Androgen Action on the Endothelium—
Clinical and preclinical evidence exists linking endothelial dysfunction to androgen deficiency (Akishita et al, 2007; Lu et al, 2007; Miller and Mulvagh, 2007; Foresta et al, 2008). In a clinical study, Akishita et al (2007) assessed 187 men with coronary risk factors and found that the percent flow-mediated vasodilation (FMD) in men in the lowest quartile of free T was 1.7 times less than percent FMD of men in the highest quartile. Total and free T were related to percent FMD, independent of other risk factors such as age, body mass index, hypertension, hyperlipidemia, and diabetes mellitus. The authors discussed mechanisms by which T might regulate vasomotor function and suggested different mechanisms depending on the mode of T administration. The authors cite studies suggesting that the benefits of short-term intracoronary administration of T and supraphysiologic doses in vitro could be mediated by smooth muscle cell membrane ion channels, and not through endothelial or androgen receptor mechanisms. Conversely, they report studies that have found that acute and chronic supplementation of T benefit patients by increasing the percent FMD without increasing basal brachial artery diameter, suggesting that T acts through an endothelium-dependent mechanism.
Testosterone might improve parameters relating to CVD through mediation of endothelial progenitor cell activity. Foresta et al (2006) investigated the effects of T on the role that endothelial progenitor cells play in endothelial repair in 10 young idiopathic patients with hypogonadotrophic hypogonadism. They observed that the number of endothelial progenitor cells in hypogonadal men was fewer than the number in healthy control subjects. The authors further found that treating idiopathic hypogonadotropic hypogonadism with T gel therapy, at 50 mg/d for 6 months, increased the number of circulating endothelial progenitor cells in these men. These findings point toward a decreased number of circulating endothelial progenitor cells as being a potential risk factor for CVD seen in patients with hypogonadism. Interestingly, Foresta et al (2008) presented clinical data demonstrating that androgens can stimulate endothelial progenitor cells. Because all of the effects were abolished after flutamide (androgen receptor blocker) pretreatment, it was concluded that the effects were mediated via the androgen receptor. The levels of T used in these studies were calculated to be in the normal physiological range (Foresta et al, 2008).
In preclinical studies, Lu et al (2007) examined the endothelium from castrated rats by transmission electron microscopy and demonstrated significant endothelium damage, in which the cell surface appeared crumpled, rough, adhesive, and ruptured. This pathology was partially restored by treatment of castrated rats with T or DHT. These observations strongly suggested that low concentrations of T or DHT were associated with ultrastructural damage to the aortic endothelium.
Liu and Dillon (2002, 2004) demonstrated through in vitro studies with endothelial cell culture that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells by a plasma membrane—dependent mechanism. This action of DHEA is mediated by a steroid-specific, G protein—coupled receptor mechanism that activates endothelial nitric oxide synthase (eNOS) in both bovine and human endothelial cells. DHEA restored aortic eNOS levels and eNOS activity, suggesting that DHEA could have direct genomic and nongenomic effects on the vascular wall. This cellular mechanism might underlie some of the cardiovascular protective effects proposed for androgens, as reviewed recently by Simoncini et al (2004) and Simoncini and Genazzani (2007).
The relationship between androgen deficiency, endothelial dysfunction, and vascular disease is very complex (Figure) and is the subject of several reviews. Insulin resistance, which is exacerbated by androgen deficiency, might mediate endothelial dysfunction and vascular disease (Traish et al, 2009b). Clinical consequences of insulin resistance include dyslipidemia (Ginsberg, 2000), hyperglycemia (Haffner, 2000; Haffner et al, 2000), hypertension, and abnormal vascular behavior (Reaven et al, 1996) and also include vascular inflammation and thrombotic risk inflammation (Calles-Escandon et al, 1998; Sobel, 1999).
Figure 1. Figure. Androgen deficiency contributes to the metabolic syndrome and vascular disease. This figure postulates that androgen deficiency “hypogonadism” is manifested in various components of the metabolic syndrome, including increased insulin resistance and glucotoxicity (Laaksonen et al, 2003; Kapoor et al, 2006; Traish et al, 2009a), increased visceral obesity and lipotoxicity (Kim et al, 2006), and increased production of inflammatory factors (Yialamas et al, 2007). These factors contribute to endothelial dysfunction (Jones et al, 2005; Kim et al, 2006). This endothelial pathology results in increased vasoconstriction, arterial sclerosis, oxidative stress, thrombosis, inflammatory cell adhesion (Fu et al, 2008), smooth muscle proliferation, and endothelial permeability (reviewed in Mombouli and Vanhoutte, 1999; Sobel, 1999; Kim et al, 2006; Yung et al, 2006; Traish et al, 2008; Higashi et al, 2009). Color figure available online at www.andrologyjournal.org.
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Endothelial dysfunction is also associated with dyslipidemia, obesity, and diabetes (McVeigh and Cohn, 2003). Mechanisms underlying lipotoxicity include oxidative stress and proinflammatory signaling, whereas the mechanisms underlying glucotoxicity include oxidative stress, advanced glycation end product formation, the hexosamine pathway, and proinflammatory signaling (Kim et al, 2006). Pharmacological intervention should target these overlapping mechanisms that contribute to the etiology of insulin resistance and endothelial dysfunction. LDL cholesterol seems to be the most important fraction that affects the endothelial function and promotes atherosclerosis. In a clinical study, Barud et al (2002) showed an inverse relationship between low T and elevated LDL antibody levels and, after multiple regression analyses, that only T was independently associated. Current drugs to improve endothelial function in patients with diabetes include folic acid, HMG-CoA reductase inhibitors, ACE inhibitors, Niaspan, l-arginine, insulin and insulin sensitizers, and possibly phosphodiesterase type 5 inhibitors (Fonseca and Jawa, 2005). Some clinical studies have also reported that simple modifications in nutrition and exercise can positively alter endothelial function and reduce inflammation (Esposito et al, 2006).
A clinical study by Malkin et al (2004b) found a reduction in proinflammatory cytokines, total cholesterol, and triglyceride levels in 27 hypogonadal men treated with T. Specifically, TNF-α and IL-1β levels were significantly reduced after 4 weeks of treatment compared with baseline measurements. The authors reported that these proinflammatory factors are associated with the development of atheromatous plaque, and the T -induced reduction of these factors is certainly beneficial to those at risk for CVD. In a different study, the authors further reported that total cholesterol and TNF-α are significantly reduced with T therapy in 10 hypogonadal men with angina (Malkin et al, 2004a). Ischemic thresholds were also improved in these men receiving T therapy.
Testosterone has been shown to produce positive effects on endothelial function, as measured via brachial arterial vasoreactivity in men with CAD (Kang et al, 2002). Men were either given placebo or 160 mg of oral T undecanoate. Those patients receiving T treatment for 12 weeks had a significantly greater percentage of flow-mediated and nitroglycerin-mediated dilation compared with the response seen in controls receiving placebo.
In animal model studies with dogs (Chou et al, 1996) and rabbits (Yue et al, 1995), the authors attributed T's positive effects on the vasculature to the direct stimulation of endothelium-derived NO or vascular smooth muscle K+ channels. Geary et al (2000) analyzed cerebral artery myogenic tone in male rats and found this to correlate with the presence of T. This effect of T was negated by removal of the endothelium or by the combined inhibition of the cyclooxygenase pathway and K+ channels. Geary et al (2000) suggests that myogenic tone is independent of NOS activity, but is an endothelium-dependent process mediated through cyclooxygenase or K+ channel inhibition, or both, in that this inhibition eliminates any differences between orchiectomized groups of animals treated with or without T.
A study by Nakao et al (1981), who used a cell culture system, suggested that T inhibits endothelial cyclooxygenase, suppressing prostacyclin production in arterial smooth muscle cells in culture. The authors suggested that “testosterone may stimulate thrombus formation and accelerate atherosclerosis by suppressing prostacyclin production in arterial smooth muscle.” These studies were performed in cultured cells that might have lost their phenotype, with T concentrations far above the physiological levels.
In a clinical study, Polderman et al (1993) suggested that plasma T levels modulate endothelin levels and these were found to be higher in men than in women, suggesting sex hormone differential regulation. Because endothelin is a powerful vasoconstrictor, it might influence myogenic tone through changes in intracellular Ca2+ ([Ca2+]i) and other second messenger systems, which could contribute to CVD. On the contrary, Kumanov et al (2002) concluded that hypogonadism significantly increased plasma endothelin levels compared with healthy male controls. Furthermore, castration of male rats also increased endothelin levels, suggesting that androgens down-regulate endothelin synthesis (Ajayi et al, 2004). Thus, the limited data on the potential role of T in regulating endothelin function does warrant definitive conclusions.
