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Canadian Journal of Cardiology

Androgen and androgen receptors as determinants of vascular sex differences across the lifespan

  • Angela K. Lucas-Herald
    Correspondence
    Correspondence: Dr Angela K Lucas-Herald MBChB, PhD, Developmental Endocrinology Research Group, School of Medicine, University of Glasgow, New South Glasgow University Hospital Campus, 1345 Govan Road, Glasgow, G51 4TF, Tel : (44) 0141-451 5885. :
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  • Rhian M. Touyz
    Correspondence
    Correspondence: Professor Rhian M Touyz MBBCh, PhD, Research Institute of the McGill University Health Centre (RI-MUHC), McGill University, 1001, Boul Décarie, ES1.5066.6, Montréal, Québec, Canada, H4A 3J1, Tel : (514) 934-1934 #71608 . :
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Open AccessPublished:September 22, 2022DOI:https://doi.org/10.1016/j.cjca.2022.09.018

      Abstract

      Androgens, including testosterone and its more potent metabolite, dihydrotestosterone, exert multiple actions in the body. Physiologically they play a critical role in male sex development. In addition, they influence vascular function including arterial vasodilation and mediation of myogenic tone. Androgens are produced from 9 weeks gestation in the human fetal testis, as well as in small amounts by the adrenal glands. Serum concentrations vary according to age and sex. The vasculature is a target for direct actions of androgens, which bind to various sex hormone receptors expressed in endothelial and vascular smooth muscle cells. Androgens exert both vasoprotective and vasoinjurious effects depending on multiple factors including sex-specific effects of androgens, heterogeneity of the vascular endothelium, differential expression of androgen and sex hormone receptors in endothelial and vascular smooth muscle cells and the chronicity of androgen administration. Chronic administration of androgens induces vasoconstriction and influences endothelial permeability, while acute administration may have opposite effects. At the cellular level, androgens stimulate endothelial cell production of nitric oxide and inhibit pro-inflammatory signaling pathways, inducing vasorelaxation and vasoprotection. However androgens also activate endothelial production of vasoconstrictors and stimulate recruitment of endothelial progenitor cells. In humans, both androgen deficiency and androgen excess are associated with increased cardiovascular morbidity and mortality. This review discusses how androgens modulate vascular sex differences across the lifespan by considering the actions and production of androgens in both sexes and describes how cardiovascular risk is altered as levels of androgens change with aging.

      Keywords

      Abbreviations:

      5-oxoETE (5-oxo-eicosatetraenoic acid), ADTRP (androgen-dependent tissue factor pathway inhibitor regulating protein), AEC (aortic endothelial cells), AR (androgen receptor), ARE (androgen response elements), BSA (bovine serum albumin), CAG (polyglutamine), CVD (cardiovascular disease), DAG (diacylglycerol), DBD (DNA binding domain), DHT (dihydrotestosterone), eNOS (endothelial nitric oxide synthase), GnRH (gonadotrophin releasing hormone), GPRC6A (G protein-coupled receptor family C group 6 member A), HAT (histone acetyltransferase), HSP (heat shock protein), IP3 (inositol triphosphate receptor), LBD (C-terminal ligand binding domain), LH (luteinizing hormone), MAPK (mitogen-activated protein kinase), NO (nitric oxide), PKA (protein kinase A), PKC (protein kinase C), RAS/MEK/ERK (rat sarcoma/map kinase kinase/extracellular signal related kinase), TBP (TATA binding protein)

      Introduction

      Androgens are steroid hormones that are critically involved in normal male sex development during the fetal period, as well as in the development of secondary sexual characteristics during puberty. Growing evidence indicates that they also have significant roles within the vasculature, including arterial vasodilation, mediation of myogenic tone and inhibition of neointimal atherosclerotic plaque formation
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      .
      Despite increasing awareness and ongoing public health measures, cardiovascular disease (CVD) remains the leading cause of death worldwide, representing over 30% of all deaths and 45% of non-communicable deaths
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      . Hypertension, in particular, occurs and progresses differently in men and women, as reviewed in Gillis et al
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      . There are several strands of evidence that demonstrate that the sex hormones, androgens and estrogens account for some of the sexual dimorphism related to hypertension and cardiovascular disease. Although estrogens also have significant effects on the vasculature, this review will focus purely on androgen production across the lifespan and the role of androgens and androgen receptors in vascular function from the fetal period to adulthood.

      Androgen production throughout the lifespan

      Testosterone is the principal male androgen and is synthesised from cholesterol primarily by the Leydig cells of the testes in males to a varying extent across the lifespan, as shown in Figure 1. Small amounts are also secreted by the zona reticularis of the adrenal glands in both sexes
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      • Cheng C.Y.
      • Liu Y.X.
      Development, function and fate of fetal Leydig cells.
      . Testosterone production by the human fetal testis begins at approximately 9 weeks, with a peak between 14-17 weeks and then a sharp decline, so that in late pregnancy the serum concentration of testosterone is similar in male and female fetuses

      Rey R, Josso N, Racine C. Sexual differentiation. Endotext [Internet]: MDText. com, Inc.; 2016.

      . In both sexes, there is also a prenatally active alternative androgen biosynthesis pathway before 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses testosterone with increased activity in congenital adrenal hyperplasia variants, resulting in virilisation in female babies
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      . From 6-10 days after birth, Gonadotrophin Releasing Hormone (GnRH) starts to rise, resulting in mini-puberty. During this time, Luteinizing Hormone (LH) reaches pubertal levels by postnatal days 16-20 7. In boys, serum testosterone levels peak between 1-3 months of age and then decline by approximately 50% per month to prepubertal levels by 9-12 months of age
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      . Although girls primarily have a peak of estrogen production during mini-puberty, serum testosterone levels also increase with some overlap between serum concentrations in both sexes
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      .
      Figure thumbnail gr1
      Figure 1Representative of androgen levels across the lifespan in men and women.
      After mini puberty there is a childhood pause of steroid production, with small irregular GnRH pulses and low gonadotrophins
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      until puberty onset. Puberty typically commences between the ages of 9-14 years in boys with a rapid rise in serum testosterone levels which are then maintained into adulthood. Beyond age 30 years, testosterone levels decrease at a rate of 1-2% per year, with some variation according to other lifestyle factors, such as adiposity, medications and chronic disease
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      . Epidemiological studies have shown that approximately 40% of men over the age of 45 years and 50% of men over 80 have low circulating testosterone levels
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      In girls, menarche is usually reached by age 13 years, with the first signs of puberty being noted from age 8 years, although there is a secular trend to these milestones occurring earlier
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      . In girls, serum testosterone levels increase during puberty to a peak between the ages of 20-25 years and then gradually decline with age, although at all ages, testosterone remains <2 nmol/L
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      Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method.
      . In adulthood, the physiological levels of testosterone in men range from 10 to 30 nmol/L with lower levels found in women (0.6–2.5 nmol/L)
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      .

