Proc. Nadl. Acad. Sci. USAVol. 91, pp. 1044-1048, February 1994Medical Sciences

Superoxide and peroxynitrite in atherosclerosisC. ROGER WHITE*, TOMMY A. BROCK*t, LING-YI CHANGt, JAMES CRAPOI, PAGE BRISCOE§, DAVID Ku¶,WILLIAM A. BRADLEY*, SANDRA H. GIANTURCO*, JERI GORE§, BRUCE A. FREEMAN§II**,AND MARGARET M. TARPEY§ttDepartments of *Medicine, Anesthesiology, lPharmacology, IBiochemistry, and **Pediatrics, University of Alabama, Birmingham, AL 35294; and*Department of Medicine, Duke University Medical Center, Durham, NC 27710

Communicated by Irwin Fridovich, July 26, 1993

ABSTRACT The role of reactive oxygen species in thevascular pathology asiated with atherosclerosis was examinedby testing the hypothesis that impaired vascular reactivityresults from the reaction of nitric oxide (NO) with superoxide(O2), yielding the oxidant peroynitrite (ONOO-). Contractilitystudies were performed on femoral arteries from rabbits fed acholesterol-supplemented diet. Cholesterol feeding shifted theEC5. for acetylcholine (ACh)-induced relaxation and impairedthe maximal response to ACh. We used pH-senitive liposomesto deliver CuZn superoxide dismutase (SOD; superoxide:super-oxide oxidoreductase, EC 1.15.1.1) to critical sites of NOreaction with O2. Intravenously Injected liposomes (3000 unitsof SOD per ml) augmented ACh-induced relaxation in thecholesterol-fed group to a greater extent than in controls.Quantitative immunocytochemistry demonstrated enhanced dis-tribution ofSOD In bothend al and vascular smooth musclecells as well as in the extr lar matrix. SOD activity in vesselhomogenates of liposome-treated rabbits was also increased.Incubation of (3 very low density lipoprotein with ONOO-resut in the rapid formation ofconjugated dienes and thiobar-bituric acid-reactive subs . Our results suggest that thereaction of °2with NO is involved in the development ofatherosclerotic disease by yielding a potent mediator of lipopro-tein oxidation, as well as by limiting NO stimulation of vascularsmooth muscle guanylate cydase activity.

Endothelium-dependent relaxation is impaired in vesselsfrom atherosclerotic patients (1, 2) and hypercholesterolemicanimal models (3-6), suggesting the functional modificationof endothelium-derived relaxing factor (EDRF) in hyperlip-idemia. The dynamic role ofthe endothelium in the regulationof vascular tone was established when it was observed thatrelaxation of isolated blood vessels by vasoactive agents,such as acetylcholine (ACh) and the calcium ionophoreA23187, was dependent on an intact endothelium and adiffusible factor (EDRF) that stimulated cGMP-dependentrelaxation of vascular smooth muscle cells (VSMCs; ref. 7).Nitric oxide (-NO) and EDRF share similar chemical andpharmacological properties (8) and are derived from theoxidation of a terminal guanidino group of L-arginine (9, 10).Numerous mechanisms have been suggested for the defect

in vascular relaxation in atherosclerosis and hypercholester-olemic animal models. They include an increased diffusionalbarrier for 'NO due to intimal cell proliferation and lipiddeposition (11), L-argifine depletion (3, 12, 13), alteredendothelial cell (EC) receptor coupling mechanisms (14), andinactivation of NO by superoxide (°2; refs. 15 and 16).Reactive oxygen species are potent pathologic mediators inatherosclerosis as well as other vascular diseases (17, 18).Both °- and hydroxyl radical ('OH) contribute to the oxi-dative modification of low density lipoproteins [i.e., low

density lipoprotein, very low density lipoprotein (LDL,VLDL); ref. 19], a critical event in development of theatherosclerotic lesion (20). In addition, reactive oxygen spe-cies have been implicated in the alterations of EC-dependentrelaxation observed in atherosclerosis (21, 22).

