Testosterone and cardiovascular risk in men.

Granata R, Isgaard J (eds): Cardiovascular Issues in Endocrinology. Front Horm Res. Basel, Karger, 2014, vol 43, pp 1–20 (DOI: 10.1159/000360553)

Testosterone and Cardiovascular Risk in Men Daniel M. Kelly a · T. Hugh Jones a, b

a

b

Department of Human Metabolism, Medical School, The University of Sheffield, Sheffield, and Centre for Diabetes and Endocrinology, Barnsley Hospital NHS Foundation Trust, Barnsley, UK

Abstract Testosterone deficiency is highly prevalent in men with cardiovascular disease (CVD) and is associated with an increased mortality. Low testosterone also has an adverse effect on several cardiovascular risk factors, which include insulin resistance, diabetes, dyslipidaemia, central adiposity and endothelial dysfunction. Male gender is a well-recognised risk factor for premature CVD and mortality. The question of whether or not testosterone deficiency is a contributory factor to atherogenesis or merely a biomarker of ill health arises. Animal studies and experiments on isolated cells indicate that many of the mechanisms intimate to the atherosclerotic process are beneficially modulated by testosterone. Epidemiological studies have shown that men with endogenous testosterone levels in the mid-upper normal range have reduced cardiovascular events and mortality compared to those with low or lower range, and with high range testosterone. Testosterone replacement in men diagnosed with hypogonadism where mid-normal range levels are achieved have shown a beneficial effect on several cardiovascular risk factors, cardiac ischaemia, functional exercise capacity and improved mortality. Yet studies where patients were either undertreated or given high-dose testosterone have been associated with an increased risk of cardiovascular-related events. Clinical monitoring and titration of testosterone dose is therefore of paramount importance. © 2014 S. Karger AG, Basel

Although cardiovascular disease (CVD) affects both sexes, more than twice as many men are affected compared to women, establishing male gender as an important risk factor. This relationship persists at all ages and is not explained by differences in the frequency of standard risk factors between men and women, such as smoking, hypertension, diabetes and hypercholesterolemia. For many years, this higher cardiovascular risk in men incited the premise that testosterone is ‘bad for the heart’ and was supported by case reports of sudden cardiovascular death amongst male athletes abusing anabolic steroids. Currently, however, a large amount of evidence suggests that low

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Epidemiology

levels of testosterone, rather than testosterone per se, are associated with cardiovascular morbidity and mortality, and testosterone deficiency has emerged as an independent cardiovascular risk factor [1]. Testosterone and Coronary Heart Disease A large number of cross-sectional epidemiological studies have been performed assessing testosterone status in men with coronary heart disease (CHD). Over the years, these studies have used different definitions of CHD and also measured various fractions of testosterone. In the majority of cases, testosterone was low in those with CHD, but a significant number did not find any correlation (table 1). Those studies which measured the biologically active components of testosterone (free or bioavailable free + albumin bound) rather than the total testosterone (includes sex hormone binding globulin-bound testosterone which is considered to be inactive) were more likely to report a correlation between low testosterone and presence of CHD. The importance of measuring a biologically active testosterone fraction was confirmed in a coronary angiographic study. This study compared men with >70% stenosis (i.e. flow limiting) of at least one artery with men who had normal angiograms. Men with CHD had significantly lower free and bioavailable testosterone whereas total testosterone approached but did not reach significance.

The MrOs (Osteoporotic Fractures in Men) study from Sweden is a large communitybased study originally set up to examine the effects of several parameters on fractures. When cardiovascular events were analysed they found that men in the higher quartile of testosterone had less events when compared to those in the lower three quartiles [2]. This study is particularly important as it measured testosterone by mass spectroscopy, which removes several questions in relation to differences and problems with immunoassays and is considered to be more accurate. A recent large (n = 3,650 men >65 years old) French study has identified a J-shaped association between both total and bioavailable testosterone and CVD events, with men in the lowest and highest total testosterone quintiles having an increased risk. In support of this finding, the HIM (Health in Men) study from Western Australia found that men with testosterone levels in the mid range had lower all-cause mortality; however, those with high normal testosterone and independently high dihydrotestosterone (DHT) had reduced CHD deaths [3]. A Japanese study of 171 middle-aged men (30–69 years) with any coronary risk factor without evidence of prior CVD followed for 77 months found those in the lower tertile of testosterone were associated with an increased risk of cardiovascular events. Furthermore, testosterone levels in an acute setting predict clinical outcomes

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Kelly · Jones Granata R, Isgaard J (eds): Cardiovascular Issues in Endocrinology. Front Horm Res. Basel, Karger, 2014, vol 43, pp 1–20 (DOI: 10.1159/000360553)


Cardiovascular Events and Mortality

Table 1. Cross-sectional studies investigating serum testosterone in men with CHD Author, year

