Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role...

Kathy K. Griendling, Dan Sorescu, Bernard Lassègue and Masuko Ushio-Fukaiand Their Role in Vascular Physiology and Pathophysiology

Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species

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Modulation of Protein Kinase Activity and Gene Expressionby Reactive Oxygen Species and Their Role in Vascular

Physiology and PathophysiologyKathy K. Griendling, Dan Sorescu, Bernard Lassegue, Masuko Ushio-Fukai

Abstract—Emerging evidence indicates that reactive oxygen species, especially superoxide and hydrogen peroxide, areimportant signaling molecules in cardiovascular cells. Their production is regulated by hormone-sensitive enzymes suchas the vascular NAD(P)H oxidases, and their metabolism is coordinated by antioxidant enzymes such as superoxidedismutase, catalase, and glutathione peroxidase. Both of these reactive oxygen species serve as second messengers toactivate multiple intracellular proteins and enzymes, including the epidermal growth factor receptor, c-Src, p38mitogen-activated protein kinase, Ras, and Akt/protein kinase B. Activation of these signaling cascades andredox-sensitive transcription factors leads to induction of many genes with important functional roles in the physiologyand pathophysiology of vascular cells. Thus, reactive oxygen species participate in vascular smooth muscle cell growthand migration; modulation of endothelial function, including endothelium-dependent relaxation and expression of aproinflammatory phenotype; and modification of the extracellular matrix. All of these events play important roles invascular diseases such as hypertension and atherosclerosis, suggesting that the sources of reactive oxygen species andthe signaling pathways that they modify may represent important therapeutic targets.(Arterioscler Thromb Vasc Biol.2000;20:2175-2183.)

Key Words: reactive oxygen speciesn vascular smooth musclen endothelial cellsn hypertensionn atherosclerosis

Reactive oxygen species (ROS) are some of the newestadditions to the family of second-messenger molecules.

Although one ROS, nitric oxide (NOz), has been known foryears to serve as a signaling molecule by activating guanylatecyclase, it has only recently become apparent that other ROS,including superoxide (O22z) and hydrogen peroxide (H2O2),can alter the function of specific proteins and enzymes aswell. In most cases, the mechanism by which these agentsinteract with their molecular targets is still unknown, but it isclear that they can mediate agonist-stimulated signaling. Inthis review, we will discuss redox-sensitive signaling cas-cades in vascular cells; their alteration by agonists, withparticular attention to angiotensin II (Ang II); and theirrelevance to cardiovascular disease.

Production and Metabolism of ROSVirtually all types of vascular cells produce O2

2z and H2O2.1

In addition to mitochondrial sources of ROS, O22z and/or

H2O2 can be derived from xanthine oxidase, cyclooxygenase,lipoxygenase, NO synthase, heme oxygenases, peroxidases,hemoproteins such as heme and hematin, and NAD(P)Hoxidases. Several investigators have shown that these latterenzymes, the membrane-associated NAD(P)H oxidase(s), arethe primary physiological producers of ROS in vascular

tissue.2–4 Of importance, the activity of these enzymes can bemodulated by vasoactive hormones and the low-molecular-weight G protein rac-1,4–7 providing a critical characteristicof any second messenger: regulation of its production. Me-tabolism of these ROS is also tightly controlled. Dismutationof O2

2z by superoxide dismutase (SOD) produces the morestable ROS H2O2, which in turn is converted to water bycatalase and glutathione peroxidase. Expression of antioxi-dant enzymes can be altered by hormones such as Ang II,tumor necrosis factor (TNF)-a, and interleukin (IL)-1b, thusprofoundly affecting ROS levels.8–11 The tight regulation ofboth production and removal of ROS makes fluctuations intheir levels transient, another requirement for second messen-gers. ROS may also act as an intracellular “rheostat,” closelymodulating the activity of a discrete set of biochemicalreactions. A schematic of the balance between oxidative andreductive states of the cell and the hormones, enzymes, andcompounds that can alter this balance and thus, the overallresponse of the cell, is presented in Figure 1.

