Sex Differences in Cardiovascular Physiology and Pathophysiology

States

Introduction to Sex Differences in Cardiovascular Physiology

Barbara T. Alexander⁎,‡,§; Babbette D. LaMarca†,‡,§    * Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, United States,

† Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, United States,

‡ Women’s Health Research Center, University of Mississippi Medical Center, Jackson, MS, United States,

§ Mississippi Center for Excellence in Perinatal Research, University of Mississippi Medical Center, Jackson, MS, United States

Abstract Sex differences between men and women have long been overlooked and underappreciated in regards to cardiovascular disease (CVD). The clinical presentation of CVD differs in women relative to men, and although sex-specific guidelines are not yet recognized, differences in sensitivity and specificity for diagnostic testing for CVD may also differ in women relative to men. Whether treatment options differ by sex following CVD is controversial and follow-up in women may involve a lack of medical management practices that are standard for men. Despite the increase in the number of women included in clinical trials, women remain underrepresented. Thus the lack of scientific attention to CVD in women has led to poorer prognoses for women with CVD when compared to men. Currently, there is an increasing recognition of fundamental sex differences in cardiovascular function, anatomy, cell signaling, and the development of pathological disease. However, further research is needed to reveal sex differences in cardiovascular risk and promote sex-specific preventative and therapeutic approaches to treat CVD.

Keywords: Sex differences; Cardiovascular disease; Hypertension; Heart disease; Kidney; Vasculature; Regnancy; Menopause; Estrogen; Testosterone; Developmental origins

Sex differences between men and women have long been overlooked and underappreciated in regards to cardiovascular disease (CVD). The clinical presentation of CVD differs in women relative to men, and although sex-specific guidelines are not yet recognized, differences in sensitivity and specificity for diagnostic testing for CVD may also differ in women relative to men (see Chapter 12). Whether treatment options differ by sex following CVD is controversial and follow-up in women may involve a lack of medical management practices that are standard for men (see Chapter 12). Despite the increase in the number of women included in clinical trials, women remain underrepresented (see Chapter 12). Thus the lack of scientific attention to CVD in women has led to poorer prognoses for women with CVD when compared to men. Currently, there is an increasing recognition of fundamental sex differences in cardiovascular function, anatomy, cell signaling, and the development of pathological disease. However, further research is needed to reveal sex differences in cardiovascular risk and promote sex-specific preventative and therapeutic approaches to treat CVD.

CVD remains the main cause of death worldwide. In general, women develop CVD about 10 years later than men [1]. The transition through menopause increases the risk for CVD and ultimately, CVD becomes the leading cause of death in women as well as in men [1]. Although men have a higher lifetime risk for CVD than women at all ages, women represent greater than 50% of the deaths from CVD [2]. A Scientific Statement from the American Heart Association published in 2018 stated that 1 in 3 deaths in women were attributed to CVD. Although numerous traditional factors including a sedentary lifestyle, Western diet, and smoking contribute to increased cardiovascular risk in men and women, sex-specific risk factors in women include early age at menarche, early age at menopause, premenopausal obesity, and hormone replacement therapy [3]. Additional female-specific risk factors include polycystic ovary syndrome and pregnancy-related disorders such as gestational diabetes and preeclampsia (see Chapter 12). Cardiovascular risk can also result from exposure to a suboptimal environment during fetal life (see Chapter 16). Numerous experimental models that mimic the etiology of impaired fetal growth and adverse developmental exposures indicate that sex alters the developmental origins of cardiovascular and renal dysfunction (see Chapter 16). The mechanisms involved are multifactorial but include structural changes in the morphology of the kidney in addition to a role for the renin-angiotensin system (RAS) and the renal nerves (see Chapter 16).

CVDs include hypertension, coronary heart disease, ischemic stroke, and heart failure. Ischemic stroke is the 5th leading cause of death in men in the United States but remains the 3rd leading cause of death in women [4]. Age, cardiovascular risk factors, stroke symptoms, and stroke care are critical to ischemic stroke outcomes (see Chapter 4). Some risk factors for stroke are unique to women and include use of oral contraceptives, preeclampsia, menopause, and hormone replacement therapy whereas other factors are found in both sexes and include atrial fibrillation which may be more prevalent in women than in men (see Chapter 4). Heart failure in women often occurs at an older age and women are more likely to survive heart failure than men (see Chapter 17). Heart failure is associated with differences in ejection fraction, preserved or reduced, and the prevalence varies in men relative to women (see Chapter 17). Hypertension and diabetes are greater risk factors for heart failure in women; sex differences in cardiac energy metabolism may also contribute to the greater prevalence of heart failure in women relative to men (see Chapter 17).

Hypertension remains a major risk factor for CVD. The incidence and severity of hypertension increases in women after menopause [5]. Despite greater medical compliance in women relative to men, blood pressure management in women with hypertension after menopause is not well controlled (see Chapter 7). Furthermore, differences in the etiology of long-term blood pressure control prior to- and postmenopause are not clear (see Chapter 7). Experimental models that adequately mimic the hormonal milieu of menopause in women are limited (see Chapter 7). Immune-mediated mechanisms may contribute (see Chapter 7), but further studies are needed to clarify the pathogenesis of hypertension in women after menopause in order to provide new treatment alternatives.

