Nitric Oxide - Oxygen Radicals Interactions in
Atherosclerosis

HOMERO RUBBO, CARLOS BATTHYANY AND RAFAEL RADI

Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo,
Uruguay

ABSTRACT

Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the ‘oxidative-modification hypothesis,’ which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (.NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and .NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of .NO. Full understanding of the physiological roles of .NO, coupled with detailed insight into .NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.

Key words: antioxidants, free radicals, lipid oxidation, low density lipoprotein oxidation, nitric oxide, peroxynitrite.

INTRODUCTION

Nitric oxide (.NO, nitrogen monoxide) is an endogenously-synthesized free radical first characterized as a component of endothelial-derived relaxation factor (Palmer et al., 1987). Nitric oxide is produced by a variety of mammalian cells including vascular endothelium, neurons, smooth muscle cells, macrophages, neutrophils, platelets and pulmonary epithelium (Moncada & Higgs, 1991). The physiological actions of .NO range from mediating vasodilation, neurotransmission, inhibition of platelet adherence/aggregation and the macrophage and neutrophil killing of pathogens (Moncada & Higgs, 1991). The high rate of production and broad distribution of sites of production of .NO, combined with its facile direct and indirect reactions with metalloproteins, thiols and various oxygen radical species, assures that .NO will play a central role in regulating vascular physiologic and cellular homeostasis as well as critical intravascular free radical and oxidant reactions. The multifaceted role that .NO plays in vascular disease will be emphasized during atherosclerosis, the largest single contributor to morbidity and mortality in Western countries.

A. The oxidative-modification hypothesis of atherosclerosis

Atherosclerosis is a complex disease of diverse ethiology, where the oxidation, increased deposition and altered metabolism of lipoproteins are key events associated with lesion development (Diaz et al., 1997, Ross, 1993). Functional responses of the vasculature become altered as well, resulting in impaired vasodilation and, in advanced stages of the disease, vasospasm (O’Brien & Chait, 1994). Recent evidence now compellingly reveals that reactive oxygen species are central mediators of the initiation and progression of the both structural and functional lesions characteristic of atherosclerosis. The relationship between hypercholesterolemia, elevated level of low-density lipoprotein (LDL) and premature atherosclerosis is now firmly established (Steinberg & Witztum, 1990). The cholesterol that accumulates in atherosclerotic lesions in vessel walls is derived primarily from lipoproteins, predominantly LDL (Steinberg et al., 1989). Postsecretory modifications in the structure of lipoproteins significantly affect their atherogenicity. Oxidative modification of LDL is probably the most important and is widely regarded as a critical event in the atherogenic process (Leake, 1993). Indeed, the past decade produced a series of remarkable studies that suggested that oxidative stress, particularly the oxidation of LDL, represents a risk factor and plays a key role at several steps of atherosclerosis, according to the oxidative-modification hypothesis of atherosclerosis (Westhuyzen, 1997; Witztum, 1994). This hypothesis suggests that oxidatively modified LDL (ox-LDL), but not native (unmodified) LDL, is taken by scavenger receptors on monocytes, smooth muscle cells and macrophages in the intima of blood vessel, by an unregulated process leading to the formation of lipid-laden foam cells. According to this hypothesis, LDL initially accumulates in the extracellular subendothelial space of arteries because of the augmented permeability of the endothelial cells secondary to any injury that leads to endothelial dysfunction. Through the action of resident vascular cells, LDL is mildly oxidized to a form known as minimally modified LDL (mm-LDL). This mm-LDL induces local vascular cells to produce monocyte chemotactic protein 1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and colony-stimulating factor 1(CSF-1), which stimulate monocyte recruitment and differentiation to macrophages in arterial walls (Parhami et al., 1993; Witztum, 1993). The accumulating activated monocytes and macrophages stimulate further peroxidation of LDL, leading to the derivatization of apolipoprotein B-100 lysine residues by products of fatty acid peroxidation (Steinbrecher et al., 1989). This completely oxidized LDL is recognized by scavenger receptors on macrophages and internalized to form foam cells (Quinn et al., 1987). In contrast to the uptake of native LDL by the LDL receptor on macrophages and other cells, the uptake of oxidized LDL by the scavenger-receptor pathway is not subject to negative-feedback regulation and thus results in massive uptake of cholesterol by macrophages (Henriksen et al., 1981).

