RESUMEN
Canine cutaneous mast cell tumors (MCT) are common, frequently malignant neoplasms that are currently graded histologically for provision of prognostic information. Continuing evidence of subsets of MCT within certain grades (with differing survival times) indicate the need for biomarkers that will facilitate better patient stratification and also provide further information on the biological processes involved in progression. We decided to investigate the expression of p62/sequestosome-1 (p62/SQSTM1), a stress-inducible "hub protein" found in all cell types that shuttles rapidly between the nucleus and cytoplasm and is known to play important roles in protein handling and tumorigenesis. The identity of canine p62/SQSTM1 was confirmed in silico and by validation of a commercial antibody using both Western blotting and functional (pharmaceutical-based) analyses in cell culture. Using immunohistochemistry, 3 patterns of p62 expression were identified based on the predominant intracellular localization, that is, nuclear, mixed (nuclear and cytoplasmic), and cytoplasmic. There was a highly significant association with the 2-tier (Kiupel) grade (P < .0001), with all p62-nuclear immunoreactivity being associated with low grade and most p62-cytoplasmic immunoreactivity (93%) with high grade. Most but not all mixed nuclear-cytoplasmic labeling occurred in low-grade MCT; in other (human) tumor types, this pattern has been interpreted as borderline malignant. These data indicate that there is a shift in protein-handling stress from the nucleus to the cytoplasm in association with increasing malignancy in MCT. Studies to identify the processes and drug-able targets involved in this progression are ongoing.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Enfermedades de los Perros/patología , Mastocitos/patología , Proteína Sequestosoma-1/metabolismo , Neoplasias Cutáneas/veterinaria , Secuencia de Aminoácidos , Animales , Carcinogénesis , Citoplasma/metabolismo , Enfermedades de los Perros/metabolismo , Perros , Inmunohistoquímica/veterinaria , Mastocitos/metabolismo , Pronóstico , Alineación de Secuencia , Proteína Sequestosoma-1/química , Proteína Sequestosoma-1/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
AIMS/HYPOTHESIS: Previous studies have implicated the glycoxidative modification of low-density lipoprotein (LDL) by glucose and aldehydes (apparently comprising both glycation and oxidation), as a causative factor in the elevated levels of atherosclerosis observed in diabetic patients. Such LDL modification can result in unregulated cellular accumulation of lipids. In previous studies we have characterized the formation of glycated, but nonoxidized, LDL by glucose and aldehydes; in this study we examine whether glycation of LDL, in the absence of oxidation, gives rise to lipid accumulation in arterial wall cell types. METHODS: Glycated LDLs were incubated with macrophage, smooth muscle, or endothelial cells. Lipid loading was assessed by HPLC analysis of cholesterol and individual esters. Oxidation was assessed by cholesterol ester loss and 7-ketocholesterol formation. Cell viability was assessed by lactate dehydrogenase release and cell protein levels. RESULTS: Glycation of LDL by glycolaldehyde and methylglyoxal, but not glucose (in either the presence or absence of copper ions), resulted in cholesterol and cholesterol ester accumulation in macrophage cells, but not smooth muscle or endothelial cells. The extent of lipid accumulation depends on the degree of glycation, with increasing aldehyde concentration or incubation time, giving rise to greater extents of particle modification and lipid accumulation. Modification of lysine residues appears to be a key determinant of cellular uptake. CONCLUSIONS/INTERPRETATION: These results are consistent with LDL glycation, in the absence of oxidation, being sufficient for rapid lipid accumulation by macrophage cells. Aldehyde-mediated "carbonyl-stress" may therefore facilitate the formation of lipid-laden (foam) cells in the artery wall.
