Androgens and Morphologic Remodeling at Penile and Cardiovascular Levels: A Common Piece in Complicated Puzzles?

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1 EUROPEAN UROLOGY 56 (2009) available at journal homepage: Review Andrology Androgens and Morphologic Remodeling at Penile and Cardiovascular Levels: A Common Piece in Complicated Puzzles? Vincenzo Mirone a, Ciro Imbimbo a, Ferdinando Fusco a, Paolo Verze a, Massimiliano Creta a, *, Gianfranco Tajana b a Department of Urology University Federico II, Naples, Italy b Department of Pharmaceutical Science-University of Salerno, Salerno, Italy Article info Article history: Accepted December 25, 2008 Published online ahead of print on January 9, 2009 Keywords: Androgens Endothelial cell Penile remodeling Smooth-muscle cell Vascular remodeling Abstract Context: Epidemiologic data demonstrate a protective role by normal androgen levels on cardiovascular health and erectile function. Low androgen levels are associated with erectile dysfunction and increased risk of cardiovascular diseases. Both conditions recognize as anatomic substrate a pathologic structural remodeling. Direct androgen effects on male external genitalia, vascular wall, and myocardium have been reported. Objective: To review current knowledge about androgen-dependent molecular signaling pathways and cellular events within penile and cardiovascular tissues involved in the homeostatic control of morphologic tissue properties and in the development of structural remodeling in presence of normal and low androgen levels, respectively. Evidence acquisition: A literature search was performed in November 2008 using the commercially available Medline online engine search to retrieve studies (from 1998 to 2008) on the mechanisms mediating the role of androgens on penile and cardiovascular morphologic homeostasis and remodeling. A combination of the following medical subject headings was used: androgens, hypogonadism, vessel tissue architecture, remodeling, cardiovascular system, and penis. Evidence synthesis: Androgens exert direct beneficial effects on both cardiovascular and penile tissues. Endothelial cells and smooth-muscle cells are the main cellular targets for direct androgen effects in both tissues and are involved in pathologic remodeling in hypogonadal models. At vascular level, androgens promote endothelial cell survival, reduce endothelial expression of proinflammatory markers, and inhibit proliferation and intimal migration of vascular smooth-muscle cells. At penile level, low androgen levels are associated with apoptosis of endothelial cells and smooth-muscle cells. Moreover, low androgen levels impair proliferation, migration, and homing of endothelial progenitor cells as well as myogenic differentiation of mesenchymal progenitor cells. Conclusions: Normal androgen levels promote vascular and penile homeostasis by direct mechanisms mainly involving endothelial cells and smooth-muscle cells. Low androgen levels are associated with impairments of such mechanisms, leading to pathologic structural remodeling. # European Association of Urology Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Urology, University Federico II, Naples, Via S. Pansini, 5, Naples, Italy. Tel ; Fax: address: mxx79@inwind.it (M. Creta) /$ see back matter # European Association of Urology Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 310 EUROPEAN UROLOGY 56 (2009) Introduction In recent years, complex interrelations have emerged between circulating androgen levels, erectile function, and cardiovascular health on the basis of epidemiologic data [1]. Low androgen levels associated with aging or androgen deprivation therapy have been reported to impair erectile function, to increase cardiovascular risk factors, and to produce marked adverse effects on cardiovascular system (ie, increased incidence of atherosclerosis and coronary heart disease, myocardial infarction, and sudden cardiac death) [1 4]. Moreover, endogenous testosterone levels have been shown to be inversely related to mortality due to cardiovascular diseases and all causes [2]. Low androgen levels have been shown to be an independent risk factor for metabolic syndrome and diabetes [5 9]. Remodeling is a general process by which tissues experience biochemical and structural changes with subsequent modifications of their functional properties. It involves cellular and extracellular events such as proliferation, apoptosis, differentiation, migration, extracellular matrix synthesis and degradation, and it is regulated by complex molecular signaling pathways. At penile and cardiovascular levels, physiologic structural rearrangements occur during embryogenesis and tissue growth, while a pathologic remodeling occurs as consequence of aging or pathology and represents the anatomic substrate for conditions such as erectile dysfunction, atherosclerosis, and