The oxidative-modification hypothesis is supported by evidence that LDL oxidation occurs in vivo. In fact, antibodies raised against oxidized LDL react with atherosclerotic lesions but not with normal arterial segments (Yla-Herttuala et al., 1989). Patients with carotid atherosclerosis have also higher levels of autoantibodies to oxidized LDL than normal subjects (Salonen et al., 1992). This link between LDL oxidation and atherogenesis provides a convenient and simple rationale for the potential beneficial effects of antioxidants on atherosclerosis disease (Gey & Puska, 1989; Riemersma et al., 1991; Rimm et al., 1993; Stampfer et al., 1993; Stephens et al., 1996).

B. Mechanisms of LDLl oxidation in vivo.

Several pathways promote LDL oxidation in vitro, but the physiologically relevant mechanisms for LDL oxidation in vivo are still to be defined, although it is clear that in vivo LDL oxidation involves free radical chain reactions (Heinecke, 1997). Next we will discuss the mechanisms that may promote LDL oxidation in the human artery wall.

B1. Metal ions: LDL oxidation by cultured arterial cells requires micromolar concentration of iron or copper, where metal chelators block LDL oxidation by most types of cells (Heinecke et al., 1984). Tissue homogenates prepared from atherosclerotic lesions contain catalitically active metal ions, suggesting that metal ions may stimulate LDL oxidation in vivo (Swain & Gutteridge, 1995). These reactions serve to modify several properties of LDL, including electrophoretic mobility, fatty acid peroxide and thiobarbituric acid-reactive material content, the extent of apoprotein amino acid oxidation, polypeptide chain scission of apolipoprotein B and ultimately, the increased uptake, degradation and accumulation of modified LDL by macrophages (Steinbrecher et al., 1984, Batthyany et al., 2000). Lipid peroxidation propagation reactions have a critical requirement for metal catalysis. Existing "seeded" lipoprotein and vascular cell lipid hydroperoxides (LOOH) play a key role in these oxidative processes and their consequences by giving rise to a variety of reactive radical species (i.e.: lipid peroxyl (LOO.) and alkoxyl (LO.) radicals) and secondary breakdown products (i.e.: reactive aldehydes) which will react with primary amines to yield fluorescent Schiff’s base products (Fruebis et al., 1992). This resultant oxidized lipoprotein product is the more anionic species, which becomes a ligand recognized by macrophage scavenger receptor(s) (Lamb et al., 1995).

B2. Lipoxygenase and myeloperoxidase: The observation that LDL may be oxidatively modified by incubation with soybean lipoxygenase and phospholipase A2 (Sparrow et al., 1988), coupled with data showing that lipoxygenase inhibitors prevent LDL oxidation by endothelial cells or macrophages (Parthasarathy et al., 1989), suggests that cellular lipoxygenases are critically involved in oxidative modification of LDL. LDL exposed to fibroblasts transfected with the gene for 15-lipoxygenase exhibited increased levels of lipid hydroperoxides and both 15-lipoxygenase mRNA and protein have been detected in human atherosclerotic lesions (Benz et al., 1995; Yla-Herttuala et al., 1990). Recent studies (Kuhn et al., 1997), have demonstrated a significant enrichment of the stereospecific isomer 13S-hydroxy-9Z,11E-octadecadienoic acid in lipids extracted from human atherosclerotic lesions, suggesting that 15-lipoxygenase may oxidize LDL in vivo.

Myeloperoxidase is a heme protein secreted in the lesion by activated neutrophiles, and catalitically active myeloperoxidase is a component of human atherosclerotic tissue, where it colocalizes with foamy macrophages in the cellular rich regions of lesions (Daugherty et al., 1994). Oxidation products of the enzyme have been detected by immunochemistry in atherosclerotic vascular lesions (Hazell et al., 1996), suggesting that myeloperoxidase promotes LDL oxidation in vivo. Recent studies demonstrate that myeloperoxidase converts tyrosine to 3-chlorotyrosine, a stable product that may therefore serve as a molecular fingerprint for the action of the enzyme (Leeuwenburgh et al., 1997). It was recently shown that the 3-chlorotyrosine level was six times greater in atherosclerotic lesion than in normal aortic tissue, and 30 fold higher than in circulating LDL (Hazen & Heinecke, 1997). These results provide evidence that myeloperoxidase constitutes a mechanism for LDL oxidation in vivo.

