p62/Sequestosome-1: Mapping Sites of Protein-Handling Stress in Canine Cutaneous Mast Cell Tumors.

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.

Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

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.

A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1.

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.

Antioxidant properties of macrophages toward low-density lipoprotein.

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.

Localizing infection with a technetium-99m-labeled peptide: initial results.

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.

Metabolism of protein-bound DOPA in mammals.

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.

Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture.

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.

Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein.

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.

Prooxidant and antioxidant activities of macrophages in metal-mediated LDL oxidation: the importance of metal sequestration.

Murine macrophages incubated in metal-supplemented RPMI could block or promote oxidation of low-density lipoprotein (LDL) depending on the degree of metal supplementation. Only at high concentrations of Cu (1 micromol/L) and Fe (30 micromol/L) were cells prooxidant, leading to an accelerated rate of LDL oxidation over that measured in comparable cell-free media. At lower concentrations of Cu and Fe in RPMI, LDL oxidation in the presence of macrophages was inhibited relative to the cell-free condition. This appeared to be dependent on a stable modification of the culture medium, because preconditioning of media by incubation with macrophages could also decrease their capacity to sustain subsequent cell-free LDL oxidation. This was due, in part, to a removal of metal from the media during preconditioning. However, resupplementation of media with metals did not fully restore oxidative capacity, indicating that other cell-dependent antioxidant modifications occurred. This did not involve significant alterations to the thiol content of the media. This study highlights the complexity of the role that cells such as macrophages have with regards to LDL oxidation in vitro and demonstrate that there are both antioxidative and prooxidative components.

Are reactive oxygen species involved in the pathogenesis of murine cerebral malaria?

To investigate the involvement of oxidative tissue damage in the pathogenesis of murine cerebral malaria (CM), brain levels of protein carbonyls, 3,4-dihydroxyphenylalanine (DOPA), o-tyrosine, and dityrosine were measured during Plasmodium berghei ANKA (PbA) and P. berghei K173 (PbK) infections. During PbA infection in a CM model, brain levels of the substances were similar to those in uninfected mice. The role of phagocyte-derived reactive oxygen species in the pathogenesis of CM was examined in gp91phox gene knockout mice. The course of CM in these mice was the same as in their wild type counterparts. To examine whether superoxide production in the central nervous system could have occurred via increased xanthine oxidase activity, brain concentrations of urate were measured in CM mice and in mice infected with PbK (which does not cause CM). Brain urate concentration increased significantly in both groups of mice, suggesting that purine breakdown is not specific to CM. These results indicate that reactive oxygen species probably do not contribute to the pathogenesis of murine CM.

Human apo-lipoprotein B from normal plasma contains oxidised peptides.

Oxidation of low density lipoprotein (LDL) may be atherogenic, but radical-initiated oxidation of its apoprotein B-100 (apoB) has been little studied. Transition metal ions iron and copper are candidates for mediating radical oxidation of LDL in vivo. Therefore, we studied the copper-ion-induced oxidation of apoB in human LDL. Using HPLC methods developed in our recent work, we studied the destruction of native and the generation of six oxidised amino acids; we also assessed the release of peptides from the LDL particle by FPLC. We observed time-dependent losses of apoB histidine, lysine and glycine. Long-lived reactive species, the reductant DOPA, and the oxidant hydroperoxides of valine and leucine (measured as hydroxides after reduction), were generated. Their relative abundance (mol/mol of parent amino acid) was DOPA > o- and m-tyrosine > dityrosine, valine-hydroxides, leucine hydroxides. Low molecular weight fragments were also released from the LDL in a time-dependent manner, contained hydroperoxides sensitive to GSH peroxidase, and generated radicals on reaction with iron-EDTA. The fragments contained peptides active in the quinone redox cycling procedure, comprising 0.25% of the supplied LDL amino acids. Characteristic peptides were present in each FPLC fraction containing the fragments, as judged by further HPLC fractionation. Some fragments were present in the unoxidised LDL preparations, and when these were largely removed by FPLC, copper oxidation could still generate fragments, suggesting that those present in the starting material might indicate prior oxidation. Concordantly, we found that fresh plasma LDL apoB contained 43% of total plasma protein-bound oxidised amino acids, and with the same relative abundance. We conclude that plasma proteins including apoB are subject to physiological oxidation, similar to that inflicted by copper ions; the latter may contribute to intimal LDL oxidation, which could be the source of oxidised plasma apoB.

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages.

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.