      The Androgen Receptor (AR)

      Approximately 7% of testosterone is converted to the more potent androgen, dihydrotestosterone (DHT) via the enzyme 5-alpha reductase. In adult men, testosterone production ranges from 3-7 mg/day
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      Metabolic clearance rate and blood production rate of testosterone and androst-4-ene-3,17-dione under basal conditions, ACTH and HCG stimulation. Comparison with urinary production rate of testosterone.
      and DHT production from 0.2-0.3 mg/day
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      Production rates of dihydrotestosterone in healthy men and women and in men with male pattern baldness: determination by stable isotope/dilution and mass spectrometry.
      . DHT is more biologically active than testosterone, with a 2-fold higher affinity for the Androgen Receptor (AR) and a 5-fold reduction in dissociation rate compared to testosterone
      • Grino P.B.
      • Griffin J.E.
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      Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone.
      . In addition, the enzyme aromatase metabolizes approximately 0.5% of testosterone to estrogen, accounting for 95% of circulating estrogen in males
      • Dickson R.
      • Clark C.
      Estrogen receptors in the male.
      . To regulate target gene transcription, testosterone and DHT can bind to the AR in a DNA binding-dependent (‘genomic’ or ‘classical’) manner resulting in new protein synthesis , or in a non-DNA binding-dependent (‘non-genomic’ or ‘non-classical’) manner leading to a rapid induction of secondary messengers to initiate cellular events, such as protein phosphorylation
      • Foradori C.D.
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      ,
      • Lucas-Herald A.K.
      • Alves-Lopes R.
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      • Ahmed S.F.
      • Touyz R.M.
      Genomic and non-genomic effects of androgens in the cardiovascular system: clinical implications.
      (Figure 2).
      Figure thumbnail gr2
      Figure 2Genomic and non-genomic actions of androgens in the vascular smooth muscle cell. Abbreviations: ADPR: ADP-ribosyl; Ang II: angiotensin II; AR: androgen receptor; AT2R: Angiotensin II type-2 receptor; CACNA1C: calcium voltage-gated channel subunit alpha1 C; eNOS: endothelial NO synthase; DAG: diacylglycerol; GPCR: G-protein coupled receptor; H2O2: hydrogen peroxide; IP3: inositol trisphosphate; MLCK: myosin light chain kinase; MLC: myosin light chain; MYPT1: myosin phosphatase target subunit 1; NADPH: nicotinamide adenine dinucleotide phosphate; NO: nitric oxide; O2- : superoxide anion; pAKT: phosphorylated protein kinase B; PARP: poly ADP-ribose polymer; peNOS: phosphorylated endothelial NO synthase; pERK 1/2: phosphorylated extracellular signal–regulated kinases 1/2; PI3K: phosphoinositide 3-kinase; PKC: protein kinase; CPLC: phospholipase C; Src: proto-oncogene tyrosine-protein kinase; TBXAR: thromboxane A2 receptor; TRPM2: transient receptor potential cation channel subfamily M member 2.
      The AR is a single copy gene located on the X chromosome at Xq11-12. Its structure is similar to that of other nuclear receptors and consists of three functional domains: 1) the N-terminal domain (NTD); 2) the DNA binding domain (DBD); and 3) the C-terminal ligand binding domain (LBD). The NTD is the most variable domain and is responsible for transcription regulation. It is encoded by exon 1 and contains a polyglutamine (CAG) sequence. This has been demonstrated to be of functional importance, because AR activity is inversely proportional to the number of CAG repeats, with the normal sequence described as 11-31 triplets
      • Choong C.
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      Trinucleotide repeats in the human androgen receptor: a molecular basis for disease.
      . Exons 2 and 3 of the AR encode the DBD, which is the most highly conserved region
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      • Grossmann M.
      Androgen Receptor Structure, Function and Biology: From Bench to Bedside.
      . The DBDs of all sex steroid receptors consist of two zinc fingers that facilitate direct DNA binding of the AR to the promoter and enhancer regions of AR-regulated genes allowing activation of the NTD and LBD for gene transcription
      • Davey R.A.
      • Grossmann M.
      Androgen Receptor Structure, Function and Biology: From Bench to Bedside.
      . Meanwhile, exons 4-8 encode the LBD
      • Werner R.
      • Holterhus P.M.
      Androgen action.
      , and this also has a similar structure to other sex steroid receptors. The LBD regulates the interactions between the AR and the heat shock and chaperone proteins and also interacts with the NTD to stabilise bound androgens
      • Davey R.A.
      • Grossmann M.
      Androgen Receptor Structure, Function and Biology: From Bench to Bedside.
      . Over 400 mutations and polymorphisms in the AR have been described
      • Gottlieb B.
      • Beitel L.K.
      • Nadarajah A.
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      • Trifiro M.
      The androgen receptor gene mutations database: 2012 update.
      and these are thought to be inversely proportional to the transcriptional response to testosterone
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      • Ahmed S.F.
      Understanding differences and disorders of sex development. Foreword.
      .

      Androgen signaling through the AR

      Without ligand binding, the AR is primarily found in its basal state in the cytoplasm. Here, it associates with heat shock proteins (HSPs), which modulate AR conformation in preparation for efficient ligand binding
      • Bennett N.C.
      • Gardiner R.A.
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      • Gobe G.C.
      Molecular cell biology of androgen receptor signalling.
      . Androgens cross the plasma membrane, enter the cytoplasm and bind to the AR. This leads to the dissociation of chaperone proteins and translocation of the complex to the nucleus where it dimerises and binds to androgen response elements (AREs) to modulate gene transcription. AR binding to specific AREs results in recruitment of histone acetyltransferase (HAT) enzymes and other essential co-regulators
      • Bennett N.C.
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      • Gobe G.C.
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      . This facilitates binding of TATA binding protein (TBP) followed by general transcription factors to begin transcription and to regulate the expression of androgen-regulated genes
      • Bennett N.C.
      • Gardiner R.A.
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      • Johnson D.W.
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      ,
      • Li J.
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      Mechanism of androgen receptor action.
      . Protein synthesis subsequently occurs
      • Grino P.B.
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      • Wilson J.D.
      Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone.
      .

