F2-isoprostanes affect macrophage migration and CSF-1 signalling.

F2-isoprostanes (F2-IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2-IsoP have been detected in atherosclerotic plaque. Colony stimulating factor-1 (CSF-1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2-IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F2t-isoprostane (15-F2t-IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F2t-IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF-1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages. We showed that 15-F2t-IsoP dose-dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF-1, with maximum effect at 10 µM. Analysis of CSF-1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15-F2t-IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15-F2t-IsoP signalling. Pyk2 silencing inhibited 15-F2t-IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F2t-IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F2t-IsoP-stimulated macrophage migration. These results demonstrate that 15-F2t-IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis.

Unexpected dose response of copper concentration on lipoprotein oxidation in serum: discovery of a unique peroxidase-like activity of urate/albumin in the presence of high copper concentrations.

Oxidative modification of low-density lipoprotein (LDL) may be an important factor in atherogenesis. The susceptibility of LDL to oxidation is usually determined in isolation by exposing LDL to oxidative stress induced by Cu ions or a free radical initiator. In these cases oxidation is carried out in the absence of water-soluble vitamins or serum proteins that may be present at the site of oxidation in vivo. We have examined the Cu2+-induced oxidation of lipoproteins in diluted serum. When oxidizing isolated LDL, there is a decrease in lag time with increasing concentration of Cu2+ until a minimum "lag time" is reached at a Cu:LDL ratio of about 50:1. In serum, we have shown an initial decrease in "lag time" with increasing Cu concentration up to 12.5 microM. However, with higher Cu concentrations "lag time" to oxidation increases, contrary to expectation, until a maximum is reached at about 50 microM Cu. This dose response observed for Cu oxidation of diluted serum was highly reproducible in a number of individual subjects. When serum was gel-filtered to remove low molecular weight compounds, the resulting filtrate behaved the same as isolated LDL. Uric acid was found to be an important component of the low molecular weight fraction responsible for the paradoxical effect of Cu concentration on serum oxidation. The same paradoxical effect was found when isolated LDL was incubated with uric acid in the presence of human serum albumin (HSA) and Cu. The incubation of HSA with reducing agents such as uric acid or bilirubin in the presence of high Cu concentrations, produces a "peroxidase-like" activity, capable of breaking down hydrogen peroxide as well as lipid hydroperoxides. The decomposition of lipid peroxides is a likely explanation for the longer serum oxidation lag times seen at higher Cu concentrations. Our study highlights the possible importance of interactions between uric acid and serum proteins in the presence of high metal ion concentrations.

The role of copper reduction by alpha-tocopherol in low-density lipoprotein oxidation.

The oxidation of lipoproteins is thought to be an important early step in atherogenesis. The measurement of lipid peroxidation in low-density lipoprotein (LDL) challenged with Cu2+ has become a widespread test to determine the "susceptibility" of LDL to oxidation. The determination of lag time to oxidation is thought to be a measure of the total antioxidant capacity of the LDL. However, we and others have failed to observe any correlation between lag time and the LDL content of its major lipid antioxidant, alpha-tocopherol. In fact, several studies now suggest a pro-oxidant role for tocopherol under some conditions of LDL oxidation. In the present study we sought to determine if there was a relationship between Cu2+ reduction by LDL and kinetic parameters of LDL oxidation. LDL (0.3 mmol/l cholesterol, approximately 0.1 mg protein/ml) was incubated at 30 degrees C with 2 microM Cu2+ and the formation of conjugated dienes measured over a 4-h period. Using neocuproine, an indicator molecule that specifically complexes Cu+ but not Cu2+, the reduction of Cu2+ by LDL was monitored. The final Cu concentration in these assays was 100 microM and neocuproine 750 microM. Cu+ formation was measured by absorbance at 454 nm. A strong negative correlation was observed between copper reduction by LDL and lag time to oxidation (r = -0.66, p < .005, n = 16). Further experiments showed that (1) LDL was able to reduce Cu2+ to Cu+ in a time and concentration-dependent manner; (2) blocking of free -SH groups on LDL apoprotein B by preincubation with dithionitrobenzoic acid (DTNB) had no significant effect on the rate and extent of Cu2+ reduction; (3) consumption of tocopherol in LDL undergoing oxidation with Cu was very rapid (rate = 6 x 10(-10) M s(-1)). When Cu+ formed during incubation with LDL was complexed with neocuproine, there was significant inhibition of LDL oxidation, as indicated by lipid peroxide formation and mobility on agarose gel electrophoresis. Surprisingly, tocopherol consumption was even more rapid in the presence of neocuproine, consistent with a shift in Cu2+/Cu+ equilibrium and faster reduction of Cu2+ by alpha-tocopherol. These results indicate that under these conditions tocopherol is a major reducing agent in LDL, converting Cu2+ to Cu+, and therefore, may play an important role in promoting LDL oxidation. However, there was no correlation between LDL tocopherol content and reduction of Cu2+. Examination of the time course of Cu2+ reduction in tocopherol enriched and depleted LDL indicates that tocopherol may determine Cu reduction at early time points but that the eventual capacity of LDL to reduce Cu may depend on more complex interactions between tocopherol and other LDL components.