Patients, n

Definition of CHD

Androgens measured

Zumoff et al., 1982 Luria et al., 1982 Labropoulos et al., 1982 Phillips et al., 1983 Heller et al., 1983 Medoza et al., 1983 Barth et al., 1983 Hromadova et al., 1985 Breier et al., 1985 Small et al., 1985 Franzen and Fex, 1986 Aksut et al., 1986 Sewdarsen et al., 1986 Chute et al., 1987 Hamalainen et al., 1987 Lichtenstein et al., 1987 Swartz and Young, 1987 Sewdarsen et al., 1988 Baumann et al., 1988 Slowinska-Srzednicka et al., 1989 Sewdarsen et al., 1990 Gray et al., 1991 Cengiz et al., 1991 Hauner et al., 1991 Rice et al., 1993 Phillips et al., 1994 Hautanen et al., 1994 Mitchell et al., 1994 Marques-Vidal et al., 1995 Feldman et al., 1998 Zhoa and Li, 1998 Kabakci et al., 1999 Schuler-Luttmann et al., 2000 English et al., 2000 Pugh et al., 2002

117 50 144 122 295 52 20 67 139 100 92 54 56 146 57 2,512 71 20 58 108 224 1,709 55 274 272 55 159 98 116 1,709 201 337 189 90 30

MI, CAD MI MI CHD CHD MI CAD ‘Coronary findings’ CAD IHD MI MI/angina MI CAD CHD IHD MI MI Atherosclerosis MI MI CAD MI, angina CAD MI CAD CAD MI MI Heart disease CAD CAD CAD CAD MI

TT TT TT TT TT TT TT TT TT TT TT TT TT/FT TT/FT TT/FT TT TT TT TT TT TT/FT TT/FT TT TT TT/FT TT/FT TT TT/FT TT TT/FT TT TT/FT TT/FT TT/FT/BT TT/BT

Androgen levels in CHD cohort

↔ ↔ ↔ ↔ ↔ ↓ ↓ ↓ ↓ ↔ ↔ ↓ ↓/↓ ↓/↓ ↓/↓ ↓ ↓ ↓ ↔ ↔ ↓/↓ ↓/↓ ↔ ↔ ↓/↓ ↓/↓ ↔ ↔/↔ ↔ ↔/↔ ↓ ↔/↔ ↔/↔ ↔/↓/↓ ↔/↓

after coronary events as men admitted with acute myocardial infarction demonstrated higher mortality after 30 days when their testosterone was low (table 2) [4]. Several community- and hospital outpatient-based studies have also reported that men with low circulating testosterone are at greater risk of all-cause mortality than those with higher levels. Specifically some studies have shown that those at

Testosterone and Cardiovascular Risk Granata R, Isgaard J (eds): Cardiovascular Issues in Endocrinology. Front Horm Res. Basel, Karger, 2014, vol 43, pp 1–20 (DOI: 10.1159/000360553)

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BT = Bioavailable testosterone; FT = free testosterone; IHD = ischemic heart disease; MI = myocardial infarction; TT = total testosterone; ↔ = no change; ↓ = decrease. Full references stated in the table are available in reference [4] and cannot be fully printed in the manuscript due to restriction to number of manuscripts that can be sited.

Table 2. Effect of baseline testosterone on CVD mortality Author, year (study name)

Patients, n Population studied

Follow-up period

CVD mortality HR/OR for TT unless indicated (95% CI)

Smith et al., 2005

2,512

Population based

16.5 years

HR 0.94 (0.80–1.11) (NS) IHD

Khaw et al., 2007 (EPIC-Norfolk)

2,314

Population based

7 years

OR quintile 2, 3, 4 vs. 1 CVD 0.89 (0.60–1.32) 0.60 (0.39–0.92) 0.53 (0.32–0.86) CHD 0.71 (0.43–1.17) 0.59 (0.39–1.00) 0.52 (0.28–0.97)

Araujo et al., 2007 (MMAS)

1,686

Population based

15.3 years

RR FT: 0.80 (0.64–0.99) p = 0.02 for trend

Laughlin et al., 2008 (Rancho Bernardo Study)

794

Population based

11.8 years

HR 1.38 (1.02–1.85)

Carrero et al., 2009

126

Haemodialysis

41 months

HR 3.19 (1.49–6.83)

Vikan et al., 2009 (Tromsö)

1,568

Population based

11.2 years

HR FT: 1.24 (1.01–1.54)

Haring et al., 2010 (SHIP)

1,954

Population based

7.2 years

HR 2.56 (1.15–6.52)

Menke et al., 2010 (NHANES III)

1,114

Population based

18 years

HR: baseline – year 9 FT: 1.53 (1.05–2.23) TT: NS Year 9–18: NS

Corona et al., 2010

1,687

Erectile dysfunction

4.3 years

HR 7.1 (1.8–28.6)

Malkin et al., 2010

930

CHD (positive angiogram)

6.9 years

HR BT: 2.2 (1.2–3.9) TT: 2.5 (1.2–5.3)

Haring et al., 2011

1,822

Men with CKD, albuminuria, kidney dysfunction

9.9 years

HR 2.01 (1.21–3.34)

Haemodialysis

37 months (median)

HR 2.92 (1.08–7.87)

Kyriazis et al., 2011

111

Lerchbaum et al., 2012

2,069

Coronary angiography referrals

7.7 years

HR 1.77 (1.23–2.55)

Hyde et al. [3], 2012 (HIM)

