Xi-Ming Yuan

Induction of cell death by the lysosomotropic detergent MSDH

Controlled lysosomal rupture was initiated in lysosome‐rich, macrophage‐like cells by the synthetic lysosomotropic detergent, O‐methyl‐serine dodecylamide hydrochloride (MSDH). When MSDH was applied at low concentrations, resulting in partial lysosomal rupture, activation of pro‐caspase‐3‐like proteases and apoptosis followed after some hours. Early during apoptosis, but clearly secondary to lysosomal destabilization, the mitochondrial transmembrane potential declined. At high concentrations, MSDH caused extensive lysosomal rupture and necrosis. It is suggested that lysosomal proteases, if released to the cytosol, may cause apoptosis directly by pro‐caspase activation and/or indirectly by mitochondrial attack with ensuing discharge of pro‐apoptotic factors.

Lysosomal destabilization in p53-induced apoptosis

The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders.

Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation

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 ofoxLDL 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-beta-glucosaminidase (NAbetaGase) 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 NAbetaGase 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.

Iron in human atheroma and LDL oxidation by macrophages following erythrophagocytosis

The oxidative modification of low density lipoprotein (LDL) has been implicated as an early step in the formation of atheromatous lesions. In vitro studies suggest it to be accelerated, or even initiated, by transition metals such as iron or copper in combination with a reducing agent. Even if such metals have been demonstrated in atheroma gruels, their origin and precise localisation within human atheroma are presently unknown. In the initial part of this study we applied Pearls method, energy dispersive X-ray microanalysis, and a modified Timm sulphide silver method (SSM) to demonstrate the occurrence of iron in early atherosclerotic lesions from a number of consecutive autopsy cases with evident, general atheromatosis. With the very sensitive SSM, but not with the other techniques, 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 where human monocyte-derived macrophages were exposed to artificially aged, UV-exposed erythrocytes. The macrophages were then exposed to LDL in serum-and iron-free RPMI medium, occasionally in the presence of the potent iron-chelator desferrioxamine. The capacity of macrophages to oxidise LDL was much enhanced following erythrophagocytosis, and the process was shown to involve secretion of iron. Consequently, LDL oxidation was greatly inhibited by desferrioxamine. We conclude that iron may be exocytosed by macrophages that previously had their lysosomal apparatus enriched with iron, e.g. due to erythrophagocytosis. Oxidation of LDL may result in ensuing foam cell-formation secondary to scavenger-receptor mediated endocytosis by macrophages.

The iron hypothesis of atherosclerosis and its clinical impact

The iron hypothesis as an alternative explanation for the gender difference in the incidence and mortality of atherosclerosis has provoked increased debates and public health concerns. In this review we summarize the historical and recent literature on the iron hypothesis and discuss several related clinical issues and their implications. Apart from misconstruction of study populations, lack of a good method to reflect the iron contents of tissues may be the major factor for causing inconsistent results from epidemi‐ological studies. Published data from 11 countries clearly indicate that the mortality from cardiovascular diseases is correlated with liver iron. We propose that redox‐active iron in tissue is the atherogenic portion of total iron stores. Recently developed magnetic resonance imaging techniques in combination with Fe chelators may allow future studies to examine this component of body iron in lesions and the whole body. Several clinical situations characterized by increased iron stores have been proposed as ‘human models’ suitable for further tests of the iron hypothesis. Patients with end‐stage renal disease may be the most unique cohort, having significant increases in their iron stores, low‐density lipoprotein (LDL) oxidation, and cardiovascular events. Other patient groups may be well suited for specific studies of different atherogenic events. With a better understanding of iron‐driven oxidative damage, well controlled and effectively designed studies on these models will finally bring us to the truth of the iron hypothesis.

Methylmercury and H2O2 provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 microM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H(2)O(2)) exposure (100 microM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H(2)O(2) preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H(2)O(2) stimulated divergent pathways, with caspases being activated only by H(2)O(2). The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H(2)O(2), suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H(2)O(2). The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.