Vascular NAD(P)H OxidasesThe major sources of ROS in the vessel wall, the vascularNAD(P)H oxidases, are similar in structure to the neutrophilNADPH oxidase, which consists of 4 major subunits: a

Received May 26, 2000; revision accepted August 10, 2000.From the Division of Cardiology, Emory University, Atlanta, Ga.Correspondence to Kathy K. Griendling, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, 319 WMB, Atlanta, GA

30322. E-mail [email protected]© 2000 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol.is available at http://www.atvbaha.org

2175

Brief Review

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cytochromeb558, comprising gp91phox and p22phox, and 2cytosolic components, p47phox and p67phox. A member ofthe low-molecular-weight G protein rac family participates inthe assembly of the active complex. Table 1 summarizes theexpression of the major phox subunits in vascular cells.Although the expression pattern of these molecules has beendemonstrated, with the exception of p22phox in vascularsmooth muscle cells (VSMCs)12 and endothelial cells13 andrac15 and p67phox in fibroblasts,14 it remains to be deter-mined which subunits participate in functional complexes inspecific cell types and/or whether as-yet-unidentified proteinstake part in O2

2z formation. If cardiovascular cells contain aneutrophil-like oxidase, it is essential to identify the electrontransport moiety of the protein. Although gp91phox mayserve this function in endothelial and adventitial cells, itsapparent absence in SMCs suggests that a substitute mustexist. Recently, several homologues of gp91phox have beencloned, and one of them, termed mox-1, formitogenicoxidase(now known as nox-1, forNADPH oxidase), has been shownto be expressed in VSMCs.15 In these cells, nox-1 mediatesthe proliferative response to serum, and nox-1 antisenseattenuates O22z production in response to platelet-derivedgrowth factor (PDGF).15 Two other nox proteins have alsobeen found: a 138-kDa protein (tox-1) that is the main, if notthe sole, component of thethyroid oxidase,16 and a 578–amino acid protein, renox, that is expressed mainly in thekidney.17 Expression of these oxidases in vascular cells andtheir interaction with other phox subunits remain to bedetermined.

Regulation of ROS Production by VasoactiveAgonists and Mechanical ForcesThere is good evidence for agonist-induced ROS productionin both SMCs and endothelial cells. One of the first reportsthat the vascular NAD(P)H oxidase was hormone sensitiveshowed that Ang II treatment of SMCs increases intracellularO2

2z production.4 Ang II–stimulated O22z is converted to H2O2

as early as 1 minute after addition of hormone.18 Superoxideproduction in response to Ang II occurs when either NADHor NADPH is used as a substrate and is inhibitable bydiphenylene iodonium (DPI), a compound that binds to andinhibits flavin-containing oxidases; Tiron, an O2

2z scavenger;N-acetylcysteine (NAC), which increases intracellular gluta-thione pools; and SOD.4 Treatment with antisense p22phox todepress NAD(P)H oxidase expression also blocks Ang II–induced O2

2z production.12 Activation of this oxidase by AngII appears to involve arachidonic acid metabolites,19 perhapsderived ultimately from phospholipase D–mediated phos-phatidylcholine hydrolysis.20 Ang II also stimulatesNAD(P)H-dependent O22z production in endothelial cells21–23

and adventitial fibroblasts.14

Other agonists and mechanical forces have also beenshown to increase ROS production in vascular cells. PDGF,thrombin, TNF-a, and lactosylceramide activate NAD(P)Hoxidase–dependent O2

2z production in SMCs.6,7,24–26Fibro-blasts exhibit increased NADH- or NADPH-driven O2

2zproduction in response to TNF-a, IL-1, and platelet-activating factor.27,28 In endothelial cells, mechanical forces,including cyclic stretch and laminar and oscillatory shearstress, stimulate NAD(P)H oxidase activity.29,30The upstreamsignals responsible for oxidase activation in each of these celltypes with each of these stimuli remain to be established.

Signal Transduction Pathways Modulated by ROSIn order for ROS to modify the response of a cell to anagonist, it must affect specific signaling cascades. Over thepast several years, many redox-sensitive proteins have beenidentified, and in some cases, it has been shown that hor-monal activation is mediated by ROS. Often, both redox-sensitive and redox-insensitive pathways contribute to acti-vation of a particular enzyme (Figure 2). The relationshipbetween signaling cascades known to respond to ROS isdepicted in Figure 2, and each pathway is discussed individ-ually below.