heart failure. Diabetes and other disorders related to the metabolic syndrome (ie, high blood pressure, impaired lipid profile, and insulin resistance) are associated with pathologic remodeling of cardiovascular and penile tissues causing structural impairments [8]. Notably, both cardiovascular and penile tissues have androgen-dependent properties. Androgen-signaling pathways are active in different cellular compartments within these tissues through both genomic and nongenomic mechanisms [10]. The classic pathway of androgen action involves the binding of the steroid to the androgen receptor (AR), a ligand-activated transcription factor regulating genome expression. Beyond genomic mechanisms, rapid, androgen-dependent, nongenomic transduction cascades have been described and are mediated by membrane AR and by intracellular signal transduction cascades [10]. This review focuses on androgen-regulated processes underlying both the physiologic structural homeostasis and the hypogonadic pathologic remodeling of penile and cardiovascular tissues that can partially explain the reported association among low androgen levels, cardiovascular diseases, and erectile dysfunction. 2. Evidence acquisition A literature search was performed in November 2008 using the commercially available Medline online search engine. A combination of the following search terms was applied to retrieve relevant articles (from 1998 to 2008): androgens, hypogonadism, vessel tissue architecture, remodeling, cardiovascular system, and penis. Review articles and basic studies describing molecular and cellular mechanisms underlying the role of androgens in regulating cardiovascular and penile structural homeostasis and their impairments in hypogonadal models responsible for pathologic remodeling were included. 3. Evidence synthesis 3.1. Androgens and vascular remodeling The vessel wall is an active, integrated organ composed of endothelial cells, vascular smooth-muscle cells (VSMC), and fibroblasts which are disposed in different layers and are coupled to each other in a complex autocrine paracrine set of interactions (Fig. 1). Endothelium is the major regulator of vascular homeostasis: it exerts vasoprotective functions such as control of vasomotor tone and local homeostasis, VSMC growth suppression, and inflammatory responses inhibition. VSMC perform biosynthetic, proliferative and contractile roles. Androgen-signaling pathways have been described in both endothelial cells and VSMC [10]. At the VSMC level, nongenomic transduction cascades are prevalently responsible for acute vasorelaxant effects and involve modulation of ion channel activity (ie, activation of large-conductance calcium-activated potassium channels and inhibition of L-type calcium channels) [10]. Vascular remodeling is defined by structural changes of the vessel wall in response to various noxious stimuli [11 14]. Hypogonadism is associated with increased risk for atherosclerotic vascular wall remodeling: clinical studies demonstrated an independent inverse association between serum testosterone levels and morphologic markers of atherosclerosis such as intima-media thickening at the aortic artery level [14]. Atherosclerotic vascular remodeling is a multistep process involving almost all vascular cellular and extracellular components and resulting in reorganization of the vessel wall architecture. Endothelial dysfunction, neointima formation, medial thickening, activation of local humoral and cellular inflammatory responses and altered extracellular matrix turnover are critical steps in the development, progression, and clinical complications of such remodeling process. Moreover, abnormal adypogenic differentiation of VSMC has been reported to take part to the process [15]. Proinflammatory cytokines activation promotes leukocyte recruitment and local inflammation in the arterial wall leading to VSMC apoptosis, degradation of the fibrin cap, and plaque rupture. In the castrated-rabbit model, replacement of physiologic testosterone levels has been reported to reduce atherosclerotic plaque area and aortic intima thickness [12]. These effects were mediate by AR and were probably related to the reduction of serum levels of tumor necrosis factor a (TNF-a), interleukin 6 (IL-6), soluble intercellular adhesion molecule-1, and matrix metalloproteinase-2 [12]. In hypogonadal men, testosterone replacement induced an anti-inflammatory shift in circulating cytokine balance by suppressing the expression of proinflammatory cytokines such as TNF-a, interleukin 1-b (IL-1b), and IL-6 and by potentiating the expression of anti-inflammatory ones such as interleukin 10

