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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
PubMed2001

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.

Apoptosis↗
PubMed2001

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.

Apoptosis↗
PubMed2001

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 7 alpha-hydroxycholesterol, cholesterol-5 beta, 6 beta-epoxide, cholesterol-5 alpha, 6 alpha-epoxide, 7 beta-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxycholesterol) 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.

Adult↗
PubMed1999

Carotenoids inhibit DNA synthesis in human aortic smooth muscle cells.

Quiescent, serum-starved human aortic smooth muscle cells were restimulated with 20% foetal calf serum in Dulbecco's modified Eagle medium, in the presence and absence of beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein or beta-cryptoxanthin, at final concentrations up to 23 microM. Concentration-dependent inhibition of DNA synthesis, measured by [methyl-3H]thymidine incorporation, was observed for the carotenoids, except for canthaxanthin and lutein which had no effect. Lycopene was the most potent of the carotenoids tested. The results suggest that antiproliferative effects of dietary carotenoids might be of significance in vivo.

Aorta↗
PubMed1999

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 microg/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.

Apoptosis↗
PubMed1999

Glutathione (GSH) and the toxicity of oxidised low-density lipoprotein to human monocyte-macrophages.

Macrophage death, believed to be an important event in the pathogenesis of human atherosclerosis, can be induced by oxidised low-density lipoprotein (LDL) in vitro. Supplementation of the culture medium with 5 mM GSH significantly protected human monocyte-macrophages in vitro against the toxicity of copper-oxidised LDL. Oxidation products of LDL include the aldehyde 4-hydroxynonenal (HNE). We present evidence that conjugation of HNE by GSH contributes to this protection. In the absence of cells, HPLC analysis showed there were marked reductions in the levels of both pure HNE and HNE in copper-oxidised LDL in the presence of GSH. However, GSH did not reverse protein modification, as judged by agarose gel electrophoresis, nor did it influence the depletion of polyunsaturated fatty acids, which were assessed using gas chromatography. The possible implications for human atherosclerosis are discussed.

Aldehydes↗
PubMed1999

Antioxidant BO-653 and human macrophage-mediated LDL oxidation.

Oxidation of LDL is now widely accepted to be involved in atherogenesis. The aim of this study was to examine the effect of BO-653, a strong radical scavenger and antioxidant, on oxidation of LDL by human macrophages in vitro. Fifty microg/ml LDL protein was incubated with macrophages in Ham's F10 medium, supplemented with additional Fe2+, for up to 48 h. Then the medium was analysed by LDL agarose gel electrophoresis, the thiobarbituric acid assay and gas chromatography. In the absence of added exogenous antioxidants, after 24h LDL oxidation produced 30.48 nmoles MDA equivalents/mg LDL protein and a relative electrophoretic mobility of 4.74. Linoleic acid (18:2), arachidonic acid (20:4) and cholesterol were depleted and 7beta-hydroxycholesterol was generated. BO-653 completely inhibited this cell-mediated oxidation of LDL in concentrations as low as 5 microM, being more effective than either alpha-tocopherol or probucol, which completely inhibited oxidation at 200 and 80 microM and only partially at 80 and 8 microM, respectively. This inhibition of cell-mediated LDL oxidation was not due to toxicity, as alpha-tocopherol, probucol and BO-653 were not toxic for the macrophages at the concentrations tested. Eighty microM alpha-tocopherol, 8 microM probucol and 5 microM BO-653 significantly reduced the toxicity to the oxidising culture caused by LDL oxidation. The results show that in this system BO-653 is a more effective antioxidant than alpha-tocopherol or probucol.

Antioxidants↗
PubMed1999

Oxysterols in cap and core of human advanced atherosclerotic lesions.

