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Title: Endothelial cell transcriptome responses to pro-apoptotic and pro-inflammatory stimuli
Author: Johnson, N. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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The aim of the first project was to test the hypothesis that ECs cultured in conditions of severe stress undergo transcriptome and glycome changes that prime them for apoptosis. This was of particular interest as, although the protein-based changes that accompany apoptosis have been extensively studied, the RNA-and carbohydrate-based changes that may prime cells for apoptosis have, until now, received little attention. These changes may play an important role in determining EC fate in occluded vessels, in tissues distal to an infarct, or in regressing vessels. I induced apoptosis in human umbilical vein EC (HUVEC) by partial survival factor deprivation (SFD), then investigated (using Affymetrix gene arrays) alterations in the transcriptome of HUVECs cultured under conditions of SFD, as well as changes in their glycome (cell-surface polysaccharide profile). The results showed that SFD-induced apoptosis of HUVEC cultures is associated with extensive changes to both the abundance of transcripts and cell surface polysaccharides. A few of these changes appeared to be protective but, surprisingly, most appeared to prepare cells for apoptosis, by decreasing the reception and transduction of pro-survival signals, increasing pro-death signals, increasing abundance of apoptotic machinery, inhibiting cellular proliferation, recruiting phagocytes to regions of cell death and promoting phagocytosis. Additional transcriptome changes appeared to alter the synthesis and modification of cell surface glycosaminoglycans. The resulting reduced abundance of sulphated cell surface glycosamioglycans may further promote cell death by inhibiting the presentation of extracellular matrix-tethered survival factors to their receptors on dying cells. These transcriptome and glycome changes appear to synergise with previously described protein-based changes to guide the apoptotic program. They reveal a novel and potentially important mechanism for the regulation of cell death. The aim of the second project was to assess the transcriptome response of ECs to inflammatory mediators. This study was designed to model the conditions that may be found in areas of inflamed tissues, and to which ECs are exposed. As transcript abundance changes are likely to contribute to the biology of inflammation, their identification is important for design of new therapeutic targets. I exposed HUVEC to a cocktail of inflammatory mediators (IL-1β, IL-8 and TNF-α) and analysed the transcriptome changes induced by these factors using Affymetrix gene arrays. The results showed that a large number of transcripts were regulated by these factors, and the transcript abundance changes described are likely to reflect the multiple roles played by EC in inflammation. These included cytoskeletal regulators, adhesion molecules, molecules involved in coagulation, chemokines, cytokines and regulators or apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available