In a clinical study, Fu et al (2008) found that in male patients with coronary heart disease, free T was inversely correlated with vascular cell adhesion molecule-1 (VCAM-1) and intima media thickness (IMT), both indicators of endothelial dysfunction. VCAM-1 is produced by endothelial cells and could be an important step in the atherosclerotic and inflammatory process because it facilitates the adherence and migration of circulating monocytes through the dysfunctional endothelium. Interestingly, however, a more recent study conducted by Webb et al (2008) did not find T treatment to influence endothelial function. The difference in global endothelial function has not been reported in men with coronary heart disease and low T compared with placebo after being treated with T undecanoate for 8 weeks. Clearly, further studies are needed to determine whether free T and VCAM-1 directly interact and the nature of any possible relationship between T and some aspects of endothelial function. Although the physiological or biochemical mechanisms remain poorly understood, the clinical and basic science evidence from the data reported to date is ample to support an association between androgen deficiency and endothelial dysfunction.
Androgen Action on Vascular Smooth Muscle Function—Clinical studies. Clinical studies have demonstrated that T could have beneficial effects in men with CVD. A series of reports by Malkin et al (2003a,b,c) suggested that men with low T levels are at increased risk of CAD and T could be a protective factor against atherosclerosis. The authors further noted that T treatment reduced the QT dispersion in men with heart failure (Malkin et al, 2003a) and advanced the hypothesis that T immunomodulating properties inhibit atheroma formation and progression to acute coronary syndrome (Malkin et al, 2004a,b). In a subsequent commentary, Malkin et al (2003c) noted that endogenous levels of T are inversely related to the severity of aortic atheroma and to the progression of aortic atheroma when assessed radiologically. Testosterone replacement therapy in hypogonadal men was shown to delay time to ischemia and improve mood and was associated with a reduction in total cholesterol and TNF-α (Malkin et al, 2004a). Furthermore, the authors (Malkin et al, 2007) presented data suggesting that physiological T therapy improved insulin sensitivity in men with moderate to severe congestive heart failure.
In clinical studies, Malkin et al (2006b) reported that T therapy in men with moderately severe heart failure is safe, with no excess of adverse events, and that T improves functional capacity and symptoms of patients with moderately severe heart failure. Similarly, Webb et al (2008) demonstrated that oral T undecanoate had selective and modest enhancing effects on perfusion in myocardium supplied by unobstructed coronary arteries and suggested that the T undecanoate—related decrease in basal arterial stiffness might partly explain the effects of exogenous T on signs of exercise-induced myocardial ischemia.
Jones et al (2004b) reviewed the role of T on vascular reactivity in men and cited studies suggesting that T replacement is associated with an improvement in vascular reactivity in men with CAD and improves endothelial FMD. The authors suggested that this might be true only in diseased vessels. Interestingly, the authors suggested that the data from animal studies are inconclusive and cited a number of references with positive, negative, and neutral results. Thus, the exact molecular mechanism of androgen action on the vasculature remains to be investigated further.
In vitro studies. Several in vitro studies investigated the physiological mechanisms of androgen-mediated vasodilation in various blood vessels from animals and humans. For instance, several studies have demonstrated vasorelaxing effects of T and 5α-DHT on vascular and nonvascular smooth muscle, probably via inhibition of L-type calcium channels (Sochorová et al, 1991; Scragg et al, 2004, 2007; Perusquía et al, 2005; Hall et al, 2006; Er et al, 2007; Montaño et al, 2008). In some studies, 5α-DHT was more potent than T in relaxing KCl-induced contractions. Testosterone at nanomolar concentrations was a powerful antagonist for L-type voltage—operated Ca2+ channels (L-VOCCs). The data showed that 5α- DHT—induced vasorelaxation is attributed to its selective blockade on L-VOCCs, but T-induced vasorelaxation involved concentration-dependent additional mechanisms involving L-VOCC antagonists at low concentrations and increasing [Ca2+]i and cAMP production at high concentrations. Similar data were noted in porcine coronary arteries in which T caused vasodilation (Hutchison et al, 2005). Similarly, Cairrão et al (2008) demonstrated that T induced relaxation of human umbilical arteries via a nongenomic-dependent mechanism. The relaxation is thought to be partially mediated by activation of large-conductance Ca(2+)-activated potassium channels (BK[Ca]) and voltage-sensitive potassium channels (Kv). The involvement of these channels in a T-relaxant mechanism is dependent on the pathways activated by the contractile agent used.