OBJECTIVE: Different parts of the advanced atherosclerotic lesion have characteristic differences in lipid content, but the distribution of lipid oxidation products has not been reported. This study provides novel data on oxysterol and hydroxyoctadecadienoic acids quantification in core versus cap. It compares the lipid composition of core and cap to assess the topographical distribution of evidence of lipid oxidation. METHODS: Lipids and oxidised lipids were analysed by gas chromatography (GC) and GC-mass spectrometry (GC-MS) in samples of human atheromatous lipid core and fibrous cap of individual advanced atherosclerotic plaques (Stary, Type V) in necropsy samples. RESULTS: The total lipid was of course massively greater in the core than in the cap. The oxidation products, cholest-5-en-3beta,26-diol (26-OH-CHOL) and cholest-5-en-3beta,7beta-diol (7beta-OH-CHOL) were detected in all the samples. 26-OH-CHOL was more abundant in the core than in the cap when related both to wet weight and to cholesterol. 7Beta-OH-CHOL levels were significantly higher in the core than in the cap when related to wet weight but not when related to cholesterol. Because the processing included a sodium borohydride reduction step, the 7beta-OH-CHOL detected could partly originate from 7-ketocholesterol or 7-hydroperoxycholesterol. Several isomeric hydroxyoctadecadienoic acids were detected in both core and cap, more in the cap when related to cholesterol content. Most of the components of the cap showed a high degree of cross-correlation on linear regression analysis, but cross-correlations were weaker for the core. The core samples contained a larger proportion of linoleate relative to oleate than the fibrous cap. CONCLUSION: The findings suggest that the different lipid and oxidised lipid contents of cap and core may be due to variations in oxidative activity in different parts of the lesion.

Aged↗
PubMed1998

Cell-mediated oxidation of LDL: comparison of different cell types of the atherosclerotic lesion.

The three major cell types of the human atherosclerotic lesion--macrophages (Mø), smooth muscle cells (SMC) and endothelial cells (EC)--were compared for their ability to oxidise low density lipoprotein (LDL) in vitro under identical conditions. Near-confluent cultures were incubated for up to 48 h with 50 microg protein/ml LDL in Ham's F10 medium supplemented with 7 microM Fe2+. All three cell types oxidised LDL readily using our culture conditions. After 24 and 48 h, the degree of LDL oxidation was in the order: Mø > SMC > EC when based on cell growth area and EC > SMC > Mo when based on cellular DNA content. However, LDL oxidation in vitro progressed more slowly between 24 and 48 h, probably due to increasing toxicity to the cells and/or depletion of polyunsaturated fatty acids. We therefore compared the time of onset of LDL oxidation. The earliest increase in LDL oxidation was always apparent with SMC. Gas chromatography revealed that LDL oxidation by all three cell types followed a similar pattern. The polyunsaturated fatty acids linoleic acid (18:2) and arachidonic acid (20:4) were depleted (to 10.3-18.1% and 4.5-24.7% respectively, compared to native LDL), whereas the content of stearic acid (18:0) and oleic acid (18:1) remained unchanged. Cholesterol was depleted (to 54.1-75.6% of native LDL) with a concomitant rise in 7 -hydroxycholesterol (to 60.6-128.1 microg/mg LDL). This corresponds to a conversion of 4.9, 9.5 and 10.4% of LDL cholesterol in EC-, SMC- and Mo-modified LDL respectively. All three cell types showed significant toxicity in the oxidising culture after 24h. The possible relevance to LDL oxidation in atherosclerosis is discussed.

Aorta↗
PubMed1997

Effect of glucose-mediated LDL oxidation on the P388D1 macrophage-like cell line.

Oxidised human low density lipoprotein (LDL) is thought to play a role in the development of atherosclerosis. Recent reports suggest that glucose-derived oxidants are capable of oxidising LDL. In this report, the effect of glucose-mediated oxidation of LDL upon the macrophage like cell line, P388D(1), was examined. Glucose-mediated oxidation of LDL was assessed by changes in the electrophoretic mobility of LDL and by analysis of lipid content using gas chromatography. The presence of Cu(II) (0.5 microM) was essential for the oxidation of LDL. The oxidation was potentiated by glucose in a dose- and time-dependent manner. At the concentration of LDL used (1 mg/ml), high concentrations of glucose (up to 500 mM) were required to oxidise LDL. The electrophoretic mobility of LDL correlated with the degree of lipid oxidation; both correlated with an inhibitory effect of oxidised LDL upon P388D(1) DNA synthesis. Diethylenetriaminepentaacetic acid (DETAPAC), a transition metal chelator, and aminoguanidine (AMG), an anti-glycation agent, inhibited the oxidation of LDL and attenuated the effects on DNA synthesis. Thus, glucose can mediate transition metal-dependent oxidation of LDL to a level that can affect P388D(1) cells, a mechanism which might have relevance to accelerated atherosclerosis in diabetic patients.

Animals↗
PubMed1997

The carotenoids beta-carotene, canthaxanthin and zeaxanthin inhibit macrophage-mediated LDL oxidation.