Asunto(s)
Acetaldehído/análogos & derivados , Acetaldehído/química , Lipoproteínas LDL/síntesis química , Lipoproteínas/sangre , Piruvaldehído/química , Animales , Productos Finales de Glicación Avanzada , Humanos , Indicadores y Reactivos , RatasRESUMEN
Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Cetocolesteroles/farmacología , Liposomas/metabolismo , Transporte Biológico , Línea Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Células Espumosas/metabolismo , Humanos , Cinética , Membrana Dobles de Lípidos/metabolismo , Macrófagos/metabolismo , Modelos Químicos , Unión Proteica , Esteroles/metabolismo , Fracciones Subcelulares , Factores de TiempoRESUMEN
Organisms produce reactive oxygen species (ROS) throughout their lives. The activities of a number of key antioxidant enzymes, such as catalase, superoxide dismutase and glutathione peroxidase, which protect against the damaging effects of ROS, have been reported to decrease with increasing age, though this is not unequivocal. In contrast, sacrificial antioxidants such as ascorbate, thiols and tocopherol do not appear to decrease with increasing age. It is also possible that ROS production increases with age as a result of poorer coupling of electron transport components, and an increased level of redox-active metal ions that could catalyse oxidant formation. As a result of this decrease in antioxidant defences, and increased rate of ROS formation, it is possible that the impact of ROS increases with age. ROS are known to oxidise biological macromolecules, with proteins an important target. If the argument that the impact of ROS increases with age is true, then proteins would be expected to accumulate oxidised materials with age, and the rate of such accumulation should increase with time, reflecting impaired inefficiency of homeostasis. Here we review the evidence for the accumulation of oxidised, or modified, extra- and intra-cellular proteins in vivo.
Asunto(s)
Envejecimiento/metabolismo , Proteínas/metabolismo , Animales , Humanos , Oxidación-ReducciónRESUMEN
The kinetics (0 to 3 h) of cholesterol efflux to delipidated apolipoprotein A-1 were investigated, and the experimental data were best fitted to a mathematical model that involves two independent pathways of cholesterol efflux. The first pathway with a rate constant of 4.6 h(-1) is fast but removes only 3-5% of total cholesterol. After preconditioning apoA-1, it was found that this pathway remains, and hence it is a property of the cholesterol-loaded cells rather than due to modification on the apolipoprotein. This fast initial efflux does not seem to contribute to cholesterol efflux at later stages (>1 h) where a second pathway predominates. However, the fast initial efflux pool can be restored if apoA-1 is withdrawn. The second slower pathway (k(membrane--media) = 0.79 h(-1)) is associated with cholesterol ester hydrolysis whose rate constant could be experimentally verified (k(cal) = 0.43, k(exp) = 0.38 +/- 0.05). The model suggests that two different plasma membrane domains are involved in the two pathways. Loading of the cells with an oxysterol, 7-ketocholesterol (7K), inhibits efflux from both pathways. The model predicts that 7K decreases the initial efflux by decreasing the available cholesterol (by possibly affecting lipid packing), while all rate constants in the second pathway are decreased. In conclusion, the kinetic model suggests that cholesterol efflux to apoA-1 is a two-step process. In the first step, some of the plasma membrane cholesterol contributes to a fast initial efflux (possibly from lipid rafts) and leads to a second pathway that mobilizes intracellular cholesterol mobilization.
Asunto(s)
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Modelos Químicos , Acetilación , Algoritmos , Transporte Biológico , Ésteres del Colesterol/metabolismo , Humanos , Hidrólisis , Cetocolesteroles/metabolismo , Cinética , Lipoproteínas LDL/metabolismo , Células Tumorales CultivadasAsunto(s)
Ácido Ascórbico/farmacología , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Ácido Ascórbico/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Monocitos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Sustancias Reductoras/farmacologíaRESUMEN
Oxidative modification of low-density lipoprotein (LDL) has been implicated in atherosclerosis. Intensive scientific efforts over the last two decades have focused on the elucidation of the mechanisms by which LDL is oxidized in vivo. A wealth of in vitro studies has demonstrated that the cell types present in atherosclerotic lesions, including monocyte/macrophages, quantitatively one of the most important cell types in plaque development, promote LDL oxidation. The mechanisms of cellular prooxidant activities have been extensively investigated. Fewer studies have addressed possible protective properties of the cells in LDL oxidation. This review summarizes recent observations of antioxidant, and potentially antiatherogenic, activities of macrophages toward LDL, including macrophage-mediated detoxification of lipid and protein hydroperoxides, metal sequestration and the generation of compounds with antioxidant properties. These activities could contribute to the net effect of macrophages on deleterious LDL oxidation and to the complex role of these cells in lesion development.
