The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes.

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 µM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.

The significance of neuroglobin in the brain.

Since its discovery in 2000, neuroglobin (Nb) has been demonstrated to have an essential and conserved function in vertebrates with the consequential discovery of a neuroprotective role. Nb is a member of the globin superfamily and is predominantly expressed in neurons of the central and peripheral nervous system. Thorough studies have been performed to elucidate the molecular structure of Nb and its ligand binding characteristics. The precise physiological function and mechanism of action of Nb is beginning to be established, with a number of hypotheses having been put forward. While Nb shares an intrinsic affinity for low-molecular weight diatomic gases similar to other globins, the relatively low level of Nb expression in cerebral neurons places limitations on its potential to function as a reservoir for oxygen, especially during periods of acute ischemia. In vitro studies have suggested that the neuroprotective role of Nb may be due to its ability to scavenge reactive oxygen (ROS) and nitrogen (RNS) species. However other studies have proposed Nb as being part of a signalling chain that transmits the redox state of the cell that is protective against oxidative stress or that inhibits apoptosis. This review is intended to summarize the structural, genomic and functional data on neuroglobin to date, thereby providing perspectives for future research on these molecules that may have substantial biomedical implications.

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Polymorphonuclear leukocyte phagocytic function increases in plasminogen knockout mice.

BACKGROUND: Mice lacking plasminogen (PG-/-) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG-/- mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type (PG+/+) mice. METHODS: Phagocytic activity of PMNs from PG-/- and PG+/+ mice was compared following exposure to Staphylococcus aureus (S. aureus) particles and the expression of Mac-1 was assessed in parallel by flow cytometric analysis. Resistance to phorbol-12-myristate-13-acetate (PMA)-induced cell death was compared between PMNs from the different genotypes. RESULTS: Stimulation of PG-/- PMNs by opsonized S. aureus diluted in PG-/- plasma significantly increased phagocytosis (15-fold) compared with stimulation of PG+/+ PMNs in PG+/+ plasma. Incubation of PG-/- PMNs with PG+/+ plasma (control) or PG-/- plasma supplemented with human PG inhibited this increased phagocytic activity. Mac-1 cell surface density increased 6.2+/-1.0-fold in PG-/- PMNs versus 2.9+/-0.6-fold in PG+/+ PMNs (P < 0.01) indicating that Mac-1 may be associated with increased phagocytic activity. Supporting this, treatment of PG-/- PMNs with an anti-Mac-1 antibody in PG-/- plasma inhibited phagocytic activity. In addition, physiologic PG blocked Mac-1 accessibility at the surface of PMNs. Addition of PMA resulted in 33% death of PMNs from PG-/- mice versus 68% in PG+/+ controls (P < 0.001). CONCLUSIONS: PMNs from PG-/- mice exhibit a Mac-1 dependent increase in phagocytic activity that is suppressed with human PG, an anti-Mac-1 antibody or the plasma from PG+/+ mice. The propensity for PMNs from PG-/- mice to be activated in response to PMA together with their relative resistance to PMA-toxicity may contribute to increased PMN half-life and enhanced fibrin(ogen) clearance in the setting of PG deficiency.

Effect of vitamin E on aortic lipid oxidation and intimal proliferation after arterial injury in cholesterol-fed rabbits.