Proximal Tyrosine KinasesGrowing evidence indicates that the epidermal growth factorreceptor (EGF-R) and the PDGF receptor (PDGF-R) serve

Figure 1. Redox “rheostat” in vascular cells. The oxidative stateof vascular cells depends on the balance between the produc-tion of oxidants and the antioxidant defenses of the cell. Extra-cellular stimulants such as Ang II and TNF-a or hypercholester-olemia can shift the balance to a pro-oxidant state, whereasexposure to extracellular chemical antioxidants (DPI, Tiron,NAC, pyrrolidine dithiocarbamate [PDTC], or probucol) orupregulation of antioxidant enzymes (SOD, catalase, or glutathi-one peroxidase) produces a more reductive environment.

TABLE 1. Expression (1) of Phagocytic Oxidase (phox) Components inVascular Cells

VSMCs Endothelial Cells Adventitial Cells

mRNA Protein mRNA Protein mRNA Protein

gp91phox 2 (13) 2 (13) 1 (13, 112, 113) 1 (13) ND 1 (114)

p22phox 1 (13, 115) 1 (13) 1 (13, 112, 113) 1 (13) ND 1 (114)

p47phox 1 (7) 1 (7) 1 (112, 113) 1 (112) ND 1 (114)

p67phox 2 (7) 2 (7) 1 (112, 113) 1 (112) 1 (14) 1 (114)

References are given in parentheses. ND indicates not determined.

2176 Arterioscler Thromb Vasc Biol. October 2000



not only as receptors for EGF and PDGF, respectively, butalso as a scaffold for assembly of signaling complexes by Gprotein–coupled receptors such as those for Ang II.31,32 It isof interest that transactivation of both of these growth factorreceptors is redox sensitive. In SMCs, H2O2 induces tyrosinephosphorylation of the EGF-R and stimulates its associationwith Shc (src homology complex)–Grb2 (growth factor re-ceptor–bound protein 2)–Sos (son-of-sevenless) complex toactivate subsequent signaling cascades (Figure 2).33 Further-more, Ang II–induced EGF-R transactivation is mediatedthrough NAD(P)H oxidase–derived ROS because it isstrongly inhibited by several antioxidants in SMCs and byNAC in cardiac fibroblasts.34,35 Heeneman et al36 have mostrecently reported that Ang II–induced phosphorylation of theShc/PDGFb-R complex is mediated by ROS.

Although phosphorylation of the EGF-R by Ang II is redoxsensitive, phosphorylation by EGF is not, suggesting that aneven more proximal kinase than the EGF-R exists. Recently, wehave shown that this kinase is c-Src.34 c-Src is an importantsignaling molecule with many functions: it phosphorylatesphospholipase C-g37; forms complexes with the EGF-R,32 pax-illin, 38 and Janus kinase (JAK)-239; and mediates activation ofmitogen-activated protein kinases (MAPKs).40 In mouse fibro-blasts, H2O2 directly activates c-Src.40 Moreover, Ang II–induced c-Src phosphorylation at both the autophosphorylationsite (Y418) and the SH2-domain (Y215) is inhibited by antioxi-dants, suggesting that in VSMCs, H2O2 is a proximal mediator ofagonist-induced c-Src activation.34

Another signaling molecule that is activated quite earlyafter receptor stimulation is the low-molecular-weight GTP-binding protein Ras. Ras has a dual role in redox-sensitivesignaling: it mediates activation of the NADH/NADPH oxi-dase to generate intracellular ROS,5 and it is also activated byROS in vivo and in vitro.41–43 ROS activate Ras via anoxidative modification of cysteine-118, leading to inhibitionof the GDP-GTP exchange.42 Moreover, ROS-triggered Rasactivation induces recruitment of phosphatidylinositol 39-kinase to Ras, an event that is required for activation ofdownstream signals such as Akt and MAPK (Figure 2 andbelow).44

Mitogen-Activated Protein KinasesThe MAPKs are a family of serine/threonine kinases thatcontrol cellular responses to growth, apoptosis, and stresssignals. There are 4 main MAPKs, including extracellularsignal–regulated kinases (ERK1/2), c-JunN-terminal kinases(JNKs, also termed SAPKs), p38 MAPKs, and big MAPK-1.These proteins are the best studied in terms of their redoxsensitivity. In SMCs, H2O2 has been shown to activate p38MAPK,45,46JNK,46 and big MAPK-1.47 Its effects on ERK1/2are controversial, with some reports showing inhibition andothers demonstrating stimulation.45,46,48,49In terms of agonist-induced activation of these enzymes, it has been clearlydemonstrated that p38 MAPK and JNK activation by Ang IIis inhibited by antioxidants (DPI, NAC), p22phox antisense,or overexpression of catalase.45,50Recently, it has been shownthat arachidonic acid stimulates JNK via Rac-1–dependentH2O2 production.51 Because arachidonic acid is produced inresponse to many vasoactive hormones, this may represent acommon mechanism of activation. Moreover, althoughPDGF-induced ERK1/2 phosphorylation is inhibited by in-cubation with catalase,25 Ang II activation of these enzymesis not.45,49,50

