This project aims to reduce animal use by adapting a new method using human cells to assess the role of oestrogen metabolites in the development of pulmonary arterial hypertension.
Pulmonary arterial hypertension (PAH) occurs when the pressure inside lung (pulmonary) arteries increases and the arteries thicken, narrow, and eventually die off, leading to heart failure. Animal models are used to examine the early stages of the disease, and have shown that it may be caused increased concentrations of oestrogen. However, current methods of measuring the presence of oestrogen and its metabolites in the lungs have limited sensitivity and require the use of large numbers of animals. The replacement of ELISA methods with HPLC and LC-MS provides much greater sensitivity and breadth permitting use of human pulmonary arterial cells (hPASMCs) for mechanistic studies and reducing the need for animal work.
Research details and methods
The project will apply the new methods to a variety of questions including whether cells from women or PAH patients have increased levels of harmful oestrogen metabolites and how testosterone metabolism differs in PAH. In addition, a method for measuring oestrogen metabolites in lung tissue will be developed and applied to the lung tissue previously harvested from experimental animals.
Pulmonary arterial hypertension (PAH) is more prevalent in women than in men yet the reasons for this are unknown. Evidence from animal models suggest that oestrogen and oestrogen metabolites may contribute to PAH. Oestrogen metabolites can be either anti-proliferative or pro-proliferative, and so it is imperative to understand how oestrogen metabolism is affected in the disease setting. ELISA approaches are used currently to measure oestrogen and oestrogen metabolites in tissues and blood from animals. This approach is, however, is severely restricted and limited. We have carried out feasibility studies for the development of a unique cellular model for examining oestrogen metabolism in human pulmonary arterial cells (hPASMCs) utilising HPLC and LC-MS techniques. This both reduces and replaces animal experiments and also increases the sensitivity and breadth of the metabolomic profile we are able to measure. This approach also increases the translational potential of the study as we can study cells from patients with PAH. Using animals, we have already shown therapeutic effects anastrozole (inhibitor of oestrogen synthesis), an ERalpha (major oestrogen receptor)-antagonist, a CYP1B1 (major metabolising enzyme of oestrogen) antagonist and ovariectomy. We wish to adapt our new methodology to measure oestrogen metabolites in lung tissue already harvested from these experimental animals. We will then determine which changes in oestrogen metabolite accumulation relates to the therapeutic effect. We will also expand our metabolic profiling to examine the synthesis and metabolism of testosterone. Using pharmacological, cell and molecular biological techniques the student will also carry out functional studies on the hPASMCs to determine the proliferative and signalling responses to key metabolic pathways that are described by these studies. The study will identify important mediators of PAH and the effects of these will be examined in animal models in vivo.
Denver N et al. (2019). Data for analysis of catechol estrogen metabolites in human plasma by liquid chromatography tandem mass spectrometry. Data Brief 23:103740. doi: 10.1016/j.dib.2019.103740
Dean A et al. (2018). Role of the Aryl Hydrocarbon Receptor in Sugen 5416-induced Experimental Pulmonary Hypertension. American Journal of Respiratory Cell and Molecular Biology 58(3):320-330. doi: 10.1165/rcmb.2017-0260OC