Oxidized low-density lipoproteins (LDL) are implicated in atherosclerosis. However, large-scale intervention studies designed to test whether antioxidants, such as vitamin E, can ameliorate cardiovascular disease have generated ambivalent results. This may relate to the fact that the mechanism whereby lipid oxidation is initiated in vivo is unknown and the lack of direct evidence for a deficiency of antioxidants in atherosclerotic lesions. Further, there is little evidence to suggest that vitamin E acts as an antioxidant for lipid peroxidation in vivo. Here we tested the antioxidant effect of dietary vitamin E (alpha-tocopherol) supplementation on intimal proliferation and lipid oxidation in balloon-injured, hypercholesterolemic rabbits. alpha-Tocopherol supplementation increased vascular content of alpha-tocopherol over 30-fold compared to nonsupplemented and alpha-tocopherol-deficient chows. Balloon injury resulted in oxidized lipid deposition in the aorta. Maximum levels of primary lipid oxidation products, measured as hydroperoxides of esterified lipid (LOOH) and oxidized linoleate (HODE), were 0.22 and 1.10 nmol/mg, representing 0.21 and 0.39% of the precursor molecule, respectively. Secondary lipid oxidation products, measured as oxysterols, were maximal at 5.60 nmol/mg or 1.48% of the precursor compound. Vascular HODE and oxysterols were significantly reduced by vitamin E supplementation. However, the intima/media ratio of aortic vessels increased with vitamin E supplementation, suggesting that the antioxidant promoted intimal proliferation. Thus, the study demonstrates a dissociation of aortic lipid oxidation and lesion development, and suggests that vitamin E does not prevent lesion development in this animal model.

A DNA oligonucleotide-hemin complex cleaves t-butyl hydroperoxide through a homolytic mechanism.

Both electron paramagnetic resonance (EPR) and electronic absorption spectroscopy have been employed to investigate the reaction of a guanine-rich DNA nucleotide-hemin complex (PS2.M-hemin complex) and organic peroxide (t-Bu-OOH). Incubation of the PS2.M-hemin complex with t-Bu-OOH resulted in the time-dependent decrease in the heme Soret with concomitant changes to the visible bands of the electronic absorbance spectrum for the PS2.M-hemin complex. Parallel EPR studies using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) combined with spectral simulation demonstrated the presence of tert-butyloxyl, carbon-centered methyl, and methyl peroxyl radicals as well as a simple nitroxide (triplet) signal. Experiments, performed by maintaining a constant ratio of t-Bu-OOH/PS2.M-hemin complex ( approximately 35 mol/mol) while varying DMPO concentration, indicated that the relative contributions of each radical adduct to the composite EPR spectrum were significantly influenced by the DMPO concentration. For example, at DMPO/PS2.M-hemin of 10-50 mol/mol, a complex mixture of radicals was consistently detected, whereas at high trapping efficiency (i.e., DMPO/PS2.M-hemin of approximately 250 mol/mol) the tert-butyloxyl-DMPO adduct was predominant. In contrast, at relatively low DMPO/PS2.M-hemin complex ratios of < or =5 mol/mol, a simple nitroxide three-line EPR signal was detected largely in the absence of all other radicals. Together, these data indicate that tert-butyloxyl radical is the primary radical likely formed from the homolytic cleavage of the O-O peroxy bond of t-Bu-OOH, while methyl and methyl peroxyl radicals result from beta-scission of the primary tert-butyloxyl radical product.

Reaction of human myoglobin and peroxynitrite: characterizing biomarkers for myoglobin-derived oxidative stress.

Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury.

Oxidants and antioxidants in atherosclerosis.

The oxidative theory suggests that LDL oxidation contributes to atherogenesis, implying that attenuation of this process by antioxidants should decrease atherosclerosis. However, a causative link between LDL oxidation and atherogenesis is not firmly established. It requires the identification of the oxidants that are responsible for the initiation of LDL oxidation, and an understanding of the modified moieties that are responsible for the proatherogenic activities of oxidized LDL. The present review summarizes recent data on potential biological oxidants for LDL in the vessel wall, and discusses the antiatherogenic role(s) of selected antioxidants.

The peroxidase activity of a hemin--DNA oligonucleotide complex: free radical damage to specific guanine bases of the DNA.

A specific DNA oligonucleotide--hemin complex (PS2.M--hemin complex) that exhibits DNA-enhanced peroxidative activity was studied by EPR and UV--visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M--hemin complex showed both a low-field g approximately 6 and a high-field g approximately 2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M--hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK(a) value for the water ligand of 8.70 +/- 0.03 (mean +/- SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M--hemin complex and hydrogen peroxide (H(2)O(2)) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H(2)O(2). These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M--hemin complex.

