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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:274.)
© 2005 American Heart Association, Inc.

Brief Reviews

Redox Mechanisms in Blood Vessels

Cornelius F.H. Mueller; Karine Laude; J. Scott McNally; David G. Harrison

From Emory University Division of Cardiology, Department of Medicine and the Atlanta Veterans Administration Hospital, Atlanta, Ga.

Correspondence to David G. Harrison, Division of Cardiology, Emory University, 1639 Pierce Drive, WMRB 319, Atlanta, GA 30322. E-mail dharr02{at}emory.edu

Series Editor: Kathy K. Griendling

	Abstract

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
Reactive oxygen species have been implicated in the pathogenesis of virtually every stage of vascular lesion formation, hypertension, and other vascular diseases. We are currently gaining insight into important sources of reactive oxygen species in the vessel wall, including the NADPH oxidases, xanthine oxidase, uncoupled nitric oxide synthase, and mitochondrial sources. Although various reactive oxygen species have pathological roles, some serve as important signaling molecules that modulate vascular tone, growth, and remodeling. In the next several months, a series of articles in Arteriosclerosis, Thrombosis, and Vascular Biology attempt to further elucidate how reactive oxygen species are produced by vascular cells and the roles of these in vascular homeostasis. This series promises to provide a valuable update on a wide variety of issues related to the biochemistry, molecular biology, and physiology of these important and fascinating molecules.

Reactive oxygen species have been implicated in the pathogenesis of virtually every stage of vascular lesion formation, hypertension, and other vascular diseases. Upcoming series of articles in Arteriosclerosis, Thrombosis, and Vascular Biology help elucidate how reactive oxygen species are produced by vascular cells and their role in vascular homeostasis.

Key Words: NADPH oxidase • superoxide • nitric oxide synthase • xanthine oxidase • mitochondria

	Introduction

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
In the next several months, Arteriosclerosis, Thrombosis, and Vascular Biology will publish a series of reviews examining in depth the source and role of reactive oxygen species (ROS) and oxidant stress in vascular disease. Disorders such as hypercholesterolemia, hypertension, diabetes, aging, and mechanical injury are associated with increased ROS production.^1–5 The fact that these conditions contribute to >33% of the deaths and 40% of the total medical expenses in industrialized countries emphasizes the importance of understanding how oxidative stress occurs and how ROS affect the vascular system.⁶ Several enzyme systems have recently been recognized to be sources of ROS in vascular cells under pathological conditions. Experimental studies in cells and experimental animals have indicated that ROS mediate or enhance virtually every aspect of atherosclerotic lesion formation. The best-characterized of these events is oxidative modification of low-density lipoprotein, which can occur via reaction of ROS with low-density lipoprotein or via direct enzymatic modification by lipoxygenases.⁷ Beyond this, ROS can promote inflammation,⁸ alter vasomotion,⁹ activate matrix metalloproteinases,¹⁰ induce apoptosis,¹¹ cause platelet aggregation,¹² and stimulate vascular smooth muscle proliferation.¹³ All of these events are active in the atherosclerotic lesion and are thought to contribute to vascular lesion formation.

See cover

It has been, only in the past decade, widely accepted that vascular cells could produce ROS, and we are still learning a great deal about the role of the various potential enzymes, how they are regulated, and the role of oxidative stress in vascular disease. A technical problem has been that vessels do not produce ROS at levels equivalent to neutrophils; therefore, the methods commonly used for neutrophils, such as cytochrome c reduction, yield values that are only slightly above the limit of detection. This problem was overcome by the use of chemiluminescence techniques, such as lucigenin-enhanced chemiluminescence, and this entire area of research was dramatically advanced by the use of such methods. In the late 1990s, several articles appeared in which the authors showed that lucigenin-enhanced chemiluminescence could artifactually produce superoxide; however, these "test tube" experiments did not reflect the assay as used in intact vessels.¹⁴ Subsequently, numerous methods including electron spin resonance, dihydroethidium oxidation, other chemiluminescence methods, and even cytochrome c reduction have been used to confirm the results observed with lucigenin-enhanced chemiluminescence. Further, assays of hydrogen peroxide often provide results that mirror these measures of superoxide production in diseased vessels. All of these have confirmed an increased ROS production by vessels in the setting of disorders like hypercholesterolemia, diabetes, and hypertension.^15–17

	Sources of ROS in Vascular Cells

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
This special series of ATVB provides an opportunity to review our current understanding of how ROS are produced in the vessel wall. Although there are numerous enzyme systems that can potentially produce ROS in the vessel wall, 4 enzyme systems seem to predominate. These include the NADPH oxidases, xanthine oxidase, uncoupled NO synthase, and mitochondrial sources. A recurring theme is that there appears to be substantial interplay between these sources, such that activation of one can enhance activity of others (Figure). This can lead to feed-forward processes, which further augment ROS production and oxidant stress. These interactions are further discussed in the following paragraphs.