Effects of Iron- and Hemoglobin-Loaded Human Monocyte–Derived Macrophages on Oxidation and Uptake of LDL

It is generally accepted that transition metals are required for cellular LDL oxidation. LDL may also be oxidized by iron and reducing agents in cell-free systems. We hypothesized that lysosomal iron may be exocytosed from macrophages that have been iron loaded by phagocytosis and degradation of iron-rich structures, eg, erythrocytes, and that such released iron may promote LDL oxidation and uptake by macrophages. Human monocyte-derived macrophages (HMDMs) were isolated and cultured for 7 days and then exposed to FeCl3, Fe-ADP, or Fe-EDTA (100 mumol/L) or hemoglobin (25 or 50 micrograms/mL) for 24 hours. After rinsing, LDL (50 to 150 micrograms/mL) was added in fresh culture medium without serum. After another 24 hours the media concentrations of iron and thiobarbituric acid-reacting substances as well as the electrophoretic mobility of LDL were increased, while the cells showed only minimal signs of decreased viability. Lipofuscin, neutral lipids, and phospholipids accumulated in a granular, lysosome-like pattern, and the cells acquired a foam cell-like morphology. There was a strong correlation (r = .87, P = .005) between the amount of iron added during the pre-exposure period and lipofuscin accumulation during the ensuing exposure to LDL in fresh, serum-free medium. Our results support our hypothesis and indicate that lysosomal iron may be exocytosed from HMDMs and promote oxidation and uptake of LDL and thus induce foam cell formation.

Cathepsin L is significantly associated with apoptosis and plaque destabilization in human atherosclerosis

OBJECTIVE Human atherosclerotic lesions overexpress elastolytic and collagenolytic cathepsins with unclear pathological implications. The aim of this study was to investigate the relationship among expression of cathepsin L, macrophage apoptosis in coronary artery disease (CAD) patients, clinical symptoms and plaque severity of human carotid atheroma. METHODS AND RESULTS Quantitative immunohistochemical analysis of human carotid atherosclerotic lesions (n=49) showed that expression of lysosomal cathepsin L was significantly increased in atherosclerotic plaques with formation of the necrotic core and rupture of the cap. In those plaques, cathepsin L was associated mainly with CD68-positive macrophages, whereas significant lower levels of smooth muscle cell actin were detected. The expression of cathepsin L in these plaques was also correlated with apoptosis and the stress protein ferritin. Plaques from symptomatic patients showed greater increased levels of cathepsin L than those from asymptomatic patients. Human monocyte-derived macrophages from CAD patients (n=7) showed significantly higher levels of cathepsin L, cellular lipids and apoptosis versus cells from matched healthy donors (n=7). 7Beta-hydroxycholesterol significantly enhanced cathepsin L in cells from healthy donors but not in cells from CAD patients. Moreover, macrophage apoptosis was significantly correlated with expression of cathepsin L in cell nuclei and membranes. CONCLUSION The results suggest that cathepsin L is involved in death of macrophages, necrotic core formation and development of atherosclerotic plaque instability. Macrophage lysosomal cathepsin L and related apoptosis may be potential targets for modulation or imaging of vulnerable plaques in human atherosclerosis.

The toxicity to macrophages of oxidized low-density lipoprotein is mediated through lysosomal damage

Oxidized low-density lipoprotein (ox-LDL) has been shown to degrade poorly within the secondary lysosomes of macrophages but its possible effect on lysosomal integrity has received less attention. The effect of ultraviolet-C oxidized LDL (UVox-LDL) on cellular viability, and lysosomal membrane stability, was examined on cultured murine J-774 cells and human monocyte-derived macrophages (HMDMs). The acridine orange (AO) relocalization test was applied to study the lysosomal integrity of living cells. UVox-LDL dramatically reduced J-774 cell proliferation at a concentration of 25 microg/ml. Incubation with 5 microM copper alone, normally used to induce LDL oxidation, was also toxic. In contrast to the effects of ox-LDL, in concentrations up to 75 microg/ml, native LDL (nLDL) rather stimulated J-774 cell replication. Incubation with UVox-LDL (25-75 microg/ml) also altered cellular AO uptake, depending on time and dose: its lysosomal accumulation decreased and its cytosolic accumulation increased. This shift indicates damaged lysosomal membranes with decreased intralysosomal, and increased cytosolic, H+ concentration. Many J-774 cells exposed to UVox-LDL initially transformed into foam cells and then assumed an apoptotic-type morphology with TUNEL-positive nuclei. We conclude that ox-LDL is cytotoxic to macrophages due to oxidative damage of lysosomal membranes, with ensuing destabilization and leakage to the cytosol of lysosomal contents, such as hydrolytic enzymes, causing degeneration of apoptotic type.

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.