The influence of gender and sex hormones in the development of translational and experimental pulmonary arterial hypertension

Wright, Audrey F. (2014) The influence of gender and sex hormones in the development of translational and experimental pulmonary arterial hypertension. PhD thesis, University of Glasgow.

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Abstract

Pulmonary arterial hypertension (PAH) is a progressive and debilitating disease characterised by increases in pulmonary vasoconstriction and excessive remodelling of the pulmonary arteries. Together, these processes lead to sustained elevations in pulmonary arterial pressure, right heart failure and eventual death if left untreated. Despite the number and variety of treatment options available, the survival rate in incident and prevalent cases of PAH remains poor. Therefore, a better understanding of the pathobiology of PAH is required to generate novel therapeutic approaches with improved efficiency in patients. In PAH there is a well described gender bias. Women are consistently reported to represent up to 75% of the total PAH population; however, the reasons for this female predominance remain unclear. Recently, estrogen has been implicated as a major risk factor, for example, elevated estrogen levels and alterations in estrogen metabolism are closely correlated with PAH development in females. The role of testosterone in PAH is currently under investigated. Effects of estrogen are mediated through two classical estrogen receptors (ER)-α and –β, or the novel G-protein-coupled estrogen receptor (GPER). Expression of all of these receptors is identified in pulmonary vasculature, including in smooth muscle and endothelial cells. The role they play in PAH pathogenesis in females is largely undetermined. Given the diverse effects of estrogen described in the pulmonary vasculature during PAH, for example, proliferative effects in pulmonary artery smooth muscle cells (PASMCs), we hypothesised that estrogen receptors play an integral role in PAH in females. To examine this, we used both translational and experimental studies to characterise ERs in PAH. Chronic hypoxic male and female mice, and mice over-expressing the serotonin transporter (SERT+ mice), which demonstrate female susceptibility, were used to investigate the effects of an ERα antagonist in vivo. GPER knockout mice were also investigated in chronic hypoxia. In situ and in vitro studies in human PASMCs with ER agonists and antagonists added clinical relevance to our findings. In addition, testosterone manipulation was investigated in male mice by castration in vivo. Immunohistochemistry, immunoblotting and qRT-PCR analysis demonstrated that ERα was increased in PASMCs and pulmonary arteries from female PAH patients and chronic hypoxic mice, respectively. On the other hand, ERβ was decreased in PAH and hypoxia. It was also observed that females expressed higher levels of ERα in PAH compared to males whereas ERβ was lower in females. PAH was assessed by measuring right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling and muscularisation. Chronic hypoxia induced-pulmonary hypertension (PH) was attenuated in female mice dosed with the ERα antagonist MPP, shown by marked reductions in RVSP and pulmonary vascular remodelling. Hypoxic male mice remained unaffected by MPP treatment. Spontaneous PH and chronic hypoxia induced-PH observed in female SERT+ mice were reversed by treatment with MPP. Immunoblotting and qRT-PCR analysis revealed that the possible mechanism involved in the beneficial effect of MPP in females in vivo involved restoring the dysfunctional bone morphogenetic protein receptor-2 (BMPR2) axis observed in PAH. This effect was only observed in female mice. In addition, chronic hypoxia induced- PH in male and female mice was unaffected by GPER deletion. Expression of GPER 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 ERα agonist, PPT stimulated proliferation of human PASMCs. Both estrogen and PPT induced proliferation was dependent on downstream PI3K/Akt and ERK MAPK activity. In males, testosterone deprivation by surgical castration had no effect on chronic-hypoxia induced PH. RVSP, RVH and pulmonary vascular remodelling were 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. Testosterone levels were approximately halved by castration. qRT-PCR analysis showed that in mouse lung there were also no difference in expression of the androgen receptor (AR) and 5α-reducatse, the testosterone metabolising enzyme. Testosterone had no effect on proliferation of human PASMCs, although its primary metabolite, dihydrotestosterone (DHT), stimulated proliferation in a dose-dependent manner. In summary of these findings, we have identified an ERα-dependent mechanism of PAH in females, but not in males. ERα is noticeably increased in female human PASMCs from PAH patients compared to male PAH patients. Additionally, ERα activation in female human PASMCs leads to proliferation driven by PI3K/Akt and ERK MAPK activation. Treatment with an ERα antagonist attenuated the development of chronic hypoxia induced-PH in females but not males, and reversed PH in SERT+ female mice. We demonstrate that the mechanism attributed to the beneficial effect of MPP in vivo involved restoration of the dysfunctional BMPR2 signalling axis. Our results suggest that increased ERα expression may drive PAH development in females. Furthermore, we demonstrate that ERα does not play a key role in the development of hypoxia induced-PH in male mice. In addition we conclude that testosterone does not contribute to chronic hypoxic-PH observed in males. We suggest that altered local 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 implicate targeting ERs as a novel therapeutic target in PAH treatment.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Pulmonary hypertension, estrogen, estrogen receptors, gender
Subjects: Q Science > Q Science (General)
Q Science > QM Human anatomy
Q Science > QP Physiology
R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences > Cardiovascular Science
Funder's Name: UNSPECIFIED
Supervisor's Name: MacLean, Professor Margaret M
Date of Award: 2014
Depositing User: Miss Audrey F Wright
Unique ID: glathesis:2014-5209
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Jun 2014 09:53
Last Modified: 27 Jun 2014 09:54
URI: http://theses.gla.ac.uk/id/eprint/5209

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