Asunto(s)
Antioxidantes/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/fisiopatología , Ésteres del Colesterol/metabolismo , Humanos , Peroxidación de Lípido , Lipoproteínas LDL/fisiología , Macrófagos/fisiología , Metales/metabolismo , Oxidación-ReducciónRESUMEN
In vitro-labeled leukocyte imaging is useful for the detection of infection, but an in vivo labeling method is preferable. This study sought to evaluate the safety and efficacy of a leukocyte-avid peptide for the detection of infection, to determine the effects of peptide dose on performance and to compare the peptide with in vitro-labeled leukocytes. A 23-amino acid peptide, P483, containing the platelet factor-4 heparin-binding sequence, was labeled with 99mTc and complexed with heparin (P483H). Thirty patients were injected with 29 microg (n = 11), 145 microg (n = 10) or 290 microg (n = 9) of labeled peptide, and imaged 15 min and 90-120 min later. Early and late images were interpreted individually and jointly. Twenty patients underwent (111)In-labeled leukocyte scintigraphy. Fourteen patients had infection: osteomyelitis (n = 7), vascular graft (n = 2), abscess (n = 2), joint replacement (n = 1), surgical wound (n = 1) and pneumonia (n = 1). There were 10 adverse events in six patients; all were mild and resolved spontaneously, and without any intervention. The sensitivity, specificity and accuracy were the same for both early and late imaging: 0.86, 0.81 and 0.83, respectively. Interpreting early and late images together did not improve the results. No relationship between peptide dose and study accuracy was found. In patients undergoing both examinations, the accuracies of the peptide and in vitro-labeled leukocyte imaging were identical: 0.80. In summary, 99mTc-P483H safely, rapidly and accurately detected focal infection, was comparable with in vitro-labeled leukocyte imaging and therefore merits further investigation.
Asunto(s)
Infecciones/diagnóstico por imagen , Compuestos de Organotecnecio , Proteínas , Radiofármacos , Adulto , Anciano , Anciano de 80 o más Años , Reacciones Falso Positivas , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Interpretación de Imagen Asistida por Computador , Leucocitos/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Compuestos de Organotecnecio/administración & dosificación , Compuestos de Organotecnecio/efectos adversos , Péptidos , Proteínas/administración & dosificación , Proteínas/efectos adversos , Cintigrafía , Radiofármacos/administración & dosificación , Radiofármacos/efectos adversosRESUMEN
Protein-bound 3,4-dihydroxyphenylalanine (DOPA) can be generated in mammalian cells by both controlled enzymatic pathways, and by uncontrolled radical reactions. Protein-bound DOPA (PB-DOPA) has reducing activity and the capacity to inflict secondary damage on other important biomolecules such as DNA. This may be mediated through replenishment of transition metals or from catechol-quinone-catechol redox cycles in the presence of cellular components such as ascorbate or cysteine, resulting in amplification of radical damaging events. The generation of PB-DOPA confers on protein the ability to chelate transition metals generating protein 'oxychelates'; this may be amongst the factors, which localise such damage. Tissue levels of PB-DOPA are increased in a number of age-related pathologies such as atherosclerosis and cataract formation. We discuss the detoxification, and the subsequent proteolysis and excretion of components of PB-DOPA. We contrast the fact that in marine organisms, and particularly in extracellular proteins, PB-DOPA and other DOPA-polymers can play important functional roles in adhesion and the provision of tensile properties.
