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Biomedical subjects

M J Mitchinson

Publications and source records attributed to M J Mitchinson.

At least 19 recordsLinked to original sources
PubMed1996

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 microM and chemotactic at 2.5 microM. 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.

Aldehydes↗
PubMed1996

Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS)

BACKGROUND: Vitamin E (alpha-tocopherol) is thought to have a role in prevention of atherosclerosis, through inhibition of oxidation of low-density lipoprotein. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of alpha-tocopherol and lower rates of ischaemic heart disease. We tested the hypothesis that treatment with a high dose of alpha-tocopherol would reduce subsequent risk of myocardial infarction (MI) and cardiovascular death in patients with established ischaemic heart disease. METHODS: In this double-blind, placebo-controlled study with stratified randomisation, 2002 patients with angiographically proven coronary atherosclerosis were enrolled and followed up for a median of 510 days (range 3-981). 1035 patients were assigned alpha-tocopherol (capsules containing 800 IU daily for first 546 patients; 400 IU daily for remainder); 967 received identical placebo capsules. The primary endpoints were a combination of cardiovascular death and non-fatal MI as well as non-fatal MI alone. FINDINGS: Plasma alpha-tocopherol concentrations (measured in subsets of patients) rose in the actively treated group (from baseline mean 34.2 micromol/L to 51.1 micromol/L with 400 IU daily and 64.5 micromol/L with 800 IU daily) but did not change in the placebo group. Alpha-tocopherol treatment significantly reduced the risk of the primary trial endpoint of cardiovascular death and non-fatal MI (41 vs 64 events; relative risk 0.53 [95% Cl 0.34-0.83; p=0.005). The beneficial effects on this composite endpoint were due to a significant reduction in the risk of non-fatal MI (14 vs 41; 0.23 [0.11-0.47]; p=0.005); however, there was a non-significant excess of cardiovascular deaths in the alpha-tocopherol group (27 vs 23; 1.18 [0.62-2.27]; p=0.61). All-cause mortality was 36 of 1035 alpha-tocopherol-treated patients and 27 of 967 placebo recipients. INTERPRETATION: We conclude that in patients with angiographically proven symptomatic coronary atherosclerosis, alpha-tocopherol treatment substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment. The effect of alpha-tocopherol treatment on cardiovascular deaths requires further study.

Coronary Artery Disease↗
PubMed1996

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+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Adenine↗
PubMed1996

Oxidation of low-density lipoprotein by human monocyte-macrophages results in toxicity to the oxidising culture.

Human monocyte-macrophage cultures were exposed to native low density lipoprotein (LDL) for up to 24 h in Ham's F10 medium and the extent of cell-mediated LDL oxidation was determined by measurement of electrophoretic mobility on agarose gels and measurement of lipids and oxidised lipids (including 7 beta-hydroxycholesterol) by GC. After an initial lag phase, which varied from 2-8 h, there was a steady increase in oxidation over 24 h. No-cell control incubations showed minimal increases in oxidation over 24 h. Significant toxicity, measured as release of radioactivity from macrophages pre-loaded with tritiated adenine, was observed in the cells when they oxidised LDL and the extent of radioactivity release correlated closely with the extent of LDL oxidation. Inhibition of oxidation using alpha-tocopherol or probucol reduced toxicity within the oxidising culture. This self-inflicted toxicity may help to explain the origin and enlargement of the lipid core of advanced atherosclerotic lesions.

Antioxidants↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Aging↗
PubMed1995

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.

Adult↗
PubMed1995

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-alpha-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-alpha-tocopherol (200 microM) and probucol (50 microM).

Adult↗
PubMed1995

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.

Cells, Cultured↗
PubMed1995

The inhibition of foam cell formation by sodium diethyldithiocarbamate.

A prominent feature of human atherosclerosis is the lipid-laden foamy macrophage, which often also contains the insoluble pigment, ceroid. The culture of macrophage-like cells, P388D1s, with artificial lipoproteins composed of cholesteryl linoleate (CL) and bovine serum albumin (BSA) results in foam cell formation with lipoprotein uptake and the intracellular accumulation of ceroid. Ceroid accumulation is accompanied by the oxidation of the cholesterol ester as monitored by gas chromatography. The sodium salt of diethyldithiocarbamic acid (DDC) at 1-5 microM effectively inhibited lipoprotein uptake, cholesteryl linoleate oxidation and ceroid accumulation in cultures of P388D1. Further studies showed that intracellular ceroid accumulation appeared to require the presence of cystine in the medium. Lipoprotein oxidation by this macrophage-like cell therefore appears to involve a mechanism dependent on cystine metabolism which is consistent with previous reports of macrophage-mediated lipoprotein oxidation. Studies on CL/BSA-induced ceroid accumulation in human monocytes also showed that DDC behaved in much the same manner. This inhibitory effect of DDC on foam cell formation, often considered a primary event of atherosclerosis, at concentrations as low as 1 microM, suggests the need for further, more comprehensive, studies on this compound's activities.

Albumins↗
PubMed1995

Metal ion release from mechanically-disrupted human arterial wall. Implications for the development of atherosclerosis.

