Paul K. Witting

Redox control of endothelial function and dysfunction: molecular mechanisms and therapeutic opportunities.

The endothelium is essential for the maintenance of vascular homeostasis. Central to this role is the production of endothelium-derived nitric oxide (EDNO), synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelial dysfunction, manifested as impaired EDNO bioactivity, is an important early event in the development of various vascular diseases, including hypertension, diabetes, and atherosclerosis. The degree of impairment of EDNO bioactivity is a determinant of future vascular complications. Accordingly, growing interest exists in defining the pathologic mechanisms involved. Considerable evidence supports a causal role for the enhanced production of reactive oxygen species (ROS) by vascular cells. ROS directly inactivate EDNO, act as cell-signaling molecules, and promote protein dysfunction, events that contribute to the initiation and progression of endothelial dysfunction. Increasing data indicate that strategies designed to limit vascular ROS production can restore endothelial function in humans with vascular complications. The purpose of this review is to outline the various ways in which ROS can influence endothelial function and dysfunction, describe the redox mechanisms involved, and discuss approaches for preventing endothelial dysfunction that may highlight future therapeutic opportunities in the treatment of cardiovascular disease.

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.

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.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here, we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-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.

Oxidation of High Density Lipoproteins I. FORMATION OF METHIONINE SULFOXIDE IN APOLIPOPROTEINS AI AND AII IS AN EARLY EVENT THAT ACCOMPANIES LIPID PEROXIDATION AND CAN BE ENHANCED BY α-TOCOPHEROL

The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL’s α-tocopherol (α-TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of α-TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with α-TOH resulted in a dose-dependent increase in the extent of peroxyl radical-induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0.92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). α-TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, α-TOH can promote this process.

Therapeutic Inflammatory Monocyte Modulation Using Immune-Modifying Microparticles

Negatively charged immune-modifying microparticles bind to the scavenger receptor MARCO on inflammatory monocytes, resulting in their apoptosis and reduced inflammatory damage in a range of diseases. A New Frontier in Immune Modulation Inflammatory monocytes markedly potentiate the immune pathology observed in many diseases, yet no therapy exists that specifically inhibits these cells. The therapeutic accessibility of monocytes in the bloodstream and their inherent propensity to engulf particulate material suggest that highly negatively charged microparticles might provide a readily translatable solution to this problem. These microparticles, referred to as immune-modifying microparticles (IMPs), may be derived from numerous compounds, including the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA-IMP), already used in humans for inter alia dissolvable sutures. Getts et al. now show that upon infusion, IMPs bind to a receptor with a positive domain on inflammatory monocytes, resulting in monocyte sequestration in the spleen and apoptosis through a similar pathway observed for senescing leukocytes. This safe monocyte clearance pathway culminated in substantially reduced inflammatory tissue damage in mouse models of West Nile virus encephalitis, experimental autoimmune encephalomyelitis, peritonitis, colitis, and myocardial infarction. Together, the data suggest that IMPs could transform the treatment of acute inflammation. Indeed, phase 1/2 testing is planned to begin in 2014, with rapid translation supported by the availability of clinical-grade PLGA. Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging

Oxidative stress is implicated in atherogenesis, yet most clinical trials with antioxidants, particularly vitamin E, have failed to protect against atherosclerotic diseases. A striking exception is probucol, which retards atherosclerosis in carotid arteries and restenosis of coronary arteries after angioplasty. Because probucol has in vitro cellular-protective effects independent of inhibiting lipid oxidation, we investigated the mode of action of probucol in vivo. We used three models of vascular disease: apolipoprotein E–deficient mice, a model of atherosclerosis; rabbit aortic balloon injury, a model of restenosis; and carotid injury in obese Zucker rats, a model of type 2 diabetes. Unexpectedly, we observed that the phenol moieties of probucol were insufficient, whereas its sulphur atoms were required for protection. Probucol and its sulphur-containing metabolite, but not a sulphur-free phenolic analogue, protected via cell-specific effects on inhibiting macrophage accumulation, stimulating reendothelialization, and inhibiting vascular smooth muscle cell proliferation. These processes were mediated via induction of heme oxygenase-1 (HO-1), an activity not shared by vitamin E. Our findings identify HO-1 as the molecular target of probucol. They indicate 2-electron rather than radical (1-electron) oxidants as important contributors to atherogenesis, and point to novel lead compounds for therapeutic intervention against atherosclerotic diseases.

Mitochondrial Targeting of Vitamin E Succinate Enhances Its Pro-apoptotic and Anti-cancer Activity via Mitochondrial Complex II

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC50 of 80 μm, whereas the electron transfer from CII to CIII was inhibited with IC50 of 1.5 μm. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser68 within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.

Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits

Antioxidants can inhibit atherosclerosis, but it is unclear how inhibition of intimal lipid oxidation relates to atherogenesis. Here we tested the effect of probucol and its metabolite bisphenol on aortic lipid (per)oxidation and atherogenesis in Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL and aortas from rabbits fed probucol contained bisphenol at concentrations comparable to those in bisphenol-treated animals. Bisphenol treatment increased plasma cholesterol slightly, and plasma and aortic alpha-tocopherol more substantially; these parameters were unaffected by probucol. Bisphenol and probucol treatment both enhanced the resistance of circulating LDL to peroxyl radical-induced lipid peroxidation; this was due to bisphenol, not probucol. Only probucol enhanced LDLs resistance to Cu(2+)-induced oxidation. Both bisphenol and probucol treatment strongly inhibited aortic accumulation of hydroperoxides and hydroxides of cholesteryl esters and triglycerides [LO(O)H]. Despite this, however, probucol had a modestly significant effect on the extent of lesion formation; bisphenol had no inhibitory effect. In addition, the extent of atherosclerosis did not correlate with amounts of aortic LO(O)H present, but, as expected, it did correlate with aortic alpha-tocopherol and cholesterol. Together, these results suggest that aortic accumulation of LO(O)H is not required for, nor is alpha-tocopherol depleted during, the initiation and progression of atherogenesis in WHHL rabbits.

Suppression of Tumor Growth In vivo by the Mitocan α-tocopheryl Succinate Requires Respiratory Complex II

Purpose: Vitamin E analogues are potent novel anticancer drugs. The purpose of this study was to elucidate the cellular target by which these agents, represented by α-tocopoheryl succinate (α-TOS), suppress tumors in vivo, with the focus on the mitochondrial complex II (CII). Experimental Design: Chinese hamster lung fibroblasts with functional, dysfunctional, and reconstituted CII were transformed using H-Ras. The cells were then used to form xenografts in immunocompromized mice, and response of the cells and the tumors to α-TOS was studied. Results: The CII-functional and CII-reconstituted cells, unlike their CII-dysfunctional counterparts, responded to α-TOS by reactive oxygen species generation and apoptosis execution. Tumors derived from these cell lines reciprocated their responses to α-TOS. Thus, growth of CII-functional and CII-reconstituted tumors was strongly suppressed by the agent, and this was accompanied by high level of apoptosis induction in the tumor cells. On the other hand, α-TOS did not inhibit the CII-dysfuntional tumors. Conclusions: We document in this report a novel paradigm, according to which the mitochondrial CII, which rarely mutates in human neoplasias, is a plausible target for anticancer drugs from the group of vitamin E analogues, providing support for their testing in clinical trials.