Autoimmune disease is the primary contributor to death in women prior to menopause [6] although cardiovascular-related mortality represents the majority of deaths in women affected by autoimmune disease [7]. The prevalence of autoimmune disease is significantly higher in women than men implicating a role for sex steroids in the pathogenesis of sex differences in autoimmune disease prevalence. Estrogen may have a direct effect on the immune system but its exact role is controversial (see Chapter 14). Estrogen, in addition to other sex steroids, regulates immune-mediated responses and exert agonistic or antagonistic role on inflammation [8]. The frequency of complications during pregnancy including preeclampsia is increased in women with autoimmune disease (see Chapter 14). Preeclampsia is associated with a chronic increase in inflammation [8]. Renal and cardiovascular functions are also impaired (see Chapter 9) and changes in the cerebrovasculature include altered blood-brain barrier, impaired autoregulation of cerebral blood flow, and cerebral ischemia (see Chapter 10). Preeclampsia is also associated with a fourfold greater prevalence of hypertension in later life [9] with a greater overall cardiovascular risk observed as early as 6 years after delivery [10]. Preeclampsia occurs in up to 10 pregnancies worldwide. It is the leading cause of maternal death and contributes to the incidence of preterm birth and low birth weight babies [8]. The diagnosis for preeclampsia is complex and treatment options to prevent or treat preeclampsia are limited (see Chapter 8). Furthermore, the link between increased cardiovascular and renal disease later in life and preeclampsia is not clear (see Chapter 9). Hence, the development of treatment measures is warranted to improve the health of women affected by this disease.

It is well established that cardiovascular disease is the major cause of morbidity in individuals with Type 1 Diabetes (T1D). T1D is an organ-specific autoimmune disease that targets the pancreas predominantly in childhood and early life [11]. The incidence of T1D is greater in females prior to puberty but is twofold greater in males after puberty (see Chapter 15). Recent studies indicate that sex hormones contribute to sex differences in the development of TID (see Chapter 15) with immunological differences in β-cell function in females and males related to sex differences in inflammatory pathways (see Chapter 15). Yet, further studies are needed to elucidate the importance of sex hormones on inflammation, immunity, and cardiovascular risk.

Patients with peripheral artery disease (PAD) have a greater vulnerability to cardiovascular events than those with CVD alone [12]. The RAS contributes to the development of atherosclerosis and abdominal aortic aneurysms (AAAs). Regulation of the RAS via sex hormones may contribute to sex differences in the development, progression, and therapy of PAD (see Chapter 11). Sex chromosomes may also play a role in the sexual dimorphism of atherosclerosis and AAAs (see Chapter 11). Regulation of blood pressure by estrogen-mediated influences on the RAS involves interactions of estrogen with the different estrogen receptors within the central neural network that controls sympathetic tone (see Chapter 3). Protective effects of estrogen mediated within the brain involve antiinflammatory pathways (see Chapter 3). However, sex differences in blood pressure regulation may also involve differences in sympathetic activity-mediated effects on vascular resistance (see Chapter 19). Loss of β-adrenergic vasodilation at menopause in addition to increased sympathetic activity contributes to the increase in blood pressure that occurs in women as they age (see Chapter 19). Sodium intake differs by sex and central-mediated mechanisms also contribute to sex differences in the effect of dietary sodium intake on cardiovascular risk (see Chapter 5).

Estrogens contribute to cardiovascular homeostasis via actions mediated through their intracellular as well as membrane-bound receptors (see Chapter 2). Estrogen signaling contributes to endothelial dependent and independent vasodilatory responses, and modulates myocardial calcium handling and apoptosis in conditions of ischemia within the heart (see Chapter 2). Regulation of the RAS via estrogen also involves modulation of its angiotensin receptors (see Chapter 6). Up-regulation of the angiotensin type 2 receptor (AT2 receptor) may contribute to blood pressure control via its actions to induce vasodilation and induce natriuretic responses (see Chapter 6). Experimental studies suggest a sex difference in AT2 receptor function and that AT2 receptor-mediated cardioprotection in females is estrogen dependent (see Chapter 6). Multiple studies have also investigated the cardioprotective effects of estrogen after menopause. Yet, the beneficial effects of estrogenic signaling remain controversial and the role of estrogen replacement therapy in CVD remains to be fully elucidated (see Chapter 2). Studies involving the sex-specific role of androgens on cardiovascular health are also conflicting. Recent studies indicate that excess androgens in women are associated with increased risk for hypertension, obesity, endothelial dysfunction, and insulin resistance, risk factors for CVD (see Chapter 1). Although the mechanisms that accentuate the negative cardiovascular effects of androgens in women are not fully understood, androgens can alter expression of the RAS, endothelin, and 20-hydroxyeicosatetraeonic acid (see Chapter 1). Testosterone deficiency (hypogonadism) is well known to have deleterious effects on men’s cardiovascular health (see Chapter 18). Thus collectively, numerous studies indicate that androgens exert sex-specific effects on cardiovascular health and although androgens may be detrimental in women, they may be beneficial in men (see Chapter 18).

Therefore this book will focus on the consideration of sex, hormonal status in men and women, pregnancy history on cardiovascular health in the mother and her offspring in later life, and how considerations related to sex correlate to the diagnosis, treatment, and management of cardiovascular health and related comorbidities. Further studies investigating sex differences in CVD are warranted in order to provide considerations for improvement in the sex-specific prevention and treatment of CVD in men and women across their lifespan.