      Actions of androgens via androgen responsive membrane receptors

      GPRC6A (G protein-coupled receptor family C group 6 member A) is a member of the Family C, G-protein coupled receptors. It is expressed in many tissues including bone marrow stromal cells, monocytes, prostate cancer cells, skeletal muscle cells, vascular smooth muscle cells, endothelial cells and Leydig cells
      • Harno E.
      • Edwards G.
      • Geraghty A.R.
      • et al.
      Evidence for the presence of GPRC6A receptors in rat mesenteric arteries.
      . It comprises a venus fly trap (VFT) motif attached to a 7-transmembrane domain and is activated by multiple ligands including calcium, zinc, magnesium, l-arginine, l-lysine, l-ornithine and the bone-derived peptide, osteocalcin
      • Pi M.
      • Nishimoto S.K.
      • Quarles L.D.
      GPRC6A: Jack of all metabolism (or master of none).
      .
      In vivo, testosterone-induced ERK phosphorylation in the bone marrow and testes is markedly attenuated in GPRC6A−/− mice, demonstrating that GPRC6A is a non-classical receptor through which androgens induce ERK activation
      • Pi M.
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      GPRC6A Mediates the Non-genomic Effects of Steroids.
      . ERK activity is rapidly stimulated by androgens in HEK-293 cells (which lack both the AR and GPRC6A receptor) transfected with GPRC6A. This rapid activation occurs in a calcium- dependent manner and does not take place in non-transfected HEK-293 controls

      Pi M, Parrill AL, Quarles LD. GPRC6A mediates the non-genomic effects of steroids. Vol 285. The Journal of Biological Chemistry2010:39953-39964.

      . Flutamide, an AR inhibitor, has no effect on testosterone-stimulated GPRC6A activation of phospho-ERK. Androgens do not stimulate ARE-luciferase activity in HEK-293 cells expressing only GPRC6A but do stimulate HEK-293 cells transfected with AR, suggesting activation of nuclear receptor signalling.
      In addition to GPRC6A, non-DNA binding-dependent actions of testosterone are associated with androgen binding to ZIP9, a membrane-integrated receptor
      • Bulldan A.
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      • Shihan M.
      • Scheiner-Bobis G.
      Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells.
      . ZIP9 acts as both a membrane AR and zinc transporter. In 93RS2 Sertoli cells, a cell line that does not express AR, testosterone at 10 nM induces ERK1/2 phosphorylation. Similar effects were also observed in CREB and ATF-1 phosphorylation, effects suppressed by ZIP9-siRNA, indicating that ZIP9 is involved in the testosterone–induced signalling pathway. Apoptosis is altered by ZIP9, with a reduction seen in cells transfected with ZIP9
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      Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: II. Role of human ZIP9 in testosterone-induced prostate and breast cancer cell apoptosis.
      .
      A third membrane receptor, which can be activated by androgens is G protein coupled oxo-eicosanoid receptor 1 OXER1 (5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor). This is a GPCR, which was first described in 2002 and which regulates the biological actions of 5-oxo-eicosatetraenoic acid (5-oxoETE), a product of the metabolism of arachidonic acid by 5-lipoxygenase (5-LOX) and peroxidase
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      Membrane androgen receptors (OXER1, GPRC6A AND ZIP9) in prostate and breast cancer: A comparative study of their expression.
      . It is expressed in liver, kidney, spleen, lung and various inflammatory cells including eosinophils, neutrophils, lymphocytes and monocytes
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      Membrane androgen receptors (OXER1, GPRC6A AND ZIP9) in prostate and breast cancer: A comparative study of their expression.
      . Testosterone acts as an antagonist of this receptor, opposing the p38 and PI3K pathways, thereby modulating cell migration and metastasis in DU-145 prostate cancer cells
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      5-Oxo-ETE analogs and the proliferation of cancer cells.
      .

