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Title: Deoxyribonucleic acid damage gene expression, ligase activity and correlation with coronary atherosclerotic plaque morphology
Author: Shah, Nikunj
ISNI:       0000 0004 7967 3012
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2019
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Despite therapeutic and interventional technological advances, the global burden of cardiovascular disease is increasing. Accumulated deoxyribonucleic acid (DNA) damage and subsequent repair pathways are now increasingly recognised as a causal factor in the initiation and progression of atherosclerosis. These molecular alterations have been shown to occur within affected vasculature, plaque microenvironment as well as in circulating cells. The DNA damage response (DDR) pathway is reliant on post-translational modification of sensing proteins which activate a signalling cascade to repair, if possible, DNA damaged sites in response to various environmental and physiological insults. Most studies to-date have focussed on animal models and cells cultured directly from atherosclerotic plaque to ascertain if altered DDR and DNA repair exist and indeed contribute to the atherosclerotic process. By using peripheral whole blood as the base for down-stream analyses, an accessible method of determining if altered expression of genes involved in DDR and enzymes involved in DNA repair could be exploited. DNA ligase is crucial in single (ssDNA) and double-stranded DNA break repair (DSBR) by facilitating the joining of DNA strands by catalysing phosphodiester bond formation. It was therefore chosen as a marker of DNA repair being a key enzyme involved in base excision repair (BER) and DSBR. This study examined the differential expression of 22 genes pertinent in the DNA damage and response pathway (DDR), in addition to DNA ligase activity, between patients with stable, unstable coronary atherosclerosis (both undergoing percutaneous coronary intervention for obstructive coronary disease) and healthy controls. In addition, correlations were performed between atherosclerotic plaque features and both DNA ligase activity and the genes of interest. To accurately analyse plaque morphology, frequency domain optical coherence tomography (FD-OCT) was used which allowed high resolution delineation of fibrous tissue, lipid accumulation, calcific deposition and fibrous cap thickness, all key features in plaque vulnerability and therefore of clinical significance. Peripheral blood mononuclear cells (PBMC) were isolated and DNA repair activity was measured from derived nuclear extracts, using a novel microplate assay examining mean apparent DNA ligase activity. A custom microarray for the 22 genes of interest was used to perform quantitative reverse transcription polymerase chain reaction for differential gene expression between all 3 cohorts of patients recruited. Data from this study demonstrated differences in DNA ligase activity and expression of genes involved in the DDR in patients with coronary atherosclerosis. DNA ligase activity correlated with the arc of lipid and cap thickness in both stable and unstable coronary patients. Differential DDR gene expression also correlated with fibrous cap thickness, predominantly in the unstable coronary group. This suggests that such alterations may contribute to the development and progression of the atherosclerotic lipid rich necrotic core as well as protective fibrous cap thickness. Although significant correlations were found between DNA ligase, genes of interest and plaque features, this study demonstrates an association but does not provide a direct mechanism by which alterations in DDR contribute to atherogenesis. Further studies are required to address differential expression across a broader spectrum of genes involved in DDR in coronary artery disease.
Supervisor: Meira, Lisie ; Mahmoudi, Michael ; de Lusignan, Simon Sponsor: Ashford and St. Peter's NHS Foundation Trust
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral