Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.     

Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages

Transglycosylation activity of endo-beta-N-acetylglucosaminidase HS (Endo HS) was investigated using native human transferrin as a donor of an asparagine-linked oligosaccharide and p-nitrophenyl-beta-d-glucose (PNP-beta-d-Glc) as an acceptor of the oligosaccharide. The amount of the product increased dependent on the concentration of the acceptors. Absorption spectrum, exoglycosidase digestion and matrix assisted laser desorption and ionization-time of flight (MALDI-TOF) mass analysis of the transglycosylation product indicated that the asialobiantennary complex type oligosaccharide of human transferrin was transferred to PNP-beta-d-Glc. Endo HS also transferred the oligosaccharide of human transferrin to PNP-alpha-d-Glc, PNP-alpha-d-Gal, PNP-beta-d-Gal, PNP-beta-d-Man, PNP-beta-d-Xyl, PNP-beta-d-GlcNAc, and PNP-glycerol at a different rate. No apparent difference in the K(m) value for human transferrin as an oligosaccharide donor was observed using different acceptors, PNP-beta-d-Glc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then very slightly decreased during the course of enzyme reaction. Endo HS was also transferred the triantennary complex type oligosaccharide of calf fetuin and the bi-, tri-, and tetrantennary complex type oligosaccharides of human alpha(1)-acid glycoprotein to PNP-beta-d-Glc. Furthermore, Endo HS transferred an asparagine-linked oligosaccharide from a hen egg glycopeptide to PNP-beta-d-Glc. The results demonstrate that Endo HS can transfer a wide variety of asparagine-linked complex type oligosaccharides to various monosaccharides. Endo HS was distinct from other enzymes in the specificity for oligosaccharide donors and acceptors.     

Variations in transmembrane Ca2+ gradient and apoptosis of macrophages induced by oxidized low density lipoprotein

While Ca²⁺ has been proposed to be a messenger in OxLDL-induced cell death, few studies have addressed the possibility that it may influence the occurrence of apoptosis and necrosis of macrophages induced by OxLDL in virtue of change of transmembrane Ca²⁺ gradient including that across plasma membrane and intracellular organelle membranes. In this paper, various lipophilic Ca²⁺ fluorescent indicators and specific organelle markers were used to study the relationship between the changes of the transmembrane Ca²⁺ gradients and the OxLDL induced apoptosis of macrophages. Our results showed that following exposure of low dose OxLDL to macrophages, the transmembrane Ca²⁺ gradient across the plasma membrane, as well as the membrane-proximal Ca²⁺ gradient, the transnuclear, and the transmitochondrial membrane Ca²⁺ gradient were all changed significantly. These data suggested that changes in transmembrane Ca²⁺ gradients might be involved in the apoptosis of macrophages induced by OxLDL.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): Protection by intracellular ascorbate against oxidant stress and apoptosis

To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.     

P-glycoprotein does not protect cells against cytolysis induced by pore-forming proteins

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR). In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response. There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation. Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different pore-forming proteins. Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp(+ve) or P-gp(-ve) cells to the pore-forming proteins complement, perforin, or pneumolysin. Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.     

Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein

Summary The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized. Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS). The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties. Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls. Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL. Cellular cholesterol content saturated at 4 hours with 50 g/ml LDL; this effect may be associated with receptor saturation. Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content. However, deposition of CE was not observed below Ac-LDL concentrations of 100 g/ml. This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM. In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS. Cholesterol esterification in response to LDL and Ac-LDL was examined by ¹⁴C-oleic acid incorporation into CE. These results confirmed the mass cellular cholesterol and CE measurements. Kinetic studies conducted with RAW264 cells treated with 50 or 100 g/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6–12 hours of incubation. Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.Abbreviations LDL Low Density Lipoprotein - Ac-LDL Acetylated Low Density Lipoprotein - FBS Fetal Bovine Serum - DM Defined Medium - PBS Phosphate Buffered Saline - CE Cholesteryl Ester     

RX871024 reduces NO production but does not protect against pancreatic beta-cell death induced by proinflammatory cytokines

The imidazoline compound RX871024 reduces IL-1beta-induced NO production thereby protecting against IL-1beta-induced beta-cell apoptosis. The aim of this study was to evaluate whether imidazolines RX871024 and efaroxan protect beta-cells against death in the presence of a combination of the cytokines IL-1beta, IFNgamma, and TNFalpha. To address this issue, experiments involving different methods for detection of cell death, different concentrations of the cytokines, and a variety of conditions of preparation and culturing of ob/ob mouse islets and beta-cells have been carried out. Thoroughly performed experiments have not been able to demonstrate a protective effect of RX871024 and efaroxan on beta-cell death induced by the combination of cytokines. However, the inhibitory effect of RX871024 on NO production in ob/ob mouse islets and beta-cells was still observed in the presence of all three cytokines and correlated with the decrease in p38 MAPK phosphorylation. Conversely, efaroxan did not affect cytokine-induced NO production. Our data indicate that a combination of pro-inflammatory cytokines IL-1beta, IFNgamma, and TNFalpha, conditions modelling those that take place in type 1 diabetes, induces pancreatic beta-cell death that does not directly correlate with NO production and cannot be counteracted with imidazoline compounds.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes

We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30–40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 μM) and 87% (0.7 μM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.     

