title: In situ and in vitro models to investigate the role of oestrogen and oestrogen metabolism in pulmonary vascular disease
Creators: name: Gregorc, Teja
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: oestrogen, pulmonary arterial hypertension, in vitro, aryl hydrocarbon receptor, hypoxia, Sugen 5416, CYP1A1, oestrogen metabolism, high-performance liquid chromatography.

description: Pulmonary arterial hypertension is an incurable vasculopathy, which affects significantly more women than men. Hence, female sex hormones may be intimately involved in the initiation and progression of disease pathogenesis. Indeed, current evidence suggests dysregulated oestrogen biosynthesis and metabolism, result in microenvironment favouring excessive proliferation of pulmonary artery smooth muscle cells, leading to significant vascular remodelling. Impairment of signalling via the bone morphogenetic protein receptor II (BMPRII) signalling pathway might also be partially accountable for increased cellular proliferation, and the sex dimorphism associated with the disease. Much of the currently available evidence regarding the pathophysiological mechanism of this vasculopathy was gained using experimental animal models of pulmonary hypertension, we aimed to develop and employ in situ and in vitro models to investigate the role of oestrogen and its metabolism in pulmonary arterial hypertension. Signalling through aryl hydrocarbon receptor results in altered expression of Phase I and II metabolising enzymes, including oestrogen metabolising enzymes. Using an in-silico approach, we demonstrated that the importance of aryl hydrocarbon receptor in experimental animal model of pulmonary hypertension induced by exposure to Sugen 5416 and chronic hypoxia. Initial quantitative real-time polymerase chain reaction analysis revealed that expression of CYP1A1 gene might be decreased in cultured pulmonary arterial smooth muscle cells from female patients, rats exposed to chronic hypoxia and Smad1 heterozygous mice. CYP1A1 gene expression was, however, greatly increased in the Sugen 5416/hypoxic experimental animal model. We further demonstrated that in this specific model several genes under the transcriptional activation of the aryl hydrocarbon receptor, such as NAD(P)H quinone dehydrogenase 1 and aryl hydrocarbon receptor repressor, are increased. In vitro stimulation of human pulmonary arterial smooth muscle cells with Sugen 5416, which is a potent agonist of the aryl hydrocarbon receptor, resulted in increased expression of CYP1A1 and 1B1. We further demonstrated by employing R.E.A.P. fractionation protocol, that stimulation of smooth muscle cells with Sugen 5416 resulted in the translocation of the aryl hydrocarbon receptor from the cytoplasm to the nucleus, where it was able to exert its transcriptional activity. The blockade of the aryl hydrocarbon receptor pathway by CH223191, resulted in attenuated expression of these oestrogen metabolising enzymes only in smooth muscle cells derived from pulmonary arteries of control subjects. In human pulmonary microvascular endothelial cells, simulation with Sugen 5416 and aryl hydrocarbon receptor inhibitor, FICZ, increased apoptosis of these cells as assessed via reduced cell number and increased cleavage of Caspase 3. Hence, Sugen 5416 may induce vascular injury, leading to initiation of pathophysiological mechanisms of experimental PH. Even though the aryl hydrocarbon receptor pathway interacts with hypoxia-inducible factor signalling, we showed that stimulation of pulmonary artery smooth muscle cells does not alter mediators involved in the latter pathway. Furthermore, we showed that sex dimorphism might exist in the basal expression of mediators involved in the hypoxia-inducible factors signalling pathway (HIFs). In smooth muscle cells derived from pulmonary arteries of female PAH patients, the expression of HIF1α was significantly increased, while the regulators of HIF1α proteasomal degradation were significantly decreased. Equally, in smooth muscle cells derived from pulmonary arteries of male PAH patients, protein expression regulators of HIF1α proteasomal degradation were significantly decreased. In vitro, we demonstrated that there a possible synergy exists between the aryl hydrocarbon receptor and hypoxia-inducible factor signalling pathways, where co-stimulation with Sugen 5416 and hypoxia resulted in increased proliferation of smooth muscle cells derived from pulmonary arteries of female PAH. We also aimed to develop and employ an in vitro model to investigate the role of oestrogen and its