Reaction of human myoglobin and H2O2. Electron transfer between tyrosine 103 phenoxyl radical and cysteine 110 yields a protein-thiyl radical.

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys(110)). Adding hydrogen peroxide (H(2)O(2)) to human Mb affords Trp(14)-peroxyl, Tyr(103)-phenoxyl, and Cys(110)-thiyl radicals and coupling of Cys(110)-thiyl radicals yields a homodimer through intermolecular disulfide bond formation (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) J. Biol. Chem. 275, 20391-20398). Treating a solution of wild type Mb and H(2)O(2) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein /= 100 mol/mol only DMPO-Tyr(103) radicals were present. The DMPO-dependent decrease in DMPO-Cys(110) was matched by a near 1:1 stoichiometric increase in DMPO-Tyr(103). In contrast, reaction of the Y103F human Mb with H(2)O(2) gave no DMPO-Cys(110) at DMPO:protein /= 100 mol/mol (i.e. conditions that consistently gave DMPO-Tyr(103) in the case of wild type Mb). No detectable homodimer was formed by incubation of the Y103F variant with H(2)O(2). However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H(2)O(2) (C110A:Y103F:H(2)O(2) 2:1:5 mol/mol/mol); the yield of this homodimer increased with increasing ratios of C110A:Y103F. Together, these data suggest that addition of H(2)O(2) to human Mb can produce Cys(110)-thiyl radicals through an intermolecular electron transfer reaction from Cys(110) to a Tyr(103)-phenoxyl radical.

Reaction of human myoglobin and nitric oxide. Heme iron or protein sulfhydryl (s) nitrosation dependence on the absence or presence of oxygen.

The amino acid sequence of human myoglobin (Mb) is similar to other mammalian Mb except for a unique cysteine residue at position 110 (Cys(110)). Anaerobic treatment of ferrous forms of wild-type human Mb, the C110A variant of human Mb or horse heart Mb, with either authentic NO or chemically derived NO in vitro yields heme-NO complexes as detected by electron paramagnetic resonance spectroscopy (EPR). By contrast, no EPR-detectable heme-NO complex was observed from the aerobic reactions of NO and either the ferric or oxy-Mb forms of wild-type human or horse heart myoglobins. Mass analyses of wild-type human Mb treated aerobically with NO indicated a mass increase of approximately 30 atomic mass units (i.e., NO/Mb = 1 mol/mol). Mass analyses of the corresponding apoprotein after heme removal showed that NO was associated with the apoprotein fraction. New electronic maxima were detected at A(333 nm) (epsilon = 3665 +/- 90 mol(-)(1) cm(-)(1); mean +/- S.D.) and A(545 nm) (epsilon = 44 +/- 3 mol(-)(1) cm(-)(1)) in solutions of S-nitrosated wild-type human Mb (similar to S-nitrosoglutathione). Importantly, the sulfhydryl S-H stretch vibration for Cys(110) measured by Fourier transform infrared (nu approximately 2552 cm(-)(1)) was absent for both holo- and apo- forms of the wild-type human protein after aerobic treatment of the protein with NO. Together, these data indicate that the reaction of wild-type human Mb and NO yields either heme-NO or a novel S-nitrosated protein dependent on the oxidation state of the heme iron and the presence or absence of dioxygen.

Anti-atherogenic effect of coenzyme Q10 in apolipoprotein E gene knockout mice.