View larger version (32K): [in this window] [in a new window]   Known sources of reactive oxygen species (ROS) and potential interactions. In vascular cells, predominant sources of ROS include the NADPH oxidase, xanthine oxidase, uncoupled nitric oxide synthase, and mitochondrial electron transport. The Nox proteins are the catalytic component of the NADPH oxidase and together with p22phox comprise the membrane cytochrome. Shown also is the oxygenase domain of eNOS, which in the absence of tetrahydrobiopterin (BH4) can produce large amounts of superoxide. ROS produced from the NADPH oxidase can promote oxidation of BH4 and increase the relative levels of xanthine oxidase in the endothelium. Hydrogen peroxide and lipid peroxides are also capable of stimulating the NADPH oxidases to further produce superoxide.

	NADPH Oxidases

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
The NADPH oxidase enzymes, also known as the Nox enzymes, represent a major source of ROS in vascular cells. The Nox proteins represent the catalytic subunits of these enzymes and vary in terms of their mode of activation and need for cofactor activation.¹⁸ Nox1 protein levels are quite low in vascular cells, but can be induced by stimuli such as PDGF, angiotensin II, and serum.¹⁸ Nox2, previously known as gp91^phox, is the large catalytic subunit of the phagocyte cytochrome b558. It is expressed in endothelial and adventitial cells of large vessels and in the vascular smooth muscle cells of smaller vessels.^19–22 Nox4 is constitutively expressed and constitutively active in vascular smooth muscle and endothelial cells.^23,24 Current understanding is that all the Nox enzymes require p22^phox, which serves as a docking protein for other subunits and stabilizes the Nox proteins.²⁵ A variety of pathological stimuli, such as angiotensin II, stretch, endothelin-1, thrombin, and catecholamines acutely activate the NADPH oxidases in vascular smooth muscle and endothelial cells. The biochemical pathway whereby angiotensin II activates the NADPH oxidase involves activation of the tyrosine kinase c-Src, transactivation of the EGF receptor, and ultimately activation and translocation of the small g protein Rac-1.²⁶ Activation of Nox1 and Nox2 also require translocation of cytoplasmic subunits, including p47^phox,²⁷ or analogues of p47^phox and p67^phox termed NoxO1 and NoxA1.^28,29 Interestingly, pathophysiological stimuli such as angiotensin II, hypercholesterolemia, growth factors, and serum can also increase expression of several of the NADPH oxidase subunits, including p22^phox, Nox1, and Nox4, further promoting an increase in ROS production.¹⁸ The biochemical pathways regulating expression of these subunits have not been elucidated. Nevertheless, activation and increased expression of these Nox-based oxidases are clearly very important in modulating oxidant stress in vascular disease. These issues are discussed in an upcoming review by Dr Francis Miller. It should be noted that Dr Miller, working in collaboration with Dr Neal Weintraub, first showed that hydrogen peroxide and lipid peroxides can stimulate activity of the NADPH oxidases in vascular smooth muscle cells, leading to a feed-forward increase in ROS production.^30,31

	Xanthine Oxidase

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
Another important source of ROS in mammalian cells is the xanthine oxidoreductase. Xanthine oxidoreductase exists in 2 forms, as xanthine dehydrogenase (XDH) and as xanthine oxidase (XO).³² XDH uses NAD⁺ to receive electrons from hypoxanthine and xanthine, yielding NADH and uric acid. In contrast, XO uses oxygen as an electron acceptor from these same substrates to form and hydrogen peroxide. The ratio of XO to XDH in the cell is therefore critical to determine the amount of ROS produced by these enzymes. Conversion of XDH to XO is stimulated by inflammatory cytokines, like tumor necrosis factor-, and also by oxidation of critical cysteine residues by oxidants, such as peroxynitrite.^33,34 Recently, we have shown in bovine and mouse aortic endothelial cells that the relative levels of these is markedly altered by the presence of a functioning NADPH oxidase, such that in cells with an absence of the NADPH oxidase, the levels of XO are extremely low.^35,36 In more recent studies, we have found that this is mediated by hydrogen peroxide released from the NADPH oxidase. Thus, this regulation represents a second situation in which cellular production of ROS from the NADPH oxidase further begets ROS production.

