Malcolm J. Mitchinson

Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma

Sections of human atherosclerotic lesions of different stages show that, in early lesions, the acellular lipid core is usually immediately adjacent to the deepest edge of a collection of macrophage foam cells. Advanced lesions with a large lipid core have variable numbers of macrophage foam cells, close to the lateral edges, or shoulders, of the core. In both early and advanced lesions, some of the macrophages nearest the core appear to be dying. Lipid cores contain two materials which in earlier lesions are found only in macrophages, namely ceroid and CD68 antigen, but do not contain recognisable smooth muscle cell actin. It is concluded that death of macrophage foam cells contributes to the origin and slow enlargement of the lipid core. The cause of macrophage death is not yet certain, but is under investigation.

LIPIDS AND OXIDISED LIPIDS IN HUMAN ATHEROSCLEROTIC LESIONS AT DIFFERENT STAGES OF DEVELOPMENT

Lipids and oxidised lipids were analysed by GC and GC-MS in human necropsy samples of normal artery and individual atherosclerotic lesions, from aorta and common carotid artery, including fatty streaks, intermediate lesions and advanced lesions. Age-related increases were seen for linoleate, oleate and cholesterol in normal artery, but not in lesions. Each category of lesion was much richer than normal artery in all the lipids measured and in oxidised lipids (oxysterols and hydroxyoctadecadienoic acids), although a degree of overlap existed between the compositions of the various categories of lesion. 26-Hydroxycholesterol and 7 beta-hydroxycholesterol levels were extremely low or undetectable in normal artery, but significantly higher in each of the categories of lesions. The generally wide variation in lipid composition of individual lesions within each category, and the fact that a few individual lesions showed no detectable 26-hydroxycholesterol or 7 beta-hydroxycholesterol, suggested that the lipid oxidation in lesions and therefore perhaps the progression of lesions may be intermittent. Fatty streaks showed the highest concentration of 7 beta-hydroxycholesterol relative to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest concentration of free radical activity. Levels of the enzymatic product 26-hydroxycholesterol were approximately proportional to cholesterol in all the categories of lesions. 26-Hydroxycholesterol was significantly more abundant in advanced lesions than in intermediate lesions or fatty streaks. 26-Hydroxycholesterol levels were higher in macrophage-rich intermediate and advanced lesions than in their fibrous counterparts. This distinction between macrophage-rich and fibrous lesions was also true for most of the other lipid components, consistent with the involvement of macrophages in lipid accumulation, lipid oxidation and lesion development.

Foam cell apoptosis and the development of the lipid core of human atherosclerosis

A characteristic feature of the advanced atherosclerotic lesion is the acellular lipid core, which appears to result at least partly from the death of macrophage foam cells. This study shows that foam cell death at the edge of the lipid core includes both necrosis and apoptosis and that remnants of apoptotic nuclei are present within the lipid core. Apoptotic cells were identified by transmission electron microscopy and by nick end‐labelling using terminal deoxynucleotidyl transferase (TUNEL). Some TUNEL‐positive cells also expressed proliferating cell nuclear antigen (PCNA). The cause of foam cell death in atherogenesis is unknown, but oxidized low‐density lipoprotein (LDL) can cause macrophage apoptosis in vitro and might therefore play a role in the formation and enlargement of the lipid core.

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.

lipids and oxidised lipids in human atheroma and normal aorta

Lipids and oxidised lipids were analysed by GC and GC-MS in samples of human atheroma (necrotic gruel from the interior of advanced atherosclerotic plaques in the aorta) and human normal aorta (lesion-free intima plus inner media) from necropsy subjects. Cholest-5-en-3 beta,26-diol and cholest-5-en-3 beta,7 beta-diol were detected in all the atheroma samples examined but not in significant amounts in normal aorta. In atheroma, cholest-5-en-3 beta,26-diol was approximately proportional to cholesterol. Several isomeric hydroxy-octadecadienoic acids were detected in atheroma, and, in smaller amounts, in normal aorta. Many of the components of atheroma showed a high degree of cross-correlation on linear regression analysis, whilst cross-correlations were somewhat weaker for normal aorta. Atheroma showed a vast accumulation of lipid, especially cholesterol, in comparison to normal aorta. The atheroma samples contained a larger proportion of linoleate relative to oleate than the normal aorta. Levels of fatty acids relative to cholesterol were lower for atheroma than for normal aorta. The chemical composition of atheroma appeared unrelated to the age of the subject, whereas age-related increases in linoleate, oleate and cholesterol content were seen in the samples of normal aorta.

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.

Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages

The death of macrophages contributes to atheroma formation. Oxidation renders low‐density lipoprotein (LDL) cytotoxic to human monocyte‐macrophages. Lipoprotein‐associated phospholipase A2 (Lp‐PLA2), also termed platelet‐activating factor acetylhydrolase, hydrolyses oxidised phospholipids. Inhibition of Lp‐PLA2 by diisopropyl fluorophosphate or Pefabloc (broad‐spectrum serine esterase/protease inhibitors), or SB222657 (a specific inhibitor of Lp‐PLA2) did not prevent LDL oxidation, but diminished the ensuing toxicity and apoptosis induction when the LDL was oxidised, and inhibited the rise in lysophosphatidylcholine levels that occurred in the inhibitors’ absence. Hydrolysis products of oxidised phospholipids thus account for over a third of the cytotoxic and apoptosis‐inducing effects of oxidised LDL on macrophages.

Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions.

Objective: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions. Methods: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples. Results: The oxysterols 7α-hydroxycholesterol, cholesterol-5β, 6β-epoxide, cholesterol-5α, 6α-epoxide, 7β-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxy-cholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol. Conclusion: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.

Transition metal ions within human atherosclerotic lesions can catalyse the oxidation of low density lipoprotein by macrophages

The oxidation of low density lipoprotein (LDL) in the arterial wall may contribute to atherogenesis. The oxidation of LDL by cells usually requires catalytically active transition metal ions. We show here some that gruel samples from human advanced atherosclerotic lesions are capable of catalysing the oxidation of LDL by macrophages as measured by thiobarbituric acid‐reactive substances, enhanced electrophoretic mobility and increased macrophage uptake. This catalysis could be inhibited by pretreatment of the gruel with Chelex‐100, which binds transition metal ions. The presence of catalytically active transition metal ions in atherosclerotic lesions may help to explain why LDL oxidation occurs at these sites.

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.