3 EUROPEAN UROLOGY 56 (2009) Fig. 1 Effects of normal and low androgen levels on endothelial cells and smooth-muscle cells in vascular tissue. y = effects mediated by dihydrotestosterone); 8 = effects mediated by dehidroepiandrosterone; * = effects mediated by testosterone. (IL-10) [9]. However, the relation between serum testosterone levels and circulating inflammatory markers is complex, and a possible suppression of testosterone secretion by increased systemic inflammation mediators has been also reported [13]. Independently of indirect effects, androgens modulate vascular wall properties by several direct mechanisms on mature endothelial cells and VSMC as well as on their precursors Effects on vascular endothelial cells Endothelial dysfunction is an early event in atherosclerotic vascular remodeling. Impaired endothelial function has been described in both animal and human models as consequence of low circulating testosterone levels. Lu et al performed an electron microscopy study on aortic endothelial cells from male rats submitted to castration or 5areductase inhibitor administration. Endothelial ultrastructural damages were noticed in both groups even if with a higher degree in the castrated group. Endothelial cells appeared severely crimpled, coarse, and protuberant, and the connections between cells appeared ruined, with many red blood cells adhering to cell surface. Interestingly, after testosterone supplementation, the endothelial structures showed a partial degree of recovery [16] Endothelial progenitor cells Studies demonstrated the existence of a circulating pool of premature progenitor cells and of a more mature pool of endothelial progenitor cells (EPC). Such cells derive from the bone marrow and represent an endogenous repair mechanism system allowing restoration of the injured endothelial monolayer thus promoting endothelial integrity and vascular homeostasis [17]. Altered EPC function may lead to accelerated vascular remodeling due to chronic impairment of endothelial maintenance. In patients with erectile dysfunction and mild intima-media thickness or plaque, the number of circulating EPC is significantly reduced thus suggesting an increased risk for endothelial dysfunction [18]. In humans, physiologic androgen concentrations act through an AR-mediated transduction pathway to promote proliferation of EPC, colony-formation activity, mobilization from the bone marrow into the peripheral circulation, incorporation into foci of neovascularization, and homing into sites of endothelial denudation, thus improving endothelial recovery [17]. In young human subjects, hypotestosteronemia is associated with a low number of circulating premature progenitor cells and EPC that increase significantly after testosterone treatment through a possible direct effect on the bone marrow [17] Endothelial cell survival and apoptosis Liu et al demonstrated that exposure of bovine aortic endothelial cells to dehydroepiandrosterone (DHEA) at concentrations found in human blood stimulated endothelial proliferation. This effect was mediated by a plasmamembrane receptor activating the extracellular regulated kinase-1 and -2 (ERK1/2) signaling pathways [19]. In vitro studies demonstrated that DHEA, at physiologic concentrations, was also able to activate critical prosurvival and antiapoptotic signaling pathways in human vascular endothelial cells [20]. This effect was independent from AR and was mediated by a functional G protein-coupled receptor located on the plasma membrane activating a signaling transduction cascade leading to enhanced antiapoptotic Bcl-2 protein expression [20] Endothelial synthesis of paracrine factors Endothelial cells exert regulatory functions on VSMC through the secretion of paracrine factors among which adrenomedullin and endothelin-1. Adrenomedullin is an autocrine and paracrine vasodilatory peptide with cytokine-like functions which has been shown to exert protective effects against pathologic vascular remodeling in the rat model by reducing neointima formation and perivascular hyperplasia, promoting re-endothelialization and inhibiting proliferation and migration of VSMC [21]. In humans, the number of aortic endothelial cells secreting adrenomedullin is positively regulated by physiologic testosterone levels [22]. Endothelin-1 is a potent vasoconstrictor peptide secreted by vascular endothelial cells which has been reported to exert a potent mitogenic effect on fibroblast and VSMC, to stimulate matrix biosynthesis and to act as a survival factor for myofibroblasts [23]. In humans, hypogonadism was associated with increased circulating levels of endothelin-1, while testosterone-replacement therapy was able to restore normal plasma levels of endothelin-1 [24].

