Shane R. Thomas

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.

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.

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis.

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.

Redox reactions related to indoleamine 2,3-dioxygenase and tryptophan metabolism along the kynurenine pathway

The heme enzyme indoleamine 2,3-dioxygenase (IDO) oxidizes the pyrrole moiety of L-tryptophan (Trp) and other indoleamines and represents the initial and rate-limiting enzyme of the kynurenine (Kyn) pathway. IDO is a unique enzyme in that it can utilize superoxide anion radical (O2*- ) as both a substrate and a co-factor. The latter role is due to the ability of O2*- to reduce inactive ferric-IDO to the active ferrous form. Nitrogen monoxide (*NO) and H2O2 inhibit the dioxygenase and various inter-relationships between the nitric oxide synthase- and IDO-initiated amino acid degradative pathways exist. Induction of IDO and metabolism of Trp along the Kyn pathway is implicated in a variety of physiological and pathophysiological processes, including anti-microbial and anti-tumor defense, neuropathology, immunoregulation and antioxidant activity. Antioxidant activity may arise from O2*- scavenging by IDO and formation of the potent radical scavengers and Kyn pathway metabolites, 3-hydroxyanthranilic acid and 3-hydroxykynurenine. Under certain conditions, these aminophenols and other Kyn pathway metabolites may exhibit pro-oxidant activities. This article reviews findings indicating that redox reactions are involved in the regulation of IDO and Trp metabolism along the Kyn pathway and also participate in the biological activities exhibited by Kyn pathway metabolites.

Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of α-Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation

There is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D-alpha-tocopherol, alpha-TOH) or dietary supplementation with D-alpha-TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of alpha-TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal-containing Hams F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.5 +/- 2 molecules of alpha-TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ10H2, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Hams F-10 medium resulted in a time-dependent loss of alpha-TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of alpha-TOH, and the rate of this oxidation decreased on alpha-TOH depletion. In vitro enrichment of LDL with alpha-TOH resulted in an LDL particle containing sixfold to sevenfold more alpha-TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo alpha-TOH-deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Hams F-10-initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patients LDL. Oral supplementation of healthy individuals for 5 days with either alpha-TOH or coenzyme Q increased the LDL levels of alpha-TOH and CoQ10H2 by two to three or three to four times, respectively. alpha-TOH-supplemented LDL was significantly more prone to oxidation, whereas CoQ10H2-enriched LDL was more resistant to oxidation initiation by Hams F-10 medium than native LDL. Cosupplementation with both alpha-TOH and coenzyme Q resulted in LDL with increased levels of alpha-TOH and CoQ10H2, and such LDL was markedly more resistant to initiation of oxidation than native or alpha-TOH-enriched LDL. These results demonstrate that oral supplementation with alpha-TOH alone results in LDL that is more prone to oxidation initiation, whereas cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.

Regulation of Angiogenesis by Glycogen Synthase Kinase-3β

Glycogen synthase kinase-3β (GSK3β) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3β signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3β was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKβ promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3β promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3β signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3β activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3β signaling enhanced capillary formation. These data suggest that GSK3β functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.

Suppression of the JNK Pathway by Induction of a Metabolic Stress Response Prevents Vascular Injury and Dysfunction

Background— Oxidative injury and dysfunction of the vascular endothelium are early and causal features of many vascular diseases. Single antioxidant strategies to prevent vascular injury have met with mixed results. Methods and Results— Here, we report that induction of a metabolic stress response with adenosine monophosphate kinase (AMPK) prevents oxidative endothelial cell injury. This response is characterized by stabilization of the mitochondrion and increased mitochondrial biogenesis, resulting in attenuation of oxidative c-Jun N-terminal kinase (JNK) activation. We report that peroxisome proliferator coactivator 1&agr; is a key downstream target of AMPK that is both necessary and sufficient for the metabolic stress response and JNK attenuation. Moreover, induction of the metabolic stress response in vivo attenuates reactive oxygen species–mediated JNK activation and endothelial dysfunction in response to angiotensin II in wild-type mice but not in animals lacking either the endothelial isoform of AMPK or peroxisome proliferator coactivator 1&agr;. Conclusion— These data highlight AMPK and peroxisome proliferator coactivator 1&agr; as potential therapeutic targets for the amelioration of endothelial dysfunction and, as a consequence, vascular disease.

Antioxidants Inhibit Indoleamine 2,3-Dioxygenase in IFN-γ-Activated Human Macrophages: Posttranslational Regulation by Pyrrolidine Dithiocarbamate

Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-γ is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-κB. Here we show that IFN-γ treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor δ-aminolevulinic acid increased IDO activity. Also, incubation of IFN-γ-activated hMDM with δ-[14C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-γ-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.

Coantioxidants make alpha-tocopherol an efficient antioxidant for low-density lipoprotein.

The oxidation of low-density lipoproteins (LDLs) is now commonly implicated as an important early event in atherogenesis. The resulting interest in LDL antioxidation has focused on alpha-tocopherol, the biologically and chemically most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. We review advances made in our understanding of the molecular action of alpha-tocopherol in radical-mediated oxidation of isolated human LDL and how the vitamins antioxidant activity is enhanced or even dependent on the presence of suitable reducing species, which are referred to as coantioxidants.