References

[1] Mosca L., Barrett-Connor E., Wenger N.K. Sex/gender differences in cardiovascular disease prevention: what a difference a decade makes. Circulation. 2011;124(19):2145–2154.

[2] Wilkins J.T., Ning H., Berry J., Zhao L., Dyer A.R., Lloyd-Jones D.M. Lifetime risk and years lived free of total cardiovascular disease. JAMA. 2012;308(17):1795–1801.

[3] Mehta L.S., Watson K.E., Barac A., Beckie T.M., Bittner V., Cruz-Flores S., Dent S., Kondapalli L., Ky B., Okwuosa T., Piña I.L., Volgman A.S., American Heart Association Cardiovascular Disease in Women and Special Populations Committee of the Council on Clinical Cardiology; Council on Cardiovascular and Stroke Nursing; and Council on Quality of Care and Outcomes Research. Cardiovascular disease and breast cancer: where these entities intersect: a scientific statement from the American Heart Association. Circulation. 2018;137(8):e30–e66.

[4] Benjamin E.J., Blaha M.J., Chiuve S.E., Cushman M., Das S.R., Deo R., de Ferranti S.D., Floyd J., Fornage M., Gillespie C., Isasi C.R., Jiménez M.C., Jordan L.C., Judd S.E., Lackland D., Lichtman J.H., Lisabeth L., Liu S., Longenecker C.T., Mackey R.H., Matsushita K., Mozaffarian D., Mussolino M.E., Nasir K., Neumar R.W., Palaniappan L., Pandey D.K., Thiagarajan R.R., Reeves M.J., Ritchey M., Rodriguez C.J., Roth G.A., Rosamond W.D., Sasson C., Towfighi A., Tsao C.W., Turner M.B., Virani S.S., Voeks J.H., Willey J.Z., Wilkins J.T., Wu J.H., Alger H.M., Wong S.S., Muntner P., American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2017 update: a report from the American Heart Association. Circulation. 2017;135(10):e146–e603.

[5] Health United States, 2010: With special feature on death and dying. National Center for Health Statistics (US). Hyattsville, MD: National Center for Health Statistics (US); 2011. Report No. 2011-1232.

[6] Fairweather D., Frisancho-Kiss S., Rose N.R. Sex differences in autoimmune disease from a pathological perspective. Am J Pathol. 2008;173(3):600–609.

[7] Durante A., Bronzato S. The increased cardiovascular risk in patients affected by autoimmune diseases: review of the various manifestations. J Clin Med Res. 2015;7(6):379–384.

[8] Corenlius. Preeclampsia: from inflammation to immunoregulation. Clin Med Insights Blood Disord. 2018;11: 1179545X17752325.

[9] Bellamy L., Casas J.P., Hingorani A.D., Williams D.J. Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ. 2007;335:974.

[10] Bergen N.E., Schalekamp-Timmermans S., Roos-Hesselink J., Roeters van Lennep J.E., Jaddoe V.V.W., Steegers E.A.P. Hypertensive disorders of pregnancy and subsequent maternal cardiovascular health. Eur J Epidemiol. 2018;33(8):763–771.

[11] Kawasaki. Type 1 diabetes and autoimmunity. Clin Pediatr Endocrinol. 2014;23(4):99–105.

[12] Grenon S.M., Vittinghoff E., Owens C.D., Conte M.S., Whooley M., Cohen B.E. Peripheral artery disease and risk of cardiovascular events in patients with coronary artery disease: insights from the Heart and Soul Study. Vasc Med. 2013;18(4):176–184.

Part I

Sex Steroid Receptors

Chapter 1

Androgens and Cardiovascular Diseases in Women

Licy L. Yanes Cardozo⁎,†; Edgar D. Torres Fernandez⁎; Damian G. Romero⁎; Jane F. Reckelhoff⁎    * Department of Cell and Molecular Biology, Mississippi Center of Excellence in Perinatal Research and Women's Health Research Center, University of Mississippi Medical Center, Jackson, MS, United States

† Department of Medicine/Endocrinology, Mississippi Center of Excellence in Perinatal Research and Women's Health Research Center, University of Mississippi Medical Center, Jackson, MS, United States

Abstract

The National Institutes of Health has recently mandated that all preclinical studies are required to use both male and female animals in their studies. While there are many opinions, both negative and positive about this mandate, it is undeniable that the data generated should vastly improve our understanding of sex differences in cardiovascular diseases. Numerous studies have evaluated how estrogens impact cardiovascular mechanisms by both genomic and nongenomic processes. Although there are studies available on how androgens impact cardiovascular functions in men, fewer studies of androgen’s effect in women are available. In this review, we will present what is currently known regarding androgen-mediated mechanisms that control cardiovascular disease and hypertension in women and female animal models, and identify the gaps in knowledge and the opportunities for future research.

Keywords

Polycystic ovary syndrome; Postmenopausal hypertension; Androgens; Cardiovascular disease; Blood pressure; Androgen receptor; Obesity

Introduction

Although there are several known sex differences in the cardiovascular system, under normal and pathological conditions, many questions still remain to be answered. Until recently, it was incorrectly assumed that men and women share the same physiological traits. Despite the increase in research leading to knowledge that this assumption is not accurate, most therapeutic guidelines have not been changed to accommodate sex differences.