In endothelial cells, H2O2 activates p38 MAPK and itsdownstream target, MAPK-activated protein (MAPKAP) ki-nase 2/3, leading to phosphorylation of heat-shock protein 27(Hsp27).52,53 ERK1/2 activation also seems to be redoxsensitive in this cell type, based on the observation that shearstress–induced ERK1/2 phosphorylation is inhibited by anti-oxidants and dominant-negative Rac-1.54 In neonatal ratventricular myocytes, all 3 MAPKs (ERK1/2, p38 MAPK,and JNK) have been demonstrated to be activated by H2O2.55

Thus, regulation of MAPK activity by ROS varies not onlyamong family members but also among cells.

Figure 2. Redox-sensitive signaling pathways in vascular cells.G protein–coupled receptor agonists, mechanical forces, andgrowth factors activate both redox-sensitive and redox-insensitive signaling pathways. Pathways linked by a solid lineare supported by experimental evidence; dotted lines depictpathways in which a relationship has been suggested but notproved. When a G protein–coupled receptor is activated, phos-pholipases produce soluble and lipid second messengers thatlead to activation of the NAD(P)H oxidase. Superoxide and H2O2

produced by this enzyme modify the activity of tyrosine kinasessuch as c-Src, Fyn, and the EGF receptor kinase, as well asserine/threonine kinases including p38MAPK, JNK, big MAPK,and Akt. Redox-sensitive and -insensitive pathways converge tostimulate downstream growth-related enzymes such as p70S6Kand p90RSK and to activate transcription factors leading toexpression of redox-sensitive genes. PAF indicates platelet-activating factor; PLC, phospholipase C; PLD, phospholipase D;DG, diacylglycerol; AA, arachidonic acid; PKC, protein kinase C;PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; andSAPK, stress-activated protein kinase. See text for explanationof other abbreviations.

Griendling et al ROS and Signal Transduction 2177



AktThe recently identified serine/threonine kinase Akt/proteinkinase B has been shown to play a key role in many cellularprocesses, including cell survival and protein synthesis.56 Aktinhibits glycogen synthase kinase 3 and activates p70S6K andthe transcription factors activator protein (AP)-1 and E2F.56

Similar to p38 MAPK, both exogenous H2O2 and Ang IIactivate Akt in SMCs.57 Ang II–induced Akt phosphorylationis inhibited by DPI or overexpression of catalase, suggestinga role for NAD(P)H oxidase–derived ROS in agonist-inducedAkt activation. H2O2 stimulation of Akt has also beenreported in other nonvascular cell types, including NIH3T3fibroblasts, human embryonic kidney 293 cells, and HeLaand Jurkat cells.58–60 It is noteworthy that Konishi et al59

demonstrated that H2O2-induced Akt activation caused asso-ciation with Hsp27, which itself is also phosphorylated byH2O2.52,61 Furthermore, MAPKAP kinase-2, a substrate ofp38 MAPK,62,63 can phosphorylate Akt in vitro,64,65 raisingthe possibility that H2O2 may phosphorylate both Akt andHsp27 by activation of p38 MAPK.

Other Candidate Redox-Sensitive EnzymesMost likely, we have only scratched the surface of the cadreof oxidant-sensitive signaling pathways. Many proteins, in-cluding phospholipase D, Fyn, proline-rich tyrosine kinase(Pyk) 2, JAK2, and signal transducer and activator of tran-scription (STAT) 1, appear to be redox sensitive, based ontheir activation by addition of exogenous ROS. For example,H2O2 and lipid hydroperoxides activate phospholipase D inendothelial cells.66 In mouse fibroblasts, H2O2 activates JAK2via Fyn kinase, resulting in the stimulation of Ras activity.67

Pyk2 has also been reported to be redox sensitive, becauseH2O2 and the strong oxidant diamide both increase Pyk2phosphorylation.68 Furthermore, PDGF-induced STAT acti-vation is inhibited by antioxidants such as NAC and DPI.69

Although, for the most part, the role of ROS in activation ofthese pathways by agonists has not been studied, their clearrelationship with ROS suggests that they are potentiallyamong the proteins that mediate redox-sensitive physiologi-cal responses.