Oxidation of low density lipoproteins (LDL) in blood vessel walls plays a significant role in the development of atherosclerosis. LDL oxidation in vitro is greatly accelerated by the presence of "catalytic" iron or copper ions, which have already been shown to be present within advanced atherosclerotic lesions. We demonstrate here that mechanical damage to human arterial wall samples (both normal and early or intermediate atherosclerotic lesions) causes release of "catalytic" iron and copper ions, to an extent increasing with the damage. It may be that traumatic (e.g. during angioplasty) or other injury to the vessel wall contributes to the generation of metal ions that can facilitate LDL oxidation and other free radical reactions, so promoting atherosclerosis.

Arteries↗
PubMed1995

Depletion of alpha-tocopherol in human atherosclerotic lesions.

Estimations of alpha-tocopherol content were made on a series of human necropsy samples of normal arterial wall and of atherosclerotic lesions. The results were compared with stage of lesion, shown by histology, and with the amounts of cholesterol and hydroxycholesterols in the same lesions. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal arterial wall but was consistently low in lesions, especially in lesions rich in macrophage foam cells. The results suggested that significant accumulation of hydroxycholesterols, found almost exclusively in lesions, only occurred when alpha-tocopherol levels were low in relation to the cholesterol content. This suggests that oxidative activity in the lesion may lead to significant oxidation of constituents of low-density lipoprotein only after alpha-tocopherol has been depleted.

Adolescent↗
PubMed1994

Lipid oxidation, lipoprotein cell-association and ceroid accumulation in P388D1 macrophage-like cells.

Flow cytometry can be used to quantify the accumulation of ceroid in macrophages, the result of cellular handling of certain lipoproteins. Using P388D1 cells, a murine-derived macrophage-like cell line, the effect of the lipophilic antioxidant, DL-alpha-tocopherol, upon the uptake and accumulation of ceroid by the cells was monitored on culture with artificial lipoproteins containing a single lipid species. Ceroid accumulation was greater for artificial lipoprotein composed of BSA complexed with cholesteryl arachidonate, than with cholesteryl linoleate. alpha-Tocopherol inhibited the ceroid accumulation, which was also dependent upon cell density. Thus, since these findings are similar to recent observations in primary cultures of murine peritoneal macrophages, it would appear that macrophage-like cell lines such as P388D1 cells are appropriate for the study of potential agonists and antagonists of lipid oxidation. Culture of P388D1 cells with oxidised human low-density lipoprotein (LDL) also resulted in ceroid formation, shown to be dependent upon the level of LDL oxidation as assessed by thiobarbituric acid-reactivity, the xylenol orange assay of peroxides and gas chromatographic analysis of cholesterol and fatty acid content. Ceroid accumulation reflected changes in the level of LDL oxidation better than did the cell association of oxidised radiolabelled LDL, monitored as that bound and retained by the cell.

Animals↗
PubMed1994

Production of oxidized lipids during modification of low-density lipoprotein by macrophages or copper.

The oxidation of low-density lipoprotein (LDL) is implicated in atherosclerosis. Lipids and oxidized lipids were analysed by gas chromatography and gas chromatography-mass spectrometry in human LDL incubated with mouse peritoneal macrophages (MPM) or copper (II) sulphate in Ham's F-10 medium or medium alone (control). MPM-modification and copper-catalysed oxidation of LDL resulted in the formation of oxysterols, mainly cholest-5-en-3 beta,7 beta-diol (7 beta-OH-CHOL); 7%-19% of the initial cholesterol was converted to 7 beta-OH-CHOL in 24 h. 7 beta-OH-CHOL levels in control LDL were very low. The increase in 7 beta-OH-CHOL in MPM and copper-oxidized LDL was accompanied by decreases in linoleate and arachidonate and increases in the electrophoretic mobility and degradation of LDL protein by 'target' macrophages. The concerted occurrence of these processes and their similarity in both MPM-modification and copper-catalysed oxidation of LDL were suggested by the highly significant cross-correlations. The fall in polyunsaturated fatty acid (PUFA) was accompanied by a directly proportional increase in electrophoretic mobility of the LDL. Production of 7 beta-OH-CHOL and protein degradation by macrophages showed modest elevations during the initial steep fall in PUFA, and showed their greatest increases as the levels of PUFA slowly approached zero. The levels of 7 beta-OH-CHOL and the degradation of LDL by macrophages were directly proportional. The degradation of LDL by macrophages increased rapidly as the electrophoretic mobility of LDL was slowly approaching its maximum level.

Animals↗
PubMed1994

Apolipoprotein oxidation in the absence of lipid peroxidation enhances LDL uptake by macrophages.

A characteristic of the antioxidant, probucol, is its inability to inhibit apolipoprotein B fragmentation in low density lipoprotein (LDL), despite a pronounced ability to inhibit lipid oxidation on relatively lengthy exposure to Cu(II). Here we show that a short exposure of LDL to hydrogen peroxide and Cu(II) leads to 125I-labelled apolipoprotein B fragmentation, the production of malondialdehyde and hydroperoxides and leads to increased uptake by macrophages on subsequent culture. However, pre-loading LDL with probucol protects LDL from lipid oxidation but not protein fragmentation or macrophage uptake. The use of probucol to conduct studies on apolipoprotein B oxidation without extensive lipid oxidation may prove useful when studying LDL apolipoprotein damage on exposure to an aqueous free radical insult.

Apolipoproteins B↗
PubMed1994

Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis.

The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.

Animals↗
PubMed1994

Measurement of ceroid accumulation in macrophages by flow cytometry.

The accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid, can be monitored semiquantitatively by staining techniques. However, such methods are crude and give little information about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, alpha-tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and flow cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell-mediated lipid oxidation and its potential antagonists.

Animals↗