Peroxynitrite (ONOO-), a product of "NO reaction withOj, has recently been defined as a potent oxidant andpotential mediator of vascular tissue injury (23-27). Wepostulate that both the independent reactions of O2 and "NOand their reaction yielding ONOO- are critical in the initia-tion and maintenance of the atherosclerotic state and con-tribute to the defect in vasorelaxation. The present studiessuggest common mechanisms underlying lipoprotein oxida-tion and impaired vascular relaxation of hypercholesterol-emic rabbits.

MATERIALS AND METHODSMaterials. Bovine CuZn superoxide dismutase (SOD; su-

peroxide:superoxide oxidoreductase, EC 1.15.1.1) was fromDiagnostic Data Inc. (Mountain View, CA). Dioleoylphos-phatidylethanolamine and dioleoylglycero-3-succinate wereobtained from Avanti Polar Lipids. L-Arginine, ACh, indo-methacin, papaverine, phenylephrine, and tetraethylammo-nium hydroxide were from Sigma.

Vessel Contraction Studies. New Zealand White rabbits(2.5-3.0 kg) were maintained on rabbit chow containing 1%cholesterol (Ralston Purina) for 6 months prior to study[cholesterol-fed (Chol-fed) group]. Age- and weight-matchedcontrols were fed a standard diet. After exsanguination underketamine/rompun anesthesia, vessels were isolated andchanges in tension were measured in femoral artery ringsegments as described (28). After maximal contraction with 70mM KCl and recovery, phenylephrine was added to achieve30% of maximal tone. Rings were then exposed to increasingdoses of ACh; relaxation is reported as the percentage de-crease in preexisting tone. After the generation of cumulativeACh dose-response curves, rings were exposed to 30 ,uMpapaverine. In some experiments, rings from control andChol-fed rabbits were incubated with 3 mM L-arginine for 30min prior to administration of ACh. In other studies, vesselswere treated with native bovine SOD (200 units/ml) beforemeasuring ACh-induced relaxation. All studies were per-formed in the presence of 5 !uM indomethacin.

Abbreviations: ACh, acetylcholine; SOD, superoxide dismutase;VLDL, very low density lipoprotein; Lip-SOD, liposome-entrappedSOD; EDRF, endothelium-derived relaxing factor; VSMC, vascularsmooth muscle cell; EC, endothelial cell; LDL, low density lipopro-tein; Chol-fed, rabbits fed a 1% cholesterol diet; TBARS, thiobar-bituric acid-reactive substances.tPresent address: Texas Biotechnology Corp., Doctor's Center,Suite 1920, 7000 Fannin Street, Houston, TX 77030.ttTo whom reprint requests should be addressed at: Department ofAnesthesiology, 952 Tinsley Harrison Tower, University of Ala-bama, Birmingham, AL 35294.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 91 (1994) 1045

-.* 1 : Chol-fed

Control

0!

-25

-50

-75-100

T Col-fedChol-fed \t T

+ L-Arg

8 7 6 5 9 8 7 6 5-log[ACh]

FIG. 1. Endothelium-dependent relaxation of femoral arterial ring segments from rabbits fed a 1% cholesterol diet for 6 months and age- andweight-matched controls. (A) Vessels from Chol-fed rabbits (e) (n = 33) demonstrate an impaired maximal response to cumulative addition ofACh as well as a shift in EC5o compared to controls (v) (n = 33). (B) Preincubation with 3 mM L-arginine (v) (n = 7) for 30 min did not alterthe response to ACh in vessels of Chol-fed animals (e) (n = 33). Data are means ± SEM. *, P < 0.01.