      Non-genomic actions of androgens

      ‘Non-genomic’ signalling is independent of the ligand-dependent transactivation function of nuclear receptors and typically occurs within a short time frame
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      . To be considered a non-genomic response, the androgen-induced response must occur in a time frame not long enough to allow gene transcription, normally within seconds to minutes. The response should be observed even when the androgen is conjugated to molecules such as bovine serum albumin (BSA) that prevent it from entering into the cytoplasm. In addition, the non-genomic response should not be blunted by inhibitors of transcription and does not require a functional nucleus or transcription/translation machinery activation
      • Foradori C.D.
      • Weiser M.J.
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      Non-genomic actions of androgens.
      . The nature of the non-genomic actions of androgens depend on the type of target cell but can include rapid Ca2+ release, activation of the RAS/MEK/ERK MAPK pathway or modification of ion channels
      • Foradori C.D.
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      Non-genomic actions of androgens.
      .
      The rapid rise of intracellular calcium concentration in response to testosterone provides the most robust evidence that androgens induce cellular effects through non-genomic signalling
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      Rapid actions of androgens.
      . Hypogonadism is associated with an increased risk of osteopenia and osteoporosis, an effect normalized by testosterone replacement
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      • Dobs A.
      Osteopenia and male hypogonadism.
      . In male rat osteoblasts, low concentrations of testosterone, 0.1-1 nmol/L increase cytosolic free calcium and membrane phospholipid metabolism rapidly (5-60s), followed by an increase in the cellular content of IP3 and DAG formation. These events are not observed in female rat osteoclasts, suggesting that this is a sex dependent effect. Androgens can also activate L-type calcium channels, thereby increasing the intracellular levels of calcium, activating PKC, and via calmodulin, activating PKA and MAPK pathways
      • Foradori C.D.
      • Weiser M.J.
      • Handa R.J.
      Non-genomic actions of androgens.
      .
      Non-genomic Ca2+ mobilization by androgens were also observed in murine macrophages. In macrophages, testosterone increases intracellular levels of Ca2+ 39. During this process, androgens interact with membrane associated AR, modulate G-protein activity and subsequently activate PLC. This results in the rapid release of intracellular calcium stores from the sarcoplasmic reticulum and consequently activation of the RAS/MEK/ERK MAPK pathway
      • Foradori C.D.
      • Weiser M.J.
      • Handa R.J.
      Non-genomic actions of androgens.
      . Likewise, through activation of plasma membrane AR associated with GPCR signalling in cardiac myocytes, stimulation with testosterone induces the release of Ca2+ from internal stores, such as endoplasmic reticulum and mitochondria
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      • Weiser M.J.
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      Non-genomic actions of androgens.
      ,
      • Bennett N.C.
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      • Gobe G.C.
      Molecular cell biology of androgen receptor signalling.
      .
      Acute testosterone-induced non-genomic vasodilatation is mediated in part via endothelium-derived nitric oxide (NO)
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      • Erol K.
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      . Aortic endothelial cells (AEC) stimulated with testosterone or non-permeable testosterone-BSA at physiological concentrations (1–100 nmol/L) present rapid (15-30 min) increases in NO level in AEC; testosterone also induces eNOS phosphorylation (Ser1177) without changing the total protein level. Activation of eNOS occurs via PI3K, caveolin-1 and c-Src binding to AR and consequently phosphorylation of AKT
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      • Touyz R.M.
      Genomic and non-genomic effects of androgens in the cardiovascular system: clinical implications.
      . AR and s-Src mediate testosterone-induced rapid eNOS phosphorylation, since pre-treatment with nilutamide or PP2, an AR and s-Src antagonist respectively, abolishes the testosterone responses. Transcriptional inhibitor, actinomycin D does not affect testosterone-induced increase in NO, which excludes the classical genomic actions
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      Androgen receptor-dependent activation of endothelial nitric oxide synthase in vascular endothelial cells: role of phosphatidylinositol 3-kinase/akt pathway.
      . Anastrozole or other estrogen receptor antagonists do not interfere in NO generation induced by testosterone, suggesting that this is not an event associated with the aromatisation of testosterone to oestradiol
      • Campelo A.E.
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      . In addition, testosterone at physiological concentrations inhibits PGF2alpha-induced Ca2+ fluxes by a non-genomic mechanism in VSMCs
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      [Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells].
      , which may contribute to testosterone-induced vasodilatation.
      Testosterone-induced vasodilatation not associated with DNA-binding is also observed in humans. A recent study demonstrated that administration of transdermal-testosterone in men with hypogonadism and severe hypotestosteronemia causes acute vasodilation and improves arterial stiffness by non-genomic mechanisms, although interestingly, the improvement is also evident after 96 hours of treatment, which would suggest a combination of genomic and non-genomic effects to reach the same response
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      Acute endothelial response to testosterone gel administration with severe hypogonadism and its relationship to androgen receptor polymorphisms: a pilot study.
      . In human aortic endothelial cells in vivo, testosterone can increase BKCa and SKCa currents leading to rapid replorisation and vasorelaxation, via the action of a surgace androgen receptor, Gi/o protein and protein kinase A
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      Testosterone rapidly increases Ca2+-activated K+ currents causing hyperpolarization in human coronary artery endothelial cells.
      .

      Vascular sex differences in the fetal period

      There are sex differences in the developmental programming of cardiovascular diseases. For example, in rats and guinea pigs, male pups subjected to perinatal hypoxia demonstrate greater susceptibility to the hypoxic insult than female pups
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      Altered renal vascular responses in the aging rat kidney.
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      Estrogen regulates angiotensin II receptor expression patterns and protects the heart from ischemic injury in female rats.
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      Maternal nutrient restriction alters Ca2+ handling properties and contractile function of isolated left ventricle bundles in male but not female juvenile rats.
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      Effects of hypoxia-induced intrauterine growth restriction on cardiopulmonary structure and function during adulthood.
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      Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart.
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      Vascular nitric oxide and superoxide anion contribute to sex-specific programmed cardiovascular physiology in mice.
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      . The underlying reasons why females and males exhibit these differences have not yet been fully elucidated, and it is not clear whether androgens have any effects on these processes.
      A newly described membrane protein called Androgen-Dependent Tissue Factor Pathway Inhibitor Regulating Protein (ADTRP) has been idenitified as having potential implications for vascular development. This protein was first described, after the gene which encodes it, the C6orf105 gene, was found to be associated with coronary artery disease during genome wide association studies of Chinese men with coronary artery disease
      • Guo C.-Y.
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      • Li L.
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      Association of SNP Rs6903956 on Chromosome 6p24.1 with Angiographical Characteristics of Coronary Atherosclerosis in a Chinese Population.
      . This gene has since been associated with cardiovascular disease in several studies
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      . ADTRP itself is a membrane protein, whose expression is upregulated by androgens and plays a major role in vascular development and function, via expression in endothelial and/or perivascular cells of Wnt-regulated genes, which control vascular stability and integrity
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      Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function.
      .

      Vascular sex differences in childhood

      Sex differences in blood pressure have been described in children as young as 8 years of age with boys being less likely to have a blood pressure within the normal reference range for age and height as defined using the age specific American Heart Association blood pressure categories than girls (86.6-88.8% vs 93.0-96.3%)
      • Hardy S.T.
      • Holliday K.M.
      • Chakladar S.
      • et al.
      Heterogeneity in blood pressure transitions over the life course: age-specific emergence of racial/ethnic and sex disparities in the united states.
      . Earlier onset of puberty and exposure to sex steroids have been shown to affect adult health outcomes, including cardiovascular health. One study of 3, 611 individuals using the ALSPAC cohort found that systolic blood pressure at age 18 years reduced by 3.9 mmHg per year of later puberty
      • Bell J.A.
      • Carslake D.
      • Wade K.H.
      • et al.
      Influence of puberty timing on adiposity and cardiometabolic traits: a Mendelian randomisation study.
      . It is therefore essential that age-, height- and sex-specific reference ranges are used in children for all clinical vascular phenotyping.
      As discussed above, the prenatal surge in testosterone during the fetal period occurs during a critical time for normal masculinisation of the genital tract
      • Welsh M.
      • Saunders P.T.K.
      • Fisken M.
      • et al.
      Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism.
      . Where there is insufficient androgen during this period, genital anomalies, such as hypospadias may develop. Hypospadias occurs in approximately 1 in 300 live births in Scotland
      • Ahmed S.F.
      • Dobbie R.
      • Finlayson A.R.
      • et al.
      Prevalence of hypospadias and other genital anomalies among singleton births, 1988–1997, in Scotland.
      and is defined as the abnormal positioning of the urethral meatus in boys. This condition therefore represents a model to investigate the effects of insufficient androgen exposure in utero on later vascular function. Vascular reactivity studies on resistance arteries from 46,XY boys with hypospadias and controls in early childhood (median age 18 months) demonstrated that arteries from boys with hypospadias had evidence of increased vasoconstriction versus controls and reduced endothelium-dependent and endothelium-independent vasorelaxation. This was in association with increased basal levels of ROS generation, hydrogen peroxide and total antioxidant capacity in VSMCs from boys with hypospadias and activation of contractile mechanisms including Rho kinase activity and myosin light chain phosphorylation. These findings suggest androgen deficiency in the fetal period may alter critical determinants of vascular function in children. Within the same study, clinical vascular phenotyping of adolescents born with hypospadias demonstrated increased systolic blood pressure and carotid intima media thickness standard deviation score (SDS) as well as increased pulse pressure compared to controls and increased rates of admission to hospital for CVD in adults born with hypospadias
      • Lucas-Herald A.K.
      • Montezano A.C.
      • Alves-Lopes R.
      • et al.
      Vascular dysfunction and increased cardiovascular risk in boys with hypospadias.
      . Whilst it is not clear what the underlying mechanism for these changes is in this cohort, it is likely that androgen signalling is altered in this group, resulting in adverse cardiovascular outcomes.