Asunto(s)
Dihidroxifenilalanina/metabolismo , Proteínas/metabolismo , Animales , Dihidroxifenilalanina/química , Humanos , Unión Proteica , Proteínas/químicaRESUMEN
Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give cross-linked adduct species which have been identified by EPR spectroscopy. HPLC analysis of the products generated on reaction of histone hydroperoxide-derived radicals with 2'-deoxyguanosine, or intact calf thymus DNA, has shown that significant levels of the mutagenic oxidized DNA base 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) are formed, with the yield dependent on the individual histone protein, the presence of hydroperoxide functions, and the concentration of metal ion. These studies demonstrate that initial oxidative damage to individual histone proteins or histone octamers can result in the transfer of oxidative damage to associated DNA via the formation and subsequent decomposition of protein hydroperoxides to reactive radicals, and provide a novel route for the formation of mutagenic lesions in DNA.
Asunto(s)
Daño del ADN , Radicales Libres/química , Histonas/metabolismo , Ácidos Nucleicos/química , Peróxidos/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Bovinos , ADN/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Relación Dosis-Respuesta en la Radiación , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Histonas/química , Histonas/efectos de la radiación , Oxidación-Reducción , Peróxidos/química , Peróxidos/efectos de la radiaciónRESUMEN
27-Hydroxycholesterol (27OH) is the major oxysterol in human atherosclerotic lesions, followed by 7-ketocholesterol (7K). Whereas 7K probably originates nonenzymically, 27OH arises by the action of sterol 27-hydroxylase, a cytochrome P450 enzyme expressed at particularly high levels in the macrophage and proposed to represent an important pathway by which macrophages eliminate excess cholesterol. We hypothesized and here show that 27-hydroxylated 7-ketocholesterol (270H-7K) is present in human lesions, probably generated by the action of sterol 27-hydroxylase on 7K. Moreover, [(3)H]27OH-7K was produced by human monocyte-derived macrophages (HMDMs) supplied with [(3)H]7K but not in HMDMs from a patient with cerebrotendinous xanthomatosis (CTX) shown to have a splice-junction mutation of sterol 27-hydroxylase. Whereas [(3)H]27OH-7K was predominantly secreted into the medium, [(3)H]-27OH formed from [(3)H]-cholesterol was mostly cell-associated. The majority of supplied [(3)H]7K was metabolized beyond 27OH-7K to aqueous-soluble products (apparently bile acids derived from the sterol 27-hydroxylase pathway). Metabolism to aqueous-soluble products was ablated by a sterol 27-hydroxylase inhibitor and absent in CTX cells. Sterol 27-hydroxylase therefore appears to represent an important pathway by which macrophages eliminate not only cholesterol but also oxysterols such as 7K. The fact that 7K (and cholesterol) still accumulates in lesions and foam cells indicates that this pathway may be perturbed in atherosclerosis and affords a new opportunity for the development of therapeutic strategies to regress atherosclerotic lesions.
Asunto(s)
Arteriosclerosis/metabolismo , Estenosis Carotídea/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Cetocolesteroles/metabolismo , Macrófagos/metabolismo , Esteroide Hidroxilasas/metabolismo , Adulto , Anciano , Empalme Alternativo , Arterias Carótidas/metabolismo , Células Cultivadas , Colestanotriol 26-Monooxigenasa , Sistema Enzimático del Citocromo P-450/genética , Femenino , Humanos , Cinética , Masculino , Persona de Mediana Edad , Esteroide Hidroxilasas/genética , Xantomatosis Cerebrotendinosa/enzimología , Xantomatosis Cerebrotendinosa/genéticaRESUMEN
Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.
Asunto(s)
Colesterol/metabolismo , Células Espumosas/metabolismo , Esteroles/metabolismo , Animales , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Línea Celular , Células Cultivadas , Ésteres del Colesterol/metabolismo , Esterificación , Humanos , Cetocolesteroles/metabolismo , Lipoproteínas LDL/metabolismo , Lisosomas/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Oxidación-Reducción , Esterol O-Aciltransferasa/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.
