Pulmonary arterial hypertension (PAH) is a progressive and debilitating diseasecharacterised by increases in pulmonary vasoconstriction and excessiveremodelling of the pulmonary arteries. Together, these processes lead tosustained elevations in pulmonary arterial pressure, right heart failure andeventual death if left untreated. Despite the number and variety of treatmentoptions available, the survival rate in incident and prevalent cases of PAHremains poor. Therefore, a better understanding of the pathobiology of PAH isrequired to generate novel therapeutic approaches with improved efficiency inpatients. In PAH there is a well described gender bias. Women are consistentlyreported to represent up to 75% of the total PAH population; however, thereasons for this female predominance remain unclear. Recently, estrogen hasbeen implicated as a major risk factor, for example, elevated estrogen levelsand alterations in estrogen metabolism are closely correlated with PAHdevelopment in females. The role of testosterone in PAH is currently underinvestigated.Effects of estrogen are mediated through two classical estrogen receptors (ER)-αand –β, or the novel G-protein-coupled estrogen receptor (GPER). Expression ofall of these receptors is identified in pulmonary vasculature, including in smoothmuscle and endothelial cells. The role they play in PAH pathogenesis in femalesis largely undetermined. Given the diverse effects of estrogen described in thepulmonary vasculature during PAH, for example, proliferative effects inpulmonary artery smooth muscle cells (PASMCs), we hypothesised that estrogenreceptors play an integral role in PAH in females. To examine this, we used bothtranslational and experimental studies to characterise ERs in PAH. Chronichypoxic male and female mice, and mice over-expressing the serotonintransporter (SERT+ mice), which demonstrate female susceptibility, were used toinvestigate the effects of an ERα antagonist in vivo. GPER knockout mice werealso investigated in chronic hypoxia. In situ and in vitro studies in humanPASMCs with ER agonists and antagonists added clinical relevance to ourfindings. In addition, testosterone manipulation was investigated in male miceby castration in vivo.Immunohistochemistry, immunoblotting and qRT-PCR analysis demonstrated thatERα was increased in PASMCs and pulmonary arteries from female PAH patientsand chronic hypoxic mice, respectively. On the other hand, ERβ was decreasedin PAH and hypoxia. It was also observed that females expressed higher levels ofERα in PAH compared to males whereas ERβ was lower in females. PAH wasassessed by measuring right ventricular systolic pressure (RVSP), right ventricularhypertrophy (RVH) and pulmonary vascular remodelling and muscularisation.Chronic hypoxia induced-pulmonary hypertension (PH) was attenuated in femalemice dosed with the ERα antagonist MPP, shown by marked reductions in RVSPand pulmonary vascular remodelling. Hypoxic male mice remained unaffectedby MPP treatment. Spontaneous PH and chronic hypoxia induced-PH observed infemale SERT+ mice were reversed by treatment with MPP. Immunoblotting andqRT-PCR analysis revealed that the possible mechanism involved in thebeneficial effect of MPP in females in vivo involved restoring the dysfunctionalbone morphogenetic protein receptor-2 (BMPR2) axis observed in PAH. Thiseffect was only observed in female mice. In addition, chronic hypoxia induced-PH in male and female mice was unaffected by GPER deletion. Expression ofGPER between female non-PAH controls and PAH patients was unchanged.In isolated human PASMCs estrogen induced proliferation was inhibited by MPP,but not PHTPP or G15, an ERβ and GPER antagonist, respectively. The ERαagonist, PPT stimulated proliferation of human PASMCs. Both estrogen and PPTinduced proliferation was dependent on downstream PI3K/Akt and ERK MAPKactivity.In males, testosterone deprivation by surgical castration had no effect onchronic-hypoxia induced PH. RVSP, RVH and pulmonary vascular remodellingwere unchanged in hypoxic castrated mice relative to sham controls.Testosterone levels, assessed by enzyme linked immunosorbent assay (ELISA)demonstrated no effects of hypoxia on plasma testosterone levels. Testosteronelevels were approximately halved by castration. qRT-PCR analysis showed thatin mouse lung there were also no difference in expression of the androgenreceptor (AR) and 5α-reducatse, the testosterone metabolising enzyme.Testosterone had no effect on proliferation of human PASMCs, although itsprimary metabolite, dihydrotestosterone (DHT), stimulated proliferation in adose-dependent manner.In summary of these findings, we have identified an ERα-dependent mechanismof PAH in females, but not in males. ERα is noticeably increased in femalehuman PASMCs from PAH patients compared to male PAH patients. Additionally,ERα activation in female human PASMCs leads to proliferation driven by PI3K/Aktand ERK MAPK activation. Treatment with an ERα antagonist attenuated thedevelopment of chronic hypoxia induced-PH in females but not males, andreversed PH in SERT+ female mice. We demonstrate that the mechanismattributed to the beneficial effect of MPP in vivo involved restoration of thedysfunctional BMPR2 signalling axis. Our results suggest that increased ERαexpression may drive PAH development in females. Furthermore, wedemonstrate that ERα does not play a key role in the development of hypoxiainduced-PH in male mice. In addition we conclude that testosterone does notcontribute to chronic hypoxic-PH observed in males. We suggest that alteredlocal synthesis and metabolism in the lung and right ventricle may however,facilitate progression of established PAH in males and worsening survival rates.Overall, our results provide evidence for ERα in PAH development and implicatetargeting ERs as a novel therapeutic target in PAH treatment.
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The influence of gender and sex hormones in the development of translational and experimental pulmonary arterial hypertension
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