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- Li, Wei, et al.
(författare)
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Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation
- 1998
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 18:2, s. 177-84
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Tidskriftsartikel (refereegranskat)abstract
- The cytotoxicity of oxidized LDL (oxLDL) to several types of artery wall cells might contribute to atherosclerosis by causing cell death, presumably by both apoptosis and necrosis. After its uptake into macrophage lysosomes by receptor-mediated endocytosis, oxLDL is poorly degraded, resulting in ceroid-containing foam cells. We studied the influence of oxLDL on lysosomal enzyme activity and, in particular, on lysosomal membrane stability and the modulation of these cellular characteristics by HDL and vitamin E (vit-E). Unexposed cells and cells exposed to acetylated LDL (AcLDL) were used as controls. The lysosomal marker enzymes cathepsin L and N-acetyl-β-glucosaminidase (NAβGase) were biochemically assayed in J-774 cells after fractionation. Lysosomal integrity in living cells was assayed by the acridine orange (AO) relocation test. Cathepsin D was immunocytochemically demonstrated in J-774 cells and human monocyte-derived macrophages. We found that the total activities of NAβGase and cathepsin L were significantly decreased, whereas their relative cytosolic activities were enhanced, after oxLDL exposure. Labilization of the lysosomal membranes was further proven by decreased lysosomal AO uptake and relocation to the cytosol of cathepsin D, as estimated by light and electron microscopic immunocytochemistry. HDL and vit-E diminished the cytotoxicity of oxLDL by decreasing the lysosomal damage. The results indicate that endocytosed oxLDL not only partially inactivates lysosomal enzymes but also destabilizes the acidic vacuolar compartment, causing relocation of lysosomal enzymes to the cytosol. Exposure to AcLDL resulted in its uptake with enlargement of the lysosomal apparatus, but the stability of the lysosomal membranes was not changed.
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- Yuan, Xi Ming, 1959-
(författare)
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Iron and macrophages in atherogenesis
- 1995
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidation of low density lipoprotein (LDL) may result in its uptake by macrophages and ensuing foam cell formation. Thus, oxidised LDL may play an important role in atherogenesis. Extensive in vitro evidences are in favour of the notion that LDL oxidation by cells present in atherosclerotic plaques requires the presence of transition metals. It has been confirmed repeatedly that atherosclerotic lesions contain significant amounts of copper and iron. The mechanism by which LDL becomes oxidised in vivo, though, remains unknown.In the first part of the present study we wanted to learn about how iron is involved in the process of LDL oxidation by human macrophages; whether iron may be exocytosed after cellular exposure to different iron compounds; and if such exocytosis would affect LDL oxidation and its uptake by macrophages. Human monocyte-derived macrophages (HMDMs) were firstly exposed to different iron compounds (100μM), or haemoglobin (25 or 50 μg/ml) for 24 hours. Following rinsing LDL (50 or 150 µg/ml) was added in fresh culture medium without serum. After another 24 hours the concentrations of iron and thiobarbituric acid-reactive substances (TBARS), as well as the electrophoretic mobility of LDL in medium, were found increased, while the cells showed only minimal signs of decreased viability. Neutral lipids and phospholipids accumulated in a granular, lysosome-like, pattern and the cells acquired a foam cell-like morphology.The second part of the study was designed (i) to establish a model of erythrophagocytosis by macrophages, and (ii) to study iron-sequestration within secondary lysosomes, and exocytosis by these cells following the degradation of erythrocytes. The binding and uptake of UV-irradiated red blood cells (UV-RBC) by human macrophages and J-774 cells were greatly stimulated compared to that of native erythrocytes. The uptake resulted in lysosomal accumulation of iron in a low-molecular-weight form, as shown by autometallography. Following the exposure to UV-RBC and ferric iron a much enhanced amount of cytosolic