Regulation of Gene Expression by ROSBecause multiple hormones and growth factors alter tissueand intracellular levels of ROS and various critical signalingpathways are activated by ROS, it is not surprising that manycardiovascular-related genes are redox sensitive. Perusal ofTable 2 indicates that ROS regulate several general classes ofgenes, including adhesion molecules and chemotactic factors,antioxidant enzymes, and vasoactive substances. Some ofthese are clearly an adaptive response, such as the inductionof SOD and catalase by H2O2.70 Most redox-sensitive geneshave been identified because they are responsive to externallyapplied oxidant stress; only a few have been demonstrated tobe downstream of an endogenous source of ROS, such as theNAD(P)H oxidase. These include TNF-a and lactosylceram-ide induction of intercellular adhesion molecule (ICAM-1)26,71 and Ang II, PDGF, and TNF-a stimulation of mono-cyte chemotactic protein (MCP)-1.24,72 In contrast,stimulation of MCP-1 by IL-1b24 in VSMCs is not affected

by antioxidants, suggesting that the control of gene expres-sion by ROS is both stimulus and tissue specific.

Induction of several genes by cytokines is inhibited by NOdonors, including vascular cell adhesion molecule (VCAM)-1,73,74 ICAM-1,73 and monocyte colony-stimulating factor(M-CSF).75 This is an interesting mechanism of regulationbecause NOzappears to act in a cGMP-independent mannerto inhibit expression at the level of transcription.76 Not onlycan NOz alter the activity and expression of transcriptionfactors, but also it scavenges O2

2z to form peroxynitrite, thusmodulating O2

2z-dependent transcription as well.Regulation of gene expression by oxidant stress occurs at

various levels. In some cases, regulation of the gene is redoxsensitive owing to the susceptibility of upstream signalingpathways to ROS. For example, induction of early growthresponse (Egr)-1 by cyclic strain has been shown to dependon redox-sensitive activation of the Ras-Raf-ERK1/2 path-way.77 Moreover, H2O2-induced AP-1 binding in porcineaortic endothelial cells requires activation of Src.78 In othercases, ROS mediate increased turnover, expression, or trans-location of specific transcription factors, thus modifying theiractivity. This mechanism has been shown to be effective forboth the nuclear factor (NF)-kB and AP-1 transcriptionfactors. Hydroperoxy fatty acids and H2O2 increase theexpression of Fos and Jun, 2 proteins that form heterodimersand activate AP-1.79 NOz increases the transcription of IkB,the inhibitory factor that binds NF-kB and causes retention ofthis transcription factor in a cytoplasmic, inactive form.73 Theturnover of IkB protein is also oxidant sensitive: antioxidantscan prevent agonist-stimulated IkB phosphorylation and deg-radation.73 Conversely, H2O2 increases nuclear translocationof NF-kB, contributing to the induction of genes responsiveto this transcription factor.78

An additional level of redox regulation of gene expressionis that the affinity of certain transcription factors for theircognate DNA-binding sites can be directly modified by ROS.This mechanism was first identified in bacteria, where excessH2O2 interacts with the oxyR regulon, and O2

2z or NOzactivates the SoxRS regulon to control the expression of asubset of genes, including MnSOD and aconitase.80 TheoxyR-binding motif has also been shown to function as aredox-sensitive transcriptional enhancer in mammaliancells.81 Since then, several mammalian transcription factorshave been shown to be directly modified by ROS or byreducing proteins that modify cysteine residues involved inDNA binding. Transcription factors in this category includeAP-1, NF-kB, and most likely hypoxia-inducible factor(HIF)-1.82,83Both Fos and Jun have a conserved cysteine in abasic motif that, when oxidized, interferes with the binding ofthese proteins to AP-1 consensus sequences. Conversely, ifFos/Jun heterodimers are bound to AP-1, they cannot beoxidized.82 The oxidation state of these important proteins iscontrolled by redox factor (REF)-1, a protein that, in coop-eration with thioredoxin, promotes the cycling of the criticalcysteines between reduced and oxidized forms.82,84 Thiore-doxin also regulates HIF-1–dependent transcription83 andmodifies the DNA binding and transcriptional activity ofNF-kB by reducing cysteine 62.85 These studies clearly




indicate the importance of the nuclear redox state in regulat-ing gene expression.

