Simon J. Hardwick

APOPTOSIS IN HUMAN MONOCYTE-MACROPHAGES EXPOSED TO OXIDIZED LOW DENSITY LIPOPROTEIN

This study has demonstrated the toxicity to human monocyte‐macrophages of low‐density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration‐ and time‐dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte‐macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end‐labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL‐positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no‐additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg2+‐activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Toxicity of oxysterols to human monocyte-macrophages

We have investigated the toxicity of the cholesterol oxidation products (oxysterols), 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol and 26-hydroxycholesterol to human monocyte-macrophages in vitro. The 7-position derivatives are present in low density lipoprotein (LDL) oxidised with copper (II) sulphate and macrophages, and in extracts of human atherosclerotic lesions, which also contain 26-hydroxycholesterol. We have also assessed 25-hydroxycholesterol for toxicity because it has often been used in studies of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition and LDL receptor down-regulation. Measurement of radioactivity release from monocyte-macrophages preloaded with tritiated adenine, as a means of assessing cytotoxicity that all the oxysterols showed time- and concentration-dependent toxicity. The cytotoxic potency of 26-hydroxycholesterol was the greatest. The 7-position derivatives also produced marked cell damage, though at higher concentrations than for 26-hydroxycholesterol. Of the oxysterols assessed, the toxicity of 25-hydroxycholesterol was the least. The cytotoxicity of 7 beta-hydroxycholesterol and 26-hydroxycholesterol was also shown using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay which confirmed that 26-hydroxycholesterol was more toxic than 7 beta-hydroxycholesterol. Incubation of monocyte-macrophages with cholesterol added to the different oxysterols gave varying results. Cholesterol, which was not itself toxic, inhibited the toxicity of 25-hydroxycholesterol and 26-hydroxycholesterol, but the toxicity of the 7-position derivatives was not affected. The possible relevance of these molecules to the death of macrophages seen in atherosclerosis is discussed.

CELL DEATH IN ATHEROSCLEROTIC PLAQUES

The investigation of the mechanisms of cell death in atherosclerosis has recently received added impetus with the realization that apoptosis is probably the predominant mechanism. This review examines the preliminary data on mechanisms of cell death in the atherosclerotic plaque.

Cytotoxic and chemotactic potencies of several aldehydic components of oxidised low density lipoprotein for human monocyte-macrophages

We have investigated the cytotoxic and chemotactic potencies of malondialdehyde (MDA), hexanal, 4‐hydroxyhexenal (HHE), 4‐hydroxynonenal (HNE) and 4‐hydroxyoctenal (HOE), which are aldehydes found in oxidised low density lipoprotein (LDL), for human monocyte‐macrophages. They were toxic in the following order: hexanal < HHE = HOE < HNE. HNE was toxic at 20 μM and chemotactic at 2.5 μM. The other aldehydes tested had no chemoattractant activity. Our results suggest that HNE arising from LDL oxidation could attract monocytes into the human atherosclerotic lesion. A direct cytotoxic role of aldehydes in foam cell death in the lesion is less likely.

Oxidized low-density lipoprotein is cytotoxic to human monocyte-macrophages: protection with lipophilic antioxidants.

Human monocyte‐macrophages were incubated for 24 h with low‐density lipoprotein (LDL) which had been previously oxidized for varying periods up to 24 h with copper ions, in the presence or absence of dl‐α‐tocopherol or probucol. The release of radioactivity from cells preloaded with tritiated adenine was used as an assay of toxicity. Toxicity of oxidized LDL increased with duration of copper oxidation and with increasing evidence of lipid oxidation, measured by assay of thiobarbituric acid‐reactive substances and by gas chromatography. Oxidation and toxicity were inhibited by dl‐α‐tocopherol (200 μM) and probucol (50 μM).

Fragmentation of DNA in P388D1 macrophages exposed to oxidised low-density lipoprotein

Exposing a macrophage‐like murine cell line to copper‐oxidised low‐density lipoprotein led to DNA fragmentation which was inhibited by the putative Ca2+/Mg2+ endonuclease inhibitor, zinc sulphate. DNA fragmentation preceded loss of membrane impermeability. These results suggest that apoptosis may be a mechanism of macrophage foam cell death in atherosclerotic lesions in the arterial wall.

Macrophage death and the role of apoptosis in human atherosclerosis

The arterial disease atherosclerosis is responsible for severe morbidity and is the most common cause of death in the Western population. The complete pathogenesis of the disease is unknown, but multiple risk factors have been identified that correlate with the development of its complications such as heart attack and stroke. Evidence suggests that atherosclerosis is an inflammatory disease and the major cell types involved are smooth muscle cells, macrophages, and T lymphocytes. In this paper, we review the function of macrophages in the context of atherosclerosis and we also discuss the role and significance of macrophage death, including apoptosis. There is much evidence, certainly in vitro, suggesting that low-density lipoprotein becomes atherogenic when it undergoes cell-mediated oxidation within the artery wall. Besides inducing apoptosis in vitro, oxidized low-density lipoprotein may also cause extensive DNA damage in intimal cells, which might presage apoptosis. We review the results of experimental and clinical studies, which may indicate how the complications of atherosclerosis could be prevented by using different therapeutical strategies including bone marrow transplantation and gene therapy.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte–macrophages committed to death by apoptosis. X‐ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 µg/ml of oxidized low‐density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase‐mediated DNA nick‐end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte–macrophages exposed to concentrations of oxidized LDL known to cause apoptosis. Copyright

Changes in vimentin in human macrophages during apoptosis induced by oxidised low density lipoprotein

Macrophage apoptosis contributes to the development of human atherosclerotic lesions. Oxidised LDL may be involved in macrophage death in vivo. We examined morphological and biochemical changes to the vimentin filament network during apoptosis of human macrophages. Only oxidised LDL, but not native or acetylated LDL, induced apoptosis, wherein vimentin was cleaved into fragments of 48-50, 46, 29 and 26 kDa. The use of caspase inhibitors suggested that caspase-6 mediates the formation of the 26 and 46 kDa fragments of vimentin. We were unable to demonstrate any significant involvement of caspase-3 in vimentin cleavage. However, caspase-3 was clearly activated during apoptosis whilst caspase-6 expression in macrophages was minimal. Vimentin filament breakdown occurred early during apoptosis and vimentin immunoreactivity was present in apoptotic bodies. However, the application of caspase inhibitors had no effect on the morphology of the vimentin network in apoptotic cells, suggesting that filament breakdown is not mediated by caspase proteolysis. Similar changes in vimentin were also seen in gliotoxin-induced apoptosis.

MACROPHAGES, LIPID OXIDATION, CEROID ACCUMULATION AND ALPHA-TOCOPHEROL DEPLETION IN HUMAN ATHEROSCLEROTIC LESIONS

Necropsy samples of atherosclerotic lesions of different histological stages have been analysed. Ceroid was present in all the lesions, within lipid-laden macrophage foam cells and extracellularly in the atheromatous core of advanced lesions. Mean levels of 7 beta-hydroxycholesterol, 26-hydroxycholesterol and hydroxyoctadecadienoic acids were all significantly greater in lesions than in normal intima. Levels of hydroxycholesterols were very low or undetectable in normal intima. Fatty streaks showed the highest ratio of 7 beta-hydroxycholesterol to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest free radical activity. Levels of 26-hydroxycholesterol, a product of the cytochrome P-450 enzyme sterol 26-hydroxylase, and the ratio of 26-hydroxycholesterol to cholesterol were significantly higher in advanced lesions than in intermediate lesions or fatty streaks. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal intima but was consistently low in lesions, especially those rich in macrophage foam cells, suggesting that oxidative activity in the lesion may lead to significant oxidation of the lesion constituents only after alpha-tocopherol has been depleted. Macrophage death was a characteristic feature of advanced lesions, with apoptotic bodies present, and occasionally, intact apoptotic cells were seen in lesions. These striking correlations between macrophages, lipid oxidation, alpha-tocopherol depletion, ceroid accumulation, and macrophage death in advanced lesions, strongly support a role for oxidative damage in atherosclerosis, and lend credence to the idea that alpha-tocopherol dietary supplementation may slow the progression of atherosclerosis in humans.