Furthermore, Navarro-Dorado et al (2008) showed that T and the nonaromatizable metabolite 5α-DHT evoked a concentration-dependent relaxation on norepinephrine precontracted small-artery aortic rings in an endothelium-independent manner. The authors suggested that T induced a direct vasodilatory action in small arteries independent of the endothelium by blockade of extracellular Ca2+ entry through L-VOCCs and non—LVOCCs. Studies in human umbilical arteries showed that T and 5α-DHT caused vasodilation via a non—androgen receptor pathway (Perusquía et al, 2007). Similarly, isolated radial arteries were relaxed by supraphysiological doses of T, presumably via activation of ATP/potassium channels (Seyrek et al, 2007). A similar vasodilation was also observed in internal mammary arteries (Yildiz et al, 2005). Testosterone induced a vasodilatory response in rabbit tracheal smooth muscle; this effect was attributed to eNOS (Kouloumenta et al, 2006).
It should be noted that T caused vasodilation in denuded vessels (Yue et al, 1995; Murphy and Khalil, 1999), suggesting an endothelium-independent mechanism. Webb et al (1999) showed that T treatment into the left coronary artery caused vasodilation and increased flow. Tep-Areenan et al (2003) investigated the effects of T on vasodilation in the rat aorta in an organ bath and demonstrated that T induced acute vasorelaxtion, which is likely mediated via inhibition of extracellular calcium influx and via the action of endothelium-derived prostanoids. Similarly, Jones et al (2004a) suggested that T-induced vasodilation via nongenomic mechanisms is independent of the androgen receptor and of the vascular endothelium. The authors postulated that the action of androgens is mediated via direct calcium antagonism in the vascular smooth muscle. Malkin et al (2006a) demonstrated that T facilitated vasodilation in subcutaneous resistance arteries from patients with heart failure in a concentration-dependent manner. Interestingly, T therapy reduced the vasodilation response to acetylcholine and sodium nitroprusside and increased contractile responses to norepinephrine. The authors postulated that the benefits of T in vascular function could be offset by a decline in vascular reactivity. However, the authors did not comment on the fact that the doses of T used in the in vitro studies are of pharmacological nature and not of physiological function, in that micromolar levels of T were used, which are far above any physiological level known in vivo. Furthermore, the resistance arteries were obtained from patients with vascular disease, which could have skewed the outcome of the study. In addition, the remodeling of tissue in response to sex steroid hormones is a long-term process and cannot be ignored in interpreting data from in vitro studies. It is our view that the data in this study do not negate the observed benefits of T in vascular function in vivo and cannot be explained purely on the contractility response data obtained in vitro.
Yildiz and Seyrek (2007) hypothesized that because denuded vasculature produced the same result in response to T as the intact blood vessels, the major effect of T is likely mediated directly by the vascular smooth muscle. The postulated mechanism suggests that T either activates K+ channels to increase efflux, inhibits Ca2+ channels, or both, causing hyperpolarization and subsequent vasodilation. Because these changes occur in seconds to minutes, it is suggested that this action is likely to be mediated via the interactions with receptors on the membrane (nongenomic effect) rather than by interaction with the nucleus (genomic effect). Furthermore, other studies have shown the presence of the androgen receptor on the membrane of vascular smooth muscle cells (Fujimoto et al, 1994; Benten et al, 1999), suggesting that the proposed mechanism is likely. The data from in vitro studies suggest that T-induced relaxation of the arterial wall was mediated via inhibition of Ca2+ entry into the smooth muscle (Crews and Khalil, 1999; Giannattasio et al, 1999). On the basis of the data reported to date, the exact mechanisms underlying the physiological effect of T on vascular reactivity remain unclear and might involve activation of K+ channels and antagonism of Ca2+ channels, are likely to be of nongenomic signaling, and might not require the endothelium.