4,249

Population based

5.1 years

HR 1.71 (1.12–2.62)

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EPIC-Norfolk = European Prospective Investigation into Cancer in Norfolk; MMAS = Massachusetts Male Aging Study; SHIP = Study of Health in Pomerania; CKD = chronic kidney disease; TT = total testosterone; BT = bioavailable testosterone; FT = free testosterone; HR = hazard ratio; OR = odds ratio; CI = confidence interval; NS = not s ignificant. Most of the references for the studies in this table are available in reference [5].

i ncreased mortality are in the lower quartile of testosterone. Some studies were sufficiently powered to identify that the major cause of death was related to CVD. Other causes in a minority of reports found a significantly increased risk of respiratory and/or cancer deaths. A meta-analysis of these studies has confirmed that low testosterone is a biomarker for a future increased risk of all-cause and cardiovascular mortality and that a decrease of 2.18 nmol/l in testosterone was associated with a 35% increased risk of all-cause mortality and a 25% increased risk of cardiovascular mortality [5]. Androgen deprivation therapy (ADT) for prostate cancer is a unique situation where the direct effects of lowering testosterone can be observed. ADT in some studies increases the risk of CHD, incident diabetes and cardiovascular death. A scientific advisory by the American Heart Association, American Urology Association and the American Cancer Society has recommended that especially for men with known CHD all secondary prevention measures should be fully optimised [6].

Cardiovascular Risk Factors

The metabolic syndrome (MetS) is a condition characterised by a clustering of commonly associated cardiovascular risk factors strongly related to the future development of CHD, myocardial infarction, stroke and sudden cardiac death. It is defined as the presence of at least three risk factors from central obesity, impaired fasting glucose or glucose tolerance or known type 2 diabetes mellitus (T2DM), hypertension, hypertriglyceridaemia and low high-density lipoprotein (HDL) cholesterol. Insulin resistance is the central biochemical defect common between MetS and T2DM, and promotes the development of many of the risk factors that make up MetS, as well as inducing vascular endothelial dysfunction and a pro-inflammatory milieu. In turn, central obesity contributes to insulin resistance and is also considered an inflammatory condition. Therefore, a negative cycle of metabolic, vascular and inflammatory dysregulation evolves with these risk factors individually and collectively perpetuating the development of atherosclerosis and CVD. The association of testosterone with these cardiovascular risk factors is discussed below.

Obesity is associated with lower levels of total, bioavailable and free testosterone levels in several epidemiological studies using traditional measures such as body mass index (BMI) and waist circumference to assess central obesity. It is important to recognise that central obesity comprises both subcutaneous abdominal fat as well as visceral fat. Low testosterone is associated with increased accumulation of both these fat depots. Indeed, increased percentage body fat is a common clinical feature of hypogonadism,


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Obesity

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and subcutaneous and visceral fat mass are negatively correlated with serum testosterone. Studies using computed tomography, magnetic resonance imaging and dualenergy X-ray absorptiometry to provide more accurate methods of assessing visceral fat volume confirm an association between visceral fat and lower testosterone levels. A selected sample of 130 community-dwelling non-smoking men from the Quebec family study also found negative correlations between total testosterone and waist circumference, body fat mass, and visceral and subcutaneous fat. Studies have shown that substantial weight loss (mainly by bariatric surgery) but also smaller weight reduction with diet and exercise can significantly raise testosterone levels [for review see ref. 7]. At the same time, low serum total testosterone predicts the development of central obesity and accumulation of intra-abdominal fat, and prostate cancer patients undergoing ADT present increased central adiposity and percentage body fat and decreased lean mass. These findings support a bidirectional relationship between serum testosterone and obesity. To explain this relationship, Cohen proposed the hypogonadal-obesity cycle hypothesis. Testosterone is converted to 17β-oestradiol by the enzymatic activity of aromatase in adipose tissue. Falling testosterone promotes an increase in adipocyte number and fat deposition, which gradually leads to further suppression of testosterone levels. Experimental studies using pluripotent stem cells have demonstrated that testosterone stimulation favours differentiation down the myocyte linage, whereas testosterone deficiency promotes adipocyte development. This leads to a further increase in adipose tissue volume and, as a consequence, increases overall aromatase activity driving the cycle forward. Adipocytokines, which include TNF-α, IL-6 and leptin, result in inhibition of the negative feedback of testosterone on the hypothalamic-pituitary-testicular axis, also blocking luteinising hormone release, further reducing testosterone levels [8, 9]. Several studies have shown that testosterone replacement therapy (TRT) in obese men reduces BMI and visceral fat mass, and increases lean body mass, and therefore significantly improves body composition. The Moscow study, a double-blind placebo-controlled trial of 30 weeks of testosterone replacement to the normal range in 184 men suffering from both MetS and hypogonadism, showed significant decreases in weight, BMI and waist circumference. Other TRT studies have also reported improved body weight, BMI and waist circumference in men with MetS, T2DM and/or hypogonadism [for review see ref. 9, 10]. In a recent registry study of 255 hypogonadal men receiving long-term testosterone treatment (≤60 months), substantial and sustained weight loss was reported. Marked weight loss was observed in approximately 95% of all patients with the magnitude of change over time most notable in the excessively obese and obese, and less noted in the group with normal BMI [11]. The direct effects of testosterone on adipose tissue at the cellular level are not fully known, but evidence for several modes of action are postulated whereby testosterone inhibits adipogenesis, decreasing triglyceride uptake and storage and reducing lipid synthesis [8]. Adipocytes express androgen receptors with their density positively regulated by testosterone.

Dyslipidaemia The balance of circulating lipids is linked with obesity as central body fat is associated with modifications in lipoprotein distribution and composition, contributing to the risk of atherosclerosis. A disruption of this balance is associated with an increased cardiovascular risk. Elevated cholesterol, especially low-density lipoprotein (LDL) cholesterol, is a powerful risk factor for the premature development of atherosclerosis. Low levels of testosterone have been associated with a pro-atherogenic lipoprotein profile, characterised by high LDL cholesterol and triglyceride levels, and a negative correlation between testosterone and both total and LDL cholesterol has been observed in numerous cross-sectional studies [for review see ref. 12, 13]. ADT causes increases in total and LDL cholesterol and triglycerides whereas HDL cholesterol falls, although some studies, however, have not confirmed these associations. HDL cholesterol is known to be atheroprotective, with its major function stimulating reverse transport of cholesterol from the periphery to the liver for excretion. While it has been reported in both healthy and diabetic men that serum testosterone correlates positively with HDL cholesterol and its major constituent, apolipoprotein A-I, the relationship is complex and other studies observe no such associations. The majority of studies investigating the effects of testosterone on HDL cholesterol have reported no alteration or a slight decrease in blood levels following replacement therapy [12, 13]. The reasons for this effect and the differences seen in the studies may be due to the duration of the treatment period. In the short term, testosterone stimulation of reverse cholesterol transport may lead to increased shuttling of cholesterol to the liver and subsequent consumption of HDL cholesterol, with stabilisation of this effect occurring over longer periods of time. Indeed, longer-term studies have shown that after an initial fall in HDL cholesterol, levels return to baseline with time. Trials using the long-acting depot testosterone undecanoate injections have reported an increase in HDL cholesterol in hypogonadal men with T2DM or MetS. Meta-analyses of clinical trials in hypogonadal men report that significant reductions in total cholesterol and LDL cholesterol are associated with intramuscular TRT [12]. Of particular interest, in two studies in elderly men with coronary artery disease (CAD) already treated with statins, testosterone replacement results in a further small reduction in cholesterol and LDL cholesterol, indicating that TRT may be therapeutically beneficial beyond statin treatment alone [13].

Diabetes is well known to be associated with an increased risk of CVD, with hypertension, obesity, atherogenic lipid profiles and a pro-inflammatory status prevailing as common co-morbidities. Over the last 3 decades, cross-sectional studies have shown that men with T2DM are more likely to exhibit low testosterone levels and more than


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Diabetes

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one third of men with T2DM have a high prevalence of hypogonadism/symptomatic testosterone deficiency ≤40% [9, 10]. This was originally considered to be related to low levels of sex hormone binding globulin that are apparent in men with insulin resistance. A meta-analysis of studies up until 2005, however, found no significant decrease in sex hormone binding globulin in diabetic men although total testosterone levels were on average 2.66 nmol/l lower than in healthy controls. Significantly, free and bioavailable testosterone levels are also lower in men with T2DM [9, 10]. NHANES III (Third National Health and Nutrition Examination Survey Mortality Study) reported a four times greater likelihood of having diabetes for men in the lower tertile of free and bioavailable testosterone concentrations, even after adjusting for age, obesity and ethnicity. As with many epidemiological findings, some consideration has been given to the cause-effect relationship of testosterone and T2DM. Indeed, evidence from animal studies has indicated that low levels of testosterone in T2DM may not depend on testosterone and may be a result of poorly controlled diabetes. In chemically induced diabetes in rats, hyperglycaemia was accompanied by significantly lower testosterone levels. Normalisation of glucose levels with insulin administration was able to restore testosterone levels to normal. This inverse relationship between glycaemic control and testosterone is evident with ADT leading to reduced insulin sensitivity and deterioration of glycaemic control in men with pre-existing T2DM [for review see ref. 9]. A review of cross-sectional studies of men undergoing long-term (>12 months) ADT over the last 20 years reveals a higher prevalence of diabetes and MetS compared with controls, predisposing ADT patients to a higher cardiovascular risk. Abdominal obesity and hyperglycaemia in relation to low testosterone were considered responsible for this higher prevalence [14]. Recent evidence has shown that men with T2DM and low testosterone are 2.3 times more likely to die from all-cause and cardiovascular mortality [15]. TRT to achieve testosterone levels in the mid-normal range improved survival rates to that expected for diabetic men with normal endogenous testosterone. The TIMES 2 study, a large, randomised, double-blind, placebo-controlled study undertaken in eight European countries, demonstrated that physiological TRT in hypogonadal men with T2DM and/or MetS improved insulin sensitivity, and reduced total and LDL cholesterol after 6 months of treatment compared with placebo [9, 10]. The Rancho Bernardo prospective population-based study found a significant inverse relationship of low baseline testosterone with follow-up fasting and post-challenge glucose and insulin levels and homeostatic model assessment for insulin resistance 8 years later. These data are of great importance in light of the UK Prospective Diabetes Study, which showed just a 1% reduction in glycated hemoglobin (HbA1c) can reduce microvascular complications, including the risk of myocardial infarction, by 16% and peripheral vascular disease and cardiovascular death by almost 50%. The mechanisms by which testosterone promotes insulin sensitivity, regulates glucose control and potentially improves T2DM is still not fully understood. Muscle,

