Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products

The Gade Institute, University of Bergen, Bergen, Hordaland, Norway
Free Radical Biology and Medicine (Impact Factor: 5.74). 02/2000; 28(2):208-18. DOI: 10.1016/S0891-5849(99)00220-8
Source: PubMed


We have previously shown that oxidized low-density lipoprotein (LDL) induces damage to the macrophage lysosomal membranes, with ensuing leakage of lysosomal contents and macrophage cell death. Cholesterol oxidation products (ChOx) have been reported to be the major cytotoxic components of oxidized LDL/LDL- and also to stimulate cholesterol accumulation in vascular cells. In the present study, we characterized the initial events during macrophage damage induced by cholesterol oxidation products (ChOx). Within 24 h of exposure, ChOx caused lysosomal destabilization, release to the cytosol of the lysosomal marker-enzyme cathepsin D, apoptosis, and postapoptotic necrosis. Enhanced autophagocytosis and chromatin margination was found 12 h after the exposure to ChOx, whereas apoptosis and postapoptotic necrosis was pronounced 24 and 48 h after the exposure. Some lysosomal vacuoles were then filled with degraded cellular organelles, indicating phagocytosis of apoptotic bodies by surviving cells. Because caspase-3 activation was detected in the ChOx-exposed cells, lysosomal destabilization may associate with the leakage of lysosomal enzymes, and activation of the caspase cascade. MnSOD mRNA levels were markedly increased after 24 h of exposure to ChOx, suggesting associated induction of mitochondrial protection repair or turnover. We conclude that ChOx-induced damage to lysosomes and mitochondria are sequelae to the cascade of oxysterol cytotoxic events. The early disruption of lysosomes induced by ChOx, with resultant autophagocytosis may be a critical event in apoptosis and/or necrosis of macrophages/foam cells during the development of atherosclerotic lesions.

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    • "In the cells treated with 7keto MDM2 levels were reduced to 56% (6 h) and 35% (16 h) of control levels (n¼ 2). 7-Oxysterols cause LMP in M1-t-p53 cells but not in p53-deficient M1 cells Our previous studies suggested that both p53 and 7-oxysterols cause apoptosis through the lysosomal–mitochondrial pathways [13] [14]. To understand the observed interactions between p53 and 7-oxysterols in the process of cell death, alterations in LMP were examined by flow cytometry after AO staining. "
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    ABSTRACT: Oxysterol accumulation and p53 expression mainly in macrophages have been associated with cell death and necrotic core formation in human atheroma progression. Oxidative stress and lysosomal membrane permeabilization (LMP) in macrophages are important causes of macrophage apoptosis in advanced lesions. However, it is not understood how p53 and oxysterols interact in the process. We show here that 7-oxysterols induce endogenous full-length p53 and phospho-p53 (p53-Ser15) in both nucleus and cytoplasm of THP1 and J774 cells, which is followed by cellular oxidative stress and apoptotic cell death. The role of p53 in 7-oxysterol-mediated cell death is further investigated in p53-transfected (M1-t-p53) and in p53-deficient (M1) cells. These results reveal that 7-oxysterols induce induction and nuclear translocation of p53 in M1-t-p53 cells, which in turn enhances LMP, mitochondrial translocation of Bax, mitochondrial membrane permeabilization, cytosolic release of cytochrome c, and cell death. Most importantly, the above effects of 7-oxysterols were not observed in p53-deficient M1 cells. The findings reveal that 7-oxysterol-induced cell death occurs via p53-dependent pathways. Subsequent p53 nuclear translocation and induction of wild-type and phosphorylated p53 are early steps in oxysterol-induced lysosomal-mitochondrial pathways.
    Free Radical Biology and Medicine 09/2012; 53(11). DOI:10.1016/j.freeradbiomed.2012.09.007 · 5.74 Impact Factor
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    • "The shift in AO fluorescence from granular red to diffusely green reflects leakage and redistribution of AO from the lysosomes, indicating impairment of the lysosomal membranes or of the ability of the lysosomes to maintain low pH. So, AO is widely used to perform lysosomal integrity measurements, and the percentage of AOnegative cells (which do not emit a red fluorescence) permits quantification of the percentage of cells with destabilized lysosomes (Yuan et al., 2000). In the present investigation, a 1- mg/mL stock solution of AO (Sigma) was prepared in distilled water (Olsson et al., 1987). "
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    ABSTRACT: X-linked adrenoleukodystrophy (X-ALD) is characterized by ABCD1 deficiency. This disease is associated with elevated concentrations of very long chain fatty acids (C24:0 and C26:0) in the plasma and tissues of patients. Under its severe form, brain demyelination and inflammation are observed. Therefore, we determined the effects of C24:0 and C26:0 on glial cells:oligodendrocytes, which synthesize myelin, and astrocytes, which participate in immune response. So, 158N murine oligodendrocytes, rat C6 glioma cells, rat primary cultures of neuronal-glial cells, and of oligodendrocytes were treated for various periods of time in the absence or presence of C24:0 and C26:0 used at plasmatic concentrations found in X-ALD patients (1-5 μM) and higher (10, 20, 40 μM). To evaluate the importance of extrinsic and intrinsic factors, the part taken by TNF-α and reduced Abcd1 level was studied. Whatever the cells considered, no effects on cell growth and/or viability were detected at 1-5 μM, more or less pronounced effects were identified at 10 μM, and an induction of cell death with increased permeability to propidium iodide and loss of transmembrane mitochondrial potential was observed at 20-40 μM. On 158N, cell death was characterized by (i) an increased superoxide anion production at the mitochondrial level; (ii) the presence of vacuoles of different sizes and shapes; a destabilization of lysosomal membrane and a cytoplasmic redistribution of lysosomes; (iii) a modulation of Abcd3/PMP70 and Acox-1 protein expression, and a decrease in catalase activity at the peroxisomal level. When TNF-α was combined with C24:0 or C26:0 and used on 158N cells, C6 cells, and on 158N cells after siRNA mediated knockdown of Abcd1, no or slight potentiation was revealed. Thus, on the different cell models used, an induction of cell death with marked cellular dysfunctions at the mitochondrial, lysosomal, and peroxisomal levels were found with C24:0 and C26:0 at 20 μM and higher. However, in our experimental conditions, plasmatic concentrations of these fatty acids were unable to induce cell death, and organelle dysfunctions on oligodendrocytes and astrocytes, and additional intrinsic and environmental factors, such as reduced Abcd1 level and/or TNF-α, were ineffective to potentiate their side effects.
    NeuroToxicology 10/2011; 33(2):212-28. DOI:10.1016/j.neuro.2011.10.007 · 3.38 Impact Factor
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    • "The accumulation of cholesterol in macrophages in the form of cytoplasmic lipid droplets is an early step in the formation of atherosclerotic lesions [28]. Continual macrophage uptake of oxLDL increases the accumulation of CE, resulting in lysosomal disturbance and ultimately apoptosis [29], [30]. In the present study we corroborated that the uptake of both lipid and protein moieties of oxLDL was dose dependent in U937 cells, and that both lipid and protein moieties of oxLDL were internalized in endosomal vesicles and localized in the lysosomal compartment. "
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    ABSTRACT: Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress. Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC. Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'.
    PLoS ONE 09/2010; 5(9). DOI:10.1371/journal.pone.0012534 · 3.23 Impact Factor
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