ferritin was demonstrated in macrophages by immunocytochemistry. Ensuing exocytosis of iron to the culture medium was demonstrated by atomic absorption spectroscopy.The third part of the study aimed to investigate oxidative stress-induced lipofuscinogenesis in human macrophages as well as in a test-tube system of mitochondria and lysosomes from rat liver. Firstly, control HMDMs, and HMDMs exposed to different iron compounds (100 µM Fe3+) or Hb (25 or 50 µg/ml), were incubated for 48 hours with LDL. Lipofuscin-specific autofluorescence was markedly increased in all LDL-exposed cells. A linear correlation was found between lipofuscin formation and the concentration of FeCl3 to which the HMDMs earlier had been pre-exposed. Secondly, endogenous iron in lysosomal-mitochondrial fraction (LMF) homogenates (545 µg/1, about 10 µM) was detected by atomic absorption spectrophotometry. After incubation of LMF with different concentrations of cystein for different periods of time a time- and dose-dependent TBARS-yield was observed. The peroxidation was completely inhibited by the addition of desferrioxamine or butylated hydroxytoluen (BHT). Under the same conditions the carbonyls of the trichloroacetic acid (TCA) precipitable protein of LMF were analysed. They were found increased, but only after a slight initial decrease. Following a sharp initial decrease, the normal tryptophan-tyrosine (protein) autofluorescence remained stable. In contrast, after a lag period of a few days, a lipofuscin-type autofluorescence was also observed.In conclusion: A. Lysosomal iron may be exocytosed from HMDMs, following a previous uptake of simple iron compounds or Hb promoting oxidation and uptake of LDL and thus induce foam cell formation. B. Macrophage erythrophagocytosis is a useful model for the study of the lysosomal sequestration of iron. Iron is accumulated within the macrophage acidic vacuolar apparatus arid subsequently exocytosed. C. Lipofuscin forms in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials D. LDL and iron may both play important roles in lipofuscinogenesis within atherosclerotic lesions.
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| 4. |
- Yuan, Xi Ming, 1959-
(författare)
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LDL oxidation, iron, lysosomes, and macrophages in early atherosclerosis
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidation of low density lipoprotein (LDL) may result in its uptake by macrophages with ensuing foam cell formation. Thus, oxidised LDL (oxLDL) may play an important role in atherogenesis. Extensive in vitro evidence is in favour of the notion that LDL oxidation by cells present in atherosclerotic plaques requires the presence of transition metals. The mechanisms by which LDL becomes oxidised in vivo and the effects of oxLDL on macrophages and foam cells, though, remain unknown.fu the first part of the present study we wanted to learn about the involvment of iron in the process of LDL oxidation by human macrophages; whether iron may be exocytosed following cellular exposure to different iron compounds; and if such exocytosis would affect LDL oxidation, and its uptake by macrophages. Human monocyte-derived macrophages (HMDMs) were exposed initially to different simple iron compounds (100 ~M), or haemoglobin (25 or 50 ~g/ml) for 24 hours. Following rinsing LDL (50 or 150 ~g/ml) was added in fresh culture medium without serum. After another 24 hours the concentrations of iron and thiobarbituric acid-reactive substances (TEARS), as well as the electrophoretic mobility of LDL, were found increased in the medium. Neutral lipids and phospholipids accumulated in a granular, lysosome-like, pattern and the cells acquired a foam cell-like morphology. Lipofuscin-specific autofluorescence was markedly increased in all iron and LDL-exposed cells. A linear correlation was found between lipofuscin formation, and the concentration of iron-complexes to which the HI\IDMs earlier had been pre-exposed.The second part of the study was designed (i) to establish a model of erythrophagocytosis by macrophages, and (ii) to study the iron-sequestration within secondary lysosomes and ironexocytosis by these cells following the degradation of erythrocytes. The binding and uptake of UV-irradiatedredblood cells (UV-RBC) by human macrophages and J-774 cells were greatly stimulated compared to that of native e1ythrocytes. The uptake resulted in