Development of E-Sense: a Flexible in vitro Platform to Detects Cardiovascular Risk

Smith, Rhys Alexander (2023) Development of E-Sense: a Flexible in vitro Platform to Detects Cardiovascular Risk. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Coronary heart disease (CHD) is one of the leading causes of mortality and morbidity worldwide. Atherosclerosis (AS), a key mechanism in the potentiation of CHD, is often defined as a chronic inflammatory disease provoked by the oxidation of lipids retained in the arterial wall which results in the formation of atherosclerotic lesions and plaques. Atherosclerotic plaques preferentially form at predilected sites of bifurcations and branch points exposed to disturbed flow patterns. In contrast, straight sectional regions of the arterial wall which are subjected to laminar flow are relatively spared, implicating the response of endothelial cells to their flow environment in the initiation of the disease. There are a variety of signalling pathways and shear responsive transcription factors known to regulate these processes such as NFκB, AP1 (inflammatory and cytokine response), IRF3 (toll-like receptor signalling), XBP1 (unfolded protein response/ER stress) and KLF2 and Nrf2 (protective signalling pathways). The understanding of these pathways is built upon decades of research using in vivo animal models. This study culminated in the development and validation of a novel endothelial cell-based in vitro platform, able to monitor the activity of atherosclerosis-related transcription factors. To facilitate this, a novel human coronary artery endothelial cell line with an extended proliferative potential, reduced senescence and mechanosensitive was created (HCAECPro) in order to circumvent one of the major drawbacks of in vitro modelling- the relative short lifespan of primary HCAECs. Transcription factor activator reporters with a novel binding response element, were incorporated into this HCAECPro cell line to create novel ‘E-Sense lines’, which upon validation, exhibited a dynamic range, several orders of magnitude greater than previously available reporters. Quantification of transcription factor reporter activity with these E-Sense lines allowed for screening of novel compounds to assess their potentiation for atherosclerosis. The use of plastics in everyday life has steadily increased over the previous few decades, in particular; the use of microplastic, Bisphenol A (BPA). The plastic has come under scrutiny in recent years, with circulating concentrations in the human body significantly higher than previously expected. Although currently considered safe for human consumption, recent studies have suggested it plays a role in the potentiation of endocrine and neurodegenerative disorders. Its potential impact on cardiovascular health is still unknown. However, the findings within this project demonstrate that exposure to microplastic, Bisphenol A (BPA), exerts a significant effect on several of the key TFARs related to cardiovascular disease progression. Indeed, these findings suggest that BPA causes a reduction of key protective signalling transcription factors KLF2 and Nrf2 under flow, whilst increasing the induction of the stress response possibly via TLR signalling.