Androgen signalling and vascular health in boys with hypospadias

Hypospadias, defined as the abnormal positioning of the urethral opening in boys, occurs in approximately 1 in 300 live births and may be associated with insufficient androgen exposure during the masculinisation programming window in utero. Testosterone is a vasoactive hormone, with effects previously described in the literature on regulation of vascular contractility and relaxation, Reactive Oxygen Species (ROS) generation, rho kinase activation and nitric oxide signalling. Men with hypogonadism are at increased risk of cardiovascular morbidity and mortality but no previous studies have investigated the link between hypospadias and vascular dysfunction. The hypothesis for this thesis was therefore that boys with hypospadias would have impaired vascular reactivity and function and evidence of increased cardiovascular risk, due to altered androgen signalling. This was investigated, firstly by studying the vascular reactivity of subcutaneous resistance arteries from boys with hypospadias (cases) compared to control boys and assessing the mechanisms underlying any differences by harvesting vascular smooth muscle cells (VSMCs). To our knowledge, these are the first experiments of this kind to be described in a paediatric population or in human penile subcutaneous arteries. Arteries from cases demonstrated increased vasoconstriction versus controls (Emax:137.9 vs 83.7, p<0.001) and reduced endothelium-dependent (Emax:77.3.4 vs 14.6, p<0.0001) and endothelium-independent (Emax:39.5 vs 24.6, p<0.0001) vasorelaxation. These data are likely representative of all systemic resistance arteries as there were no differences in vascular reactivity between arteries obtained from penile skin and those from abdominal fat. VSMCs from cases produced more oxidative stress as measured by increased levels of ROS generation (4.3 fold, p<0.01), hydrogen peroxide (1.2 fold, p<0.01) and total antioxidant capacity (34.4 fold, p<0.0001). These changes were likely driven by Nox5 and resulted in subsequent increased Rho kinase activity (9.7 fold, p<0.01) and myosin light chain phosphorylation (11 fold, p<0.0001). In addition, cases had increased DNA methyltransferase activity (1.2 fold, p<0.05) suggesting epigenetic change. Due to the known effects of testosterone on the vasculature and the fact that boys with hypospadias may be deficient in testosterone at a critical period of foetal development, the effects of sex steroids were investigated on arteries and VSMCs from boys with hypospadias and controls, demonstrating differential sex steroid responses and aberrant androgen and oestrogen signalling. Non-invasive vascular phenotyping of adolescents aged 12.0-18.9 years with hypospadias identified that cases had increased systolic blood pressure standard deviation scores (SDS) compared to healthy age- and physical activity matched- controls (median (range) 1.4 (-0.7, 2.7) vs 0.2 (-0.7, 0.8), p<0.01) but no difference in diastolic blood pressure SDS (median (range) 0.3 (-2.1, 1.6) vs 0.1 (-0.7, 0.8), p=0.8). In addition, cases had increased CIMT SDS (median (range) 1.6 (0.7, 2.0) vs 1.2 (-1.2, 2.0), p<0.05) and increased pulse pressure (median (range) 40 (20, 52) mmHg vs to (21, 64) mmHg, p<0.05). Analysis of routinely collected hospital data from throughout Scotland from 1981-2019 revealed that men born with hypospadias had a 3-fold higher risk of arrhythmia (OR [95% CI] 2.8[1.4-5.6], p<0.001); 3-fold higher risk of hypertension (OR [95% CI] 4.2[1.5-11.9], p<0.05) and 2-fold higher risk of heart failure (OR [95% CI] 1.9 [1.7-114.3], p