Mass spectrometry imaging of lung in pulmonary arterial hypertension
Creators: name: Weatherill, James William
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: Mass Spectrometry Imaging
Lung
Pulmonary arterial hypertension (PAH)
liquid chromatography (LC)-MS method
ion mobility (IM)-MS
solid phase extraction (SPE)

description: Pulmonary arterial hypertension (PAH) is a rare, progressive, and ultimately deadly cardiovascular disease. A large disparity in the prevalence and progression of PAH between the sexes exists. Females are ~4-times more likely to develop PAH, whereas males are ~2-times more likely to die from the disease. PAH is characterised by muscularisation and thickening of pulmonary arterial walls. This increases blood pressure, puts strain on the heart, and ultimately leads to the development of right-ventricular hypertrophy, RV failure, and death. Oestrogens are the primary female sex hormones. Downstream metabolites of oestrogen can possess both protective and harmful effects in health and disease. In PAH, some metabolites are pro-proliferative while others anti-angiogenic. The delicate balance of these molecules greatly influences cardiovascular health, and therefore understanding their molecular profiles within the body is vital. There is a further need to explore and identify molecular pathways at tissue level underpinning PAH. It was hypothesised that the application of mass spectrometry (MS)-based analytical methods can be used to assess differential dysregulation of oestrogen and lipid metabolism between tissues from control and rodent models of pulmonary hypertension (PH). The primary aims were to develop a liquid chromatography (LC)-MS method for measuring oestrogens in lung tissue, explore an alternative separation approach for oestrogens using ion mobility (IM)-MS, and undertake an untargeted assessment of the small molecule “omic” profiles of PAH lungs. A Sugen/Hypoxic rodent animal model (n = 6 /group) was used to represent a PAH phenotype in both sexes. This thesis demonstrates a novel approach to the analysis of oestrogens in PAH. Building on an existing plasma LC-MS/MS method, a new protocol was developed, optimised, and validated for a panel of 12 oestrogen metabolites in lung tissue, using solid phase extraction (SPE) and derivatisation with 1-(5-fluoro-2,4-dinitrophenyl)-4,4-dimethylpiperazine (MPPZ). Acceptable ranges for recovery (90-104%) and ion suppression (91-109%) of oestrogens extracted from tissue were achieved. Method validation showed LoD of 10 pg/mL and LoQ of 33 pg/mL for most analytes. The finalised method was applied to rodent tissue samples for comparison of sex effects in control and PAH, where four analytes were detected in lung tissue. There was no difference in the lung expression of oestrogen metabolites between males and females or after the induction of PH. There was, however, evidence of increased estradiol (E2) and estrone (E1) in the female control rats compared with males. To enable tissue sampling, IM-MS was verified as an orthogonal separation method for oestrogens. In IM, separation is based on a molecules three-dimensional shape, size, and charge, denoted as collision cross section (CCS). Separation of isomeric oestrogens was compared using two IM technologies (travelling wave (TW)- and cyclic (c)-IMS). Separation of derivatised oestrogens (MPPZ, dansyl, 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ)) was also tested by TWIMS. PPZ derivatisation allowed separation of a group of isomeric metabolites (11βOHE2, 16αOHE2, 16βOHE2), increasing CCSN2 difference from &lt; 2 Å2 to &gt; 10 Å2. Further work evaluated CCS prediction tools. There was good agreement between the measured CCSN2 values (mean: 7.5% difference), as well as in comparison to predicted values (means: 4%-12% difference) for underivatised molecules. There was less agreement for derivatised analytes. A MS imaging (MSI) approach was developed to visualise changes in the distribution of lipids and metabolites within the lung, as well as interrogate the changes in the wider “omic” profile of PAH. A specialised sample collection, preparation, acquisition, and data processing method was developed. Using a gelatine lung inflation, 6 different matrices (3 positive, 3 negative) and their deposition methods were evaluated. The optimised approach was applied to male and female control and PAH tissue samples. Differentially regulated lipids from a range of classes were putatively identified (phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, phosphatidylcholines etc.) and related back to PAH biology and oestrogen metabolism. This thesis demonstrates successful development of three analytical approaches. Firstly, the optimisation of an extraction method for the analysis of 12 oestrogens in rat lung tissue, which showed the presence of oestrogenic metabolites when applied in control and PAH model lung tissues. Next, an assessment of computational modelling and CCS prediction tools was undertaken and related to the separation of oestrogens and their derivatives by novel IMS methods. Clear separation of isomeric species was achieved by derivatisation in TWIMS and increasing pass number in cIMS. Finally, a new end-to-end MSI pipeline (tissue collection, sample preparation, sample analysis, data processing and interpretation) for lung tissue imaging was developed, culminating in the analysis of 20 rodent samples for comparison of control and PAH and the sexes. Significantly up- and down-regulated analytes were identified, and their functions related to PAH.