Roland Stocker

Presence of hypochlorite-modified proteins in human atherosclerotic lesions.

Oxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl-oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo.

Insulin resistance is a cellular antioxidant defense mechanism

We know a great deal about the cellular response to starvation via AMPK, but less is known about the reaction to nutrient excess. Insulin resistance may be an appropriate response to nutrient excess, but the cellular sensors that link these parameters remain poorly defined. In the present study we provide evidence that mitochondrial superoxide production is a common feature of many different models of insulin resistance in adipocytes, myotubes, and mice. In particular, insulin resistance was rapidly reversible upon exposure to agents that act as mitochondrial uncouplers, ETC inhibitors, or mitochondrial superoxide dismutase (MnSOD) mimetics. Similar effects were observed with overexpression of mitochondrial MnSOD. Furthermore, acute induction of mitochondrial superoxide production using the complex III antagonist antimycin A caused rapid attenuation of insulin action independently of changes in the canonical PI3K/Akt pathway. These results were validated in vivo in that MnSOD transgenic mice were partially protected against HFD induced insulin resistance and MnSOD+/− mice were glucose intolerant on a standard chow diet. These data place mitochondrial superoxide at the nexus between intracellular metabolism and the control of insulin action potentially defining this as a metabolic sensor of energy excess.

Human Atherosclerotic Plaque Contains Both Oxidized Lipids and Relatively Large Amounts of α-Tocopherol and Ascorbate

We assessed the antioxidant status and contents of unoxidized and oxidized lipids in freshly obtained, homogenized samples of both normal human iliac arteries and carotid and femoral atherosclerotic plaque. Optimal sample preparation involved homogenization of human atherosclerotic plaque for 5 minutes, which resulted in recovery of most of the unoxidized and oxidized lipids without substantial destruction of endogenous vitamins C and E and 87% and 43% recoveries of added standards of alpha-tocotrienol and isoascorbate, respectively. The total protein, lipid, and antioxidant levels obtained from human plaque varied among donors, although the reproducibility of replicates from a single sample was within 3%, except for ubiquinone-10 and ascorbate, which varied by 20% and 25%, respectively. Plaque samples contained significantly more ascorbate and urate than control arteries, with no discernible difference in the vitamin C redox status between plaque and control materials. The concentrations of alpha-tocopherol and ubiquinone-10 were comparable in plaque samples and control arteries. However, approximately 9 mol percent of plaque alpha-tocopherol was present as alpha-tocopherylquinone, whereas this oxidation product of vitamin E was not detectable in control arteries. Coenzyme Q10 in plaque and control arteries was only detected in the oxidized form ubiquinone-10, although coenzyme Q10 oxidation may have occurred during processing. The most abundant of all studied lipids in plaque samples was free cholesterol, followed by cholesteryl oleate and cholesteryl linoleate (Ch18:2). Approximately 30% of plaque Ch18:2 was oxidized, with 17%, 12%, and 1% present as fatty acyl hydroxides, ketones, and hydroperoxides, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of High Density Lipoproteins II. EVIDENCE FOR DIRECT REDUCTION OF LIPID HYDROPEROXIDES BY METHIONINE RESIDUES OF APOLIPOPROTEINS AI AND AII

Human high density lipoproteins (HDL) can reduce cholesteryl ester hydroperoxides to the corresponding hydroxides (Sattler W., Christison J. K., and Stocker, R. (1995) Free Radical Biol. & Med. 18, 421–429). Here we demonstrate that this reducing activity extended to hydroperoxides of phosphatidylcholine, was similar in HDL2 and HDL3, was independent of arylesterase and lecithin:cholesteryl acyltransferase activity, was unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing apoAI or apoAII only, as well as isolated human apoAI. Concomitant with the reduction of lipid hydroperoxides specific oxidized forms of apoAI and apoAII formed in blood-derived and reconstituted HDL. Similarly, specific oxidized forms of apoAI accumulated upon treatment of isolated apoAI with authentic cholesteryl linoleate hydroperoxide. These specific oxidized forms of apoAI and apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction and the associated formation of specific oxidized forms of apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was dose-dependent and accompanied by the conversion of specific Met residues of apoAI and apoAII into Met(O). Canine HDL, which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker lipid hydroperoxide reducing activity and lower extents of formation of oxidized forms of apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and phosphatidylcholine associated with human HDL.

Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.

Publisher Summary Peroxidation of polyunsaturated lipids proceeds via a free radical chain reaction with consumption of molecular oxygen and concomitant formation of lipid hydroperoxides as the primary reaction products. These lipid hydroperoxides may undergo a variety of secondary reactions that result in the formation of reactive carbonyl products. Lipid peroxidation is commonly assessed by measurement of lipid hydroperoxides, diene conjugates, oxygen consumption, or secondary reaction products (example, malonylaldehyde and 4-hydroxynonenal. The different methods available vary greatly in their requirement for specialized (and expensive) equipment and, at least for biological systems, in their limitations and number of potential interferences and/or artifacts. This chapter discusses developments in the use of the high-performance liquid chromatography (HPLC) postcolumn chemiluminescence (CL) method 5 for the assessment of lipoprotein lipid peroxidation. Lipoprotein oxidation, particularly that of low-density lipoproteins (LDL), as oxidative LDL modification is thought to represent an early and important step in the development of atherosclerosis. In the HPLC postcolumn CL method, the different classes of unoxidized and oxidized lipids are extracted, separated (from one another and from lipophilic antioxidants) by HPLC, and detected sequentially by UV absorption and CL, respectively.

Induction of Haem Oxygenase as a Defence Against Oxidative Stress

Cells respond to metabolic perturbations by producing specific stress proteins. Exposure of mammalian cells to various forms of oxidative stress induces haem oxygenase, the rate-limiting enzyme in haem degradation. This response is proposed to represent an antioxidant defence operating at two different stages simultaneously. It (i) decreases the levels of the potential pro-oxidants haem and haem proteins such as cytochrome P-450 and protoporphyrinogen oxidase, and (ii) increases the tissue concentrations of antioxidatively active bile pigments.

Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement.

The ‘oxidation theory’ of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although little direct evidence for a causative role of ‘oxidized LDL’ in atherogenesis exists, several studies show that, in vitro, oxidized LDL exhibits potentially proatherogenic activities and lipoproteins isolated from atherosclerotic lesions are oxidized. As a consequence, the molecular mechanisms of LDL oxidation and the actions of α‐tocopherol (α‐TOH, vitamin E), the major lipid‐soluble lipoprotein anti‐oxidant, have been studied in detail. Based on the known antioxidant action of α‐TOH and epidemiological evidence, vitamin E is generally considered to be beneficial in coronary artery disease. However, intervention studies overall show a null effect of vitamin E on atherosclerosis. This confounding outcome can be rationalized by the recently discovered diverse role for α‐TOH in lipoprotein oxidation; that is, α‐TOH displays neutral, anti‐, or, indeed, prooxidant activity under various conditions. This review describes the latter, novel action of α‐TOH, termed tocopherol‐mediated peroxidation, and discusses the benefits of vitamin E supplementation alone or together with other antioxidants that work in concert with α‐TOH in ameliorating lipoprotein lipid peroxidation in the artery wall and, hence, atherosclerosis.—Upston, J. M., Terentis, A. C., Stocker, R. Tocopherol‐mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J. 13, 977–994 (1999)

Kynurenine is an endothelium-derived relaxing factor produced during inflammation.

