Two Distinct Calcium-Dependent Mitochondrial Pathways Are Involved in Oxidized LDL-Induced Apoptosis

Abstract

Objective-Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular endothelial cells may contribute to plaque erosion and rupture. We aimed to clarify the relationship between the oxLDL-induced calcium signal and induction of apoptotic pathways. Methods and Results-Apoptosis was evaluated by biochemical methods, including studies of enzyme activities, protein processing, release of proapoptotic factors, chromatin cleavage, and especially by morphological methods that evaluate apoptosis/necrosis by SYTO-13/propidium iodide fluorescent labeling. The oxLDL-induced sustained calcium rise activated 2 distinct calcium-dependent mitochondrial apoptotic pathways in human microvascular endothelial cells. OxLDLs induced calpain activation and subsequent Bid cleavage and cytochrome C release, which were blocked by calpeptin. Cyclosporin-A inhibited cytochrome C release, possibly by inhibiting the opening of the mitochondrial permeability transition pore (mPTP). Calcineurin, another cyclosporin-sensitive step, was not implicated, because oxLDLs inhibited calcineurin and FK-506 treatment was ineffective. Cytochrome C release in turn induced caspase-3 activation. In addition, oxLDLs triggered release and nuclear translocation of mitochondrial apoptosis-inducing factor through a mechanism dependent on calcium but independent of calpains, mPTP, and caspases. Conclusions-OxLDL-induced apoptosis involves 2 distinct calcium-dependent pathways, the first mediated by calpain/ mPTP/cytochrome C/caspase-3 and the second mediated by apoptosis-inducing factor, which is cyclosporin-insensitive and caspase-independent. Key Words: calpain Ⅲ caspase Ⅲ mitochondria Ⅲ apoptosis-inducing factor Ⅲ oxidized low-density lipoprotein Ⅲ atherosclerosis A therogenesis is characterized by lipid deposition, a chronic inflammatory response, and chronic wound healing processes. 1,2 Apoptosis may play a role in endothelial cell lining defects, necrotic core formation, or plaque erosion and rupture. [3][4][5] Among the variety of proapoptotic factors present in atherosclerotic plaques, oxidized low-density lipoproteins (oxLDLs) are thought to play a crucial role by concomitantly inducing lipid storage, local inflammation, and toxic events. 4,[5][6][7][8] OxLDLs trigger apoptosis or necrosis of cultured vascular cells 7-9 and may therefore participate in vascular wall injury, plaque erosion/rupture, and subsequent athero-thrombotic events. 4,5 The proapoptotic effects of oxLDLs are mediated through a complex sequence of signaling events that lead to activation of several caspase-dependent or -independent apoptotic pathways. 8,9 Two separate caspase-dependent apoptotic pathways have been implicated in oxLDL-induced apoptosis. 7-9 The extrinsic apoptotic pathway, mediated by death receptors, Fas, and/or tumor necrosis factor receptor (TNFR) and downstream by caspase-8/caspase-3, is involved in oxLDLinduced apoptosis in endothelial cells. 8,10,11 However, a recent report contests this hypothesis. 12 The intrinsic mitochondrial apoptotic pathway, ...

HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs

“…This suggests that HDLs may act upstream by inhibiting a signaling pathway common to apoptosis and autophagy. We, therefore, hypothesized that Ca 2 þ signaling may constitute this critical trigger, as Ca 2 þ deregulation is involved in apoptosis 6 and ER stress 9 induced by oxLDLs, and is also implicated in autophagy. 14,38 The reported data show, for the first time, that HDLs completely blocked ER stress and autophagy induced by oxLDLs by preventing the cytosolic Ca 2 þ rise.…”

“…Apoptotic and necrotic cells were counted after fluorescent staining by two fluorescent dyes, the permeant DNA intercalating green fluorescent probe SYTO-13 (0.6 mM) and the non-permeant DNA intercalating red fluorescent probe PI (15 mM) using an inverted fluorescence microscope (Fluovert FU, Leitz, Leica, Wetzlar, Germany). 6 Normal nuclei exhibit a loose green-colored chromatin. Nuclei of primary necrotic cells exhibit loose redcolored chromatin.…”

“…1 Oxidized LDLs (oxLDLs) exhibit a variety of atherogenic properties including foam cell formation, inflammatory response, cell proliferation at low concentration and apoptosis at higher concentration 1,2 OxLDLs may alter the fragile balance between survival and death of vascular cells, thereby, leading to plaque instability and finally to atherothrombotic events. 3,4 The proapoptotic effects of oxLDLs are mediated through a complex sequence of signaling events that lead to the activation of several caspase-dependent or caspase-independent apoptotic pathways 2,5,6 Previously, we reported that treatment of human vascular endothelial and smooth muscle cells with oxLDLs or 7-ketocholesterol (a major oxysterol in oxLDLs) causes an increase in cytosolic Ca 2 þ through TRPC1 channels that triggers a proteolytic cascade involving Ca 2 þ -dependent calpains, Bid cleavage and cytochrome c release, leading finally to caspase-3 activation and apoptosis. [7][8][9] Recently, we have reported that oxLDLs induced the unfolded protein response (UPR) and triggered endoplasmic reticulum (ER) stress.…”

Signalisation apototique induite par les LDL oxydées Implication dans l’athérosclérose

“…fmk, Z-DVED. fmk) réduisent partiellement l'apoptose induite par les LDL oxydées [79][80][81]. L'apoptose induite par les LDL oxydées est inhibée par l'expression de Bcl2, mais les cellules meurent alors par nécrose [18].…”

“…Les calpaïnes peuvent cliver Bid qui induit également l'ouverture du PTP [86,87]. Les mitochondries sont également impliquées dans le relargage de l'AIF (apoptosis inducing factor), qui transloque dans le noyau et induit un clivage de l'ADN en gros fragments [81]. …”