An investigation of epigenetic effects in Atherosclerosis

Abstract

Epigenetic mechanisms may explain some of the missing heritability associated with CVD and how external factors such as diet, environment and lifestyle contribute to disease development and progression. This thesis aimed to investigate the role of epigenetics in atherosclerosis- from initiation to plaque development and its subsequent outcomes.

Injury to the endothelium and subsequent inflammation represent a critical starting point for atherosclerosis. A primary objective of this study was to evaluate the use of epigenetic targeting agents SFN and SAHA (Vorinostat) on the expression of CXCL16, IL8, IL18 and miR210 and miR145, in TNFα stimulated endothelial (EA.hy926) and macrophage (THP-1) cell lines. The findings of this thesis suggest that the expression of CXCL16, IL8, IL18 and the indicated miRNAs can be altered by histone deacetylase inhibitors SFN and SAHA. However, the amount of HDACi (SFN and SAHA) and duration of treatment may have disparate effects and depend on the pre-existing inflammatory background in this case cytokine levels. This could limit potential use in the clinic.

As CXCL16 plays a multifaceted role in all stages of atherosclerosis, genetic variation which influences gene expression is of particular interest. An AEI investigation of the CXCL16 SNP rs2277680 was undertaken to determine allelic expression imbalance in the presence and absence of epigenetic modifying agents. These experiments found that AEI can be induced following treatment with various ETAs, proposing an epigenetic mechanism for AEI. The question remains as to whether all cells produce mRNA from both alleles, albeit at different levels, or one allele is silenced completely (monoallelic expression) and the observations are the result of heterogeneous mixtures of cells with either one or the other allele silenced. However, monoallelic expression is unlikely as AEI can be observed following 6 h treatment, a time point which is too short to be attributed to cell turnover. This study also investigated the stability of AEI over multiple passages during cell culture. These results suggest that AEI is predominately stable, with considerable noise, over cell culture but becomes less predictable with increasing passage number. This study also raises the question as to the effectiveness and reproducibility of using cells which have been frozen in DMSO due to its potential for altering allelic expression imbalance.

Finally, the expression of miRNAs 145 and 210 has previously been shown to be reduced in patients with plaques which are prone to rupture. However, an association in stroke recurrence prediction from an initial cardiac event has not been examined. This is the first study to demonstrate an inverse relationship between miR145 and CD68 macrophages in stroke recurrence in a clinical data set. This finding confirms the role of miR145 in CVD, and as a biomarker of decreased stroke recurrence, a potential therapeutic target and predictive marker. Data generated in this study suggest that decreased miR145 expression is associated with a greater risk of stroke recurrence. Levels of miR145 were significantly increased in non-recurrent patients when compared against those who had stroke recurrence after initial stroke/TIA, but before carotid endarterectomy (P=0.0360). Furthermore, miR145 expression was also shown to be significantly associated with histological features of plaque inflammation and instability, such as increased cap and plaque macrophage count, evidence of intraplaque haemorrhage, luminal thrombus with lipid nanocapsules and the degree of plaque stability (P=0.0207, 0.0258, 0.0447, respectively).

The work detailed in this thesis describes different methodologies that can be used to help elucidate and alter the epigenetic mechanisms affecting gene expression in atherosclerosis. The results unequivocally show an influence of histone acetylation and RNA interference in the development and stability of atherosclerotic plaque.