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Title: The role of shear stress and NF-κB in regulating endothelial dysfunction and atherosclerosis
Author: Bowden, Neil P.
ISNI:       0000 0004 6347 1854
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Atherosclerotic plaques are predominantly localised to bends and branches of arteries, where low shear stress causes altered gene expression in endothelial cells, leading to enhanced proliferation, apoptosis, inflammation and lesion development. A central regulator of these events is NF-κB signalling, comprising a family of transcription factors which can be subdivided into two pathways - canonical and non-canonical. While the canonical pathway is known to promote atherogenesis, investigation of the role of its negative regulator Cezanne (OTUD7B), has not been undertaken. Cezanne also inhibits the non-canonical NF-κB pathway, which has not been studied in the context of ECs exposed to shear stress. The hypothesis that Cezanne expression was regulated by shear stress and consequently modify NF-κB activity and lesion development was tested. In addition, the expression and role of non-canonical NF-κB was investigated in endothelial cells exposed to shear stress, and the contribution of Cezanne to this pathway also tested. Immunofluorescence staining for Cezanne in the murine aortic arch revealed elevated expression at an atheroprone site. Exposure of human cells to shear stress in two complimentary flow systems in vitro showed enhanced expression under low shear stress. Following silencing of Cezanne, NF-κB phosphorylation was found to be enhanced, but no effect was detected on NF-κB target genes, or on non-canonical NF-κB. Otud7b-/- mice were also found to have no difference in NF-κB target gene expression or proliferation as measured by immunofluorescence staining; moreover, lesion size in Otud7b-/-Ldlr-/- animals was unaltered. The expression of non-canonical NF-κB subunits p100/p52 and RelB were also found be enhanced by low shear stress, in RNA isolated from porcine aortas as well as in vitro. Silencing and immunofluorescence staining revealed that the pathway promoted proliferation specifically at sites of low shear stress in vitro, via repression of p21. Analysis of proliferation in the aortic arches of NF-κB2-/- recapitulated this finding, with decreased proliferation at the inner curvature of the aortic arch, measured by Ki-67 positivity. The conclusions were that Cezanne expression was elevated by low shear stress, and is responsible for inhibition of NF-κB phosphorylation, but does not affect target gene expression or the development of atherosclerosis. The non-canonical NF-κB pathway is also elevated by low shear stress. Cezanne did not alter its activity, which was to promote proliferation by inhibiting the expression of p21. This reveals a novel mechanism for the control of low shear stress proliferation, as well as identifying the role of non-canonical NF-κB in endothelial cells.
Supervisor: Evans, Paul C. ; Francis, Sheila E. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available