Limiting macrophages through blocking chemokines and studying vascular disease as potential therapies for chronic kidney disease

Cardiovascular disease (CVD) and chronic kidney disease (CKD) are causes of major morbidity and mortality globally. In Australia alone, one person dies of CVD every 12 minutes and it accounts for 30% of all deaths. Similarly CKD is common, affecting more than 1.7 million Australians. Within the CKD population CVD is a major complication and patients are 20 times more likely to die from cardio-vascular events than the general population. Atherosclerosis and arterial vascular calcification are contributory factors for the CVD. Both CVD and CKD inflict a major economic burden on Australian society, with CVD costing $1.7 billion and CKD costing $4.1 billion in 2012. It is now recognised that the immune system plays an important role in the progression of atherosclerosis and vascular calcification. Monocyte/macrophage infiltration plays a crucial role in the pathogenesis of atherosclerosis. Monocytes use a variety of cell surface expressed chemokines such as CCL2 and CX3CL1 to infiltrate into atherosclerotic plaques. CX3CL1 and its receptor, CX3CR1 which is expressed on monocytes, have been identified as one of the chemokine/receptor pairs that have an important role in the proliferation, migration, adhesion and recruitment of monocytes during the pathogenesis atherosclerosis. Macrophages also play a role in the osteogenic change of vascular smooth muscle cells (VSMCs) in vascular calcification. The toll like receptor-4 (TLR4) signalling pathway in particular, is involved during the pathogenesis of atherosclerosis and vascular calcification. The first aim of these studies was to establish a model of blockade of the CX3CL1/CX3CR1 pathway in the mouse that could potentially be developed for use in humans. This was achieved using DNA vaccination against CX3CR1 in ApoE-/- mice which are known to develop atherosclerosis. Successful blockade of the CX3CL1/CX3CR1 pathway then allowed evaluation the possible mechanisms of action of CX3CR1 in the pathogenesis of atherosclerosis. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. This was associated with less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. In addition, there was less lipid deposition in the lesions, but blocking CX3CR1 had no effect on smooth muscle cell migration. The second aim of these studies was to establish a model of dietary adenine induced CKD to examine the role of macrophages in CKD induced vascular calcification. An excess adenine diet induced renal fibrotic injury and tubule-interstitial injury in both ApoE-/- and C57BL/6 mice, however, it did not induce vascular calcification in either the ApoE-/- or C57BL/6 mice. There was no significant aortic macrophage infiltration found. The third aim of these studies was to establish a model of vascular calcification using high dose vitamin D, and to then examine the role of macrophages and the TLR4 signalling pathway in the pathogenesis of vascular calcification. High dose vitamin D caused significant vascular calcification and aortic macrophage infiltration in C57BL/6 mice. TLR4-/- mice demonstrated a significant reduction in vascular calcification and aortic macrophage infiltration suggesting TLR4 may play a role in mediating macrophage activation and recruitment during vascular calcification. The work within this thesis demonstrates firstly that DNA vaccination against the CX3CR1 chemokine pathway potentially offers a therapeutic option for the treatment or prevention of atherosclerosis by reducing the lipid content and more importantly the macrophage infiltration into atherosclerotic plaques. Secondly, the model of vascular calcification suggests a role for the TLR-4 pathway in the recruitment of macrophages during the calcification process and targeting this pathway could offer potential therapeutic benefit