      Vascular sex differences in adulthood

      Hypertension is more common in men until the age of ∼60 years, at which point women are more likely to be hypertensive
      • Dubey R.K.
      • Oparil S.
      • Imthurn B.
      • Jackson E.K.
      Sex hormones and hypertension.
      . Post menopause, the average systolic blood pressure of women is 5mmHg higher than that demonstrated pre- or peri-menopause
      • Dubey R.K.
      • Oparil S.
      • Imthurn B.
      • Jackson E.K.
      Sex hormones and hypertension.
      . Altered estrogen:androgen ratios, activation of the renin-angiotensin-system (RAS), endothelin-1 (ET-1), sympathetic nervous system activation, increased inflammation, increased vasoconstrictor eicosanoids and anxiety and depression have all been postulated as mechanisms for the increase in blood pressure seen in women post menopause
      • Song J.-J.
      • Ma Z.
      • Wang J.
      • Chen L.-X.
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      Gender Differences in Hypertension.
      ,
      • Maranon R.O.
      • Lima R.
      • Mathbout M.
      • et al.
      Postmenopausal hypertension: role of the sympathetic nervous system in an animal model.
      .
      Atherosclerosis has also been demonstrated to present differently according to biological sex. Women are more likely to have diffuse atherosclerotic disease, with stable plaques which gradually erode, compared to men who often experience more acute plaque rupture, as recently reviewed by Man et al
      • Man J.J.
      • Beckman J.A.
      • Jaffe I.Z.
      Sex as a Biological Variable in Atherosclerosis.
      . Differences in the development of atherosclerosis have been attributed to sex-specific gene regulatory networks involved in smooth muscle cell phenotype switching
      • Hartman R.J.G.
      • Owsiany K.
      • Ma L.
      • et al.
      Sex-Stratified Gene Regulatory Networks Reveal Female Key Driver Genes of Atherosclerosis Involved in Smooth Muscle Cell Phenotype Switching.
      , differences in vascular inflammation
      • Moss M.E.
      • Lu Q.
      • Iyer S.L.
      • et al.
      Endothelial Mineralocorticoid Receptors Contribute to Vascular Inflammation in Atherosclerosis in a Sex-Specific Manner.
      and sexually dimorphic body composition
      • Liao Y.-Y.
      • Chu C.
      • Wang Y.
      • et al.
      Sex differences in impact of long-term burden and trends of body mass index and blood pressure from childhood to adulthood on arterial stiffness in adults: A 30-year cohort study.
      . Differences in AR mediated vascular remodelling, resulting in altered angiogenesis and increased risk of atherosclerosis have been identified
      • Takov K.
      • Wu J.
      • Denvir M.A.
      • Smith L.B.
      • Hadoke P.W.F.
      The role of androgen receptors in atherosclerosis.
      . In animal models, atherosclerosis and atherogenic lipids are increased in animals with an XX karyotype, regardless of hormone status, suggesting that there may be other mechanisms underpinning these differences outwith androgens and estrogens
      • AlSiraj Y.
      • Chen X.
      • Thatcher S.E.
      • et al.
      XX sex chromosome complement promotes atherosclerosis in mice.
      .

      Specific circumstances in adulthood

      Cardiovascular risk in androgen excess

      Polycystic ovarian syndrome (PCOS), a condition characterised by oligo/anovulation, hyperandrogenism and polycystic ovaries on ultrasound, is a useful clinical paradigm for the consideration of cardiovascular effects of androgen excess. Metabolic complications, including dyslipidaemia and raised body mass index (BMI) are common in affected women, which may also exacerbate cardiovascular risk. Comparison of the cardiovascular events in PCOS patients of reproductive age and in menopausal/ageing women are higher than in healthy controls (pooled hazard ratio (HR): 1.38, 95% confidence interval (CI): 1.12-1.171 and 1.53, 95% CI: 1.15-2.04) respectively
      • Ramezani Tehrani F.
      • Amiri M.
      • Behboudi-Gandevani S.
      • Bidhendi-Yarandi R.
      • Carmina E.
      Cardiovascular events among reproductive and menopausal age women with polycystic ovary syndrome: a systematic review and meta-analysis.
      . One small study of 151 Taiwanese women demonstrated that in women with PCOS, high testosterone levels, as measured by a raised free androgen index ≥19% was associated with raised blood pressure (SBP ≥130mmHg ± DBP ≥85mmHg) (odds ratio (OR) 3.817, CI 1.14-12.74, p=0.029) independent of age, insulin resistance, obesity or dyslipidaemia
      • Chen M.J.
      • Yang W.S.
      • Yang J.H.
      • et al.
      Relationship between androgen levels and blood pressure in young women with polycystic ovary syndrome.
      . However, a prospective study including 8,612 women from the Australian Longitudinal Study of Women’s Health suggests that although women with PCOS are more likely to be hypertensive than controls, this is not associated with raised BMI
      • Joham A.E.
      • Boyle J.A.
      • Zoungas S.
      • Teede H.J.
      Hypertension in reproductive-aged women with polycystic ovary syndrome and association with obesity.
      , again implying that high androgen levels play a greater role in the development of hypertension than other cardiovascular risk factors. Androgens seem to mediate this cardiovascular risk in PCOS via suppression of NO leading to reduced endothelin-1-induced vasodilatation both at the cellular level and demonstrated using doppler flowmetry to assess microvascular endothelial function
      • Usselman C.W.
      • Yarovinsky T.O.
      • Steele F.E.
      • et al.
      Androgens drive microvascular endothelial dysfunction in women with polycystic ovary syndrome: role of the endothelin B receptor.
      .
      During pregnancy, androgens increase with gestation
      • Mizuno M.
      • Lobotsky J.
      • Lloyd C.
      • Kobayashi T.
      • Murasawa Y.
      Plasma androstenedione and testerone during pregnancy and in the newborn.
      . Pregnant women with PCOS have increased risk of pregnancy-induced hypertension and it is not clear whether this is secondary to elevated androgen levels. In one study recruiting women with no prevous history of PCOS, those with higher androgen levels in the 3rd trimester were more likely to develop pre-eclampsia
      • Salamalekis E.
      • Bakas P.
      • Vitoratos N.
      • Eleptheriadis M.
      • Creatsas G.
      Androgen levels in the third trimester of pregnancy in patients with preeclampsia.
      . In addition, increased placental AR expression has been described in pre-eclampsia, as reviewed by Kumar et al
      • Kumar S.
      • Gordon G.H.
      • Abbott D.H.
      • Mishra J.S.
      Androgens in maternal vascular and placental function: implications for preeclampsia pathogenesis.
      . As such, the role of androgens during pregnancy should also be an area for future research.
      Another example of androgen excess can be seen in athletes who abuse anabolic steroids to increase muscle mass. Excess exogenous testosterone in this way, has however been associated with an increase in cardiovascular related deaths, secondary to cardiac hypertrophy, thrombus formation and arterial vasospasm
      • Hernández-Guerra A.I.
      • Tapia J.
      • Menéndez-Quintanal L.M.
      • Lucena J.S.
      Sudden cardiac death in anabolic androgenic steroids abuse: case report and literature review.
      . Interestingly, however the administration of testosterone supplementation to female-to-male transgender patients results in only modest increases in blood pressure, which resolve after cessation of therapy
      • Velho I.
      • Fighera T.M.
      • Ziegelmann P.K.
      • Spritzer P.M.
      Effects of testosterone therapy on BMI, blood pressure, and laboratory profile of transgender men: a systematic review.
      .
      Transgender individuals receiving gender affirming hormones should also be considered regarding the potential effects of exogenous androgen intake. As reviewed by Connelly et al., there is some evidence that transgender individuals may have increased risk of myocardial infarction, without associated increase in likelihood of angina or coronary artery disease, although data from studies remain conflicting
      • Connelly P.J.
      • Marie Freel E.
      • Perry C.
      • et al.
      Gender-Affirming Hormone Therapy, Vascular Health and Cardiovascular Disease in Transgender Adults.
      . More recent reviews such as have demonstrated that whilst the risk of CVD remained low overall, transgender individuals were at increased risk of CV events when receiving gender affirming treatment compared to cisgender individuals, although the mechanisms for this remain unclear
      • Karalexi M.A.
      • Frisell T.
      • Cnattingius S.
      • et al.
      Cardiovascular outcomes in transgender individuals in Sweden after initiation of gender-affirming hormone therapy.
      .