B3. Reactive nitrogen species: Nitric oxide reacts with superoxide (O₂^.) to form peroxynitrite (ONOO^- ), a reactive nitrogen species that promotes LDL oxidation. The radical-radical reaction between O₂.- and .NO is extremely fast and almost diffusionally limited in rate (~1010 M-1.s-1) (Kissner et al., 1997), giving rise to significant quantities of a molecule with strong oxidizing properties. Peroxynitrite is also a nitrating agent: this is the addition of a nitro group (-NO₂) to a biomolecule. It reacts with tyrosine in vitro to yield the stable product 3-nitrotyrosine, which can then be used as a fingerprint for ONOO^- reaction in tissues. Monoclonal and polyclonal antibodies to nitrotyrosine formation show immunoreactivity in fatty streaks of coronary arteries of young autopsy subjects (Beckmann et al., 1994). In older patients, nitrotyrosine immunoreactivity is found in close association with foam cells, vascular endothelium and in the neointima of advanced atherosclerotic lesions. We should note that myeloperoxidase may also play a role in nitrotyrosine formation, because it can convert nitrite, the autoxidation product of .NO, to a reactive intermediate that nitrates the aromatic ring of tyrosine and proteins in vitro (Eiserich et al., 1998). In fact, the enzyme catalyzes aromatic nitration in media containing nitrite concentrations approximating those found in biologic fluids, confirming that this major .NO metabolite can serve as a physiological substrate for myeloperoxidase in the presence of plasma levels of chloride (Eiserich et al., 1998). It has been demonstrated that reactive nitrogen species generated by the myeloperoxidase/hydrogen peroxide/nitrite system of monocytes convert LDL to a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation (Podrez et al., 1999).

C) Antioxidant properties of nitric oxide in atherosclerosis

Nitric oxide has many physiological actions that can be interpreted to be potentially antiatherosclerotic (Boger et al., 1998; Bult, 1996). It inhibits 1) platelet aggregation and adherence to endothelial cells, 2) monocyte adherence to endothelial cells, 3) the expression of the monocyte chemoattractant protein, 4) vascular smooth muscle cell migration and proliferation and 5) the in vivo intimal proliferative response to ballon injury. Nitric oxide reduces oxidant stress in the vascular wall, which in turn may lower the rate of LDL oxidation and the expression of redox-sensitive genes that contribute to atherogenesis. In fact, vascular .NO either suppresses the expression of adhesion molecules by endothelial cells or the generation of products that are chemotactic for monocytes such as oxidized LDL. There is accumulating evidence that the salutary effects of ^.NO are diminished in atherosclerotic vessels due to its reactions with reactive oxygen species. In particular, the reaction of .NO with O₂^•-, as well as its reaction with LO^. and LOO^. to inhibit lipid oxidation, suggests that ^.NO can both enhance and inhibit lipoprotein oxidation in the vessel wall. The removal of ^.NO from the vascular compartment by its rapid reactions with these free radical species will concomitantly lower its steady state concentration, thus increasing platelet and inflammatory cell adhesion to the vessel wall and impairing endothelial-dependent mechanisms of relaxation. The following sections develop these concepts in more detail.

C1. Nitric oxide reaction with lipid peroxyl radicals. Nitric oxide has been observed to play a critical role in regulating lipid oxidation induced by reactive oxygen and nitrogen species (Rubbo et al., 1994; Rubbo et al., 2000). Lipid reactions of ^.NO are an important area of focus for multiple reasons. First, this reactive species significantly concentrates in lipophilic cell compartments, with an n-octanol:water partition coefficient of 6-8:1. This property will further enhance the ability of ^.NO to regulate oxidant-induced membrane lipid oxidation. Second, ^.NO reacts with LO^. and LOO^. at near diffusion-limited rates (Padmaja & Huie, 1993), inferring that both lipid peroxidation processes and reactions of lipophilic antioxidants will be influenced by local .NO concentrations.