From a simplistic point of view, biological sex differences at any levels may be explained by genetic factors, such as chromosomal effects, and/or sex hormones. While we acknowledge that environmental factors may also contribute to sex differences, for the purposes of this review, we will focus on hormones and genetics. The main sex hormone in men are androgens and in women, estrogens. Interestingly, the plasma concentration of testosterone in women is higher than estradiol; however, whether and how androgens may affect the cardiovascular system in women remains unclear. There are several clinical scenarios, both common and uncommon, in women where plasma androgen levels are elevated. These can include Polycystic Ovarian Syndrome (PCOS), Congenital Adrenal Hyperplasia, adrenal and ovarian tumors, hormone replacement therapy, and androgen anabolic use in recreational and professional bodybuilders, female-to-male transsexual individuals, menopause, and the undesired side effects of some medications, such as valproic acid. Therefore it is imperative to understand the physiological and pathophysiological effects of elevated levels of androgens in women.

Importantly, excess androgens in women are correlated with increased risk for developing major risk factors for cardiovascular disease including hypertension, obesity, endothelial dysfunction, insulin resistance, inflammation, and fatty liver and cardiac hypertrophy. Furthermore, the mechanisms that underline the negative cardiometabolic effects of androgens in women are not fully understood.

We have extensively characterized the cardiometabolic profile of two experimental models whereby androgens are elevated in female rats: the aging female spontaneously hypertensive rat (SHR), a model of postmenopausal hypertension (referred to in this review as the PMR), and the hyperandrogenemic Sprague-Dawley female rat (HAF), a model of PCOS. Based on our data and a review of the literature, this chapter will highlight the fundamental role that androgens play in mediating the increase in cardiometabolic risk factors in women, discuss some of the mechanisms that may underline the negative androgenic effects, and relate unanswered questions and opportunities for additional research. Results from these studies may lead to more effective and safer therapeutic agents to treat androgen-mediated cardiometabolic abnormalities in women.

Androgens and Cardiovascular Disease in Women

There are sex differences in cardiovascular disease and major cardiovascular risk factors. The concept that the prevalence of coronary artery disease (CAD) in premenopausal women is less than in men has had a major impact on the current management of CAD in women. Although less prevalent, once initiated, the morbidity and mortality of CAD in women is much worse than in men, making gender-specific clinical management important. In support of this concept, there is exciting new data from a prospective survey that aimed to determine sex differences in the management and clinical outcomes in acute coronary syndromes in Israel. This study showed that women, aged 55 years or younger, received less invasive therapy during hospitalization for acute coronary syndrome; at discharge had underprescription of ACEIs, statins, and aspirin by caregivers; and had a 5-year mortality rate that was higher than in men [1]. Similar observations were reported in the EUROASPIRE III study that was carried out in 22 European countries, showing that despite similarities in medication exposure, women were less likely than men to achieve blood pressure (BP), LDL-cholesterol, and HbA1c targets after a coronary event [2]. A follow-up study, EUROASPIRE IV by the European Society of Cardiology, reported not only that the clustering of cardiovascular risk factors was higher in females, but also that on individual risk factors, women performed worse than men. Importantly, the gender gap decreased with increasing educational level, but increased with patients’ age [3]. These data suggest that the pathophysiology underlying cardiovascular diseases in men and women is different, and thus therapeutic guidelines need to be changed to provide the best possible care for women.

Diabetes Mellitus is a major cardiovascular risk factor. It is well known that female patients with Diabetes Mellitus have a higher cardiovascular risk than do male patients [4]. In addition, women with Type 2 Diabetes Mellitus present higher cardiovascular risk factors, myocardial infarction, and stroke mortality than men, compared with nondiabetic subjects. Diabetes Mellitus appears to attenuate the protective effect of female sex in the development of cardiac diseases and nephropathy [5]. Patients with Type 2 Diabetes Mellitus have a higher level of circulating free testosterone that may be related to a defect in aromatase activity or an increase in the production of androgens [5]. Taking all of these studies into consideration, it is clear that more research is needed to elucidate the role of androgens in mediating cardiometabolic diseases in women.

Androgens and Blood Pressure Regulation in Women

PCOS is the most common endocrine disorder that affects women in their reproductive years. The definition of PCOS and diagnosis remain controversial as three different diagnostic criteria are available for clinicians and researchers [6–8]. Each criterion is based on a different combination of hyperandrogenism and/or hyperandrogenemia, ovulatory dysfunction, and polycystic ovarian morphology. About 80% of women with PCOS have hyperandrogenemia and the level of testosterone is about 1.5-fold higher compared to normal cycling women. Interestingly, PCOS women with elevated androgen levels have a worse cardiometabolic profile compared to women with PCOS with normal levels of androgens [6, 9–13].

Hypertension is a major risk factor for cardiovascular disease [14, 15]. Several clinical studies showed that increased BP or the incidence of hypertension is significantly elevated in PCOS women [16–22]. Moreover, the higher prevalence of hypertension in PCOS women is observed across multiple ethnic groups [16, 19]. The level of androgens positively correlates with BP in a cohort of PCOS women [23]. Recently, a large case-control study, including 1550 PCOS women and 447 control women, showed that PCOS women present both systolic and diastolic BP increases [24].