4 312 EUROPEAN UROLOGY 56 (2009) Endothelial expression of inflammatory markers Androgens can reduce the expression of inflammatory markers by endothelial cells through direct mechanisms. Norata et al demonstrated that incubation of human endothelial cells with dihydrotestosterone (DHT) reduced the inflammatory response induced by TNF-a and lipopolysaccharide. This effect was mediated by AR and resulted in decreased expression of adhesion molecules (ie, vascular cell adhesion molecule-1 [VCAM-1], and intercellular adhesion molecule-1 [ICAM-1]), chemokines (ie, [IL-6] and monocyte chemoattractant protein-1 [MCP-1]) and proteases [25]. Similar results were reported by Hatakeyama et al: investigators incubated human aortic endothelial cells with testosterone and found a dose-dependent reduction of the expression of VCAM-1 [26]. Both DHT and testosterone induced the reported effects through the inhibition of the nuclear factor-kb (NF-kB) signaling pathway [25,26]. DHEA induced similar anti-inflammatory effects through the activation of the peroxisome proliferator-activated receptor (PPAR)-a [27] Effects on vascular smooth-muscle cells Proliferation and migration of VSMC promote neointimal proliferation and media thickness [28]. Preclinical data demonstrate that physiologic androgen levels contribute to the preservation of vascular-wall structural homeostasis through direct mechanisms on mature VSMC Inhibition of vascular smooth-muscle cell proliferation and migration In a study conduced by Bowles et al, testosterone was able to exert antiproliferative, proapoptotic effects on porcine coronary VSMC in a dose-dependent manner [28]. Specifically, testosterone induced cell-cycle arrest at G 0 /G1 phase by reducing cyclin D 1 and cyclin E levels, by decreasing CDK2 and CDK6 activities, and by increasing both p21 cip1 and p27 kip1 protein expression [28]. While the effects of testosterone on cyclin D 1, cyclin E, and p21 cip1 were dependent on protein kinase C (PKC) d overexpression, the effects on p27 kip1 were PKC d independent [28]. Furthermore, testosterone increased VSMC apoptosis in a dose-dependent manner through PKC d dependent stimulation of caspase 3 expression [28]. Williams et al provided evidence that DHEA was able to attenuate proliferation of human VSMC in a dose-dependent manner and by mechanisms independent of either androgen or estrogen receptors but possibly via a DHEA-specific receptor activating ERK1 pathway [29]. Furutama et al reported in rat aortic VSMC line an inhibitory effect by physiologic levels of DHEA on migration and proliferative behaviors. These effects were the result of inhibition of VSMC attachment to fibronectin and were mediated by a DHEAspecific receptor [30] Androgens and myocardial remodeling Recent preclinical data suggest a role for androgens in preventing myocardial remodeling. To date, however, cellular and molecular mechanisms are poorly understood. Initial reports suggest that androgens could promote a physiologic remodeling process by mechanisms such as inhibition of local and systemic proinflammatory cytokine patterns or stimulation of local expression of insulin-like growth gactor-1 [31,32] Androgens and penile remodeling The penis is a complex morphofunctional system whose structural and functional integrity are essential for a normal erectile function and are dependent on proper vascular supply, neural integrity, and androgen stimulation (Fig. 2). In mammalians, induction and differentiation of the genital tubercle into the external male genitalia, as well as fetal, early neonatal, and pubescent phallic growth, are dependent on serum testosterone levels, tissue conversion to DHT, and functional AR. Abnormalities in testosterone synthesis, it s conversion to DHT or AR mutations may lead to incomplete masculinization or to the development of an abnormally small but otherwise anatomically normal penis (micropenis). At the conclusion of puberty, penile growth slows and is then arrested at the end point of phallic development despite continuing testosterone production. In adulthood, a normogonadal status contributes to maintain penile morphofunctional homeostasis. Pathologic penile remodeling refers to molecular and histomorphologic alterations involving cellular and extracellular compartments and leading to hemodynamic and mechanical impairments responsible for organic erectile dysfunction. Penile remodeling can occur as a consequence of aging process, neurovascular injury, or androgen insufficiency. Hypogonadism promotes pathologic penile remodeling through direct and indirect mechanisms, the last involving impairment of penile innervation or vascular supply Androgens and structural remodeling of cavernous trabecular tissue Penile corpora cavernosa are specialized vascular beds characterized by a complex trabecular angioarchitecture composed of different cellular types which strictly interact within an extracellular matrix whose main components are collagen, elastic fibers, and hyaluronan. Main cellular types include cavernous smooth-muscle cells (CSMC), endothelial cells, neuronal cells, and fibroblasts. CSMC are disposed in clusters within the trabeculae of the cavernous spaces and, similar to VSMC, they interact with endothelial cells regulating blood flow within cavernous spaces owing to their contractile properties. Testosterone modulates the proliferation of CSMC and fibroblasts through an ARdependent signaling pathway [33]. In experimental animal models, testosterone deprivation was reported to induce significant histologic alterations within cavernous trabecular structures such as apoptosis of endothelial cells lining the sinusoidal spaces, and of CSMC accompanied by increased connective tissue deposition leading to a gradual loss of cavernosal compliance [3]. Ultrastructural evaluations demonstrated spatial disorganization of CSMC, with a large number of cytoplasmic vacuoles containing flocculent

