7,8-dihydroneopterin inhibition of oxidised low density lipoprotein-induced cellular death
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The death of monocyte and macrophage cells in the atherosclerotic plaque is thought to be due principally to the toxicity of oxidised LDL (oxLDL). This makes an important contribution to the development of the lipid core of advanced plaque and also to the overall stability of the plaque. Under inflammatory conditions in which IFNγ is present, human monocytes and macrophages produce the partially reduced pteridine 7,8-dihydroneopterin (78NP). This molecule has been shown to be a very efficient free radical scavenger and to protect some cell types from damage during incubation with reactive oxygen species. The possibility of 78NP reducing loss of cell viability of monocytes and macrophages in the atherosclerotic plaque was examined in this research. THP-1 and U937 monocytes and THP-1 macrophage-like cells were incubated with AAPH or oxidised LDL, and loss of cell viability and total reduced thiols measured. In monocytes, both AAPH and oxLDL caused concentration-dependent reduction in viability. The loss of reduced thiols was also proportional to AAPH and oxLDL concentration, except in U937 monocytes with oxLDL, where the levels dropped suddenly. Viability loss in THP-1 macrophage-like cells was found to be small and not dependent on the concentration of the AAPH or oxLDL. The development of apoptotic features, including nuclear morphological changes, caspase enzyme activation and phosphatidylserine exposure was examined in monocytes incubated with oxLDL. Both cell types had characteristics typical of apoptosis, with the exception of low levels of caspase enzyme activity in U937 monocytes, which decreased even further during incubation with oxLDL. Evidence is presented that this may be due to the oxidation of the crucial cysteine residue on the caspase enzymes. During incubations with AAPH, 78NP significantly reduced cell viability and thiol loss in U937 cells and decreased cell viability loss by a small amount in THP-1 monocytes when the incubations were carried out in EBSS media. 78NP had a similar effect in U937 cells with oxLDL, but no protective effect was observed in THP-7 monocytes. 78NP had no effect on viability loss with AAPH or oxLDL in THP-1 macrophage-like cells. The protective effect of78NP may be dependent on its ability to inhibit thiol loss. All three cell types were shown to produce 78NP after stimulation with IFNγ. However, inclusion of IFNγ did not in most cases prevent cell viability loss during exposure to oxLDL. 78NP was found in inflammatory material, but only at levels a hundred-fold less than those required to provide an antioxidant effect. 78NP is able to protect cells from viability loss generated by free radicals and the reactive oxygen species in oxLDL in some circumstances. However, since the levels in vivo are likely to be far lower than those required for antioxidant activity, the evidence presented does not support the hypothesis that 78NP would be able to protect monocyte and macrophage cells from cell death in the atherosclerotic plaque.