      Cardiovascular risk in androgen deficiency

      Androgen deficiency, or hypogonadism, is defined as low levels of circulating testosterone.
      In animal models, androgen deprivation via orchiectomy results in function and structural changes to the internal pudendal arteries, resulting in vascular endothelial dysfunction as well as reduced nitric oxide (NO) activity, which is reversed by testosterone replacement
      • Alves-Lopes R.
      • Neves K.B.
      • Silva M.A.
      • et al.
      Functional and structural changes in internal pudendal arteries underlie erectile dysfunction induced by androgen deprivation.
      . In humans, studies analysing the effects of androgen deprivation therapy for prostate cancer have demonstrated increased risk of incident diabetes, myocardial infarction, CVD
      • Keating N.L.
      • O'Malley A.J.
      • Smith M.R.
      Diabetes and cardiovascular disease during androgen deprivation therapy for prostate cancer.
      and vascular stiffness
      • Smith J.C.
      • Bennett S.
      • Evans L.M.
      • et al.
      The effects of induced hypogonadism on arterial stiffness, body composition, and metabolic parameters in males with prostate cancer.
      , across the range of different androgen deprivation modalities including gonadtrophin-releasing hormone agonists, antagonists, androgen blockage and CYP17 inhibitors, as discussed in a recent meta-analysis by Hu et al
      • Hu J.-R.
      • Duncan M.S.
      • Morgans A.K.
      • et al.
      Cardiovascular Effects of Androgen Deprivation Therapy in Prostate Cancer: Contemporary Meta-Analyses.
      .
      Basal testosterone levels are inversely related to mortality due to CVD in adult men
      • Khaw K.T.
      • 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.
      . All-cause mortality is increased in hypogonadal men and testosterone therapy reduces mortality to 8.4% compared with 19.2% in untreated groups
      • Muraleedharan V.
      • Marsh H.
      • Kapoor D.
      • Channer K.S.
      • Jones T.H.
      Testosterone deficiency is associated with increased risk of mortality and testosterone replacement improves survival in men with type 2 diabetes.
      . A large French study (n=3,650) has identified a J-shaped association between total and bioavailable testosterone levels, as measured by mass spectroscopy, and CVD events, with men in the lowest and highest total testosterone quintiles having an increased risk
      • Soisson V.
      • Brailly-Tabard S.
      • Helmer C.
      • et al.
      A J-shaped association between plasma testosterone and risk of ischemic arterial event in elderly men: the French 3C cohort study.
      . In addition, some studies including a 4-year follow-up study found that atherosclerosis progression is inversely associated with testosterone levels
      • Svartberg J.
      • von Muhlen D.
      • Mathiesen E.
      • et al.
      Low testosterone levels are associated with carotid atherosclerosis in men.
      ,
      • Muller M.
      • van den Beld A.W.
      • Bots M.L.
      • et al.
      Endogenous sex hormones and progression of carotid atherosclerosis in elderly men.
      .
      The mechanisms whereby hypogonadism exerts increased cardiovascular risk are likely multifactorial due to the many effects of testosterone on the vasculature. Hypogonadism has been associated with arrhythmia including long QT syndrome
      • Pecori Giraldi F.
      • Toja P.
      • Filippini B.
      • et al.
      Increased prevalence of prolonged QT interval in males with primary or secondary hypogonadism: a pilot study.
      and torsades de pointes
      • Salem J.-E.
      • Waintraub X.
      • Courtillot C.
      • et al.
      Hypogonadism as a reversible cause of torsades de pointes in men.
      . In addition, hypertension has been associated with low testosterone levels in men undergoing investigation for erectile dysfunction
      • Garcia-Cruz E.
      • Piqueras M.
      • Huguet J.
      • et al.
      Hypertension, dyslipidemia and overweight are related to lower testosterone levels in a cohort of men undergoing prostate biopsy.
      and 25% of heart failure patients are reported to have biochemical hypogonadism
      • Giagulli V.A.
      • Guastamacchia E.
      • Pergola G.D.
      • Iacoviello M.
      • Triggiani V.
      Testosterone deficiency in male: a risk factor for heart failure.
      . Testosterone can reduce body fat and regulate metabolism, which is also corroborated by the fact that adult men with hypogonadism have increased rates of metabolic syndrome
      • Sonmez A.
      • Haymana C.
      • Bolu E.
      • et al.
      Metabolic syndrome and the effect of testosterone treatment in young men with congenital hypogonadotropic hypogonadism.
      . Some key studies reporting risk of cardiovascular consequences in androgen deficiency (defined as circulating testosterone levels under the lower limit of the local reference range) in men are summarised in Table 191, 99-110.
      Table 1Studies demonstrating a link between low endogenous testosterone level (hypogonadism) and cardiovascular disease.
      ReferenceNumber of patientsOutcome measureHazard ratio (95% CI)p
      (Zhang, Huang et al. 2021)602Framingham CV risk score1.8 (1.64-2.12)NA
      (Corona, Rastrelli et al. 2011)12,566CVD diagnosis2.55 (-3.39, -1.71)<0.0001
      (Corona, Monami et al. 2010)1697Major adverse cardiovascular event7.1 (1.6-28.6)<0.001
      (Soisson, Brailly-Tabard et al. 2013)3650First ischaemic arterial disease event2.23 (1.02-4.88)<0.01
      (Farias, Tinetti et al. 2014)115CIMT8.43 (2.5-25.8)<0.0001
      (Yeap, Hyde et al. 2009)3433Stroke1.99 (1.33-2.99)0.01
      (Laughlin, Barrett-Connor et al. 2008)794CV death1.38 (1.02, 1.85)NA
      (Araujo, Dixon et al. 2011)17,091CV death1.54 (1.28-1.85)0.02
      (Malkin, Pugh et al. 2010)930CV death2.2 (1.4-3.6)<0.0001
      (Haring, Volzke et al. 2010)1954CV death2.56 (1.15-6.52)<0.05
      (Hyde, Norman et al. 2012)3637CV death1.71 (1.12-2.62)0.024
      (Pye, Huhtaniemi et al. 2014)2599CV death5.2 (2.0-13.7)NA
      (Boden, Miller et al. 2020)2,118CVD diagnosis1.370.03
      Abbreviations: CI: confidence interval; CIMT: carotid intima media thickness; CV: cardiovascular; CVD: cardiovascular disease; NA: not available.
      As above, testosterone levels naturally decrease in men with ageing. This effect has also been seen in women post-menopause, although other descriptions of women with androgen deficiency are scarce, as reviewed by Traish and Morgentaler recently
      • Traish A.M.
      • Morgentaler A.
      Androgen Therapy in Women with Testosterone Insufficiency: Looking Back and Looking Ahead.
      . The cardiovascular benefits of testosterone replacement are controversial. Testosterone can reduce diastolic and systolic blood pressure in some cases but increase it in others, with no clear mechanisms underlying these differential effects
      • Jones T.
      Testosterone deficiency: a risk factor for cardiovascular disease?.
      . Testosterone replacement in hypogonadal men has been demonstrated to reduce obesity, fat mass, waist circumference and mortality as well as improve glycaemic control and overall cardiometabolic status compared to placebo

      Morgentaler A, Miner MM, Caliber M, et al. Testosterone therapy and cardiovascular risk: advances and controversies. Mayo Clinic Proceedings. Vol 90: Elsevier; 2015:224-251.

      . Other studies have demonstrated that testosterone can modulate the response of subcutaneous resistance arteries to vasoconstrictors and vasodilators in different vascular beds, including rabbit coronary arteries and aorta, mouse iliac arteries and rat coronary, mesenteric and pulmonary arteries
      • Kelly D.M.
      • Jones T.H.
      Testosterone: a vascular hormone in health and disease.
      . Endothelial progenitor cells (EPCs) are essential for neovascularisation and androgens have been shown to increase EPC function resulting in coronary collateralisation in men with hypogonadism
      • Lam Y.T.
      • Hsu C.-J.
      • Simpson P.J.L.
      • et al.
      Androgens Stimulate EPC-Mediated Neovascularization and Are Associated with Increased Coronary Collateralization.
      , as well as enhancing post-ischemic vascular repair by promoting angiogenesis
      • Lam Y.T.
      • Lecce L.
      • Yuen G.S.C.
      • et al.
      Androgen action augments ischemia-induced, bone marrow progenitor cell-mediated vasculogenesis.
      .
      However, testosterone supplementation also increases haematocrit levels and reduces high density lipoprotein (HDL) cholesterol levels, which can result in increased cardiovascular morbidity and mortality
      • Basaria S.
      • Coviello A.D.
      • Travison T.G.
      • et al.
      Adverse events associated with testosterone administration.
      . Isolated clinical trials have suggested that testosterone replacement therapy (TRT) may result in adverse outcomes but a recent meta-analysis of 39 randomised controlled trials and 10 observational studies, spanning trials of TRT of between 6 weeks and 3 years duration and including 5,451 men (3,230 receiving TRT and 2,221 placebo) demonstrated no significant association between testosterone therapy and major adverse cardiovascular events, where testosterone was replaced to the normal healthy range
      • Alexander G.C.
      • Iyer G.
      • Lucas E.
      • Lin D.
      • Singh S.
      Cardiovascular risks of exogenous testosterone use among men: a systematic review and meta-analysis.
      . It is possible therefore that the excess in cardiovascular morbidity and mortality, seen in athletes who abuse testosterone, is secondary to the supraphysiological levels of testosterone ingested, which can be as high as 5-29 times greater than usual physiological replacement doses in men with previously normal androgen levels
      • Perry P.J.
      • Lund B.C.
      • Deninger M.J.
      • Kutscher E.C.
      • Schneider J.
      Anabolic steroid use in weightlifters and bodybuilders: an internet survey of drug utilization.
      .
      Different routes of administration of testosterone may, however, be associated with variable cardiovascular risk
      • Borst S.E.
      • Shuster J.J.
      • Zou B.
      • et al.
      Cardiovascular risks and elevation of serum DHT vary by route of testosterone administration: a systematic review and meta-analysis.
      , which may alter depending on duration of treatment. The mechanisms underlying the differential effects of exogenous testosterone supplementation remain unclear. A summary of studies investigating the effects of testosterone replacement therapy on CVD has been published recently by Gagliano-Juca and colleagues
      • Gagliano-Jucá T.
      • Basaria S.
      Testosterone replacement therapy and cardiovascular risk.
      .