5 EUROPEAN UROLOGY 56 (2009) Fig. 2 Effects of low androgen levels and of androgen replacement on cellular and extracellular penile compartments. material and a decreased amount of cytoplasmic myofilaments [3]. Moreover, penile tissues from orchiectomized rabbits exhibited accumulation of fat-containing cells (adipocytes) in the subtunical region of the corpora cavernosa; a differentiation of stromal precursor cells into an adipogenic lineage producing fat-containing cells or a transdifferentiation process involving CSMC have been hypothesized as possible mechanisms [3]. Even if not directly demonstrated in the penile tissue, both testosterone and DHT were reported to regulate lineage determination in mouse C3H 10T1/2 mesenchymal pluripotent cells by promoting their commitment to the myogenic lineage and inhibiting differentiation toward adipogenic lineage [34]. These effects were mediated by AR that activated intracytoplasmic and nuclear pathways leading to increased expression of myogenic proteins (ie, MyoD and MHC II), and that inhibited expression of adipogenic differentiation factors (ie, C/EBP-a) in a dose-dependent manner [35]. An intense remodeling process characterized by a decrease of CSMC amount and an increase in collagen synthesis was described in animal and human models as consequence of cavernous nerve damage or ischemic conditions too [36]. In a study by Yamamoto et al, apoptosis of endothelial cells in cavernous tissues from castrated rats was associated with increased expression of p53 protein [37]. Zhang et al proposed that increased calcium influx into cavernosal cells after castration and subsequent activation of endogenous endonucleases could represent a possible mechanism for rat penile apoptosis after castration [38]. Vascular endothelial growth factor (VEGF) is a paracrine mediator involved in penile remodeling. VEGF-signaling pathways are reported in endothelial cells and CSMC from rat and human penile tissues [39]. VEGF acts via a paracrine mechanism promoting CSMC integrity, proliferation, and migration to proper sites in the cavernous spaces during vasculogenic developmental processes [40]. Age-related differences in VEGF expression exist: CSMC from rats of different ages express different VEGF levels and respond to VEGF at different rates [40]. Intracavernous VEGF injection in the rat model inhibits apoptosis by restoring CSMC integrity [40]. Interestingly, Rogers et al reported impaired VEGF synthesis leading to decrease smooth muscle fibers in androgen-deficient models [39]. Transforming growth factor b1 (TGF-b1) is a pleiotropic cytokine secreted by smoothmuscle cells in vitro and by endothelial cells in vivo that, under normal conditions, regulates penile homeostasis by directly inhibiting cell proliferation and extracellular matrix synthesis [41]. Enhanced TGF-b1 signaling was reported to stimulate human fibroblasts to increase collagen synthesis within both corpora cavernosa and tunica albuginea [41 42]. Tissue hypoxia is a leading cause for increased TGF-b1 expression, with consequent corpora cavernosa fibrosis following bilateral cavernous nerve ablation or atherosclerotic vascular impairment [41]. Furthermore, in the animal model, TGF-b1 causes a dose-dependent decrease in the percentage of CSMC when administered intracorporally [41]. In the rat model, the in-vivo expression of TGF-b1 in the penis seems to be downregulated by androgens, while androgen deficiency could promote TGF-b1 synthesis [43]. Structural cavernosal impairments following castration are partially reversible: Shabsigh et al demonstrated that testosterone replacement therapy in castrated rats was able to stimulate new DNA synthesis in both CSMC and endothelial cells [44]. One critical aspect of concern was the difference existing between circulating and tissue androgen levels: Becker et al demonstrated differences between serum and cavernous androgen levels in different penile conditions (ie, flaccidity or erection) [45].