Liposome-Entrapped SOD (Lip-SOD). Liposomes were

composed of dioleoylphosphatidylethanolamine and dio-leoylglycero-3-succinate (1:1). Lipids were dried under N2and hydrated 36 hr in 210 mM sucrose/7 mM Hepes. Duringhydration, pH 8.5 was maintained with tetraethylammoniumhydroxide. Lipids were added to SOD and dissolved insucrose/Hepes buffer, and the mixture was extruded througha 600-nm filter under N2 pressure (Extruder, Lipex Biomem-branes, Vancouver, BC Canada); mean liposome diameterwas 217 nm. Final SOD concentration was 3000 units/ml.Lip-SOD was injected daily (1500 units/kg) via the marginalear vein for 5 days before sacrifice.

Analytical Procedures. Plasma cholesterol levels were de-termined by an enzymatic method (29) modified for 96-wellplates. Aortic SOD activity was assayed in a 1o homoge-nate in 50mM KPi/0.1 mM EDTA/0.1% 3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonate, pH 7.8. Aftercentrifugation at 10,000 x g for 10 min at 40C, supernatantSOD activity was measured by inhibition of xanthine oxi-dase-mediated reduction of cytochrome c (30). Tissue DNAwas measured by fluorescence (31). Aortic segments wereprepared for quantitative electron microscopic immunocyto-chemistry as described (32). Ultrathin cryosections wereincubated with rabbit anti-bovine CuZn SOD (1:100 dilution)and 10-nm gold colloid conjugated to protein A. Distributionof SOD labeling was measured by counting gold granules andtissue points of randomly selected photographic fields. Be-cause of variable loss of endothelium during cryosectioning,EC SOD density was quantitated in selected segments withintact endothelium.

13-VLDL Oxidation. Peroxynitrite was synthesized as de-scribed (23). 3-VLDL, isolated as described (33), was ex-

posed to ONOO- or 5 9M CUS04. Lipid oxidation wasassessed by measurement ofthiobarbituric acid-reactive sub-stances (TBARS) and formation of conjugated dienes (25).

0

14

0

co

2501'

-25-50-75

-100

-AChol-fed

\ ControlControl

+Native SOD

8 7 6

RESULTSPlasma cholesterol was markedly elevated in rabbits fed a 1%cholesterol diet for 6 months, from 43 ± 6 to 2696 ± 292mg/dl. Intimal thickening was apparent, with extensiveplaque deposition, -=50% of the luminal surface of the fem-oral artery.

Control and Chol-fed groups exhibited no differences in themaximal tone generated with 70 mM KCl or in the phenyl-ephrine concentrations required to achieve submaximal con-traction of vessels. Chol-fed rabbits manifested a significantshift in the EC50 to ACh-induced relaxation: 1.73 pM vs.0.0521 gM (P < 0.01) as well as a 33% reduction in maximalresponse (Fig. 1A). Incubation of ring segments with L-argi-nine failed to augment ACh-induced relaxation in Chol-fedrabbits (Fig. 1B). Pretreatment with native CuZn SOD had aminor effect on the ACh dose-response profile in control butnot Chol-fed rabbits (Fig. 2A). Due to the limited cellularuptake of native SOD, we delivered SOD in vivo via pH-sensitive liposomes. Vessels isolated from both control andChol-fed rabbits treated for 5 days with Lip-SOD demon-strated enhanced ACh-induced relaxation (Fig. 2B). Lip-SOD restored ACh-induced relaxation in Chol-fed rabbitsclose to control values (EC50, 0.165 gM). In addition, theAEC50 for ACh-induced relaxation was greater (P < 0.01) inthe Chol-fed group than in the control group. Furthermore,Lip-SOD treatment enhanced the maximum relaxation ofsegments to ACh in Chol-fed rabbits. Treatment of bothcontrol and Chol-fed rabbits with empty pH-sensitive lipo-somes had no effect on ACh responses.There was no difference between groups in the response to

papaverine, an endothelial-independent vasodilator; vesselsrelaxed below the initial vessel tone measured before phen-ylephrine administration, 114.52% 3.13% (control) vs.