3-Hydroxylysine, a potential marker for studying radical-induced protein oxidation.

gamma-Irradiation of several amino acids (Val, Leu, Ile, Lys, Pro, and Glu) in the presence of O2 generates hydroperoxides. We have previously isolated and characterized valine and leucine hydroperoxides, and hydroxides, and have detected these products in both isolated systems [e.g., bovine serum albumin (BSA) and human low-density lipoprotein (LDL)] and diseased human tissues (atherosclerotic plaques and lens cataractous proteins). This work was aimed at investigating oxidized lysine as a sensitive marker for protein oxidation, as such residues are present on protein surfaces, and are therefore likely to be particularly susceptible to oxidation by radicals in bulk solution. HO* attack on lysine in the presence of oxygen, followed by NaBH4 reduction, is shown to give rise to (2S)-3-hydroxylysine [(2S)-2,6-diamino-3-hydroxyhexanoic acid], (2S)-4-hydroxylysine [(2S)-2,6-diamino-4-hydroxyhexanoic acid], (2S, 5R)-5-hydroxylysine [(2S,5R)-2,6-diamino-5-hydroxyhexanoic acid], and (2S,5S)-5-hydroxylysine [(2S,5S)-2,6-diamino-5-hydroxyhexanoic acid]. 5-Hydroxylysines are natural products formed by lysyl oxidase and are therefore not good markers of radical-mediated oxidation. The other hydroxylysines are however useful markers, with HPLC analysis of 9-fluorenylmethyl chloroformate (FMOC) derivatives providing a sensitive and accurate method for quantitative measurement. Hydroxylysines have been detected in the hydrolysates of peptides (Gly-Lys-Gly and Lys-Val-Ile-Leu-Phe) and proteins (BSA and histone H1) exposed to HO./O2, and subsequently treated with NaBH4. Quantification of the hydroxylysines yields, and comparison with hydroxyvalines and hydroxyleucines, supports the hypothesis that surface residues give higher yields of oxidized products than the hydrophobic leucines and valines, at least with globular proteins such as BSA. Hydroxylysines, and particularly 3-hydroxylysine, may therefore be sensitive and useful markers of radical-mediated protein oxidation in biological systems.

Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: a potential anti-atherogenic function?

Apolipoprotein J (apo J) is a secreted glycoprotein of which the exact function remains a matter for speculation. Apo J has been implicated in such diverse processes as sperm maturation, regulation of complement activation, programmed cell death, tissue remodelling and lipid transport. In this study a possible role for apo J in lipid transport was explored. Mouse peritoneal macrophages were incubated with acetylated low-density lipoprotein (AcLDL) to produce foam cells containing cholesterol and cholesteryl esters. Incubation of the foam cells with physiological concentrations of purified apo J led to a dose-dependent export of cholesterol. The appearance of cholesterol in the medium was associated predominantly with a decline in intracellular cholesteryl esters rather than intracellular free cholesterol. The kinetics of cholesterol release to apo J were similar to apo A-I, an established promoter of cholesterol efflux. Apo J was also shown to induce phospholipid efflux from cells, whereas the cholesterol exported to the medium was associated with the apo J. Studies using foam cells from apo E-null mice showed that the cholesterol exported to the medium was independent of apo E production by the cells. These results present the first evidence that apo J can promote cholesterol efflux from foam cells and indicates that it might have a function in cellular cholesterol homoeostasis in both normal and pathological situations.

Alpha-tocopherol supplementation of macrophages does not influence their ability to oxidize LDL.

We have investigated the effect of alpha-tocopherol-loading of mouse peritoneal macrophages and human monocytes on their ability to oxidize human low density lipoprotein (LDL). Mouse peritoneal macrophages incorporated alpha-tocopherol (alpha-TOH) from culture medium supplemented with the vitamin in a time- and concentration-dependent manner. Subcellular fractionation by density gradient ultracentrifugation showed that the distribution of incorporated alpha-TOH within the cell was similar to that of free cholesterol. Most (approximately 88%) of alpha-TOH partitioned into the membrane fractions (plasma membrane approximately 41%, mitochondria and lysosomes approximately 26%, and endosomes plus endoplasmic reticulum approximately 21%). Cellular alpha-TOH was stable for at least 24 h in serum- or LDL-free media whether permissive (Ham's F-10) or non-permissive (Dulbecco's minimum essential medium, DMEM) for LDL oxidation. When incubated with LDL in DMEM, alpha-TOH-preloaded cells transferred small amounts of alpha-TOH (approximately 1 nmol/mg LDL protein after 9 h) to the lipoprotein. However, enrichment of the cells with alpha-TOH did not change the kinetics of oxidation of either normal or TOH-depleted LDL in Ham's F-10 medium compared with non-loaded cells, as assessed by alpha-TOH consumption, cholesteryl ester degradation, and cholesteryl ester hydroperoxide and 7-ketocholesterol accumulation. Nor did it alter superoxide release by the cells or their ability to reduce extracellular copper(II). Similar to mouse macrophages, enrichment of human monocytes with alpha-TOH did not change the kinetics of cell-mediated LDL oxidation. We conclude that elevated cellular levels of alpha-TOH in mouse peritoneal macrophages and in human monocytes do not affect their ability to oxidize LDL lipids in vitro. This suggests that either cell-mediated oxidation of LDL under the conditions of this study is not dependent on cell-derived radical species or that cellular alpha-TOH is unable to affect their formation.

Pharmacokinetic considerations in the development of peptide-based imaging agents.

Pharmacokinetic factors to be considered in the development of peptide-based imaging agents are reviewed. These include size, plasma protein binding, lipophilicity, resistance or susceptibility to proteolysis and radiolabel integrity. These factors are discussed in the context of several examples of thrombus and tumor imaging agents either commercially available or in development.