6 314 EUROPEAN UROLOGY 56 (2009) Androgens and remodeling of the tunica albuginea In rats, androgens are essential for maintaining the physiologic structure of penile tunica albuginea. In experimental castrated rat models, within 4 wk from castration, tunica albuginea appeared thinner with fewer elastic fibers and increased density of irregular arranged collagen fibers at electron microscopy scanning [46]. Similar alterations were described in noncastrated rats of advancing age, accounting for veno-occlusive dysfunction. Mechanisms by which androgens promote penile tunica albuginea integrity remain underinvestigated Androgens and remodeling of penile neural network In the rat model, physiologically circulating testosterone levels are critical for neuronal maturation and homeostasis within pelvic autonomic pathways as well as for preserving structural and functional integrity of peripheral autonomic and sensory penile innervations [47]. In cavernous nerves from animal models, castration impaired nerve structure and axonal regeneration following nerve graft. The number of nitrous oxide synthase containing nerve fibers within both the dorsal nerves of the rat penis and the corpora cavernosa was found to be decreased after castration compared to controls [47]. Main ultrastructural degenerative alterations were myelin stealth degeneration and diameter reduction of myelinized and nonmyelinized nerve bundles, with increased number of nucleated Schwann cells [3]. Interestingly, testosterone-replacement therapy was able to restore nerve fibers and myelin stealth structure [3,48]. VEGF administration to castrated animals is able to restore neural ultrastructure, suggesting that androgens may act on neural tissue via a paracrine mechanism [39]. Neuronal remodeling and regeneration are critical events in erectile dysfunction following nerve-sparing pelvic surgery [36,49] and may play an important role also in erectile dysfunction caused by testosterone deficiency. The time necessary to obtain neuronal regeneration may explain the reported long latency time (12 24 wk) between testosterone treatment onset and clinical benefits on sexual function [50]. Fig. 3 Effects of normal androgen levels on endothelial cells (EC), smooth-muscle cells (SMC), endothelial progenitor cells (EPC), and stromal precursor cells (SPC) presumably involved in both penile and vascular structural homeostasis. AR+ = mechanisms that are androgenreceptor dependent; ARS = mechanisms that are androgen-receptor independent; AD = adipocytes. proinflammatory markers expression and endothelin-1 synthesis as well as the increased expression of adrenomedullin have been reported to occur in the presence of normal androgen levels only in vascular endothelial cells. Furthermore, the effects on smooth-muscle cells vary between vessel and penile tissue: androgens seem to exert antiapoptotic effects on CSMC and antiproliferative effects on VSMC. The latter mechanism is consistent with the beneficial role of testosterone in the atherosclerotic remodeling. Obviously, the effects on tunica albuginea and on penile dorsal nerves are organ-specific. Conversely, a low androgen status is associated with pathologic remodeling of vessels and penis due to impairment of the reported homeostatic processes (Fig. 4). At the penile level, hypoandrogenemia is associated with apoptosis of endothelial cells and VSMC, with abnormal deposition of collagen fibers within corpora cavernosa, fibrosis of the tunica albuginea, and impaired neural supply. At vascular level, low androgen levels cause endothelial damage, 4. Conclusions Androgens are key factors in preserving penile and cardiovascular structural homeostasis by direct mechanisms involving mainly endothelial cells and smooth-muscle cells (Fig. 3). Even if some molecular and cellular events are common or potentially common, clear differences exist on the basis of different homeostatic requirements and anatomic properties of systemic vessels and corpora cavernosa. Endothelial cell survival is a critical process at both penile and cardiovascular level and is mediated through proliferative and migratory effects exerted by androgens on EPC in bone marrow, providing endothelium stability and recovery in both penis and vessels. Moreover, the ability of androgens to promote myogenic differentiation of stromal precursor cells, rather than adipogenic, is a further mechanism potentially involved in both cardiovascular and penile homeostasis. However, the inhibition of Fig. 4 Effects of low androgen levels on endothelial cells (EC), smoothmuscle cells (SMC), endothelial progenitor cells (EPC) and stromal precursor cells (SPC) presumably involved in both penile and vascular remodeling. AR+ = mechanisms that are androgen-receptor dependent; ARS = mechanisms that are androgen-receptor independent; AD = adipocytes.