Women with PCOS are not always treated with antihypertensive drugs since their BP may not reach the levels required by the guidelines (JNC VIII). However, longitudinal data obtained from the Framingham Heart Study indicated that BP values between 130–139/85–89 mmHg, are associated with more than a twofold increase in relative risk for cardiovascular disease, as compared with BP levels below 120/80 mmHg, suggesting that an elevation in BP, even within the upper normal range, increases the risk of cardiovascular disease [25]. Because many women with PCOS desire to become pregnant, the treatment of hypertension is challenging, as antihypertensive agents, such as renin-angiotensin system blockers, are teratogens. The development of novel and effective therapeutic approaches that can be safely used by PCOS women desiring to become pregnant is desperately needed.

Postmenopausal women make up one of the fastest growing populations in the United States. Various studies report that serum testosterone is increased in some, but not all, postmenopausal women [26]. BP is typically lower in premenopausal women than in men [27]. However, after menopause, the prevalence of hypertension in women is higher than it is in men. Hypertension is a major risk factor for cardiovascular disease in women and men. Furthermore, there is evidence that BP may not be as well controlled in women as in men, despite the fact that most women adhere better to their therapeutic regimens and medications than do men, and have their BP measured more frequently than do men. The Women’s Health Initiative (WHI) report was based on nearly 100,000 women, ages 50–79 years, in the United States. This report showed that although older hypertensive women (aged 70–79 years) were as likely to be treated for hypertension (63.2%) as younger women (64.2%), a substantially smaller percentage of them had their BP under control (i.e., < 140/90 mmHg; 29.3% versus 41.3% for the older versus younger women, respectively) [28]. Similar findings were observed when the results from the National Health and Nutrition Estimation Survey (NHANES) III data set (ending 1994) were compared with NHANES IV data set (ending 2004) [29]. A better understanding of the complex pathogenesis of postmenopausal hypertension and the effect of androgens is needed in order to offer the best therapeutic options to better treat hypertension and cardiovascular diseases in women.

The Hyperandrogenemic Female Rat, a Model of Polycystic Ovary Syndrome

As mentioned before, PCOS is the most common endocrine disorder that affects young women during their reproductive years. The etiology of the syndrome is unknown. Several epidemiological studies show a positive correlation between the plasma levels of androgens and BP [23], obesity [30], insulin resistance [31], and endothelial dysfunction [32] in PCOS women. Moreover, a reduction in testosterone is associated with improvement in dyslipidemia [33], endothelial dysfunction, body weight, and insulin resistance in PCOS women [34]. The effect of an elevated level of androgens in female rats was described by pioneer studies done by Manneras and colleagues [35]. In this model, female normotensive Sprague-Dawley rats are implanted with dihydrotestosterone (DHT), a nonaromatizable androgen (7.5 mg pellet/90 d) beginning at 4 weeks of age. They reported that the administration of DHT caused many symptoms found in PCOS women, such as gain weight, irregular estrous cycle, insulin resistance, and an increase in adiposity. We further demonstrated that administration of DHT with the same protocol causes an approximate three- to four-fold increase in plasma DHT with no change in estradiol levels compared with placebo-treated controls, an increase in food intake (∼ 3 g of food/day), and the development of body weight ∼ 20%–30% heavier than placebo controls [36]. The increase in body weight is associated with an increase in lean and fat mass. In addition, hyperandrogenemic female (HAF) rats exhibit dyslipidemia and increases in leptin [36]. We also reported that DHT administration causes a mild but consistent increase of BP (10 mmHg), measured by radiotelemetry in freely moving conscious animals, and renal injury.

Excessive hair growth or hirsutism is a common clinical manifestation among PCOS women [37]. Androgen receptor blockers ameliorate hirsutism in PCOS women although they are considered as second line of therapy for hirsutism after oral contraceptives and insulin sensitizers. In the United States, the androgen receptor blocker most commonly used in the clinic is spironolactone [38]. Spironolactone is a nonspecific mineralocorticoid receptor blocker that also binds to the progesterone receptor. Usually, a high dose of spironolactone is necessary to block the androgen receptor, which potentially could lead to higher incidence of undesired side effects. Other potent antiandrogens are flutamide and cyproterone acetate. At present, cyproterone acetate is not available in the United States for therapeutic use, and flutamide is associated with severe hepatoxicity in hyperandrogenemic women [39] and is not currently FDA approved for use in PCOS women in the United States. The development of more efficient and specific androgen receptor blockers will positively impact the management of the cardiometabolic risk factors in women with PCOS.

The HAF rat thus provides a means to study the different mechanisms by which androgens mediate cardiovascular diseases, and could provide the preclinical basis for the development of better therapeutic agents that are desperately needed to treat cardiometabolic abnormalities in PCOS women, and also in other clinical conditions where androgens are elevated.

The Aging Female Spontaneously Hypertensive Female Rat (PMR)

Premenopausal women experience a lower prevalence of cardiovascular disease (CVD) compared with age-matched men [40, 41]. Whether this protection is due to the presence of female sex hormone, estrogen, or a lower concentration of androgens compared to age-matched men is unknown. On the contrary, after menopause, the prevalence of CVD and mortality in women is higher than in men [42].