Oxidation of low-density lipoprotein (LDL) lipid is implicated in atherogenesis and certain antioxidants inhibit atherosclerosis. Ubiquinol-10 (CoQ10H2) inhibits LDL lipid peroxidation in vitro although it is not known whether such activity occurs in vivo, and, if so, whether this is anti-atherogenic. We therefore tested the effect of ubiquinone-10 (CoQ10) supplemented at 1% (w/w) on aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet. Hydroperoxides of cholesteryl esters and triacylglycerols (together referred to as LOOH) and their corresponding alcohols were used as the marker for lipoprotein lipid oxidation. Atherosclerosis was assessed by morphometry at the aortic root, proximal and distal arch, and the descending thoracic and abdominal aorta. Compared to controls, CoQ10-treatment increased plasma coenzyme Q, ascorbate, and the CoQ10H2:CoQ10 + CoQ10H2 ratio, decreased plasma alpha-tocopherol (alpha-TOH), and had no effect on cholesterol and cholesterylester alcohols (CE-OH). Plasma from CoQ10-supplemented mice was more resistant to ex vivo lipid peroxidation. CoQ10 treatment increased aortic coenzyme Q and alpha-TOH and decreased the absolute concentration of LOOH, whereas tissue cholesterol, cholesteryl esters, CE-OH, and LOOH expressed per bisallylic hydrogen-containing lipids were not significantly different. CoQ10-treatment significantly decreased lesion size in the aortic root and the ascending and the descending aorta. Together these data show that CoQ10 decreases the absolute concentration of aortic LOOH and atherosclerosis in apoE-/- mice.

Reaction of human myoglobin and H2O2. Involvement of a thiyl radical produced at cysteine 110.

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H(2)O(2) (protein/H(2)O(2) = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO( small middle dot)) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g approximately 2. 036) was detected after decay of Trp-OO( small middle dot) that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios /=1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H(2)O(2) demonstrated the formation of a homodimer (mass of 34,107 +/- 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H(2)O(2) differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.

Lipid oxidation in human low-density lipoprotein induced by metmyoglobin/H2O2: involvement of alpha-tocopheroxyl and phosphatidylcholine alkoxyl radicals.

Metmyoglobin (metMb) and H(2)O(2) can oxidize low-density lipoprotein (LDL) in vitro, and oxidized LDL may be atherogenic. The role of alpha-tocopherol (alpha-TOH) in LDL oxidation by peroxidases such as metMb is unclear. Herein, we show that during metMb/H(2)O(2)-induced oxidation of native LDL, alpha-tocopheroxyl radical (alpha-TO(*)) and hydroperoxides and alcohols of cholesteryl esters [CE-O(O)H] and phosphatidylcholine [PC-O(O)H] accumulate concomitantly with alpha-TOH consumption. The ratio of accumulating CE-O(O)H to PC-O(O)H remains constant as long as alpha-TOH is present. Accumulation of CE-O(O)H is dependent on, and correlates with, LDL's alpha-TOH content, yet does not require preformed lipid hydroperoxides or H(2)O(2). This indicates that in native LDL alpha-TOH can act as a phase-transfer agent and alpha-TO(*) as a chain-transfer agent propagating LDL lipid peroxidation via tocopherol-mediated peroxidation (TMP). After alpha-TOH depletion, CE-O(O)H continues to accumulate, albeit at a slower rate than in the presence of alpha-TOH. This second phase of LDL oxidation is accompanied by depletion of PC-OOH, a rapid increase in the CE-O(O)H/PC-O(O)H ratio, formation of lipid-derived alkoxyl radicals and phosphatidylcholine hydroxides (PC-OH), and accumulation of a second organic radical, characterized by a broad singlet EPR signal. The latter persists for several hours at 37 degrees C. We conclude that metMb/H(2)O(2)-induced peroxidation of LDL lipids occurs initially via TMP. After alpha-TOH depletion, cholesteryl esters peroxidize at higher fractional rates than surface phospholipids, and this appears to be mediated at least in part via reactions involving alkoxyl radicals derived from the peroxidatic activity of metMb on PC-OOH.

A role for reduced coenzyme Q in atherosclerosis?