liver and adipose tissue are the three tissues primarily involved in glucose regulation and are responsible for the majority of the body’s insulin sensitivity. Investigation into the action of testosterone in these tissues offers some mechanistic insight [8]. Insulin-stimulated glucose uptake into muscle and adipose tissue is largely mediated by the Glut4 glucose transporter iso-form, which resides in membrane vesicles inside the cell until insulin receptor activation and signalling promote translocation to the membrane. Glut4 and/or insulin receptor signalling molecules were up-regulated in adipocytes and skeletal muscle cells following testosterone treatment in vitro, and insulin receptor expression was increased in liver cells stimulated with testosterone. Additionally, testosterone increased phosphorylation of Akt and protein kinase C, key steps in the insulin receptor signalling pathways for the regulation of Glut4 translocation in muscle. Glut4 expression in muscle, adipose tissue and liver is restored to normal levels in castrated rats following testosterone replacement and glucose uptake returned to wild-type levels. Beyond glucose uptake to reduce blood glucose levels, testosterone also builds up muscle glycogen content by increasing glycolysis and decreases glycogen breakdown in animal studies. This action may potentially be via enhanced activity of hexokinase and phosphofructokinase, key enzymes involved in glycolysis as well as glycogen synthase. Some experimental studies, however, have reported conflicting results on the regulation of glycolysis in different muscle groups, indicating that testosterone actions may be location specific [8]. Another potential mechanism for the beneficial effect of testosterone in diabetic men may be to increase the metabolic rate in skeletal muscle. A study of 60 men using the hyperinsulinaemic-euglycaemic clamp compared insulin resistance with testosterone status. Testosterone levels correlated positively with insulin sensitivity and aerobic capacity (VO2max). In skeletal muscle biopsies, the expression of genes involved in mitochondrial oxidative phosphorylation were related to testosterone levels, impaired glucose tolerance and the presence of T2DM. The largest difference in mitochondrial gene expression was found with ubiquinol cytochrome c reductase binding protein, showing a 20% reduction in tissue from men with T2DM and impaired glucose tolerance [8].

A negative correlation between testosterone and hypertension has been demonstrated in men. Some studies have found that testosterone reduces diastolic and, less frequently, systolic blood pressure [1]. Castration of male animals in models of diabetes has been shown to decrease blood pressure, with testosterone administration reversing this effect. Testosterone treatment has also been shown to increase blood pressure in some human studies [1]. Contradictory results in clinical trials may be due to heterogeneous study groups regarding age and prevalence of CVD, differences in the duration of androgen treatment and the type of androgenic preparation being used.


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Hypertension

An effect of testosterone on the renin-angiotensin system may also influence systemic blood pressure, yet a direct correlation between androgens and the renal-vascular system still needs to be shown in large clinical studies.

Endothelial dysfunction is characterised by a shift in the actions of the endothelium toward reduced vasodilation, and pro-inflammatory and prothrombic states, as key early events in the development of atherosclerosis. Impaired flow-mediated dilation (FMD) represents endothelial dysfunction and precedes the development of clinically apparent atherosclerosis; FMD predicts cardiovascular events independently of established atherosclerosis. Alterations in brachial artery diameter by ultrasound imaging allow non-invasive assessment of FMD and vascular reactivity. Current knowledge on the effects of testosterone on endothelial dysfunction is conflicting with the majority of data derived from small studies in individuals during exogenous testosterone administration. Testosterone treatment of hypogonadal men led to a reduction in FMD in some studies, and assessment of anabolic steroid use in male bodybuilders has yielded equivalent worsening of FMD. These individuals were otherwise relatively healthy allowing a positive influence of testosterone treatment that may only be observed when vascular reactivity is sufficiently impaired, as observed in CAD patients. Indeed, both high-dose intravenous testosterone and oral physiological testosterone treatment of men with CAD resulted in a significant increase in brachial artery FMD. Furthermore, hypogonadal men without CVD exhibit decreased flow- and nitrate-mediated brachial artery vasodilation, or no change following testosterone treatment or transdermal DHT administration [for review see ref. 16]. Although epidemiological studies are lacking, there are two studies which have demonstrated that lower total and free testosterone levels are associated with impaired FMD, findings that were independent of major cardiovascular confounders. In contrast, prostate carcinoma patients undergoing therapeutic or surgical castration demonstrate increased FMD compared with the controls who were healthy men or patients with no prostate cancers. Testosterone also influences endothelial function through the modulation of nitric oxide (NO) release. Endothelium-derived NO has been shown to modulate a variety of vascular functions, including vasodilation, inhibition of cell death, inhibition of platelet aggregation and attenuation of leukocyte infiltration. In vitro, physiological concentrations of testosterone have been shown to increase NO production via activation of intracellular signalling pathways and increased endothelial NO synthase expression. Whether via influence on NO or not, testosterone is known to have an effect on endothelial cell inflammation. In vitro studies, however, have produced conflicting results with androgen treatment both augmenting and inhibiting the expression of cell adhesion molecules, and cytokine and chemokine release in response to inflammatory stimuli [16].

