Background: Endogenous and exogenous reactive oxygen species (ROS) inducers cause intracellular metabolic modifications leading eventually to serious diseases.Overproduction of ROS is associated with many diseases including diabetes, diabetic retinopathy and cardiovascular diseases (CVD). Exposing of different cell types to oxidative agents impair the cell redox status by primarily inducing the excessive generation of superoxide that is enzymatically converted to other forms of oxidative agents causing cell damage. The hyperglycaemia that occurs due to diabetes mellitus causes high ROS production. Identification of reliable markers that indicate metabolic alteration due to oxidative stress could enable a better understanding of many diseases in terms of diagnosis and treatment with therapeutic targets.Aim: To identify the main metabolic markers that are influenced by ROS in pulmonary artery smooth muscle cells (PASMCs) and retinal pigment epithelial cells(RPE) in-vitro. To investigate the impact of anti-oxidant agents in attenuating the modifications that may appear due to the high production of ROS. Study design and methodology: biological cell samples were prepared and collected for each of the four included studies based on the intervention of each study whether it is the impact of hyperglycaemia with or without vitamin as antioxidants and the impact of TSPO protein deletion. In the first study, PASMCs were exposed to diabetic-like conditions and either supplemented or not supplemented by anti-oxidant agents. In the following study, PASMCs were also examined under hyperglycaemia in normoxic or hypoxic conditions. In the two other following studies, we investigated the impact of oxidised low density lipoprotein (OxLDL) and hyperglycaemia on the TSPO knockout and wildtype RPE cells.All studies were carried out utilizing high-performance liquid chromatography-mass spectrometry. We used untargeted metabolic profiling to detect comprehensive metabolic alterations that occurred intracellularly due to our interventions. MzMatch software was used to identify the metabolites present;multivariate and univariate analyses were employed to determine the most reliable metabolites as biomarkers for oxidative stress.Results: The outcomes established that high glucose influences cell proliferation and enhances oxidative stress. The general metabolomics results show the induction of carbohydrate metabolism accompanied by activation of the pentose phosphate pathway (PPP) to boost the generation of NADPH as an antioxidant product. Amino acids were also disturbed while some of them were up-regulated and some others were down-regulated. Lipids were frequently the most influenced pathway suggesting that the cell membrane was damaged by ROS induction.Purines and pyrimidines, in addition, were influenced consequently due to the changes occurring in PPP metabolism.Conclusion: These hypothesis-free metabolomics profiling studies have identified intracellular metabolites in PASMCs, and RPE cells which are differentially sensitiveto the hyperglycaemia or oxidative stress inducers and may be useful to initiate hypothesises about markers that can help to prevent or cure diseases where oxidative stress plays a role.
|Date of Award||28 Aug 2019|
- University Of Strathclyde
|Supervisor||David Watson (Supervisor) & Ruangelie Edrada-Ebel (Supervisor)|