7 EUROPEAN UROLOGY 56 (2009) neointimal proliferation and intima thickness thus promoting atherosclerotic remodeling. Data available support a crucial homeostatic role of testosterone in vascular and penile districts. When feasible, the restoration of a normal gonadal status could be efficacious in preventing or reversing pathologic remodeling occurring in both penile and cardiovascular system in hypogonadic patients. Further clinical experience on humans is warranted to confirm and validate this therapeutic approach. Author contributions: Massimiliano Creta had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Mirone, Fusco, Tajana. Acquisition of data: Creta, Imbimbo. Analysis and interpretation of data: Fusco, Verze. Drafting of the manuscript: Mirone, Creta. Critical revision of the manuscript for important intellectual content: Verze, Fusco, Tajana. Statistical analysis: None. Obtaining funding: None. Administrative, technical, or material support: Imbimbo, Verze, Creta. Supervision: Mirone, Imbimbo, Tajana. Other (specify): None. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None. Funding/Support and role of the sponsor: None. References [1] Jones TH. Testosterone associations with erectile dysfunction, diabetes, and metabolic syndrome. Eur Urol Suppl 2007;6: [2] Khaw KT, Dowsett M, Folkerd E, et al. Endogenous testosterone and mortality due to all causes, cardiovascular disease, and cancer in men: European prospective investigation into cancer in Norfolk (EPIC-Norfolk) Prospective Population Study. Circulation 2007;116: [3] Traish AM, Guay AT. Are androgens critical for penile erections in humans? Examining the clinical and preclinical evidence. J Sex Med 2006;3: [4] Martínez-Jabaloyas JM, Queipo-Zaragozá A, Rodríguez-Navarro R, Queipo-Zaragozá JA, Gil-Salom M, Chuan-Nuez P. Relationship between the Saint Louis University ADAM questionnaire and sexual hormonal levels in a male outpatient population over 50 years of age. Eur Urol 2007;52: [5] Hatzimouratidis K, Hatzichristou D. Testosterone and erectile function: an unresolved enigma. Eur Urol 2007;52:26 8. [6] Osuna JA, Gómez-Pérez R, Arata-Bellabarba G, Villaroel V. Relationship between BMI, total testosterone, sex hormone-binding-globulin, leptin, insulin and insulin resistance in obese men. Arch Androl 2006;52: [7] Traish AM, Goldstein I, Kim NN. Testosterone and erectile function: from basic research to a new clinical paradigm for managing men with androgen insufficiency and erectile dysfunction. Eur Urol 2007;52: [8] Wang D, Xie H, Wang G. Apoptosis and the expression of Bax and Bcl-2 in the penis of diabetic rats. Zhonghua Nan Ke Xue 2004;10: [9] Malkin CJ, Pugh PJ, Jones RD, Kapoor D, Channer KS, Jones TH. The effect of testosterone replacement on endogenous inflammatory cytokines and lipid profiles in hypogonadal men. J Clin Endocrinol Metab 2004;89: [10] Rahman F, Christian HC. Non-classical actions of testosterone: an update. Trends Endocrinol Metab 2007;18: [11] Schober A, Zernecke A. Chemokines in vascular remodelling. Thromb Haemost 2007;97: [12] Li S, Li X, Li Y. Regulation of atherosclerotic plaque growth and stability by testosterone and its receptor via influence of inflammatory reaction. Vascul Pharmacol 2008;49:14 8. [13] Cannon JG. Adaptive interactions between cytokines and the hypothalamic-pituitary-gonadal axis. Ann N Y Acad Sci 1998;856: [14] Demirbag R, Yilmaz R, Ulucay A, Unlu D. The inverse relationship between thoracic aortic intima media thickness and testosterone level. Endocr Res 2005;31: [15] Davies JD, Carpenter KL, Challis IR. Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth-muscle cells. J Biol Chem 2005;280: [16] Lu YL, Kuang L, Zhu H, et al. Changes in aortic endothelium ultrastructure in male rats following castration, replacement with testosterone and administration of 5alpha-reductase inhibitor. Asian J Androl 2007;9: [17] Foresta C, Zuccarello D, De Toni L, Garolla A, Caretta N, Ferlin A. Androgens stimulate endothelial progenitor cells through an androgen receptor-mediated pathway. Clin Endocrinol 2008;68: [18] Foresta C, Caretta N, Lana A, et al. Relationship between vascular damage degrees and endothelial progenitor cells in patients with erectile dysfunction: effect of vardenafil administration and PDE5 expression in the bone marrow. Eur Urol 2007;51: [19] Liu D, Iruthayanathan M, Homan LL, et al. Dehydroepiandrosterone stimulates endothelial proliferation and angiogenesis through extracellular signal-regulated kinase 1/2-mediated mechanisms. Endocrinology 2008;149: [20] Liu D, Si H, Reynolds KA, Zhen W, Jia Z, Dillon JS. Dehydroepiandrosterone protects vascular endothelial cells against apoptosis through a Galphai protein-dependent activation of phosphatidylinositol 3-kinase/Akt and regulation of antiapoptotic Bcl-2 expression. Endocrinology 2007;148: [21] Rauma-Pinola T, Pääkkö P, Ilves M, et al. Adrenomedullin gene transfer induces neointimal apoptosis and inhibits neointimal hyperplasia in injured rat artery. J Gene Med 2006;8: [22] Pearson LJ, Rait C, Nicholls MG, Yandle TG, Evans JJ. Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-ii. J Endocrinol 2006;191: [23] Garncarczyk A, Jurzak M, Gojniczek K. Characteristic of the endogenous peptides endothelins and their role in the connective tissue fibrosis. Wiad Lek 2008;61: [24] Kumanov P, Tomova A, Kirilov G. Testosterone replacement therapy in male hypogonadism is not associated with increase of endothelin-1 levels. Int J Androl 2007;30:41 7. [25] Norata GD, Tibolla G, Seccomandi PM, Poletti A, Catapano AL. Dihydrotestosterone decreases tumor necrosis factor-alpha and lipopolysaccharide-induced inflammatory response in human endothelial cells. J Clin Endocrinol Metab 2006;91: [26] Hatakeyama H, Nishizawa M, Nakagawa A, Nakano S, Kigoshi T, Uchida K. Testosterone inhibits tumor necrosis factor-alphainduced vascular cell adhesion molecule-1 expression in human aortic endothelial cells. FEBS Lett 2002;530:

8 316 EUROPEAN UROLOGY 56 (2009) [27] Altman R, Motton DD, Kota RS, Rutledge JC. Inhibition of vascular inflammation by dehydroepiandrosterone sulfate in human aortic endothelial cells: roles of PPARalpha and NF-kappaB. Vascul Pharmacol 2008;48: [28] Bowles DK, Maddali KK, Dhulipala VC, Korzick DH. PKC delta mediates anti-proliferative, pro-apoptic effects of testosterone on coronary smooth muscle. Am J Physiol Cell Physiol 2007;293: C [29] Williams MR, Ling S, Dawood T, et al. Dehydroepiandrosterone inhibits human vascular smooth-muscle cell proliferation independent of ARs and ERs. J Clin Endocrinol Metab 2002;87: [30] Furutama D, Fukui R, Amakawa M, Ohsawa N. Inhibition of migration and proliferation of vascular smooth-muscle cells by dehydroepiandrosterone sulfate. Biochim Biophys Acta 1998;1406: [31] Zhang YZ, Xing XW, He B, Wang LX. Effects of testosterone on cytokines and left ventricular remodeling following heart failure. Cell Physiol Biochem 2007;20: [32] Nahrendorf M, Frantz S, Hu K, et al. Effect of testosterone on postmyocardial infarction remodeling and function. Cardiovasc Res 2003;57: [33] Liu J, Tsang S, Wong TM. Testosterone is required for delayed cardioprotection and enhanced heat shock protein 70 expression induced by preconditioning. Endocrinology 2006;147: [34] Singh R, Artaza JN, Taylor WE, Gonzalez-Cadavid NF, Bhasin S. Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology 2003;144: [35] Singh R, Artaza JN, Taylor WE, et al. Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors. Endocrinology 2006;147: [36] Iacono F, Giannella R, Somma P, Manno G, Fusco F, Mirone V. Histological alterations in cavernous tissue after radical prostatectomy. J Urol 2005;173: [37] Yamamoto H, Sasaki S, Tatsura H. Penile apoptosis in association with p53 under lack of testosterone. Urol Res 2004;32:9 13. [38] Zhang XH, Hu LQ, Zheng XM, Li SW. Apoptosis in rat erectile tissue induced by castration. Asian J Androl 1999;1: [39] Rogers RS, Graziottin TM, Lin CS, Kan YW, Lue TF. Intracavernosal vascular endothelial growth factor (VEGF) injection and adenoassociated virus-mediated VEGF gene therapy prevent and reverse venogenic erectile dysfunction in rats. Int J Impot Res 2003;15: [40] Rajasekaran M, Kasyan A, Allilain W, Monga M. Ex vivo expression of angiogenic growth factors and their receptors in human penile cavernosal cells. J Androl 2003;24: [41] Ryu JK, Han JY, Chu YC, et al. Expression of cavernous transforming growth factor-beta1 and its type II receptor in patients with erectile dysfunction. Int J Androl 2004;27:42 9. [42] Haag SM, Hauck EW, Szardening-Kirchner C, et al. Alterations in the transforming growth factor (TGF)-b pathway as a potential factor in the pathogenesis of Peyronie s disease. Eur Urol 2007;51: [43] Gelman J, Garbán H, Shen R, et al. Transforming growth factorbeta1 (TGF-beta1) in penile and prostate growth in the rat during sexual maturation. J Androl 1998;19:50 7. [44] Shabsigh R, Raymond JF, Olsson CA, O Toole K, Buttyan R. Androgen induction of DNA synthesis in the rat penis. Urology 1998;52: [45] Becker AJ, Uckert S, Stief CG, et al. Cavernous and systemic testosterone plasma levels during different penile conditions in healthy males and patients with erectile dysfunction. Urology 2001;58: [46] Shen ZJ, Zhou XL, Lu YL, Chen ZD. Effect of androgen deprivation on penile ultrastructure. Asian J Androl 2003;5:33 6. [47] Armagan A, Hatsushi K, Toselli P. The effects of testosterone deficiency on the structural integrity of the penile dorsal nerve in the rat. Int J Impot Res 2008;20:73 8. [48] Baba K, Yajima M, Carrier S, et al. Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis. BJU Int 2000;85: [49] Syme DB, Corcoran NM, Bouchier-Hayes DM, Morrison WA, Costello AJ. The effect of androgen status on the structural and functional success of cavernous nerve grafting in an experimental rat model. J Urol 2007;177: [50] Yassin AA, Saad F. Improvement of sexual function in men with late-onset hypogonadism treated with testosterone only. J Sex Med 2007;4: