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Title: Transcriptome-profiling in porcine arteries to identify shear-responsive regulators of endothelial apoptosis
Author: De Luca, Amalia
ISNI:       0000 0004 5989 4379
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Atherosclerosis develops predominantly at regions of the arterial tree exposed to disturbed blood flow, which generates low, oscillatory shear stress (WSS) at the lumen. Of note, endothelial cells (EC) at lesion-prone regions are characterised by an increased rate of apoptosis, thus providing a potential explanation for the distinct spatial localisation of atherosclerosis. To understand the interaction between flow and apoptosis we used microarray technology coupled to computational fluid dynamics (CFD) to identify genes differentially expressed at high or low WSS regions of the porcine aorta. We examined whether putative regulators of apoptosis can be activated by flow in vitro and studied their function using cultured EC. In this study, we employed magnetic resonance imaging and CFD to model blood flow in the porcine aortic arch and generate WSS maps that served as guidelines for the isolation of EC for subsequent transcriptional analysis. Furthermore we characterised the flow in stenosed carotid arteries where a constrictive extravascular device was surgically applied. The influence of WSS on the expression of putative regulators of apoptosis was studied using in vitro flow assays and gene function was analysed using siRNA-based approaches. Computed WSS maps revealed great spatial heterogeneity and challenged common assumptions about the mechanical conditions at susceptible and protected regions. In addition, microarray analysis of ECs isolated from the aortic arches of 5 pigs identified 764 differentially expressed genes that influence diverse physiological activities. Functional annotation of these transcripts highlighted the presence of 41 molecules with an inferred or known role in the regulation of apoptosis. We selected two candidates for functional screening in vitro: PERP and PDCD2L. Staining for active caspase-3 and DNA fragmentation revealed that EC apoptosis was significantly enhanced in EC exposed to oscillatory shear stress compared to cells exposed to uniform flow. Silencing of PERP reduced apoptosis in EC exposed to oscillatory shear stress, while silencing PDCD2L did not have a significant effect. We conclude that shear stress influences EC viability through transcriptional mechanisms that might involve the novel apoptosis regulator PERP. Our observations illuminate the molecular mechanisms that regulate the focal nature of vascular injury and atherosclerosis and provide a large genetic dataset to use in future studies.
Supervisor: Mason, Justin ; Sherwin, Spencer ; Evans, Paul Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral