Identification of oxLDL-induced oxidative stress sources in cardiovascular disease.
Thesis DisciplineBiological Sciences
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Atherosclerosis is characterized by chronic inflammation of the arterial wall through the accumulation of lipid-loaded macrophages. Cell death within this region causes the progression of the fatty streak into an advanced atherosclerotic lesion with a characteristic necrotic core, consisting of cholesterol, calcium deposits, dead cells and other cellular debris. The oxidation of low-density lipoprotein (LDL) to form oxidized LDL (oxLDL) is a crucial event in the development of the atherosclerotic lesion resulting in death to a range of cell types within the intima. OxLDL is cytotoxic to macrophages through a rapid induction of intracellular oxidative stress, resulting in oxidative damage to essential biomolecules causing cell death that contributes to the development of the necrotic core. This study investigated the source of the cytotoxic oxidative stress triggered by the presence of oxLDL in U937 monocyte like cells. The research investigated whether NADPH oxidase, myeloperoxidase or uncoupled mitochondria generated intracellular oxidants in response to oxLDL. Specific NOX inhibitors, (gp91ds-tat and apocynin), provided insight on the involvement of NOX in the oxLDL-induced oxidative stress mechanism. Increasing concentrations of gp91ds-tat significantly decreased intracellular superoxide generation in U937 cells but did not prevent cell viability loss. The competitive inhibitor, gp91ds-tat, did not completely inhibit the complex, allowing for residual superoxide to be generated. Apocynin treated plaque sections showed a significant reduction in 7,8-dihydroneopterin (7,8-NP) oxidation. This study also measured the U937 intracellular 7,8-NP oxidation by cellular oxidants in response to oxLDL. The addition of apocynin significant reduced the oxLDL-induced 7,8-NP oxidation. This suggested that NOX was the major source of intracellular oxidative stress in both in vivo and in vitro research. Myeloperoxidase was shown to not be involved in the in vitro mechanism, as inhibition of hypochlorite production through MPO inhibitors (ABAH & thiocyanate) did not restore cell viability, nor did it reduce the intracellular oxidative stress when U937 cells were incubated with oxLDL. This suggests that MPO is not involved in the U937 cell death mechanism as a result of oxLDL exposure. OxLDL induced a significant depolarization of the mitochondria membrane potential after 6 hours exposure and the addition of 7,8-NP did not protect the membrane depolarization. However, the data that was collected was deemed inconclusive due to fundamental problems occurring within the JC-1 assay. Our results suggest that NOX is the major source of oxidative stress causing significant cell death and progression of the plaque necrotic core. It was shown that myeloperoxidase has no involvement in the oxidative stress mechanism and that the mitochondria appears to play a role.