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Endothelial Dysfunction

When the endothelium is damaged, cell senescence and cell death can occur leading to further vascular disruption. Circulating endothelial cells derive directly from the damaged vasculature, and therefore act as a marker of endothelial damage. This cell population is shown to be significantly increased in hypogonadal men with an exponential correlation between testosterone concentration and circulating endothelial cell count. In addition to promoting endothelial cell growth, physiological testosterone concentrations induce proliferation, migration and colony formation activity of endothelial progenitor cells as a mechanism to restore the lining of damaged blood vessels. Further research is needed in this area to determine whether testosterone has beneficial or adverse effects on endothelial dysfunction.

Inflammation is involved in all stages of atherosclerosis: from early lesion development to plaque rupture. Inflammatory proteins, or markers of inflammation, can be elevated in the circulation of patients with atherosclerosis. It has been suggested that plasma cytokine levels and low-grade systemic inflammation correlate negatively with androgen levels in men [13]. A significant inverse correlation between serum testosterone levels and IL-6, soluble intracellular adhesion molecule and C-reactive protein (CRP) has also been described, and elderly hypogonadal men exhibit raised serum levels of TNF-α and IL-6 [13, 16]. Indeed, an inverse relationship was seen between serum IL-1β and endogenous testosterone in CAD patients, with the increase in IL-1β significantly related to disease severity in a stepwise manner. Many studies have focussed on elderly men, and therefore the causality of the relationship may be complicated by both the age-related increase in low-grade systemic inflammation and progressive testosterone decline. A recent study, however, in young men without manifestations of systemic disease has demonstrated a negative association between testosterone concentration and inflammatory markers (TNF-α and macrophage inflammatory proteins 1α and 1β). Furthermore, ADT is associated with increased levels of pro-inflammatory factors and decreased levels of anti-inflammatory cytokines. Although androgens are recognised to have anti-inflammatory actions, evidence from testosterone treatment studies has not been consistent. In hypogonadal men with cardiovascular symptoms, TRT has shown a significant reduction in TNF-α and an elevation in circulating anti-inflammatory IL-10. Of note, the Moscow Study demonstrated that 30 weeks of testosterone treatment to normalise levels in hypogonadal men with MetS resulted in a significant reduction in circulating CRP, IL-1β and TNF-α, with a trend towards lower IL-6 compared with placebo. In other studies, testosterone administration had no effect on TNF-α, IL-6 or CRP. Likewise, in a prospective study in older men, the administration of DHT or human chorionic


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Inflammation

g onadotrophin (to stimulate testosterone synthesis from Leydig cells) did not significantly alter the high-sensitivity CRP, soluble vascular cell adhesion molecule 1 or soluble intracellular adhesion molecule levels [for review see ref. 16]. In addition to influencing the systemic markers of inflammation, in vitro studies have provided some evidence of testosterone action on the inflammatory activation of leukocytes. Although only relatively few studies have investigated the influence of androgens on monocyte and macrophage function relevant to atherosclerosis, the majority of these indicate anti-inflammatory actions. In culture, testosterone inhibits TNF-α, IL-1β and IL-6 release from peripheral blood monocytes isolated from androgen-deficient men with T2DM and from a CHD age-relevant population or a healthy male population. Testosterone also stimulates the production of anti-inflammatory cytokines such as IL-10 from lymphocytes. Testosterone down-regulates inducible NO synthase expression in mouse macrophages, thus reducing inflammatory oxidative damage, and was found to reduce inducible NO synthase and IL-1β expression in a human macrophage cell line. No effects were observed on IL-6 and CRP expression in monocytes in other studies, however, and no differences were detected in cytokine release before or after treatment from lipopolysaccharide- or interferon-stimulated monocytes isolated from men with T2DM [16]. Coagulation Evidence suggests that testosterone deficiency is associated with a pro-coaguable state [for review see ref. 13, 16]. The anti-coagulation agent tissue plasminogen activator is positively correlated to serum testosterone levels, while the pro-thrombotic mediators fibrinogen, clotting factor VII and plasminogen activator inhibitor 1 demonstrate a negative correlation. Importantly, in the Tromsö study, a long-term, prospective, population-based, clinical investigation, endogenous testosterone concentration was not associated with the risk of venous thromboembolism. TRT in testosterone-deficient men and androgen treatment in healthy men leads to reduced plasminogen activator inhibitor-1 and fibrinogen levels in some studies. This beneficial action on coagulation status appears to be diminished in men with CAD as testosterone treatment in men with stable angina, which included eugonadal as well as hypogonadal patients, showed no change in tissue plasminogen activator, plasminogen activator inhibitor-1 or fibrinogen.