Asunto(s)
Ésteres del Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Recuento de Células , Línea Celular , Células Cultivadas , Quelantes/metabolismo , Cromatografía Líquida de Alta Presión , Medios de Cultivo/metabolismo , Cistina/farmacología , Humanos , Hidróxidos/metabolismo , Ratones , Monocitos/metabolismo , Factores de Tiempo , Vitamina E/metabolismoRESUMEN
Exposure of amino acids, peptides and proteins to radicals, in the presence of oxygen, gives high yields of hydroperoxides. These materials are readily decomposed by transition metal ions to give further radicals. We hypothesized that hydroperoxide formation on nuclear proteins, and subsequent decomposition of these hydroperoxides to radicals, might result in oxidative damage to associated DNA. We demonstrate here that exposure of histone H1 and model compounds to gamma-radiation in the presence of oxygen gives hydroperoxides in a dose-dependent manner. These hydroperoxides decompose to oxygen- and carbon-centred radicals (detected by electron paramagnetic resonance spectroscopy) on exposure to Cu(+) and other transition metal ions. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to give adduct species (i.e. protein-DNA base cross-links). Product analysis has demonstrated that radicals from histone H1-hydroperoxides, and other protein and amino acid hydroperoxides, can also oxidize both free 2'-deoxyguanosine and intact calf thymus DNA to give the mutagenic oxidized base 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine, 8-oxodG). The yield of 7,8-dihydro-8-oxo-2'-deoxyguanosine is proportional to the initial protein-hydroperoxide concentration, and corresponds (for histone H1-hydroperoxide, 280 microM) to approx. 1. 4% conversion for free 2'-deoxyguanosine (200 microM), and 0.14% for 2'-deoxyguanosine in DNA (70 microgram/ml). Evidence has also been obtained with DNA for reaction at cytosine and thymine, but not adenine; the lack of damage to the latter may result from damage transfer to 2'-deoxyguanosine residues. These studies demonstrate that initial radical-induced damage to nuclear proteins can give rise to subsequent DNA damage; the latter includes both DNA-protein cross-links and formation of oxidized DNA bases.
Asunto(s)
ADN/metabolismo , Histonas/metabolismo , Peróxido de Hidrógeno/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Aminoácidos/metabolismo , Animales , Bovinos , ADN/química , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Técnicas In Vitro , Lisina/metabolismo , Meliteno/metabolismo , Oxidación-Reducción , Proteínas/metabolismoRESUMEN
Apolipoprotein A-I (apoA-I) overexpression inhibits atherogenesis in mice, and apolipoprotein E (apoE) secreted by foam cell macrophages may exert antiatherogenic effects within the arterial wall. We hypothesized that interaction between apoA-I and apoE contributed to the antiatherogenic properties of apoA-I, and therefore investigated whether apoA-I stimulated secretion of apoE by foam cell macrophages. Cholesterol enrichment of primary murine and human macrophages increased spontaneous apoE secretion 2-fold, as quantified by Western blot and chemiluminescence detection. Human apoA-I caused a further marked increase of apoE secretion from both murine (3.8-fold, p < 0.01) and human (3.2-fold, p = 0.01) foam cells in a time- and concentration- dependent manner, and this increase was confirmed by immunoprecipitation of [(35)S]methionine-labeled macrophage apoE. The protein synthesis inhibitor cycloheximide, but not the transcription inhibitor actinomycin D, markedly inhibited apoE secretion to apoA-I (73.1 +/- 9.8% inhibition at 4 h) and completely suppressed apoE secretion beyond 4 h. Pretreatment of macrophages with Pronase inhibited initial apoA-I-mediated apoE secretion by 70.5 +/- 6.5% at 2 h, but by 8 h apoA-I-induced apoE secretion was the same in Pronase-pretreated and non-pretreated cells. Non-apolipoprotein-mediated cholesterol efflux induced by trimethyl-beta cyclodextrin did not enhance apoE secretion, whereas phospholipid vesicles inducing the same degree of cholesterol efflux substantially enhanced apoE secretion, and apoA-I and phospholipid vesicles in combination demonstrated additive induction of apoE secretion. We conclude that apoA-I concurrently stimulates apoE secretion and cholesterol efflux from foam cell macrophages and that lipoprotein-derived apoA-I may enhance local secretion and accumulation of apoE in atherosclerotic lesions.