Control of blood vessel tone is central to vascular homeostasis. Here we show that metabolism of tryptophan to kynurenine by indoleamine 2,3-dioxygenase (Ido) expressed in endothelial cells contributes to arterial vessel relaxation and the control of blood pressure. Infection of mice with malarial parasites (Plasmodium berghei) or induction of endotoxemia in mice led to endothelial expression of Ido, decreased plasma tryptophan concentration, increased kynurenine concentration and hypotension. Pharmacological inhibition of Ido increased blood pressure in systemically inflamed mice but not in mice deficient in Ido or interferon-γ, which is required for Ido induction. Both tryptophan and kynurenine dilated preconstricted porcine coronary arteries; the dilating effect of tryptophan required the presence of active Ido and an intact endothelium, whereas the effect of kynurenine was endothelium independent. The arterial relaxation induced by kynurenine was mediated by activation of the adenylate and soluble guanylate cyclase pathways. Kynurenine administration decreased blood pressure in a dose-dependent manner in spontaneously hypertensive rats. Our results identify tryptophan metabolism by Ido as a new pathway contributing to the regulation of vascular tone.

Requirement for, promotion, or inhibition by alpha-tocopherol of radical-induced initiation of plasma lipoprotein lipid peroxidation.

alpha-Tocopherol (alpha-TOH), generally regarded as the most important lipid-soluble, chain-breaking antioxidant in human plasma, can also be a pro-oxidant in isolated low-density lipoprotein (LDL) (Bowry V. W.; Stocker R. J. Am. Chem. Soc. 115:6029-6044; 1993). Here we examined whether this pro-oxidant activity of alpha-TOH is of more general relevance. We compared the oxidizability of lipid hydroperoxide-free, in vivo or in vitro alpha-TOH-depleted LDL and high-density lipoprotein (HDL), as well as plasma reconstituted with alpha-TOH-depleted lipoproteins, with that of the corresponding native and alpha-TOH-supplemented samples, using water- and lipid-soluble peroxyl radicals (ROO.), hydroxyl radicals (.OH), Cu2+, the transition metal-containing Hams F-10 medium, soybean 15-lipoxygenase, and horseradish peroxidase as oxidants. Lipoprotein and plasma oxidizability was assessed by the loss of cholesteryl esters and alpha-TOH and the accumulation of hydroperoxides of cholesteryl esters and phospholipids. Compared to native LDL, HDL, and plasma, the in vivo and in vitro alpha-TOH-depleted counterparts were highly resistant to peroxidation initiation by all oxidants when used at mild radical flux conditions. Wherever tested, the oxidizability of isolated LDL decreased proportionally with decreasing alpha-TOH content. Initiation of LDL lipid oxidation by lipoxygenase and Cu2+ (even up to Cu2+:LDL ratio of 20:1) had an absolute requirement for alpha-TOH. Oxidation of reconstituted plasma with ROO. showed that in the absence of the vitamin, plasma lipids were largely resistant to oxidation, whereas bilirubin and urate oxidized more rapidly. Replenishing the in vitro depleted LDL with alpha-TOH, but not with alpha-tocopherol acetate, fully restored its original content of vitamin E and its oxidizability. Similarly, dietary supplementation with alpha-TOH restored the vitamin content and oxidizability of the in vivo alpha-TOH-depleted lipoproteins and plasma obtained from a patient with familial isolated vitamin E deficiency. Under high fluxes of ROO. and .OH, the activity of alpha-TOH in LDL switched from pro- to anti-oxidant, with the switching point for .OH observed at a lower radical flux than that for ROO.. Together, our results show that alpha-TOH generally makes lipoproteins more reactive towards radical oxidants; this can result in a pro-oxidant activity depending on the specific oxidation conditions.

Ascorbate: The Most Effective Antioxidant in Human Blood Plasma

Ascorbate is the only endogenous antioxidant in plasma that can completely protect the lipoproteins from detectable peroxidative damage induced by aqueous peroxyl radicals and the oxidants released from activated PMNs. In contrast to aqueous oxidants, lipid-soluble peroxyl radicals unsparingly induce detectable peroxidative damage to plasma lipids. However, under these conditions, too, ascorbate appears to belong to the first line of antioxidant defense. Our findings strongly suggest that pathologically relevant lipid hydroperoxide formation consequent to acute or chronic leukocyte activation can be prevented by ascorbate supplementation, provided no free metal catalysts are present. Ascorbate should prove very helpful in the treatment and prevention of diseases and degenerative processes caused by oxidative stress.