The aged postestrous cycling postmenopausal female SHR (PMR) [43] is a very useful model to study mechanisms involved in postmenopausal hypertension. This unique model presents with two main features of menopause, aging and a gradual decline of plasma estradiol. At 4 months of age, male SHR have BP that is 25–30 mmHg higher than aged-matched females. PMR stop cycling at 10–12 months of age, as defined by 8 weeks of continuous estrus-looking cells on vaginal smears. At 18 months of age BP is significantly higher in PMR compared to young female SHR. Thus by 18 months of age, the sex difference in BP in SHR is no longer present as the BP is not different between PMRs and age-matched male SHR. BP does not change significantly in male SHR from 8 to 18 months. Renal hemodynamics are also altered in the experimental postmenopausal rodent model of the aged SHR (PMR) [43]. Glomerular filtration rate (GFR) and renal plasma flow (RPF) are reduced in association with an increase in renal vascular resistance (RVR) in the PMR suggesting the presence of an increase in renal vasoconstrictors or decrease in vasodilators. Therefore the loss of the sex difference in BP is not due to a reduction in BP with age in males but rather to an increase in BP in aging females, similar to what is reported in human studies.

In association with the increase in BP in PMR, plasma levels of testosterone are significantly elevated compared to their younger counterparts [43]. Serum testosterone is shown to be increased in some, but not all, studies of postmenopausal women [26]. The kidney of postmenopausal women can synthesize testosterone and dihydrotestosterone since it contains the cytochrome P450 enzymes that are necessary for its biosynthesis [44]. Whether tissue-derived androgen production contributes to plasma levels of androgens after menopause remains unknown.

Following we will describe possible mechanisms by which androgens could elevate BP in female rats, and potentially in women, and subsequently mediate cardiovascular diseases.

Mechanisms by Which Androgens Can Increase BP in Women

Renin-Angiotensin-Aldosterone System

A major system that regulates BP and body fluid volume is the renin-angiotensin system (RAS). The biological effects of Angiotensin II (Ang II) are mediated through the angiotensin type 1 receptor (AT1R) causing vasoconstriction, aldosterone and vasopressin release, salt and water retention, sympathetic activation, and oxidative stress, or after conversion to Ang (1–7) and via angiotensin type II receptors (AT2R), vasodilation and, salt and water excretion.

Sex differences in the RAS are well described for humans and in animal models [45, 46]. Women with PCOS have elevated levels of renin that positively correlate with hyperandrogenemia [47]. We have shown that androgen replacement in castrated male rats increases renin and angiotensinogen synthesis [48]. Moreover, androgens also mediate a portion of the salt-sensitive hypertension in Dahl salt-sensitive rats [48]. If renin enzyme activity is below Vmax, as has been shown in both humans and rats, then an increase in angiotensinogen will cause an increase in the conversion of angiotensinogen to angiotensin I, leading to an increase in Ang II production, since renin, not angiotensin-converting enzyme (ACE), is the rate-limiting enzyme for Ang II production [49].

Testosterone stimulates renal angiotensinogen expression in rat kidney [50] and DHT is also shown to stimulate renin expression in adrenal glands from mice [51]. We also found that renal angiotensinogen is upregulated in the HAF rat, suggesting that the intrarenal RAS is activated in this model [36]. Whether activation of the RAS plays a role mediating hypertension in PCOS women is unknown, but Telmisartan, an Angiotensin type 1 receptor (AT1R) antagonist, significantly reduces BP in PCOS patients [52], and AT1R blockers or ACE inhibitors are widely used and are effective antihypertensive drugs in the general hypertensive population. Women should be advised about the potential teratogenic risks of ACE inhibitors or AT1R blockers if they become pregnant. Novel and tissue-selective RAS inhibitors that do not cross the placental barrier are warranted to ameliorate the increases in BP in women with PCOS in the future.

We previously reported that physiological concentrations of sex steroids do not modify aldosterone secretion by human adrenal cells [53]. However, supraphysiological concentrations of DHT stimulate aldosterone secretion by human adrenal via calmodulin/calmodulin-dependent protein kinase (CaMK) and protein kinase C pathways but independently of the classical androgen receptor since the DHT-mediated effect is not altered by flutamide. Whether physiological or supraphysiological concentration of plasma androgens has any effect on synthesis of Ang(1–7), ACE2, or the AT2R is not known.

In aging female SHR (PMR), as mentioned previously, BP is similar or higher than in age-matched males [43]. Blockade of the RAS with ACEI normalizes BP in aging males, but reduces BP by only 30% in PMR [54], suggesting that multiple mechanisms are responsible for the elevated BP in PMR.

Endothelin System

Endothelin-1 is a potent vasoactive agent that mediates vasoconstriction and vasodilation via Endothelin type A (ETA-R) and Endothelin type B (ETB-R) receptors, respectively. Insights into the mechanisms by which androgens may promote endothelial dysfunction in PCOS women came from elegant studies performed by Wenner and colleagues. This group demonstrated that women with PCOS have lower ETB-R-mediated vasodilation in skin compared to BMI-matched control subjects [55]. In follow-up studies, this group demonstrated that suppression of the chronic elevation of testosterone improves microvascular function in PCOS women [56]. In short, these data demonstrate the key role of testosterone in mediating endothelial dysfunction in PCOS women.

