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.

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 > School of Cardiovascular & Metabolic Health > Cardiovascular & Metabolic Health
• 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: https://theses.gla.ac.uk/id/eprint/5209