On the other hand, adult male plasma testosterone levels have been found to inversely correlate with levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and the thrombogenic factor fibrinogen,26 potentially atheroprotective effects. However, these correlations between endogenous androgens and coronary risk factors in men are confounded by even stronger inverse relationships that exist between serum testosterone levels and risk factors for the metabolic syndrome, including body mass index, amount of visceral fat, waist-hip ratio and serum levels of insulin, leptin and free fatty acids.20,27 These findings indicate that low serum testosterone in men may be a component of a metabolic syndrome, characterised by obesity, insulin resistance, hypertension, hypertriglyceridaemia, low HDL-C, and a procoagulatory and antifibrinolytic state. In contrast to men, women suffering from polycystic ovary syndrome (PCOS), the most common cause of
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hyperandrogenism in women, have elevated testosterone levels, ‘atherogenic’ lipoprotein profiles (e.g. low levels of HDL-C and increased ratios of LDL to HDL-C28 ), elevated fibrinogen and PAI-1 and an increased risk for myocardial infarction.29 Furthermore, female hyperandrogenism, rather than hypoandrogenism, is associated with high body mass index, elevated waist-hip ratio and high levels of leptin and insulin and low levels of HDLC.30 In total, these data suggest that androgens exert gender-dependent effects on a myriad of cardiovascular risk factors, with implications for gender differences in cardiovascular disease.
Animal Studies—Androgens and Atherosclerosis Animal studies evaluating the role of androgens in atherogenesis have yielded differing results, in a variety of different animal models. Testosterone has been found to exacerbate atherogenesis in male chicks31 and female cynomolgus monkeys,32 whilst experiments in rabbits and mice have yielded conflicting results with proatherogenic, neutral or anti-atherogenic effects reported depending on the specific animal model and hormone intervention involved.13,33,34 Nevertheless, sex-specific effects of androgens in atherogenesis have also been reported. In a study by Bruck et al.,35 testosterone was found to have an inhibitory effect on aortic arch intimal thickening in male but not female rabbits. By contrast, in a study using apoEdeficient mice, von Dehn et al. reported a proatherogenic effect of testosterone in males but an anti-atherogenic effect in females.33 Administration of a gonadotropinreleasing hormone (GnRH) antagonist Cetrorelix in this study produced a moderate anti-atherogenic effect in both sexes. The androgen effect in any in vivo model may depend on variations in the balance between male and female hormones in each animal species as well as the extent of aromatisation of testosterone to oestrogen metabolites. The diverse results from the animal studies also reflect the fact that many different mechanistic events in atherogenesis are potentially influenced by androgen exposure depending on the animal model, as well as gender-specific responses to androgens. Findings from animal studies evaluating gender differences in atherogenesis must be interpreted with some caution, for (with the exception of the non-human primate) the human gender difference in atherosclerosis is not found in most conventional animal models.32,36,37
Gender-specific Effects of Androgens in Vascular Biology Effects of Androgens on Macrophage Foam Cell Formation A prominent early event in atherogenesis is the adherence of monocytes to endothelial cells and their subsequent transmigration into the subendothelial space to differentiate into macrophages.38 The macrophages then accumulate very large amounts of cholesteryl esters (CE), giving them a foamy appearance (hence ‘foam’ cell). In recent in vitro studies, we reported that exposure to
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either dihydrotestosterone or the adrenal androgen dehydroepiandrosterone produced dose-dependent, androgen receptor-mediated increases in CE accumulation in human male-donor monocyte-derived macrophages.15,39 By contrast, androgen exposure had no effect on foam cell formation in female-donor monocyte-derived macrophages, a finding in keeping with gender differences in androgen receptor expression in macrophages. In subsequent studies using cDNA arrays, androgen exposure was found to exert widespread effects on the expression of genes involved in lipoprotein metabolism in male but not female monocyte-derived macrophages.11 In male macrophages, androgen effects included upregulation of genes involved in: (1) lysosomal lipoprotein processing (lysosomal acid lipase) and; (2) post lysosomal processes including intracytoplasmic cholesterol esterification (acyl coA:cholesterol acyl transferase (ACAT) and cholesteryl ester hydrolysis (hormone-sensitive lipase). The functional significance of these findings were validated by lipoprotein metabolism studies demonstrating androgen-dependent upregulation of lysosomal acid lipase activity in male-donor monocyte-derived macrophages.11 These findings suggest that androgens may be involved in gender-dependent regulation of atherogenic processes within the vessel wall. Whilst macrophage CE accumulation (i.e. foam cell formation) has been historically viewed as a key proatherogenic event, recent data suggests that macrophage CE accumulation in atherosclerotic lesions functions as a key defensive mechanism to protect the macrophage against the cytotoxic effects of excess intracellular free cholesterol as a result of phagocytosis of modified lipoproteins in the subintimal space.40 Attempts at inhibiting atherosclerosis by inhibition of macrophage cholesteryl esterification by gene knockdown of ACAT in mice or by pharmacological inhibition of ACAT in man have both failed and have resulted in paradoxical increases in atherosclerotic lesion size, probably as a consequence of increased macrophage death due to toxicity from unesterified cholesterol.40,41 In the light of these recent data, the androgenic promotion of macrophage CE accumulation in males may be viewed a ‘plaque stabilising’ event rather than as a proatherogenic one.