Nitric oxide has been reported to have contrasting effects on LDL oxidation, with the pro-oxidant versus antioxidant outcome of ^.NO extremely dependent on relative concentrations of individual reactive species. For both macrophage and endothelial cell model systems, increased rates of cell ^.NO production via cytokine-mediated stimulation of inducible macrophage nitric oxide synthase gene expression and activity or exogenous addition of ^.N have been shown to inhibit cell and O2.--mediated lipoprotein oxidation (Hogg et al., 1993; Jessup et al., 1992; Rubbo et al., 1995). In contrast to these examples, the simultaneous production of ^.NO and O2.- by 1,3-morpholino-sydnonimine-HCl (SIN-1) or the direct addition of ONOO- has been shown to oxidize lipoproteins to potentially atherogenic forms (Darley-Usmar et al., 1992). It has also been shown that either ONOO- or the myeloperoxidase/hydrogen peroxide/nitrite system depletes LDL of native antioxidants and converts the LDL to a form readily recognized by macrophage scavenger receptors (Graham et al., 1993; Podrez et al., 1999, Panasenko et al., 2000). We have learned that LDL oxidation is inhibited by ^.NO via the termination of lipid radical-mediated chain propagation reactions (Rubbo et al., 1995). This reaction is now shown to occur both in vitro and in lipid extracts of atherosclerotic vascular lesions, in the major oxidizable lipid in LDL, cholesteryl linoleate, yielding nitrogen-containing oxidized lipid derivatives (unpublished data). Some nitrogen-containing lipid intermediates appear to be highly unstable and may decompose to reinitiate radical processes. In particular, the product of the LOO·/^.NO combination reaction (LOONO) may be cleaved by homolysis to LO. and ^.NO₂ with rearrangement of LO. to an epoxyallylic acid radical L(O). followed by recombination of L(O). with ^.NO₂ (Odonnell et al, 1999). These observations taken together with the fact that ^.NO can diffuse into the hydrophobic core of the LDL particle (unpublished data) are in agreement with our hypothesis that ^.NO can represent the major lipophilic antioxidant in LDL.

C2. Nitric oxide and a-tocopherol inhibition of lipid oxidation. a-Tocopherol (a-TH), a lipophilic chain-breaking antioxidant in biological membranes and lipoproteins, acts by donating hydrogen atoms to chain-propagating LOO·to form the corresponding LOOH (Liebler, 1993). Since the reaction of LOO^.with a-TH occurs at a rate three orders of magnitude less than for the reaction of LOO^.with ^.NO, ^.NO could act more readily than or in concert with a-TH, lycopene, retinyl derivatives and ß-carotene as an antioxidant defense against oxygen radical-derived oxidized lipid species. In fact, based on a comparison of relative rate constants, it is predicted that the termination of LOO by NO (k=2.0 x 10⁹ M^-1. s^-1) will be significantly more facile than the reaction of LOO^.with a-tocopherol (k=2.5 x 106 M^-1. s^-1), thus protecting a-TH from oxidation (Rubbo et al., 2000). Because of a high reactivity with other radical species, a relatively lower reactivity of lipid radical-^.NO termination products and an ability of ^.NO to readily traverse membranes and lipoproteins, ^.NO can effectively terminate radical species throughout all aspects of membrane and lipoprotein microenvironments. This can also help spare other tissue antioxidant defenses as well during periods of oxidant stress.

Figure 1. Pro- and antioxidant fates of nitric oxide on low density lipoprotein oxidation

C3. Nitric oxide and mechanisms underlying impaired vasomotor responses in atherosclerosis. Endothelium plays an important role in maintaining vascular integrity by the synthesis and release of vasoactive substances such as ^.NO. The changes that occur during atherosclerosis include the loss of the control of vascular tone, a ^.NO-dependent event. The mechanisms accounting for endothelial dysfunction in hypercholesterolemia have not been completely elucidated, but may be explained by decreased bioavailability of ^.NO due to either decreased expression of the eNOS, decreased substrate availability, presence of an endogenous eNOS inhibitor or increased ^.NO degradation by reactive oxygen-and nitrogen species (Busse & Fleming, 1996).