116.35% ± 6.51% (Chol-fed). Lip-SOD had no effect onresponses to papaverine in either group.

250*

-25

-50

-75-100

5 9-log[ACh

[B

ControlControl+Lip-S'OD ODho-fd

8 7 6 5

FIG. 2. Cumulative dose-response curves for ACh in femoral artery rings from Chol-fed rabbits (.) (n = 33) and controls (n) (n = 33). (A)Separate groups of vessels from control (o) (n = 21) and Chol-fed (o) (n = 15) rabbits were incubated with CuZn bovine SOD (200 units/ml)before addition of ACh. Native SOD failed to improve EC-dependent relaxation in segments from Chol-fed animals. (B) Control (o) (n = 20)and Chol-fed (o) (n = 30) rabbits were treated with Lip-SOD for 5 days prior to study. Lip-SOD enhanced ACh-induced relaxation in both groups.The AEC50 for ACh-induced relaxation as well as the change in maximal relaxation was greater in the Chol-fed group. Data are means ± SEM.*, P < 0.01; **, P < 0.05.

o 0'0

0

- 25

) -50bo

.= -75

e -100

Medical Sciences: White et al.

I

1046 Medical Sciences: White et al.

Table 1. Vascular enzymatic activity and tissue distribution of CuZn SOD delivered viapH-sensitive liposomes

- SOD liposomes + SOD liposomes

Measurement Control Chol-fed Control Chol-fedEnzymatic activityt 0.92 ± 0.13 1.06 ± 0.43 1.73 ± 0.46* 1.73 ± 0.55*Tissue distributionsEndothelium 0.70 ± 0.02 1.53 ± 0.27**Smooth muscle 0.99 ± 0.08 1.88 ± 0.11**Extracellular matrix 0.40 ± 0.12 1.18 ± 0.14**Collagen 0.31 ± 0.06 1.16 ± 0.32**Elastin 1.95 ± 0.16 2.43 ± 0.33

SOD activity is expressed as mean ± SD ofaortae from three animals from each group. Morphometricdata are means ± SD from analysis of four sites from each treatment group. Because there was nodifference in SOD distribution between control and Chol-fed groups, the groups were combined andanalyzed for treatment with and without Lip-SOD. *, P < 0.01; **, P < 0.05.tUnit(s) of SOD per jg of DNA.*Point density of colloidal gold particles x 10-2 (relative ratio of no. of immunogold particles per tissuepoint).

Quantitative electron microscopic immunocytochemistryassessed the distribution of SOD in the blood vessel wall.Lip-SOD localized not only in the endothelium but also in thesubendothelial space and in underlying VSMCs (Table 1; Fig.3). A small degree of crossreactivity was evident between

a

rabbit anti-bovine SOD and endogenous rabbit SOD. How-ever, multivariate analysis of labeling densities showed sig-nificant increases in labeling in sections from Lip-SOD-injected rabbit vessels compared with controls (Table 1).SOD activity was similar in vessel homogenates from control

..i~~~~~~~~~~~~~~~K.Z.W.XJ!2~~~~~~~~~~~~~~~~~~~~~~~~. ,,ea

Col .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..

1

FIG. 3. Electron micrographsof aortic segments from controland Lip-SOD-treated rabbits. (a)A small degree of crossreaction ofthe anti-bovine CuZn SOD to rab-bit CuZn SOD was observed in theECs from control rabbits. (b) La-beling of ultrathin cryosectionswith anti-bovine SOD (arrow-heads) demonstrates enhancedSOD distribution in the endothe-lium, smooth muscle, and extra-cellular matrix of the Lip-SOD-treated animal. m, Mitochondria;d, desmosome; Col, collagen fi-brils; El, elastin. (Bars = 0.5 gm.)