Atherosclerosis is a complex disease of the arteries characterised by endothelial dysfunction, vascular inflammation and the build-up of lipids within the intima of the vessel wall, and it is the major underlying cause of CVD. It is a progressive disease

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Atherosclerosis


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that starts with the formation of early ‘fatty streaks’ composed of lipid and inflammatory immune cells in the vessel wall. As the accumulation of lipid and inflammatory cell recruitment continues, early lesions progress to the formation of an atherosclerotic plaque, which can cause stenosis of the artery, reduced blood flow and increased blood pressure, further disrupting normal vascular dynamics. Without resolution, atherosclerotic plaques may rupture, exposing thrombotic contents and promoting clot formation. The assessment of atherogenesis and plaque-specific mechanisms in the development of atherosclerosis is limited by the ability to obtain sufficient supply of pathological human tissue, and therefore clinical studies are limited to the assessment of subclinical surrogate markers. Carotid intima media thickness (CIMT) is one of the most commonly used surrogate end points of atherosclerosis. A large cross-sectional population study of >1,400 men demonstrated a negative correlation between total testosterone and CIMT. Other studies also support this association between testosterone and carotid atherosclerosis, and indicate that men with low levels of testosterone develop greater increases in CIMT. Similarly, a recent Chinese population-based study demonstrated that men with testosterone levels in the highest tertile had significantly less severe coronary atherosclerosis determined by angiography. Long-term testosterone treatment has also been shown to reduce CIMT in men with low baseline testosterone levels. Animal studies have allowed a more direct investigation of testosterone and atherosclerotic plaque development. A number of animal models demonstrates that castration or hypogonadism in mice or rabbits fed a pro-atherogenic diet results in increased atherosclerosis [16]. Testosterone supplementation to achieve levels within the physiological range, either by intramuscular injection or orally, inhibits plaque formation. Some studies highlight the role of the oestrogen receptor in this process as simultaneous inhibition of aromatase activity (and therefore testosterone conversion to oestrogen) or oestrogen receptor blockade reduced or abrogated the beneficial effects of testosterone treatment. Indeed, androgen receptor defunct mice with low testosterone develop increased aortic atherosclerosis, and physiological testosterone treatment significantly inhibits atherogenesis, although aromatase and oestrogen receptor blockade partially but not completely abolished this effect. This suggests an action mediated in part through oestradiol and in part by a direct effect of testosterone per se. Other studies, however, have shown that the non-aromatisable androgen DHT was able to reduce aortic fatty streak formation, and androgen receptor blockade with flutamide prevented the protective effect of testosterone on aortic plaque growth in castrated rabbits. The mechanisms underpinning the effect of atherosclerosis, therefore, remain controversial. DHT has been shown to inhibit induction of foam cells by oxidised LDL in cultured macrophages. Furthermore, human monocyte-derived macrophages treated with testosterone in vitro inhibit cholesterol accumulation. Recently, we have shown that testosterone stimulates cholesterol efflux from the THP-1 monocyte/

macrophage cell line, which is mediated by increased expression of ATP binding cassette cholesterol transporter type 1 and its translocation to the cell membrane [unpubl. data]. Macrophages provide the predominant cellular infiltrate in atherosclerotic plaques and are considered to be the ‘driving force’ behind atherogenesis; they contribute to apoptosis and the destabilisation of the plaque [16].

Vascular Reactivity

The vascular system is a direct target for androgen action, and beneficial effects of testosterone on symptoms of angina, blood pressure and erectile dysfunction are observed in many studies, with the mode of action considered to be due to a vasodilatory effect of testosterone and restoration of normal vascular function.

Altogether, there have been 12 studies of the use of testosterone therapy in men with angina that have produced consistently positive results. An overview of these trials has shown that the extent of the clinical benefit is greater in men with the lowest baseline levels of testosterone (50) to adjust the data, this finding was reversed, with greater events in those receiving therapy. Furthermore, there is doubt that the treated patients were receiving adequate TRT as 17.3% only filled one prescription and the mean testosterone level on treatment was 11.8 nmol/l, which strongly suggests that many subjects were undertreated. Two thirds of patients received 2.5-mg testosterone patches, which is half the usual dose. It is also known that one third of patients are intolerant of the patches as a result


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Testosterone Replacement Therapy and Cardiovascular Safety

of skin irritation or allergy. This particular study fails to provide any clarity on testosterone safety in men with CHD. Two studies have reported that survival over 6 years is improved in men with hypogonadism who are treated compared to those who were not in an endocrine clinic and a diabetes registry study [14, 26]. In each study, testosterone was carefully titrated to achieve mid-normal range levels, as recommended by international guidelines. Men with endogenous testosterone levels in the mid to upper normal range have reduced cardiovascular events as well as mortality, whereas those with low or low normal and high normal levels have increased events. Evidence may, therefore, suggest that either low testosterone or testosterone above the normal range has an adverse effect on CVD whereas testosterone levels titrated to within the mid-upper normal range have either at least a neutral effect or, taking into account the knowledge of the beneficial effects of testosterone on a series of cardiovascular risk factors, there may be a cardio-protective action. Until we have the result of a large long-term ideally 5-year placebo-controlled outcome study, a definitive answer to the question of testosterone replacement regarding benefit versus risk is lacking. Currently, the available evidence does suggest that when there is careful titration and monitoring of testosterone levels and haematocrit, even in men with proven CVD, there are no reports of adverse effects but potential benefits. However, if patients are under- or over-treated, then this may increase risk. This is not surprising as endocrinologists understand that with any hormone replacement, levels should be titrated to normal: for example in the case of thyroxine.