Role of ROS in Vascular Physiologyand Pathophysiology

The intracellular and extracellular production of ROS and theconsequent activation of specific signaling pathways andinduction of redox-sensitive genes coordinate several inte-grated physiological responses in cardiovascular tissue, in-cluding growth of smooth muscle, induction of an inflamma-tory response, impairment of endothelium-dependentrelaxation, and cardiac hypertrophy. Each of these responses,when uncontrolled, contributes to vascular disease.

Vascular Smooth Muscle Growth, Hypertrophy,and ApoptosisA characteristic of hypertension is hypertrophy of largevessels.86 We have demonstrated that Ang II–induced hyper-trophy of SMCs is dependent on intracellularly producedH2O2, which is derived, at least in part, from an NAD(P)Hoxidase.4,12,18 Ang II–induced hypertrophy can be inhibitedby DPI,4 attenuation of NAD(P)H oxidase activity by trans-fection of antisense p22phox,12 and catalase overexpression.18

Similar findings were reported for cardiac myocytes, in whichAng II–induced hypertrophy was associated with intracellularproduction of ROS and was blocked by antioxidants.87

Other vascular disorders such as restenosis have a signif-icant proliferative component, resulting from SMC and/orfibroblast migration and multiplication in the neointima.88

TABLE 2. Redox Sensitivity of Gene Expression in Cardiovascular Cells

Gene Cell Type Stimulus Reference

VCAM-1 Endothelial cells TNF-a, IL-1a, IL-1b, IL-4 116, 117

ICAM-1 Endothelial cells TNF-a, NO, lactosylceramide 26, 71, 73, 116

E-selectin Endothelial cells IL-1a, LPS, PMA, TNF-a 73, 117, 118

MCP-1 Mesangial cells TNF-a 24, 72, 106, 119

VSMCs PDGF

VSMCs Ang II

VSMCs TNF-a

MCSF Endothelial cells TNF-a, ox-LDL 75, 76, 119

Endothelial cells H2O2, TNF-a

Mesangial cells TNF-a

eNOS Endothelial cells Xanthine/xanthine oxidase 120

iNOS Mesangial cells IL-1b 121

Cu/Zn-SOD Endothelial cells H2O2 70

Catalase Endothelial cells H2O2 70

Glutathione peroxidase Endothelial cells H2O2 70

Mn-SOD Endothelial cells Thioredoxin 122

HO-1 Endothelial cells H2O2, shear stress 30, 123, 124

Macrophages Ox-LDL

VSMCs PDTC

COX-2 Mesangial cells IL-1b 89, 121

VSMCs Catalase overexpression

HSP-70 Endothelial cells H2O2 70, 125

Xanthine/xanthine oxidase

Scavenger receptor VSMCs PMA, H2O2/vanadate 126

Macrophages

IL-8 Microvascularendothelial cells

H2O2 127

HB-EGF Endothelial cells H2O2 128, 129

VSMCs Methylglyoxal

Atrial natriuretic factor Cardiacmyocytes

Ouabain 130

VEGF Endothelial cells H2O2 131, 132

VSMCs H2O2, 4-hydroxynonenal

e/i NOS indicates endothelial/inducible nitric oxide synthase; HO-1, heme oxygenase-1; COX-2, cyclooxygenase-2;HB, heparin binding; LPS, lipopolysaccharide; PMA, phorbol myristate acetate; ox, oxidized; and PDTC, pyrrolidinedithiocarbamate.