Effects of Androgens on Cellular and Systemic Inflammation Local cellular and systemic inflammation are key biologic processes which promote atherogenic risk.42 Elevated C-reactive protein (CRP) levels, a systemic marker of inflammation, have been shown to predict adverse cardiovascular outcomes in men and women.43,44 At a cellular level, the binding and recruitment of circulating monocytes to the vascular endothelium, key processes in early atherogenesis, are facilitated by a family of cellular mediators of inflammation or cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Elevated concentrations of soluble ICAM-1 predict the risk of myocardial infarction in men.45 Furthermore, high cir-
culating levels of VCAM-1 have been associated with increased carotid intimal-medial thickness (a marker of subclinical atherosclerosis),46 with the extent of peripheral vascular disease as evaluated by angiography47 and with future risk of cardiovascular death in patients with angiographically documented coronary artery disease.48 In in vitro experiments using male-donor human umbilical vein endothelial cells (HUVECs), McCrohon et al. found that dihydrotestosterone increased monocyteendothelial adhesion via androgen receptor-dependent upregulation of VCAM-1.49 By contrast, dihyrotestosterone had no effect on VCAM-1 expression in femaledonor HUVECs,50 whilst testosterone attenuated TNF∝induced VCAM-1 expression by aromatase-dependent conversion to oestradiol. In subsequent mechanistic studies by Death et al., androgen effects on endothelial cell VCAM-1 expression found to be due to gender-specific activation of the key inflammatory transcription factor, nuclear factor-B.14 Consistent with these findings in endothelial cells, we found by cDNA array analysis that androgen exposure in macrophages produced a sexspecific upregulation of genes implicated in promoting the adherence of monocytes to vascular endothelium (including CD40, leukotriene B4 receptor and cadherin 19). CD40 ligation in human macrophages has been shown to trigger the expression of adhesion molecules including lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1.51 Leukotriene B4, a potent chemoattractant and proinflammatory mediator, has also been implicated in monocyte recruitment, activation and adhesion. These results suggest that androgens exert strongly gender-dependent effects on adhesion and cellular inflammation. As androgens exert gender-specific effects on cellular adhesion and inflammation, we studied the effects of androgen replacement therapy on serum inflammatory markers, including high sensitivity C-reactive protein (CRP), soluble VCAM-1 (sVCAM-1) and soluble ICAM-1 (sICAM-1), in healthy older men with partial androgen deficiency (serum testosterone levels <15 nmol/L) in two randomised double-blind placebo-controlled trials.52 One trial evaluated the effects of a transdermal gel of the non-aromatisable androgen dihydrotestosterone whilst a second trial evaluated the effects of subcutaneous recombinant human chorionic gonadotropin (rhCG), a placental hormone which is a close analog of pituitary luteinising hormone (LH) and acts upon the same Leydig cell receptor to increase testosterone production and thereby also estradiol formation, via aromatisation of testosterone. Therefore treatment with rhCG, unlike dihydrotestosterone, produces increases in both serum testosterone and oestradiol levels. We found that androgen replacement in older men to eugonadal levels, with either dihyrotestosterone or rHCG, for three months had no significant effects on the serum levels of hsCRP, sICAM-1 or sVCAM-1. Our findings are in contrast to those for oestrogen hormone replacement therapy in postmenopausal women, which produces a sustained increase in hsCRP levels.53,54 Oestrogen therapy also reduces circulating levels of cell adhesion molecules in postmenopausal women such as
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sICAM-1, sVCAM-1 and E-Selectin in.55,56 In view of the absence of an effect on inflammatory markers with DHT, a pure androgen, results from our rHCG study suggest that oestrogens may not have a significant effect on circulating ICAM-1, VCAM-1 or CRP in older men, unlike their effects in postmenopausal women. Our observations suggest that a gender difference may exist with regard to the effects of oestrogens on serum inflammatory markers.