metabolism in the pathophysiological mechanism of pulmonary arterial hypertension, and to quantitatively assess oestrogen metabolism in this model. We showed that stimulation with 2-methoxyoestrogens increased expression of prostacyclin synthase in pulmonary microvascular endothelial cells from female control subject. Using immunoblotting technique, we further demonstrated that in smooth muscle cells derived from pulmonary arteries of male control subjects 2-methoxyoestradiol increased protein expression of Id3 and had no significant effect in smooth muscle cells derived from pulmonary arteries of female control subjects. It appears that in female pulmonary artery smooth muscle cells 2-methoxyoestradiol increased the expression of p27/Kip1 in a concentration-dependent manner. In fact, in vitro stimulation with 2-methoxyoestradiol attenuated serum-induced proliferation in smooth muscle cells derived from pulmonary arteries of control subjects of both sexes. In smooth muscle cells derived from pulmonary arteries of PAH patients, 2-methoxyoestradiol only reduced cellular proliferation in female cells, while having no effect in male cells. We have also examined the effects of 2- and 4-hydroxyoestradiol on serum-induced proliferation in pulmonary artery smooth muscle cells from male control subjects. Here we found both metabolites mediated attenuation of cellular proliferation, with 2-hydroxyoestradiol mediating its effects through its biotransformation into 2-methoxyoestradiol, as catechol-O-methyl transferase inhibition restored cellular proliferation. Inhibition of catechol-O-methyl transferase did not have an effect on 4-hydroxyoestradiol-mediated reduction of serum-induced proliferation. Using the in vitro model of pulmonary arterial hypertension employing pulmonary artery smooth muscle cells, we have assessed the effects of treprostinil on oestrogen metabolism in these cells using high performance liquid chromatography/flux analysis. We demonstrated that pulmonary artery smooth muscle cells derived from female and male PAH patients might metabolise 17β-oestradiol differently than those derived from control subjects. Moreover, treprostinil might affect the metabolism of 17β-oestradiol to oestrone, probably by affecting the activity of 17β-hydroxysteroid dehydrogenase type 2. Basal protein expression level as assessed by immunoblotting technique indicated that the latter enzyme might be increased in pulmonary artery smooth muscle cells derived from male PAH patients. To overcome certain shortcomings of high performance liquid chromatography/flux analysis for investigation of oestrogen metabolism, we aimed to develop and optimise a liquid chromatography tandem mass spectrometry approach. We have established the technique, and optimised separation of methoxyoestrogens and wash steps. The novel approach was used to assess oestrogen metabolism in in vitro model of pulmonary arterial hypertension, showing that smooth muscle cells derived from female PAH patients produce significantly more oestrone and 17α-isomer of oestradiol. Moreover, smooth muscle cells derived from male PAH patients metabolised the least 17β-oestradiol in comparison with female patient cells. Although the technique is now established, further optimisation is required to achieve reliable quantification of oestrogen metabolites in reasonable concentration ranges, as currently the method appears unreliable at concentration ranges for some oestrogen metabolites. Using a hypothesis-free metabolomic screen we also demonstrated that certain metabolic pathways associated with energy reactive oxygen species and L-tryptophan metabolism might be affected in pulmonary artery smooth muscle cells. Using in situ model we showed that induction of the aryl hydrocarbon receptor by Sugen 5416 leads to alterations in the expression of oestrogen metabolising enzymes, such as CYP1B1 and CYP1A1. Thereby possibly affecting the pathogenic mechanisms by introducing an imbalance of oestrogen metabolism. By using in vitro model of pulmonary arterial hypertension, we also showed methoxyoestrogen the role of methoxyoestrogens, 2-and 4-hydroxyoestrogens in the attenuation of cellular proliferation in pulmonary artery smooth muscle cells. Furthermore, we established oestrogen metabolic profile in pulmonary artery smooth muscle cells derived from controls and patients of both sexes and quantified these metabolites. We have therefore successfully applied in situ and in vitro approaches to investigate the role of oestrogen and oestrogen metabolism in the pathogenesis of pulmonary arterial hypertension.