      Conclusions

      The investigation of the effects of sex steroids on the vasculature is subject to a number of different confounders, making it a challenging field to be able to truly determine what the underlying molecular mechanisms are whereby sex hormones influence vascular function. As discussed, androgens have multiple actions on the vasculature, some of which are vasoprotective and others that induce vascular injury. Both androgen excess and androgen deficiency can result in adverse cardiovascular outcomes and there are many gaps in the scientific literature regarding the mechanisms for these differences, including the roles of sex steroid receptors and proteins, such as AR, GPRC6A and ADTRP in these processes. The need to replace testosterone in adulthood has been of significant research interest, but it still remains unclear what the implications are of altered androgen signalling in early life, such as in children and young people with hypospadias. Understanding the physiological basis behind these changes may allow for the mitigation of cardiovascular risk later on.
      In summary, although this review focuses on androgens, estrogens are also key players in the sexual dimorphism associated with vascular disease. The roles of the sex hormones may alter throughout the lifespan. Further research is however required to better understand the physiological basis behind these changes to allow for the mitigation of cardiovascular risk later on.
      Araujo, A.B., Dixon, J.M., Suarez, E.A., Murad, M.H., Guey, L.T. and Wittert, G.A. (2011). "Endogenous testosterone and mortality in men: a systematic review and meta-analysis." The Journal of Clinical Endocrinology and Metabolism 96(10): 3007-3019.
      Boden, W.E., Miller, M.G., Mcbride, R., Harvey, C., Snabes, M.C., Schmidt, J., Mcgovern, M.E., Fleg, J.L., Desvigne-Nickens, P., Anderson, T., Kashyap, M. and Probstfield, J.L. (2020). "Testosterone concentrations and risk of cardiovascular events in androgen-deficient men with atherosclerotic cardiovascular disease." American Heart Journal 224: 65-76.
      Corona, G., Monami, M., Boddi, V., Cameron-Smith, M., Fisher, A.D., De Vita, G., Melani, C., Balzi, D., Sforza, A., Forti, G., Mannucci, E. and Maggi, M. (2010). "Low testosterone is associated with an increased risk of MACE lethality in subjects with erectile dysfunction." Journal of Sexual Medicine 7(4 Pt 1): 1557-1564.
      Corona, G., Rastrelli, G., Monami, M., Guay, A., Buvat, J., Sforza, A., Forti, G., Mannucci, E. and Maggi, M. (2011). "Hypogonadism as a risk factor for cardiovascular mortality in men: a meta-analytic study." European Journal of Endocrinology 165(5): 687-701.
      Farias, J.M., Tinetti, M., Khoury, M. and Umpierrez, G.E. (2014). "Low testosterone concentration and atherosclerotic disease markers in male patients with type 2 diabetes." The Journal of Clinical Endocrinology and Metabolism 99(12): 4698-4703.
      Haring, R., Volzke, H., Steveling, A., Krebs, A., Felix, S.B., Schofl, C., Dorr, M., Nauck, M. and Wallaschofski, H. (2010). "Low serum testosterone levels are associated with increased risk of mortality in a population-based cohort of men aged 20-79." European Heart Journal 31(12): 1494-1501.
      Hyde, Z., Norman, P.E., Flicker, L., Hankey, G.J., Almeida, O.P., Mccaul, K.A., Chubb, S.A. and Yeap, B.B. (2012). "Low free testosterone predicts mortality from cardiovascular disease but not other causes: the Health in Men Study." The Journal of Clinical Endocrinology and Metabolism 97(1): 179-189.
      Laughlin, G.A., Barrett-Connor, E. and Bergstrom, J. (2008). "Low serum testosterone and mortality in older men." The Journal of Clinical Endocrinology and Metabolism 93(1): 68-75.
      Malkin, C.J., Pugh, P.J., Morris, P.D., Asif, S., Jones, T.H. and Channer, K.S. (2010). "Low serum testosterone and increased mortality in men with coronary heart disease." Heart 96(22): 1821-1825.
      Pye, S.R., Huhtaniemi, I.T., Finn, J.D., Lee, D.M., O'neill, T.W., Tajar, A., Bartfai, G., Boonen, S., Casanueva, F.F., Forti, G., Giwercman, A., Han, T.S., Kula, K., Lean, M.E., Pendleton, N., Punab, M., Rutter, M.K., Vanderschueren, D. and Wu, F.C. (2014). "Late-onset hypogonadism and mortality in aging men." The Journal of Clinical Endocrinology and Metabolism 99(4): 1357-1366.
      Soisson, V., Brailly-Tabard, S., Helmer, C., Rouaud, O., Ancelin, M.L., Zerhouni, C., Guiochon-Mantel, A. and Scarabin, P.Y. (2013). "A J-shaped association between plasma testosterone and risk of ischemic arterial event in elderly men: the French 3C cohort study." Maturitas 75(3): 282-288.
      Yeap, B.B., Hyde, Z., Almeida, O.P., Norman, P.E., Chubb, S.A., Jamrozik, K., Flicker, L. and Hankey, G.J. (2009). "Lower testosterone levels predict incident stroke and transient ischemic attack in older men." The Journal of Clinical Endocrinology and Metabolism 94(7): 2353-2359.
      Zhang, X., Huang, K., Saad, F., Haider, K.S., Haider, A. and Xu, X. (2021). "Testosterone Therapy Reduces Cardiovascular Risk Among Hypogonadal Men: A Prospective Cohort Study in Germany." Androgens: Clinical Research and Therapeutics 2(1): 64-72.

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