Effects of Androgens on Platelet Function Platelet aggregation is a prerequisite for thrombosis, leading to acute coronary syndromes. Ajayi et al. reported that testosterone administration in eugonadal men is associated with increased ex vivo platelet aggregation in response to the thromboxane analogue, I-BOP.57 Furthermore, Higashiura et al. reported that dihydrotestosterone upregulated thromboxane A2 receptor density on rat aortic smooth muscle cells in a genderdependent fashion—with male cells having a two-fold greater response in comparison to female cells.16 The authors also reported that male-derived rat aortic smooth muscle cells expressed significantly higher levels of androgen receptor protein than female-derived cells—thereby implicating the androgen receptor in mediating genderdependent platelet responses.
Androgens and Angiogenesis Angiogenesis is a fundamental process by which new blood vessels are formed.58 It is a prerequisite for development and plays a critical role in postnatal physiological processes such as wound healing and in tissue neovascularisation in response to ischaemia. There is considerable evidence for a direct effect of oestrogens in the modulation of angiogenesis.59 Such evidence includes the recurrent hormone-regulated neovascularisation of the female reproductive tract, impaired angiogenesis in oestrogen receptor knockout mice, and a positive correlation between oestrogen receptor expression and the degree of angiogenic activity and breast tumour invasiveness. In vitro, oestrogen has been demonstrated to promote key cellular activities necessary for angiogenesis including endothelial proliferation and migration. Whilst far less studied than oestrogen, multiple lines of evidence point to a role for androgens in the modulation of angiogenesis. It is well known that androgens promote tumour neovascularisation and tumour growth via androgen receptor activation in the context of prostate cancer. However, to date, the role of androgens in angiogenesis outside this neoplastic context has been largely ignored. Nevertheless, there is some evidence for a physiologic role of androgens in angiogenesis in a variety of tissues. We have shown that androgens upregulate expression of vascular endothelial growth factor (VEGF) 165 receptors 1 and 2 in male but not female monocytederived macrophages.11 In rats, testosterone stimulates pulmonary vascular endothelial cell proliferation in malebut not female-donor cells.60 In avian higher vocal centres, testosterone strongly stimulates endothelial cell proliferation and angiogenesis, an effect associated with
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upregulation of VEGF and VEGF receptor-2.61 Consistent with this, dihydrotestosterone upregulates the expression of VEGF mRNA and VEGF biological activity in adult rat prostates62 and testosterone increases VEGF expression in an AR-dependent fashion in an immortalised cell line S115.63 In total, these data suggest that a pathway for androgen receptor-mediated VEGF-dependent angiogenesis exists in a variety of tissues, including vascular endothelial cells.
Conclusion There is an emerging body of evidence that suggests that gender-specific effects of androgens in the vasculature may contribute to the striking gender differences in the pathophysiology, prevalence and severity of atherosclerosis. A critical feature of androgen action is the function of the ‘classic’ androgen receptor as a nuclear transcription factor that regulates the transcriptional activity of androgen-sensitive genes. Recent studies show that gender differences exist in androgen receptor expression in key cells and tissues in the human vasculature with consistently higher levels in men than women. As a consequence there appear to be gender differences in genomic responses to androgen exposure in male compared to female cells. Indeed, androgens exert complex effects on a multiplicity of vascular biological processes, often in a sex-specific fashion. Some androgen effects appear to be proatherogenic whilst others potentially atheroprotective, making a global assessment of androgen effects complex and difficult. Current evidence suggests that the long held assumption that the gender gap in atherogenesis is attributable to favourable vascular effects of oestrogens in contrast to the assumed unfavourable effects of androgens is, at best, an inaccurate oversimplification. In fact, some emerging data challenge this notion and suggest that androgens may instead be cardioprotective in men but not in women. There is a need to better address the possibility of differential gender effects in future research in the field of sex hormones and atherogenesis. Given the inherently genomic nature of androgen and oestrogen action, further functional genomic and proteomic characterisation of sex steroid actions on a variety of male and female vascular cells and tissues will substantially enhance our understanding of the role of sex steroids in atherogenesis. Such observations may eventually lead to the novel concept of ‘gender specific’ treatments for cardiovascular disease.
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