The sub-endothelial retention of lipoproteins is one of the key events that trigger the atherosclerosis process. Low-density lipoprotein (LDL) particles trapped within the arterial wall are prone to progressive oxidation by monocytes/macrophages. Oxidized LDL (oxLDL) is present in atherosclerotic lesions, and has been suggested to play a significant role in atherogenesis ( Nishi et al., 2002). The pathophysiology of atherosclerosis involves both apoptosis and proliferation at different stages of the vessel lesion. In advanced atherosclerotic plaques, up to 50% of the apoptotic cells are macrophages, which may promote core expansion and plaque instability ( Tabas et al., 2009).
In our study, we defined the mechanism by which oxLDL induces apoptotic cell death in the J774A.1 macrophage cell line, a widely used in vitro model for evaluating the mechanisms underlying the adverse effects of oxidized lipids and oxLDL with native LDL (nLDL) as the control ( Supplementary Materials and methods ).
First, we demonstrated that oxLDL induced apoptosis in J774A.1 by activating caspase-9 and caspase-3, but not caspase-8 using the specific inhibitors, and determining the active form of caspases ( Supplementary Figure S1 ). The first toxic effect of oxLDL was an early and progressive increase in reactive oxygen species (ROSs) production, showing a biphasic trend with the highest, non-reversible increase after 6 h, which corresponded to a significant depletion of glutathione (GSH) ( Supplementary Figure S2 ). The onset of oxidative stress clearly indicated that the antioxidant system could no longer buffer the overproduction of ROSs. ROSs are involved in various biological events that are mediated by different signaling pathways, such as MAPK, NF-κB, Akt, and PKC ( Irani, 2000; Cichon and Radisky, 2010), some controlling the level and nuclear accumulation of the tumor suppressor protein p53.