Sundaresan et al25 demonstrated a clear requirement for H2O2

in PDGF-induced proliferation. Migration in response to thisagonist is also inhibited by catalase, suggesting that it, too, ismediated by ROS. Similar results were found by Brown etal,89 who showed that overexpression of catalase in SMCs notonly inhibited serum-induced [3H]thymidine incorporationand proliferation but also promoted apoptosis. Phenylephrine-induced proliferation of rabbit aortic SMCs has also beenshown to require H2O2.90 Proof that balloon angioplastyincreases oxidant stress has been provided in 2 studies.Within 30 minutes after injury, glutathione levels fall by63%, coincident with medial smooth muscle apoptosis, sug-gesting that this early step in the response to injury isassociated with severe oxidant stress. Importantly, adminis-tration of NAC or pyrrolidine dithiocarbamate prevents theglutathione loss and the smooth muscle apoptosis.91 Inanother study, Nunes et al92 showed that vascular O22z wasincreased 2.5-fold in injured arteries compared with uninjuredcontrols. Moreover, treatment with either probucol or thecombination of vitamins C and E normalized O2

2z levels andpartially suppressed neointimal formation.93 Davies et al94

have recently reported that p38 MAPK is upregulated afterinjury, suggesting that this signaling pathway might also be aredox-sensitive target in vivo.

Endothelial DysfunctionEndothelial dysfunction is a hallmark of multiple vasculardiseases, including hypertension, atherosclerosis, and diabe-tes mellitus. Impaired endothelial function has several con-sequences, the most important of which is decreased endo-thelium-dependent vasodilation. The endothelial cell redoxrheostat is primarily regulated by the dynamic production ofand interaction between NOz and O2

2z. NOz is the most potentendogenous vasodilator and inhibits smooth muscle prolifer-ation and migration, adhesion of leukocytes to the endothe-lium, and platelet aggregation.95 In cholesterol-fed rabbits,O2

2z is increased in the aorta,96 and treatment with polyeth-ylene glycol–SOD reverses the impairment in endothelium-dependent relaxation.97 In the same animal model, treatmentwith probucol (a lipid-lowering agent with potent antioxidantproperties) corrects endothelial dysfunction and lowersO2

2z.98 Impaired endothelium-dependent vasodilation alsooccurs in hypertension, such as that produced by infusion ofrats with Ang II,3 restriction of blood flow to 1 kidney,99 andadministration of deoxycorticosterone acetate-salt.100 Theendothelial dysfunction that accompanies Ang II infusion ordeoxycorticosterone acetate-salt can be corrected by admin-istration of liposomal or matrix-targeted SOD,100–102provid-ing further proof that ROS, and specifically O2

2z, are involvedin this response.

The Inflammatory ResponseAnother consequence of endothelial dysfunction and SMCactivation is increased monocyte adhesion, foam cell forma-tion, and thrombosis. As noted above, pro-oxidant agonistssuch as Ang II and TNF-a induce the expression of proin-flammatory molecules such as VCAM-1, MCP-1, and thethrombin receptor.6,72,103–106Each of these molecules is inturn redox sensitive,72,104,107and in the case of MCP-1 and the

thrombin receptor, a role for ROS in Ang II–mediated geneexpression has been demonstrated.72,104

Matrix RemodelingCollagen degradation depends on the activity of enzymesknown as metalloproteinases (MMPs). MMP-2 (gelatinase A,which degrades collagen IV from the basal membrane) andMMP-9 (gelatinase B, which acts on collagen I fibers) aresecreted by macrophages and vascular myocytes in an inac-tive form.108 MMP-9 expression is increased in the shoulderregion of atherosclerotic plaques; ie, in the sites prone toplaque rupture.109 Rajagopalan et al110 demonstrated thatpro–MMP-9 and pro–MMP2 secreted into the medium ofcultured human SMCs are activated by ROS. Moreover, NACtreatment prevents MMP-9 expression and activation inhypercholesterolemic rabbits,111 suggesting a mechanism forhow antioxidants may contribute to plaque stabilization.

Conclusions and Future DirectionsMuch remains to be learned concerning the signaling path-ways and genes that are regulated by ROS. Because redox-sensitive responses appear at times to be cell specific, it willbe important to identify the sources of oxidant stress in eachcell, the mechanism of regulation of antioxidant enzymes,and the effect of ROS on signaling pathways specific to thefunction of that particular cell and to gain further insight intothe physiological responses affected by oxidant stress. Anunderstanding of these events will enable us to devisetherapeutic strategies to target specific cellular events con-tributing to vascular disease.

AcknowledgmentsThis review was supported by NIH grants HL38206, HL58000, andHL58863. The authors thank Carolyn Morris for excellentsecretarial assistance.

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Modulation of Protein Kinase Activity and Gene Expression by Reactive Oxygen Species and Their Role...

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