Dietary L-arginine and other strategies for the enhancement of vessel wall ^.NO synthesis have been shown to be antiatherogenic in this vascular disease associated with excess production and reactions of reactive oxygen species. In fact, both animal models and clinical studies show that chronic administration of L-arginine restores endogenous ^.NO production, improves endothelial dependent relaxation, decreases inflammatory cell accumulation at the vessel wall and reduces intimal hyperplasia, all hallmarks of atherosclerotic disease (Boger et al., 1998; Clarkson et al., 1996; Cooke, 1998; Davies et al., 1995; Hayward & Lefer, 1998; Wang et al., 1994). Furthermore, balloon angioplasty is often used to treat atherosclerotic vaso occlusive problems. Both the administration of .NO donors and the transfection of constitutive nitric oxide synthase to balloon-injured vessels reduce intimal cell hyperplasia, often the cause for repeat angioplasty, aortocoronary bypass graft surgery or myocardial infarction (Hoshida et al., 1996).

Antioxidants can improve endothelium-dependent vasodilation in humans with atherosclerosis documented by coronary angiography. In fact, acute administration of superoxide dismutase or a combination of lovastatin with probucol for one year reverts the abnormal vasoconstrictor response of the coronary arteries to acetylcholine in atherosclerotic patients (Anderson et al., 1995). In essential hypertensive patients, impaired endothelial vasodilation can also be improved by intrabrachial administration of ascorbic acid (vitamin C), the main water-soluble antioxidant in human plasma, an effect that can be reversed by nitric oxide synthase inhibitors, supporting the hypothesis that ^.NO inactivation by oxidant species contributes to endothelial dysfunction (Taddei et al., 1998). In addition, the administration of ascorbic acid improves endothelium-dependent vasodilatation in patients with hypercholesterolaemia in the absence of clinical evidence of atherosclerosis (Ting et al., 1997). Intracellular ascorbic acid is able to enhance .NO synthesis in endothelial cells, and this may explain, in part, its beneficial vascular effects (Heller et al., 1999; Huang et al., 2000).

CONCLUSIONS

The recent observations of a) the potent inhibitory effects of ^.NO towards platelet function and neutrophil margination on the vessel wall, b) the extremely fast and direct reactivity of^.NO with oxidizing lipids and O₂^.-, c) the tenuous balance between O₂^.-, oxidized lipoproteins and ^.NO in regulating endothelial-dependent relaxation, and d) the diversity of pro-atherogenic oxidizing events that occur in the vascular compartment all strongly support a central role for ^.NO in regulating vascular atherogenic processes. The reaction between ^.NO and O₂^.- in the vasculature has the combined effect of eliminating a putative antioxidant (^.NO) while at the same time generating a potent oxidant (ONOO^-). Since the rate constant for ^.NO reaction with LOO^. is greater than that for a-TH reaction with LOO^. and the ability of ^.NO to concentrate in membranes, ^.NO can act more readily than or in concert with lipophilic antioxidants as an adjunct antioxidant defense against oxygen radical-derived oxidized lipid species. The anti-atherogenic and endothelial-dependent relaxation restoring effects observed following L-arginine dietary supplementation provides strong support for these concepts. Future directions may include the development of novel pharmacological strategies against atherosclerosis, investigating the antioxidant capacity of various compounds. Special attention will be paid to the direct reactions and indirect protective effects that antioxidants exert toward both lipids and lipoproteins through different mechanisms, such as ^.NO donors, ONOO^- scavengers, and superoxide dismutase mimetics.

ACKNOWLEDGMENTS

This work was supported from CONICYT, Parke-Davies Laboratory; Fundación Manuel Pérez, Facultad de Medicina and PEDECIBA

Corresponding Author:Dr. Homero Rubbo. Departamento de Bioquímica, Facultad de Medicina. General Flores 2125, 11800 Montevideo, Uruguay. Phone: 5982-9249561 Fax: 5982-9249563 E-mail: hrubbo@fmed.edu.uy

Received: October 1, 1999. Accepted: January 10, 2000

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