Human monocyte-macrophages were incubated for 24 h in Ham's F-10 medium with human low-density lipoprotein (LDL) in the presence or absence of beta-carotene, canthaxanthin or zeaxanthin, at final concentrations of 2.5, 12.5 and 25 mg/l. LDL oxidation, measured by agarose gel electrophoresis, the thiobarbituric acid assay and gas chromatography, was inhibited by each of the carotenoids in a concentration-dependent manner. Canthaxanthin was more effective when incorporated into LDL before addition to the cultures whereas beta-carotene and zeaxanthin were more effective when added simultaneously with LDL. The results suggest that dietary carotenoids might help slow atherosclerosis progression.

Antioxidants↗
PubMed1997

Factors affecting events during oxidation of low density lipoprotein: correlation of multiple parameters of oxidation.

The present study shows that copper oxidation of LDL is a tightly-ordered process which can be finely controlled by appropriate selection of duration of oxidation and of concentrations of LDL and copper. Oxidation of LDL (0.1-2.0 mg LDL protein/ml) was carried out by copper catalysis (in the ratio of 2.5 microM Cu2+ to 0.1 mg LDL protein/ml) in phosphate-buffered saline, and was monitored by agarose gel electro-phoresis, gas chromatography (GC), anion exchange fast protein liquid chromatography (FPLC), fluorescence spectroscopy and dynamic light scattering. Analysis of the data showed strong cross correlations between many of the parameters of oxidation. Oxidation was more rapid for lower concentrations than for higher concentrations of LDL, despite the same ratio of copper to LDL being employed. Chemical kinetics analysis of the GC data suggested that 7beta-hydroxycholesterol formation occurred as a first order (or pseudo first order) consecutive reaction to the oxidation of linoleate. The first order rate constants for decomposition of linoleate and production of 7beta-hydroxycholesterol correlated closely with the theoretically-calculated times between collision of LDL particles. LDL particle diameter, measured by dynamic light scattering, increased by ca. 50% over 24 h oxidation, suggesting unfolding of apo B-100. Prolonged oxidation of LDL at low concentration suggested that the radical chain reaction was able to propagate, albeit slowly, on cholesterol after all the polyunsaturated fatty acid was consumed. For higher concentrations of LDL, prolonged oxidation resulted in partial aggregation. These findings are applicable to preparing oxidised LDL with different degrees of oxidation, under controlled conditions, for studying its biological properties.

Arachidonic Acid↗
PubMed1997

Toxicity of polyunsaturated fatty acid esters for human monocyte-macrophages: the anomalous behaviour of cholesteryl linolenate.

We have investigated the toxicity to human monocytemacrophages, and susceptibility to oxidation, of different individual dietary fatty acids in cholesterol esters and triglycerides, added to the cell cultures as coacervates with bovine serum albumin. Toxicity was assessed using release of radioactivity from cells preloaded with tritiated adenine. Lipid oxidation was measured by gas chromatography (GC). The triglycerides showed a direct relationship between toxicity and increasing unsaturation, which in turn correlated with increasing susceptibility to oxidation. Triolein (18:1; omega-9) and trilinolein (18:2; omega-6) were non-toxic. Trilinolenin (18:3; omega-3) was toxic only after prolonged incubation. Triarachidonin (20:4; omega-6), trieicosapentaenoin (20:5; omega-3) and tridocosahexaenoin (22:6; omega-3) were profoundly and rapidly toxic. There was a similar relationship between toxicity and increasing unsaturation for most of the cholesterol esters, but cholesteryl linolenate was apparently anomalous, being non-toxic in spite of possessing three double bonds and being extensively oxidised. Probucol and DL-alpha-tocopherol conferred protection against the toxicity of cholesteryl arachidonate and triarachidonin. The oxidation in these experiments was largely independent of the presence of cells. GC indicated that formation of 7-oxysterols might contribute to the toxicity of cholesteryl linoleate. The toxicity of triglycerides suggests that polyunsaturated fatty acid peroxidation products are also toxic. Possible mechanisms of cytotoxicity and relevance to atherosclerosis are discussed.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1996

Do human atherosclerotic lesions contain nitrotyrosine?

Nitrotyrosine has been widely used as a marker of peroxynitrite formation in normal and diseased tissues. However, studies of normal human aortic intima and atherosclerotic lesions at various stages of development failed to reveal its presence except in a very few specimens of both normal and diseased tissues. The techniques used to detect nitrotyrosine-HPLC with photodiode array detection and Western blotting- were able to identify nitrotyrosine after chemical nitration. Hence peroxynitrite may not be as important in the initiation of atherosclerosis as has commonly been proposed.

Antibody Specificity↗
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↗