Activation of the endothelin system is present in postmenopausal hypertension, and in one study, a positive correlation was found between endothelin and testosterone levels. Along the same line, in female-to-male transsexuals, testosterone treatment increases plasma endothelin levels [57]. In support of this notion, we previously reported that a portion of the hypertension in PMR is mediated by activation of the endothelin system, since the endothelin type A receptor antagonist, ABT627, decreases BP [58]. We found that the levels of endothelin are elevated in the kidney in PMR compared to young females. However, the use of ABT627 only reduced BP about 20 mmHg, and BP remained significantly higher in the aged PMR than in young female rats. Ang II is shown to increase expression of preproendothelin in the kidney [59]. Since androgens increase angiotensinogen and PRA, it is possible that androgens could increase Ang II leading to increased endothelin, and subsequent hypertension and renal injury. Alternatively, since estradiol can downregulate AT1R expression [60], estrogen replacement therapy may also prevent an increase in endothelin and protect against renal injury.

Sympathetic Nervous System

The sympathetic nervous system is activated in women with PCOS [61, 62]. The increase in sympathetic drive and impaired endothelial function seems to be independent of obesity and metabolic disturbances [63]. There is also evidence that the renal sympathetic nervous system may also be upregulated. Schlaich and colleagues reported that radiofrequency renal nerve ablation reduced BP in two hypertensive young women with PCOS [61]. We recently showed that elevated BP in HAF rats is due, in part, to activation of the sympathetic nervous system since administration of an α1,β1,2-adrenergic receptor antagonists reduces their BP [64]. Moreover, renal denervation in HAF rats significantly attenuated the androgen-mediated increase in BP suggesting a critical role of the renal nerves in BP increase in this animal experimental model [64]. The mechanism by which the sympathetic nervous system is activated in women with PCOS or in HAF rats is not clear. Hall and colleagues reported that obesity-related hypertension and the concomitant sympathetic activation are mediated, in part, by the melanocortin-4-receptor (MC4R) in the brain, since intracerebroventricular (icv) infusion of the MC4R antagonist, SHU-9119, reduces BP [65–67]. We found that MC4R receptor expression in the hypothalamus is significantly upregulated in the PCOS model compared to controls, and that icv administration of SHU 9119 reduces their BP to normotensive range without any effect on BP in control rats [64]. Moreover, the critical role of the MC4R in mediating the increase in BP by androgens was confirmed when the administration of DHT to MC4R KO rats failed to increase BP, despite increases in body weight [64]. Therefore targeting the MC4R could be a novel therapeutic tool to ameliorate the cardiometabolic risk factors in PCOS.

The sympathetic nervous system is also activated in postmenopausal hypertension. We recently reported in the aged SHR model of postmenopausal hypertension, the PMR, that renal denervation significantly reduces BP in the aged female SHR compared to sham animals [68]. Activation of the RAS in this model is independent of the sympathetic nervous system as administration of losartan, an AT1R blocker, after renal denervation further reduced BP in the aged female SHR, despite BP remaining significantly elevated in the aged female SHR [69]. These data suggest that both the renal sympathetic nervous system and the RAS play independent roles in mediating hypertension in the aged female SHR. The mechanism responsible for the activation of the sympathetic nervous system is different in the aged female SHR than in the HAF rats since icv blockade of the MC4R in the aged female SHR model of PMR has no effect on their BP, but does reduce the BP in HAF rats [64]. Thus future studies into the mechanisms responsible for sympathetic nervous system activation in aging women and in women with PCOS are warranted.

20-HETE

The effect of 20-hydroxyeicosatetraeonic acid (20-HETE) on BP is well established [70, 71]. 20-HETE is produced from arachidonic acid conversion by cytochrome P450 ω-hydroxylase. 20-HETE has differential effects on the kidney depending on the location of synthesis. In the renal microvasculature, 20-HETE promotes hypertension, acting as a vasoconstrictor, whereas in the renal tubules, 20-HETE attenuates Na+ reabsorption, promoting natriuresis and thus is antihypertensive [72].

20-HETE plays a role in androgen-mediated hypertension (Miki Schwartzman and Capdevila). We recently demonstrated that in DHT-treated HAF rats, the well-established rodent model of PCOS, the cytochrome P450 (CYP) ω-hydroxylase isoform CYP4A2 is upregulated in association with an increase in endogenous renal microvascular 20-HETE [73]. Supporting a role for 20-HETE in androgen-mediated increases in BP, DHT fails to increase BP in 20-HETE-deficient Dahl salt-sensitive (SS) female rats, whereas DHT does increase BP in Dahl salt-resistant (SR) females [73]. Thus an intact 20-HETE system is necessary for the androgen-mediated increases in BP in the PCOS model. The enzyme CYP4A2 ω-hydroxylase seems to be a critical component to the increase in BP since the lack of this enzyme prevents the DHT-mediated pressor response in CYP4A2 null female rats. Taken together, these data suggest that 20-HETE contributes to the DHT-mediated elevation in BP in the PCOS model, and that the CYP4A2 isoform is partially responsible for the increase in BP. These data also suggest that metabolites of arachidonic acid constitute a novel pathway that may be involved in mediating the BP elevation in androgen-mediated hypertension in women, as observed in the PCOS model, and may thus provide a novel therapeutic pathway for future development.

