Molecular mechanisms underlying the development of atherosclerosis

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

Atherosclerosis is a chronic inflammatory condition, resulting in the formation of plaques within the walls of large arteries. Over time, plaque development and rupture can cause cardiovascular complications representing the leading causes of death worldwide. Part one of this thesis (Chapters 1 and 2) focused on determining the contribution of the enzyme heparanase to the development of atherosclerosis and diabetes-accelerated atherosclerosis. Heparanase mediates inflammation by regulating several key processes including immune cell activation and migration. Although inflammation is central to the development of atherosclerosis, atherosclerotic plaque development in the absence of heparanase has not been addressed. The aim of this project was to determine the extent of atherosclerotic and diabetes-accelerated atherosclerotic disease development in the absence of heparanase using mouse models. Previously generated heparanase knockout mice (HPSE-/-) were crossed with atherosclerosis-prone apolipoprotein E knockout mice (ApoE-/-) to generate a novel ApoE-/- x HPSE-/- double knockout strain (AExH-/-). Mice were placed on a high fat diet for 6- or 14-weeks, or administered streptozotocin to induce diabetes, and the extent of plaque development was assessed with Oil Red O staining of the aorta along with haematoxylin and eosin staining of the aortic sinus. In the absence of heparanase, atherosclerotic and diabetes-accelerated atherosclerotic plaque development was reduced, suggesting that heparanase is a potential therapeutic target for the treatment of atherosclerosis. Part two of this thesis (Chapters 3, 4 and 5) explored the role of endothelial cell death and disassembly in the development of atherosclerosis. A form of programmed cell death known as apoptosis can be induced in endothelial cells in response to pro-atherosclerotic factors, which leads to aberrant functioning of the endothelium and is thought to contribute to the initial stages of plaque development. During apoptosis, dying cells undergo apoptotic cell disassembly resulting in fragmentation of the cell to release apoptotic bodies, which function in intercellular communication. Although apoptosis has been identified in the endothelium of atherosclerotic plaques, the role of endothelial cell ApoBDs has not been investigated. The aim of this project was to characterise endothelial cell death and disassembly along with the possible roles of endothelial cell ApoBDs in atherosclerosis. It was demonstrated that the formation of endothelial cell ApoBDs is regulated by the pannexin 1 channel and vesicle trafficking. Further investigation into the characteristics of ApoBDs showed that surface markers present on the cell of origin can be used to distinguish endothelial cell ApoBDs from those of other cell types. Additionally, RNA deep sequencing of highly pure endothelial cell ApoBDs revealed the presence of microRNA, which can be transferred to phagocytes upon uptake of endothelial cell apoptotic bodies. Taken together, data highlight that the formation of endothelial cell ApoBDs is an important consideration in the development of atherosclerosis due to the potential contribution to intercellular communication.

This thesis was a recipient of the Nancy Millis Award for theses of exceptional merit.