Conclusion

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There is compelling evidence that testosterone deficiency is associated with the presence of CVD, adversely affects several key cardiovascular risk factors and increased the risk of cardiovascular mortality. There is little doubt that testosterone deficiency is a biomarker of ill health, but there is some evidence to support the notion that it contributes to the atherogenesis and may accelerate the disease process. Whether or not testosterone replacement improves cardiovascular outcomes or not, or indeed has adverse effects, is not fully clear and is currently a controversial subject. However, careful review of the medical literature does not show any increased risk and indeed benefits when a diagnosis of hypogonadism is made according to clinical guidelines, testosterone is replaced to the mid-normal range and monitored carefully. The studies that have reported a potential adverse effect are those where diagnosis of hypogonadism is unclear, patients are under-treated or that they have been given a higher than recommended dose of testosterone. There is certainly a need now for a well-designed, large, placebo-controlled trial to investigate this further and establish those parameters required for benefit and safety.

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1 Jones TH: Testosterone deficiency: a risk factor for cardiovascular disease? Trends Endocrinol Metab 2010;21:496–503. 2 Ohlsson C, Barrett-Connor E, Bhasin S, Orwoll E, Labrie F, Karlsson MK, Ljunggren O, Vandenput L, Mellström D, Tivesten A: High serum testosterone is associated with reduced risk of cardiovascular events in elderly men. The MrOS (Osteoporotic Fractures in Men) Study in Sweden. J Am Coll Cardiol 2011;58: 1674–1681. 3 Hyde Z, Norman PE, Flicker L, Hankey GJ, Almeida OP, McCaul KA, Chubb SA, Yeap BB: Low free testosterone predicts mortality from cardiovascular disease but not other causes: the Health in Men Study. J Clin Endocrinol Metab 2012;97:179–189. 4 Jones RD, Nettleship JE, Kapoor D, Jones HT, Channer KS: Testosterone and atherosclerosis in aging men: purported association and clinical implications. Am J Cardiovasc Drugs 2005;5:141–154. 5 Araujo AB, Dixon JM, Suarez EA, Murad MH, Guey LT, Wittert GA: Endogenous testosterone and mortality in men: a systematic review and meta-analysis. J Clin Endocrinol Metab 2011;96:3007–3019. 6 Levine GN, D’Amico AV, Berger P, Clark PE, Eckel RH, Keating NL, Milani RV, Sagalowsky AI, Smith MR, Zakai N; American Heart Association Council on Clinical Cardiology and Council on Epidemiology and Prevention, the American Cancer Society, and the American Urological Association: Androgen-deprivation therapy in prostate cancer and cardiovascular risk: a science advisory from the American Heart Association, American Cancer Society, and American Urological Association endorsed by the American Society for Radiation Oncology. Circulation 2010;121:833–840. 7 Grossmann M: Low testosterone in men with type 2 diabetes: significance and treatment. J Clin Endocrinol Metab 2011;96:2341–2353. 8 Kelly DM, Jones TH: Testosterone: a metabolic hormone in health and disease. J Endocrinol 2013; 217: R25–R45. 9 Rao PM, Kelly DM, Jones TH: Testosterone and insulin resistance in the metabolic syndrome and T2DM in men. Nat Rev Endocrinol 2013;9:479–493. 10 Wang C, Jackson G, Jones TH, Matsumoto AM, Nehra A, Perelman MA, Swerdloff RS, Traish A, Zitzmann M, Cunningham G: Low testosterone associated with obesity and the metabolic syndrome contributes to sexual dysfunction and cardiovascular disease risk in men with type 2 diabetes. Diabetes Care 2011;34:1669–1675. 11 Saad F, Haider A, Doros G, Traish A: Long-term treatment of hypogonadal men with testosterone produces substantial and sustained weight loss. Obesity (Silver Spring) 2013;21:1975–1981.

25 Vigen R, O’Donnell CI, Barón AE, Grunwald GK, Maddox TM, Bradley SM, Barqawi A, Woning G, Wierman ME, Plomondon ME, Rumsfeld JS, Ho PM: Association of testosterone therapy with mortality, myocardial infarction, and stroke in men with low testosterone levels. JAMA 2013;310:1829–1836. 26 Shores MM, Smith NL, Forsberg CW, Anawalt BD, Matsumoto AM: Testosterone treatment and mortality in men with low testosterone levels. J Clin Endocrinol Metab 2012;97:2050–2058.

Prof. Thomas Hugh Jones Centre for Diabetes and Endocrinology, Barnsley Hospital NHS Foundation Trust Gawber Road Barnsley S75 2EP (UK) E-Mail [email protected]

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