PGF2-isoprostanes formed during copper-induced oxidation of low-density lipoproteins are the prostaglandins that cross-react with PGE2 antibodies.

It has been previously shown that LDL oxidation can produce a prostaglandin artifact(s) which cross-reacts with PGE2 antibodies. The nature of this cross-reacting material had not been identified. In this study we have examined the products of LDL oxidation and found substances cross-reacting with both PGE2 and LTB4 antibodies. Lipid extracts of copper oxidised LDL were separated by reverse-phase HPLC and fractions cross-reacting with PGE2 antibody identified. Fractions were collected and examined by GC-MS as methyl ester TMS ether derivatives. 8-epi PGF2 alpha (8-isoprostane) was identified as one of the major metabolites eluting in fractions cross-reacting with PGE2. 8-epi-PGF2 alpha showed 4% cross-reactivity with a PGE2 antibody but very little with LTB4 or thromboxane B2 antibodies. Treatment of oxidised LDL with a specific 8-isoprostane antibody removed any detectable PGE2 levels in subsequent PGE2 RIA. The generation of cross-reacting material during LDL oxidation was not influenced by indomethacin. The generation of 8-isoprostanes by free radical oxidation of arachidonic acid in LDL may explain much of the cross-reactivity seen with PGE2 antibodies. The identity of substances cross-reacting with LTB4 have yet to be identified.

Phenolic content of various beverages determines the extent of inhibition of human serum and low-density lipoprotein oxidation in vitro: identification and mechanism of action of some cinnamic acid derivatives from red wine.

1. An antioxidant effect of phenolic substances in red wine to reduce oxidizability of low-density lipoprotein has been proposed as the basis for a relatively lower incidence of coronary disease in populations with high red wine intake. We have now investigated the possible antioxidant effects of various beverages, including red wines, white wines, beers and red grape juices (diluted 1:500), on metal ion-dependent (copper) and -independent (aqueous peroxyl radicals) oxidation of isolated human low-density lipoprotein. We also tested the effects of these beverages on copper-initiated oxidation of lipoproteins in serum. 2. The higher the polyphenolic content of a beverage, the greater was its antioxidative effect measured as change in lag time in the different oxidation systems. Upon stripping the polyphenolics from the drinks, the lag times returned to control levels in isolated low-density lipoprotein; however, the low concentrations of phenolics remaining after stripping had a lesser but still significant effect on oxidation of lipoproteins in serum. The inhibitory effect of these phenolics appeared to be more pronounced for metal ion (copper)-induced oxidation than for those induced by aqueous peroxyl radicals, suggesting that both copper-binding and free radical-trapping activities may be involved. A mixture of the carboxylic acids representative of those present in red wine exhibited no significant effect on lag time of metal ion-dependent and -independent low-density lipoprotein oxidations. Ethanol, at concentrations of 0.1-0.5%, had no effect on either copper-induced or aqueous peroxyl radical oxidations. 3. Extracts of acid-hydrolysed red wine were separated by thin-layer chromatography and the most active antioxidant fractions identified. GC-MS and HPLC analysis of these fractions resulted in the identification of several cinnamic acid derivatives, such as coumaric acid, caffeic acid and protocatechuic acid. Dose-response studies using the pure compounds indicated that caffeic acid was the most active antioxidant with an IC50 < 1 mumol/l for copper-initiated low-density lipoprotein oxidation. Caffeic acid (1 mumol/l) significantly inhibited lipid hydroperoxide formation while sparing alpha-tocopherol consumption. Caffeic acid at the same concentration also inhibits aqueous peroxyl radical-induced oxidation of low-density lipoprotein, sparing alpha-tocopherol. There was no evidence of caffeic acid preventing the binding of copper to low-density lipoprotein. 4. We conclude that phenolics in both alcoholic and non-alcoholic beverages can give dose-dependent protection against oxidation of low-density lipoprotein. Caffeic acid and protocatechuic acid are two compounds likely to contribute to these effects. These findings may be relevant to the putative cardiovascular-protective effects of high phenolic content alcoholic beverages such as red wine; however, the widespread occurrence of antioxidants such as caffeic acid in fruits and vegetables suggests that these protective principles are not limited to red wine.