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.
Linköping: Linköpings universitet , 1997. , 63 p.
1997-11-22, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Papers, included in the Ph.D. thesis, are not registered and included in the posts from 1999 and backwards.