20-HETE also plays a role in hypertension in PMR model. In this model the extra renal 20-HETE concentration is increased in PMR compared to young females. Omega-hydroxylase activity is also higher in cerebral vessels of PMR compared with young females. Finally, 20- HETE blockade decreases but does not normalize BP in the PMR model [74] suggesting that 20-HETE is a component, but not the major mediator, of the mechanisms responsible for the elevated BP in the aging female SHR. Our data emphasize that multiple mechanisms are involved in postmenopausal hypertension.

Oxidative stress

Studies indicate that oxidative stress is increased in postmenopausal women [75]. We previously reported that plasma F2-isoprostanes are elevated in PMRs compared with young females, but is similar in old males [43]. However, PMRs excrete 200% more F2-isoprostanes than do old males, suggesting either differences in renal handling of F2-isoprostanes between PMRs and old males or more renal production of F2-isoprostanes in old females than males. Where and how oxidative stress impacts BP in postmenopausal hypertension is unclear since antioxidants do not reduce BP in old female SHR [43]. There is a clear sex difference in oxidative stress-mediated control of BP in the SHR since antioxidants do reduce BP in old and young males [43, 76]. Studies in hypertensive humans show an inconsistency to demonstrate a reduction in BP with antioxidants [77] calling into question the role of oxidative stress in mediating hypertension in humans. However, it is not clear whether those studies evaluated the data separately for men and women. Therefore it is possible that antioxidants reduce BP in men, but not in women which would be consistent with our published studies.

Androgens and Obesity in Women

The menopause transition, as well as the early postmenopausal period, is associated with an increase in total and central obesity. Increased visceral fat is associated with insulin resistance, and this preferential storage of abdominal fat may contribute to cardiovascular disease and Type 2 Diabetes Mellitus that is often present in postmenopausal women [78]. Weight gain and obesity may be the driving factor by which BP and metabolic syndrome increased after menopause. Plasma levels of bioavailable testosterone are positively correlated with increased visceral fat during perimenopausal transition [79]. We found there is a significant increase in fat deposition in the abdominal cavity of the PMR model [80]. Adiposity is also increased in the PCOS model [36]. Serum androgens are positively correlated with BMI not only in PCOS, but also in simple obesity in women [81]. Weight loss via lifestyle modification, pharmacotherapy, or bariatric surgery reduces androgen levels in women with PCOS [82, 83]. Furthermore, the concentrations of androstenedione, DHEA, and testosterone in female adipose tissue are several folds higher than in plasma [84–87].

The adipose tissue is able to activate androgens via the 17β-hydroxysteroid dehydrogenase (17β-HSD). This enzyme plays a critical role in the biological activity of estrogens and androgens by catalyzing the reduction of 17-ketosteroids or the oxidation of 17β-hydroxysteroids, using NAD(P)H or NAD(P) as cofactors [88]. Currently, fourteen 17β-HSD isoenzymes are identified in mammals. The enzymatic activity of each isoform is tissue specific and varies from steroidogenic to steroid-inactivating properties. The 17β-HSD type 3 and 5 are the main isoforms that can convert androstenedione (a weak androgen) to testosterone (a potent androgen). Both isoforms are highly expressed in subcutaneous adipose tissue. 17β-HSD type 5 (AKR1C3) is more highly expressed in subcutaneous than visceral adipose tissue [89]. Since adipocyte size and differentiation significantly increase the expression of 17β-HSD type 5, one could speculate that weight gain likely results in an intraadipose increase in androgen levels [90]. Enhancement in the steroidogenic activity of subcutaneous adipose tissue in PCOS is demonstrated in some recent studies [91, 92]. We have found that subcutaneous fat is significantly increased in our PCOS model both when the rats are young and with aging when visceral adiposity also increases [93]. The kidney of postmenopausal women also has all the enzymes needed to synthesize testosterone and is thought by some to be a major source of androgens after menopause [44]. However, the role of locally synthesized androgens in mediating cardiovascular disease and hypertension in women is unknown.

Androgens and Androgen Receptor Action in Women

Androgens exert both genomic and nongenomic actions [94, 95]. Genomic actions of androgens are mediated through the classic androgen receptor, which is a 110-kDa protein composed of multiple domains for ligand (i.e., androgen) binding, DNA binding, and transactivation. The ligand-bound classic androgen receptor mainly functions as a transcription factor modulating the expression of androgen receptor target genes via androgen response elements (AREs). On the other hand, the nongenomic actions of androgens include an acute increase in intracellular calcium, and activation of protein kinases, such as Src tyrosine kinase (c-Src), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphatidylinositol 3-kinase (PI3K) [96–101]. While there have been a significant number of studies on how estrogens impact both genomic and nongenomic cardiovascular events and processes, there are relatively few studies on genomic and nongenomic effects of androgens in males, much less in females, and this provides a wide-open area of research that would make significant progress toward our understanding of the role of androgens in mediating cardiovascular disease and hypertension in women.

Summary

There is a current trend and mandate by National Institutes of Health to include both sexes in basic and clinical research [102–106]. However, in clinical practice, hypertension in women is treated identically as men. Consequently, there is a big gap between what we know in research and what is applied at the bedside. Research may shed light into better understanding of the role of androgens in mediating cardiovascular diseases in women. Challenges are also opportunities, and one thing is clear, there are plenty of opportunities to provide better medical care to women.

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