Low density lipoprotein induction of intracellular oxidants production
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Atherosclerosis is a complex cardiovascular disease characterized by chronic progressive inflammation of the arteries. The progression of atherosclerosis from fatty streak to advance atherosclerotic plaque involves the development of a necrotic core region consists of cholesterol, lipids, calcium (Ca²⁺), dead cells and other cellular debris. Macrophage infiltrations occurred in all stages of atherosclerotic progression and they are abundantly found in atherosclerotic plaques. Oxidised low density lipoprotein (oxLDL) plays a vital role in the initiation and development of atherosclerosis. OxLDL is present within atherosclerotic plaque and has been shown to be cytotoxic to various types of cells including macrophages. This research initially examined the cytotoxic effects of copper oxidised LDL on U937, human monocytes and HMDM cells. As expected oxLDL was cytotoxic; causing rapid, concentration and time dependent cell viability loss in all types of cells examined. Examination of the cell morphology showed that oxLDL caused a necrotic like cell death characterised by cell swelling and lysis. Flow cytometric assay coupled with propidium iodide (PI) staining of necrotic cells was compared to MTT reduction assays of cell viability. The flow cytometric technique had the advantage over the MTT reduction assay of being rapid and showing both the live and dead cell levels at an individual cell level. The progression of oxLDL-induced cell death correlated with the rapid increased in intracellular ROS production in the cytosol and the mitochondria. Immunoblotting results showed that oxLDL induced NADPH oxidase (NOX) activation and increased p47phox expression. This suggests NOX as the generator of reactive oxygen species (ROS) induced by oxLDL in these cells. However, apocynin and VAS2870, the two NOX inhibitors used in this study, were unable to inhibit the ROS generation caused by the oxLDL. This suggests that either these inhibitors are unable to inhibit the targeted NOX or other sources of ROS might exist and contributed to the overall increase in oxidative flux. OxLDL caused a rapid increase in cytosolic Ca²⁺ level. This was contributed by the extracellular Ca²⁺ source as well as Ca²⁺ mobilisation from the intracellular stores such as endoplasmic reticulum (ER). OxLDL-induced intracellular Ca²⁺ increase also correlated with the increase in intracellular ROS. Nevertheless, blocking of oxLDL-induced intracellular Ca²⁺ elevation by Ca²⁺ chelator, EGTA, did not reduce intracellular ROS generation. Accordingly, this suggests that oxLDL-induced intracellular Ca²⁺ increase is not the cause for oxLDL-induced cell death. Additionally, oxLDL may also initiate a Ca²⁺-independent cell death pathway. The excess cytosolic Ca²⁺ taken up by the mitochondria may be detrimental and could result in mitochondrial Ca²⁺ overload. This will increase mitochondrial ROS production and initiate mitochondrial permeability transition (MPT) pores opening. Consequently, this could collapse the mitochondrial membrane potential (𝚫𝚿m) due to the rupture of outer mitochondrial membrane (OMM) and resulted in cell death. Blocking of Ca²⁺ uptake into the mitochondria by ruthenium red protected cells from oxLDL-mediated cell death, possibly by reducing ROS production and preventing MPT activation. This study also demonstrated the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP protected cells from oxLDL-induced intracellular ROS generation and cell viability loss. Intracellular Ca²⁺ increase was also reduced by 7,8-NP in particular after 3 hours incubation with oxLDL. The action of 7,8-NP was better than that of apocynin in protecting U937 cells from oxLDL suggests its potential ability to scavenge ROS from various sources. Studies have implicated the involvement of H₂S in various biological processes including atherosclerosis. Thus, the disruption of H₂S homeostasis may contribute to the progression of atherosclerosis. Slow releasing H₂S molecules (H₂S donors) have been developed for a controlled and stable delivery of H₂S to cells. In this study, specific H₂S donors, including one which targets the mitochondria, were found to be protective against oxLDL-induced cell death in U937, human monocytes and HMDM cells. Although the exact mechanism is yet to be elucidated, these H₂S donors were shown to block the elevation of intracellular Ca²⁺ and ROS production mediated by oxLDL. Therefore, these H₂S donors could be the potential candidates for future development of therapeutics in treating atherosclerosis.