Asunto(s)
Apolipoproteína A-I/farmacología , Apolipoproteínas E/metabolismo , Células Espumosas/metabolismo , Animales , Apolipoproteína A-I/sangre , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/genética , Células Cultivadas , Colesterol/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Células Espumosas/efectos de los fármacos , Humanos , Cinética , Mediciones Luminiscentes , Metionina/metabolismo , Ratones , Ratones Endogámicos C57BL , Especificidad de la Especie , Radioisótopos de AzufreRESUMEN
Oxidized forms of cholesterol (oxysterols) are present in atherosclerotic lesions and may play an active role in lesion development. For example, 7-ketocholesterol (7KC) inhibits cholesterol efflux from macrophage foam cells induced by apolipoprotein A-I (apoA-I). Such oxysterols may promote foam cell formation in atherosclerotic lesions by preventing effective clearance of excess cholesterol. ApoA-I also induces phospholipid (PL) export from foam cells and it has been suggested that cholesterol efflux is dependent upon PL association with the apolipoprotein. In the current study, the effect of oxysterol enrichment of foam cells on phospholipid efflux was measured. Export of cellular PL to apoA-I from 7KC-enriched foam cells was inhibited to the same extent as cholesterol, indicating that the reduced cholesterol export may be a consequence of a decline in the capacity of the foam cells to generate PL/apoA-I particles capable of accepting cellular cholesterol. Incubation of foam cells with pre-formed PL/apoA-I discs increased cholesterol export from 7KC-enriched cells to levels seen in 7KC-free cells. Foam cells produced by uptake of oxidized LDL, which contain similar amounts of 7KC plus other oxidation products, expressed a more profound inhibition of PL export to apoA-I. Cholesterol efflux from these cells improved only partially by provision of PL-containing acceptors. Efflux of 7KC from both foam cell types occurred to PL/apoA-I discs but was only minimal to lipid-free apoA-I, indicating that export of this oxysterol is more dependent than cholesterol upon the presence of extracellular phospholipid.
Asunto(s)
Células Espumosas/metabolismo , Fosfolípidos/metabolismo , Esteroles/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Transporte Biológico Activo , Humanos , Técnicas In Vitro , Cetocolesteroles/metabolismo , Cinética , Ratones , Fosfatidilcolinas/metabolismoRESUMEN
After investigation of the contents and redox status of antioxidants and lipids in homogenates of both normal artery and atherosclerotic plaque, we now investigated them in the density fractions (very low, low, high, and protein fractions) of atherosclerotic plaque freshly obtained from carotid endarterectomy. By using the optimum extraction method (homogenization in carbonate buffer) and after density gradient ultracentrifugation, we isolated and characterized density fractions of plaque for apolipoproteins, size and contents of alpha-tocopherol (alpha-TOH), unesterified cholesterol, cholesteryl linoleate (Ch18:2), and hydroxides and hydroperoxides of Ch18:2, ie, Ch18:2-O(O)H. The distribution of apolipoproteins was more heterogeneous than that in the corresponding lipoproteins isolated from blood, and the majority of material in all plaque density fractions was present in large particles eluting in the void volume of gel-filtration columns. The content of unesterified cholesterol per unit of protein in low- and high-density fractions was 10-fold that in corresponding plasma lipoproteins. Low- and very-low-density fractions contained most of the lesion lipids and alpha-TOH. Two to five percent of lesion Ch18:2 was present as Ch18:2-O(O)H and distributed more or less equally among all density fractions, yet the content of alpha-TOH per unit of Ch18:2 was higher than that in corresponding plasma lipoproteins. These results demonstrate that alpha-TOH and oxidized lipids coexist in all lesion density fractions, further supporting the notion that large proportions of lipids in lipoproteins of advanced stages of atherosclerosis are oxidized. However, although not ruling it out, our results do not support the suggestion that advanced stages of atherosclerosis are associated with gross deficiencies in the lipoproteins' vitamin E content.