The role of oxidised low-density lipoprotein in initiating cell death and inflammation

Atherosclerosis accounts for a significant amount of mortality and morbidity in the developed countries. Oxidised low-density lipoprotein (oxLDL) is widely believed to be the key pro- atherogenic factor triggering the development of cardiovascular diseases. OxLDL mediated cytotoxicity contributes to the progression of atherosclerosis by triggering the formation of necrotic core through the death of macrophages within the arterial wall. Previous studies have shown that the activation of Extracellular signal-regulated kinase (ERK)1/2 is implicated in oxLDL mediated cytotoxicity in U937 cells by the activation of β-nicotinamide adenine dinucleotide phosphate oxidase (NOX), a superoxide generating enzyme. This study will investigate the mechanisms by which oxLDL induces cytotoxicity in U937 cells by examining ERK1/2 activation and the kinetics of intracellular superoxide generation. This study will also investigate whether oxLDL triggers an inflammatory response in peripheral blood mononuclear cells (PBMC). Low density lipoprotein (LDL) was isolated from the plasma donated by fasting volunteers using ultracentrifugation. The LDL preparation was subsequently oxidised by copper ions using a standardised protocol. U937 cells were incubated with increasing concentrations of oxLDL and the level of intracellular superoxide was measured by DHE staining followed by flow cytometry analysis. The timing and magnitude of ERK1/2 activation was measured by western blot. OxLDL induced cell death was measured by propidium iodide staining followed by flow cytometry analysis. The inflammatory status of oxLDL treated PBMC was evaluated by measuring the levels of neopterin and 7,8-dihydroneopterin (7,8-NP) in the cell culture media by High Performance Liquid Chromatography (HPLC). Neopterin and 7,8-NP are markers of inflammation. Significant variations in the timing and level of intracellular superoxide in U937 cells were observed in response to oxLDL. This level of variation had not been observed in previous studies within the laboratory. Significant variation in cytotoxicity of the oxLDL preparations was also measured. The level of conjugated dienes in oxLDL showed moderate correlation with the cytotoxicity of oxLDL and indicated there was a significant level of variations in the oxidation status between different oxLDL preparations. The causes of this variability were not determined. OxLDL caused a concentration and time dependent increase in the level of phosphorylated ERK1/2 but no change in the overall level of ERK1/2 expression. OxLDL induced no significant rise in the extracellular release of 7,8-NP by PBMCs or its oxidation product neopterin. OxLDL alone does not appear to be able to induce macrophage inflammation. This research confirmed the cytotoxicity of oxLDL against U937 cells and suggested that phosphorylated ERK1/2 was the upstream regulator of NOX and its activation by oxLDL leads to the release of superoxide. The activation of ERK1/2 in U937 after exposure to oxLDL was highly dynamic and unpredictable, this was at least partly due to the varying oxidation status of the oxLDL preparation. OxLDL does not trigger macrophage inflammation as shown by the lack of neopterin release by PBMC