There is substantial debate as to whether XDH is expressed. Immunohistochemical studies of normal human tissues have failed to demonstrate XDH in endothelial cells or other cardiovascular tissues.³⁶ In contrast, there is evidence that XO can produce ROS and affect endothelial function in humans in the setting of pathology,^15,37–39 possibly caused by stimulation of XO expression by inflammatory cytokines or other stimuli in these conditions. For example, Sohn et al have shown that hypoxia induces XO activity in human umbilical vein endothelial cells.⁴⁰ There is substantial interest in the concept that the XO present in endothelial cells originates from other organs and that the enzyme is probably taken-up via heparin binding sites.^15,41,42 Whatever the source of XO, its vascular activity correlates inversely with endothelial function in patients with heart failure and in subjects with atherosclerosis.^15,43 Dr Margaret Tarpey discusses these concepts in greater depth in her upcoming review of xanthine oxidoreductase.

	Uncoupled Endothelial Nitric Oxide Synthase

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
A third major contributor to vascular ROS generation is uncoupled endothelial nitric oxide synthase (eNOS). The normal product of this enzyme is nitric oxide (NO); however, in the absence of either L-arginine or tetrahydrobiopterin (BH₄), the NO synthases are incapable of transferring electrons to L-arginine and begin to use oxygen as a substrate for formation.⁴⁴ Uncoupling of eNOS has been demonstrated in various pathophysiological conditions including diabetes,¹⁷ hypercholesterolemia,⁴⁵ and hypertension.⁴⁶ In DOCA-salt hypertension, oxidation of BH₄ occurs as a result of ROS (particularly peroxynitrite) produced by the NADPH oxidase. Oral treatment of mice that have DOCA-salt hypertension with BH₄"re-couples" eNOS, leading to increased vascular NO production, decreased production, and lowering of blood pressure.⁴⁶ Thus, uncoupling of eNOS caused by oxidation of BH₄ represents a third mechanism whereby ROS produced by the NADPH oxidase can stimulate another enzyme to produce additional ROS in a self-perpetuating fashion.

Related to this situation, eNOS is subject to regulation by H₂O₂, which acutely activates the enzyme and, over the longer-term, increases its expression. Thus, increased expression of eNOS in the setting of oxidant stress can lead to a situation in which high levels of the uncoupled enzyme can generate even greater amounts of . It should be stressed that not all eNOS becomes uncoupled, such that some of the enzyme continues to produce NO, leading to a condition favoring production of peroxynitrite. Dr Thomas Münzel discusses NOS uncoupling in much greater detail in his upcoming review.

	Mitochondrial Electron Transport

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
In other organs and tissues, the mitochondrial electron transport chain represents the predominant source of and consequently H₂O₂. It has been estimated that between 1% and 4% of the oxygen reacting with the respiratory chain is incompletely reduced to . Defects in mitochondria DNA can be inherited or can develop as a result of disease, and have been proposed to augment ROS production by these organelles.^47,48 Interestingly, exposure of endothelial or vascular smooth muscle cells to exogenous peroxynitrite or H₂O₂ leads to mitochondrial DNA damage.⁴⁹ This represents yet another mechanism whereby oxidant stress could beget further oxidant stress.

In the vessel wall, the precise contribution of mitochondria to the total ROS production, however, remains unclear. Part of this problem relates to the fact that specific antagonists are not well-characterized. For example, rotenone is often used to inhibit mitochondrial radical production^50,51 but, in fact, is capable of having the opposite effect.^31,52 Furthermore, inhibition of mitochondrial function can dramatically alter many other aspects of cell metabolism, making results from such interventions difficult to interpret. Nevertheless, there is ample evidence that mitochondria play an important role in vascular ROS production. Stimuli such as high glucose, cyclic strain, leptin, and cigarette smoking have been shown to damage aortic mitochondrial DNA and alter mitochondrial enzyme activity. Dr Paul Schumacker’s review examines the role of mitochondria in vascular oxidant stress further.

