Atherosclerosis is a disorder that involves the build-up of plaque in medium and large arteries. Atherosclerotic plaques are made from a range of cellular and extracellular components including lipid-filled immune cells, smooth muscle cells (SMC), T-cells and fibroblasts. The collection of these immune cells within the artery wall restricts the blood flow which leads to a stroke or heart attack. Macrophages and SMCs have specific receptors for the uptake of oxidised low-density lipoproteins (oxLDL) and CD36 is one of the main scavenger receptors which leads to foam cell formation of macrophages. The formation of foam cells and atherosclerotic plaque growth can be inhibited by the control of CD36. One potential protective mechanism against the formation of foam cells via CD36 downregulation is by human macrophage-derived 7,8-dihydroneopterin (7,8-NP). This thesis examines the effect and mechanism of 7,8-dihydroneopterin in the downregulation of CD36 in U937 monocyte-like cells.

This study characterized the effect of 7,8-dihydroneopterin on CD36 protein levels and explored how foam cell formation is inhibited through CD36 downregulation. Western blot analysis showed that in U937 cells, 7,8-dihydroneopterin concentrations of up to 150 μm down- regulated CD36 significantly. The effect observed here was a lot stronger than the previously seen in human monocyte-derived macrophages (HMDM). This research also confirmed that pterins other than 7,8-dihydroneopterin have no effect on CD36 expression in U937 cells. The inhibition of foam cell formation by 7,8-dihydroneopterin was determined by Oil-Red O staining and HPLC analysis. In situ results also confirmed that 7,8-dihydroneopterin significantly decreased CD36 protein levels in plaque tissue using direct tissue blotting.

The core of this study was to explore the mechanism of CD36 downregulation by 7,8- dihydroneopterin. CD36 is mainly regulated by the transcriptional factor Peroxisome Proliferator-Activated Receptor (PPAR-γ). Using PPAR-γ agonists including rosiglitazone and Azelaoyl-PAF, it was suggested that CD36 downregulation by 7,8-dihydroneopterin is not due to the alteration of the PPAR-γ expression, but because of PPAR-γ lipid-binding site not being activated.

Using a spectrophotometry based lipid oxidation assay, it was shown that 7,8-dihydroneopterin inhibited soya bean lipoxygenase. This suggested that 7,8-dihydroneopterin was inhibiting 15- lipoxygenase, the proposed source of the oxidised fatty acids responsible for the PPAR-γ activation. HPLC-TBARS analysis of 7,8-dihydroneopterin treated U937 cells confirmed that 7,8-dihydroneopterin was reducing the intracellular lipid oxides levels. Upregulation of CD36 by oxLDL was found to be reversed by 7,8-dihydroneopterin in U937 cells. IFN-γ, which is an essential factor for 7,8-dihydroneopterin production, was found to decrease the levels of CD36 in U937 cells, but the addition of 7,8-dihydroneopterin had no extra effect on CD36 downregulation.

ATP-binding cassette transporter (ABCA1), is preliminary responsible for cholesterol efflux from the macrophage cell. Its levels were not altered by 7,8-dihydroneopterin incubation in U937 cells.

This research has showed that 7,8-dihydroneopterin is a potential regulator of cellular CD36 levels within atherosclerotic plaques through inhibition of cellular lipoxygenase. 7,8- dihydroneopterin released during inflammation may be able to inhibit or slow plaque progress within the artery wall.