Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/130122
Type: Thesis
Title: Molecular Mediators of Macrophage Foam Cell Formation and Atherosclerosis and their Pharmacological Modification
Author: Fernando, Weerahennedige Sanuja Lakshani
Issue Date: 2020
School/Discipline: Adelaide Medical School
Abstract: Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of death and morbidity around the world. It results from build-up of cholesterol-rich, inflamed plaques in the walls of arteries. Inflammation is central to atherosclerosis. A key driver of inflammation in plaque is the accumulation of cholesterol-laden macrophages, called foam cells. Uncontrolled inflammation in plaques makes them “unstable” and vulnerable to rupture and thrombosis, which causes complications, such as myocardial infarction (MI) in the case of atherosclerotic coronary artery disease (CAD). Current therapies, such as lipid-lowering statins, do not adequately control plaque inflammation, leaving an unacceptably high residual risk of adverse cardiovascular events in patients with subclinical and clinical ASCVD. Consequently, new anti-atherosclerotic therapies are needed, especially those that can better target plaque inflammation. This thesis set out to mechanistically investigate the regulation of macrophage functions in the context of atherosclerosis using two therapeutic approaches: (1) the repurposing of the well-established, anti-gout drug, colchicine; and (2) the genetic knock-out and pharmacological inhibition of eukaryotic elongation factor 2 kinase (eEF2K), a structurally unique enzyme known to be important for protein translation in cells under different stress conditions. Colchicine is a broad-acting anti-inflammatory agent that has attracted considerable interest for repurposing in ASCVD. Despite evidence for outcome benefits in stable CAD and after recent MI, the mechanistic basis for colchicine’s actions in ASCVD remains speculative and poorly defined. Chapter 3 of this thesis aimed to dive deep into colchicine's actions on one of the key immune cells involved in atherosclerosis, namely macrophages. This in vitro and ex vivo body of work was supported by collaborative in vivo studies, which explored whether colchicine reduces plaque burden and stabilises plaque composition in two murine models of atherosclerosis. These studies showed for the first time that colchicine inhibits macrophage-to-foam cell transformation in the presence of oxidised low-density lipoprotein cholesterol (oxLDL), associated with reduction in the glycosylated form of the CD36 cholesterol uptake receptor, and its reduced expression on the cell surface membrane. Colchicine also increases the efflux capacity of cholesterol to high-density lipoproteins (HDL), accompanied by a selective increase in the surface expression of the cholesterol transporter, ATP Binding Cassette Subfamily G Member 1 (ABCG1), whose mechanistic basis requires deeper evaluation. Moreover, it mitigates cholesterol crystal (CC)-induced inflammation, dampening activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. Colchicine was also found to have an anti-priming effect on the NLRP3 inflammasome components by downregulating their transcriptional expression. This is likely due to its anti-phagocytic effects as it reduces CC ingestion by macrophages and the subsequent intracellular accumulation of lysosomes. Apart from repurposing established anti-inflammatory drugs like colchicine, there is substantial work being undertaken to identify novel therapies against atherosclerosis that target new mechanisms to more effectively mitigate plaque inflammation. Chapters 4 and 5 tackled one such novel target, eEF2K, by exploring its effects on macrophage responses to atherogenic stimuli in vitro and in a murine model of atherosclerosis in vivo, respectively. Although previous studies had suggested a role for eEF2K in atherosclerosis, they had thus far provided little mechanistic insight. In Chapter 4, the disabling of eEF2K by pharmacological inhibition or genetic knock-out, was found to cause a consistent inhibition of oxLDL-induced foam cell formation. As with colchicine, this is associated with selective, post-transcriptional downregulation of CD36, although through a different mechanism. In keeping with eEF2K’s known regulation of the translation of certain other mRNAs, its inhibition specifically reduces the translational efficiency of Cd36 mRNA, as seen by reduced ribosomal occupation, thus decreasing the synthesis of CD36 protein. This is a novel regulatory mechanism for modulating CD36 expression that has not been reported before. For the first time, we also determined that eEF2K inhibition and knock-out also increase cholesterol efflux and inhibit the proliferation of oxLDL-treated macrophages, while having no effect on other macrophage properties, such as polarisation, viability, phagocytosis, efferocytosis or CC-induced activation of the NLRP3 inflammasome. Chapter 5 explored the effect of knocking out eEF2K in a mouse model of atherosclerosis, induced by high cholesterol diet feeding on the pro-atherogenic Ldlr-/- background. We first established that eEF2K is active within macrophages in murine plaques. Eef2k(-/-)-Ldlr(-/-) mice developed smaller atherosclerotic lesions in the aortic sinus than their Eef2k(+/+)-Ldlr(-/-) counterparts. However, surprisingly this was not accompanied by a decrease in plaque lipid content, despite confirmation that Eef2k(-/-)-Ldlr(-/-) mice not only had lower CD36 expression in both plaque itself and on circulating monocytes, but also reduced capacity to form foam cells from their bone marrow-derived and peritoneal macrophages when exposed to oxLDL. Finally, pharmacological inhibition of eEF2K was shown to have a particularly striking effect on reducing CD36 expression and foam cell formation in human monocyte-derived macrophages. This was supported by the novel observation that EEF2K mRNA expression was higher in blood mononuclear cells from patients with unstable, advanced CAD, as defined by a recent MI, compared to those with chronic stable CAD. This in turn was associated with higher CD36 expression at both mRNA and surface membrane level in monocytes from this high-risk cohort. These findings provide a crucial early step towards better understanding eEF2K’s role in human atherosclerosis. In summary, this thesis provides new evidence in the search for novel mechanisms and therapeutic targets that could lead to the mitigation of ASCVD, by firstly providing new insights into the mechanistic basis for colchicine’s anti-atherosclerotic properties, and secondly by revealing eEF2K to be a unique mediator of CD36 translation and expression in macrophages, thereby regulating foam cell transformation.
Advisor: Psaltis, Peter
Proud, Christopher
Nicholls, Stephen
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020
Keywords: Atherosclerosis
plaque
macrophages
foam cells
eEF2K
colchicine
cardiovascular
Provenance: This thesis is currently under Embargo and not available.
Appears in Collections:Research Theses

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