lysosomal accumulation of iron in a low-molecular-weight form, as shown by autometallography. Following the exposure to UV -RBC or ferric iron a much enhanced amount of cytosolic ferritin was demonstrated in macrophages by immunocytochemistry. Ensuing exocytosis of iron to the culture medium was demonstrated by atomic absorption spectroscopy.The third part of the study aimed to localise the occurrence of iron in early atherosclerotic lesions from a number of consecutive autopsy cases with evident, general atheromatosis. With the SSM, we found foam cells to contain heavy metals with a mainly lysosomal localization. On the basis of the hypothesis that such a lysosomal accumulation of iron might be due to erythrophagocytosis by migrating tissue-bound macrophages that later develop into foam cells, we designed an in vitro model system in which human monocyte-derived macrophages were exposed to artificially aged, UV -exposed erythrocytes. The capacity of macrophages to oxidise LDL was found to be much enhanced following erythrophagocytosis, and the process was shown to involve secretion of iron. Consequently, LDL oxidation was greatly inhibited by desferrioxamine.The effect of oxLDL on cellular viability and lysosomal membrane stability was examined on cultured murine J -77 4 cells and human monocyte-derived macrophages (HMDMs) in the fourth part of this study. The acridine orange (AO) relocalisation test was applied to study the lysosomal integrity of living cells. UVoxLDL dramatically reduced cell proliferation at a concentration of 25 Jlglml. Incubation with 5 JlM copper alone, normally used to induce LDL oxidation, was also toxic. In contrast to the effects of oxLDL, in concentrations up to 75 J-Lg/ml, native LDL (nLDL) stimulated J-774 cell replication. Incubation with UVoxLDL (25-75 j.ig/nal) altered the cellular AO-uptake, depending on time and dose; the lysosomal accumulation decreased while the cytosolic accumulation increased. This shift indicates damaged lysosomal membranes with decreased intralysosomal and increased cytosolic proton (H+) concentration. Cells initially exposed to UVoxLDL changed into foam cells and subsequently assumed an apoptotic-type morphology.The fifth part of this study aimed to investigate the nature of accumulated iron, and its possible relation to the apoptotic process in human atherosclerotic lesions. Pronounced fenitinaccumulation, occurrence of low-molecular-weight iron, and apoptosis concerned mainlyCD68-positive iron-rich cells (macrophages) within the atherosclerotic lesions. No ferritin- or CD 68-positivity was found in normal coronary arteries from young forensic-autopsy cases, while a moderate number of such cells were observed in the intima of normal-looking vessel areas from the clinical cases with general atherosclerosis. In the atheroma intima, ferritin and iron were found in many CD68-positive macrophages which frequently were surrounded by erythrocytes. A substantial number of apoptotic cells within the intima, media, and adventitia were registered in all atherosclerotic lesions examined, although mainly in the macrophageenriched area of the atheroma shoulder.In conclusion: A. Lysosomal iron may be exocytosed from HMDMs, following a previous uptake of simple iron compounds or Hb, promoting oxidation, uptake of LDL, and foam cell formation. B. Macrophage erythrophagocytosis is a useful model for the study of the lysosomal sequestration of iron in cell-mediated LDL oxidation. Iron is accumulated within the macrophage acidic vacuolar apparatus and subsequently exocytosed. C. Iron promotes lipofuscin formation in the LDL-macrophage system, supporting the concept that lipofuscin accumulates in lysosomes as a result of iron-catalyzed lipid peroxidation. D. Iron, stored as ferritin, may occur in macrophages, and macrophage-derived foam cells as a consequence of erythrophagocytosis or phagocytosis of apoptotic cells. E. OxLDL, but not native LDL, is cytotoxic to macrophages. The cytotoxic effect of oxLDL may result from oxidative damage of lysosomal membranes, with ensuing destabilisation and leakage into the cytosol of lysosomal contents, such as hydrolytic enzymes. F. Dysregulated iron- and ferritin-metabolism within macrophage/foam cells suggest that iron/ferritin may be associated with ongoing apoptosis in vivo, contributing to the instability of atherosclerotic plaques.
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