	Diverse Properties of ROS

Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 
It is important to note that one should not lump all ROS together when considering the topic of oxidant stress and vascular disease. Some ROS, such as superoxide the hydroxyl radical (HO), peroxy-radicals (ROO·), and NO have unpaired electrons in their outer orbital. Others, such as H₂O₂ and peroxynitrite, do not have unpaired electrons but are oxidants. These can have very different roles. Superoxide reacts with NO in a diffusion limited fashion, leading to formation of peroxynitrite and loss of the beneficial actions of NO. This phenomenon is thought to be responsible for reduced endothelium-dependent vasodilatation in many conditions, and likely contributes to hypertension by removing the vasodilator effect of NO. Peroxynitrite is a very potent oxidant and can oxidize lipids and thiols and react with iron sulfur centers in a variety of enzymes. Whereas scavenging improves endothelium-dependent vasodilatation, it might not reduce atherosclerosis, because scavenging does not eliminate other ROS and, in fact, can enhance production of H₂O₂ (Scheme 1).⁵³

View larger version (6K): [in this window] [in a new window]   Scheme 1:

Scheme 1 is important because there is increasing evidence that H₂O₂ plays an important role in atherosclerosis. As an example, Tribble et al showed that lesion formation was not diminished in mice overexpressing Cu/Zn SOD and, in fact, was increased in direct relation to aortic Cu/Zn SOD activity.⁵⁴

Another aspect of ROS is that they are not uniformly deleterious and can have important signaling properties within the vessel wall.⁵⁵ Hydrogen peroxide, in particular, is relatively stable and uncharged so that it can easily diffuse between cells. Recently, it has become clear that endogenously produced H₂O₂ can act as an endogenous hyperpolarizing factor in both human and mouse resistance vessels.^56,57 Dr Gutterman discusses this further in his upcoming review. Hydrogen peroxide has been shown to inhibit phosphatases, and activate guanylate cyclase, and to stimulate NO production and alter gene expression. The mechanisms involved in these signaling events are varied. An important reaction is oxidation of critical cysteines to sulfenic acids, which in turn can serve as precursors to sulfonic acid and/or disulfide formation. These modifications can alter function of a number of enzymes, including phosphatases, glyceraldehyde-3-phosphate dehydrogenase, and peroxiredoxin.^58–60 Recent evidence suggests that interactions between H₂O₂ and peroxidases, especially myeloperoxidase, lead to formation of oxidizing and nitrosating species that are particularly atherogenic. Importantly, modification of proteins by these radicals alters their enzymatic function. Nitration of apolipoprotein A1 dramatically impairs its ability to participate in reverse cholesterol transport from lipid-laden macrophages, and thus reduces atheroprotective properties of high-density lipoprotein.⁶¹ Dr Hazen discusses this in detail in his review of myeloperoxidase.

Finally, it is essential that we develop a greater understanding of all the compensatory responses to prolonged oxidant stress. Recent studies from our groups have supported the concept that increased ROS production has minimal effects on vascular function and hemodynamics at baseline but can augment the response pathogenic stimuli. We have produced mice with smooth muscle-targeted overexpression of the NADPH oxidase subunit p22^phox. Overexpression of this subunit resulted in a concomitant increase in expression of Nox1 and enhanced vascular and H₂O₂ production.⁶² At baseline, these mice were normotensive and had normal vascular reactivity and histology. In response to angiotensin II, however, augmented hypertension and increased thickness of the vascular media develop in these animals.⁶³ In addition, vascular injury enhances atheroma formation in these animals.⁶⁴ Further characterization of these mice indicated that they have increased endothelial NO synthase expression, NO production, and expression of the extracellular superoxide dismutase, which likely compensate for their enhanced vascular ROS production. We have concluded from these studies that increased vascular ROS production has minimal effects in the absence of stimuli such as angiotensin II or mechanical injury, but the presence of such stimuli markedly augments pathological responses. In this regard, the compensatory responses to oxidative stress are likely critically important, and disease probably does not occur until these mechanisms fail. It remains a challenge to understand all of these compensatory mechanisms, why they fail, and what can be done to preserve their function.

Received September 27, 2004; accepted October 20, 2004.

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Top Abstract Introduction Sources of ROS in... NADPH Oxidases Xanthine Oxidase Uncoupled Endothelial Nitric... Mitochondrial Electron Transport Diverse Properties of ROS References

 

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