Substantial evidence implicates oxidative modification of low density lipoprotein (LDL) as an important event contributing to atherogenesis. As a result, the elucidation of the molecular mechanisms by which LDL is oxidized and how such oxidation is prevented by antioxidants has been a significant research focus. Studies on the antioxidation of LDL lipids have focused primarily on alpha-tocopherol (alpha-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to alpha-TOH, plasma LDL also contains low levels of ubiquinol-10 (CoQ10H2; the reduced form of coenzyme Q10). Recent studies have shown that in oxidizing plasma lipoproteins alpha-TOH can exhibit anti- or pro-oxidant activities for the lipoprotein's lipids exposed to a vast array of oxidants. This article reviews the molecular action of alpha-TOH in LDL undergoing "mild" radical-initiated lipid peroxidation, and discusses how small levels of CoQ10H2 can represent an efficient antioxidant defence for lipoprotein lipids. We also comment on the levels alpha-TOH, CoQ10H2 and lipid oxidation products in the intima of patients with coronary artery disease and report on preliminary studies examining the effect of coenzyme Q10 supplementation on atherogenesis in apolipoprotein E knockout mice.

Time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E knockout mice.

Oxidation of lipoproteins is thought to be an early event in atherogenesis. To evaluate whether aortic lipoprotein lipid (per)oxidation contributes to atherosclerosis, we investigated the time-dependent changes to lipids and antioxidants in plasma and aortas of apolipoprotein E gene knockout (apoE-/-) mice receiving a high fat diet, and compared these changes with lesion development. Circulating buoyant lipoproteins and associated cholesterol (C), cholesteryl esters (CE), and alpha-tocopherol (alpha-TOH) increased within 1 month then remained largely constant up to 6 months. Coenzyme Q (CoQ) remained unchanged for the first 3 months and increased marginally after 6 months. With increasing duration of the diet, plasma lipids showed an increased propensity to undergo peroxyl radical-induced (per)oxidation. Absolute concentrations of aortic C, hydroperoxides and hydroxides of CE (CE-O(O)H) and alpha-TOH increased gradually while aortic CE increased more markedly with changes to cholesteryl linoleate being most pronounced. Aortic CoQ remained largely unchanged. Overall, the extent of aortic CE (per)oxidation remained low (

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.

Secretory phospholipase A2 and lipoprotein lipase enhance 15-lipoxygenase-induced enzymic and nonenzymic lipid peroxidation in low-density lipoproteins.

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis. 15-Lipoxygenase (15LO) induces LDL oxidation, and phospholipase A2 enhances this process [Sparrow, C. P. , Parthasarathy, S., and Steinberg, D. (1988) J. LipidRes. 29, 745-753]. As the underlying mechanism of the enhancing effect has not been investigated previously, we here show that in the presence of soybean 15LO (SLO) or human 15LO (rhLO), the addition of lipoprotein lipase, porcine pancreatic, or human type IIa secretory phospholipase A2 (sPLA2) greatly enhanced the accumulation of hydro(pero)xides of all major classes of LDL's lipids. Hydroperoxides of free fatty acids accumulated exclusively as enzymic products with kinetics reflecting both the formation of free fatty acids and the initial 'build-up' of alpha-tocopheroxyl radical. In contrast, hydroperoxides of cholesteryl esters and phosphatidylcholine accumulated linearly over comparatively longer periods of time and, in the case of rhLO, well beyond inactivation of the oxygenase. With SLO, formation of oxidized esterified lipids occurred nonenzymically, independent of the presence of lipase and despite the oxygenase remaining active until the end of the incubation. Enhancement of rhLO-induced LDL lipid peroxidation by sPLA2 was eliminated by a neutralizing anti-sPLA2 antibody, indicating that lipolytic activity was required for this effect. LDL depleted of alpha-tocopherol was resistant to oxidation by 15LO alone, whereas lipase overcame this resistance, demonstrating that lipases enhance 15LO-induced enzymic and nonenzymic peroxidation of LDL lipids. This is likely due to provision of free fatty acid substrate, resulting in an enhanced rate of free radical formation which itself causes nonenzymic peroxidation of esterified lipids. As lipases and 15LO are present in atherosclerotic lesions, our findings could be of pathophysiological significance.