Inhibition of human low density lipoprotein oxidation by active principles from spices

Spice components and their active principles are potential antioxidants. In this study we examined the effect of phenolic and non-phenolic active principles of common spices on copper ion-induced lipid peroxidation of human low density lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive substance (TBARS) and relative electrophoretic mobility (REM) of LDL on agarose gel. Curcurriin, capsaicin, quercetin, piperine, eugenol and allyl sulfide inhibited the formation of TBARS effectively through out the incubation period of 12 h and decreased the REM of LDL. Spice phenolic active principles viz. curcumin, quercetin and capsaicin at 10 M produced 40–85% inhibition of LDL oxidation at different time intervals while non-phenolic antioxidant allyl sulfide was less potent in inhibiting oxidation of LDL. However, allyl sulfide, eugenol and ascorbic acid showed pro-oxidant activity at lower concentrations (10 M) and antioxidant activity at higher concentrations (50 M) only. Among the spice principles tested quercetin and curcumin showed the highest inhibitory activity while piperine showed least antioxidant activity at equimolar concentration during initiation phase of oxidation of LDL. The inhibitory effect of curcumin, quercetin and capsaicin was comparable to that of BHA, but relatively more potent than ascorbic acid. Further, the effect of curcurnin, quercetin, capsaicin and BHA on initiation and propagation phases of LDL oxidation showed that curcurnin significantly inhibited both initiation and propagation phases of LDL oxidation, while quercetin was found to be ineffective at propagation phase. These data suggest that the above spice active principles, which constitute about 1–4% of above spices, are effective antioxidants and offer protection against oxidation of human LDL.     

Exogenous catalase introduced in CHO cells by electroporation does not protect against chromosome damage induced by ionizing radiation.

The relative importance of hydrogen peroxide generated as a consequence of irradiation with X-rays for the production of chromosomal aberrations has been studied in cultured CHO cells. Catalase introduced into cells by electroporation protected DNA from strand breakage induced by hydrogen peroxide given 4h later, and the yield of chromosome aberrations was also reduced. Nevertheless, when the cells were irradiated after treatment with catalase following a similar protocol and the yield of chromosomal aberrations analyzed at metaphase, no protective effect was observed as compared with cells treated with X-rays alone. These observations seem to support the hypothesis that hydroxyl radicals generated from hydrogen peroxide are not a major factor responsible for chromosome damage induced by ionizing radiation.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure

Long-chain saturated fatty acids are cytotoxic to pancreatic β-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 β-cells to serum withdrawal or a combination of interleukin-1β plus interferon-γ. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic β-cells.     

Identification of a potential HIV-induced source of bystander-mediated apoptosis in T cells: Upregulation of TRAIL in primary human macrophages by HIV-1 tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.     

ROS production by adrenodoxin does not cause apoptosis in fission yeast

We previously showed that production of reactive oxygen species (ROS) caused by overexpression of the mitochondrial electron transfer protein adrenodoxin (Adx) induces apoptosis in mammalian cells. In the fission yeast Schizosaccharomyces pombe, ROS are also produced in cells that undergo an apoptotic-like cell death, but it is not yet clear whether they are actually causative for this phenomenon or whether they are merely produced as a by-product. Therefore, the purpose of this study was to trigger mitochondrial ROS production in fission yeast by overexpression of either wildtype Adx (Adx-WT) or of several activated Adx mutants and to investigate its consequences. It was found that strong expression of either Adx-WT or Adx-S112W did not produce any ROS, while Adx-D113Y caused a twofold and Adx^1–108 a threefold increase in ROS formation as compared to basal levels. However, no typical apoptotic markers or decreased viability could be observed in these strains. Since we previously observed that an increase in mitochondrial ROS formation of about 60% above basal levels is sufficient to strongly induce apoptosis in mammalian cells, we conclude that S. pombe is either very robust to mitochondrial ROS production or does not undergo apoptotic cell death in response to mitochondrial ROS at all.