Proc. Natl. Acad Sci. USA 91 (1994)

R!4:, ;-

I-i:, 1,s:,:;,.:::........ :z

Proc. Natl. Acad. Sci. USA 91 (1994) 1047

600 B

2500 ONOO- _r.o 400

3300

0 200-

p 100

ONOO-

2.25 / ~~~~~~~~~~~.............................../ * ~~CU2+

0.75 ..,.................

000-I*0 200 400 600 800 1000Time, min

u 30 300Time, min

FIG. 4. Peroxynitrite-induced lipid oxidation in /3VLDL. (A) Conjugated diene formation: 3-VLDL (10 pg of protein per ml in 50mM KPi,pH 7.4) was incubated with 5 1LM Cu2+ (...*) or 100 ILM ONOO- (-) and the AA234 was recorded. Tracings are representative of lipoproteinsfrom three rabbits. (B) TBARS formation: (3-VLDL (100 jg of protein per ml in 50 mM KP1, pH 7.4, 370C) was incubated with ONOO- (n) orCu2+ (o). A 30-min exposure to ONOO- resulted in a 4-fold increase in TBARS formation, while 5 A&M Cu2+ did not increase TBARS for 300min. Data are presented as average percentage control ± SD (n = 8). *, P < 0.01.

and Chol-fed rabbits (Table 1). Lip-SOD led to an almost2-fold increase in vessel SOD activity in both groups.Exposure of f-VLDL to ONOO- resulted in rapid gener-

ation of lipid oxidation products as indicated by both TBARSand conjugated diene formation (Fig. 4). Control experimentsdemonstrated that up to 100 ,uM "NO alone did not stimulatelipid peroxidation.

DISCUSSIONThe present studies suggest that defective EC-dependentrelaxation in a rabbit model of hypercholesterolemia is due inpart to the reaction of EDRF/NO with 02. Numerousmechanisms have been postulated for the impaired EC-dependent relaxation of hypercholesterolemic blood vessels.Intimal thickening as a barrier to EDRF does not appear tobe crucial, as cessation of a high cholesterol diet restoresEC-dependent relaxation in Cynomolgus monkeys despitethe continued presence of thickened intima (34).

Vessels from Chol-fed rabbits did not differ in their re-sponse to an EC-independent vasodilator, in agreement withother investigators (21), suggesting that the ability ofVSMCsto relax is intact. Other reports propose that impaired EC-dependent relaxation is due to L-arginine depletion, thesubstrate for NO synthesis (3, 12, 13). However, we andothers (35) were unable to restore vascular relaxation inarterial segments pretreated with arginine. Further evidenceagainst L-arginine depletion includes chemiluminescent tech-niques demonstrating that the basal release of NO and itsmetabolites are increased in the aorta of hypercholesterol-emic rabbits (36). This suggests that EDRF/NO synthesis iselevated and that impaired relaxation may be due to en-hanced inactivation of EDRF/'NO, leading to a reduction inits vasoactive properties.

Reactive oxygen species have been implicated in both thepathogenesis and altered physiologic responses of athero-sclerosis. Oxidation of LDL, a critical event in atheromaformation, is associated with enhanced cellular production of0° (37, 38). The efficacy of probucol in the prevention ofintimal lesion development in Watanabe hyperlipidemic rab-bits is related to its antioxidant properties (39). In addition,the presence of oxidized LDL in atheromatous plaquescorrelates with progression of atherosclerotic carotid disease(40). Oxidized LDL has also been shown to directly inhibitEC-dependent vasorelaxation (41, 42). Generation of 0° insitu reduces endothelial-dependent relaxation in normal ves-sels (16, 43) and may be involved in the abnormal EC-dependent relaxation in atherosclerosis (22). Indirect mea-sures of excess production of reactive oxygen species inhypercholesterolemia have been reported, including elevatedlevels of cholesterol oxides and oxidant-modified proteins

(44, 45). However, the precise role of oxidants and theircritical reaction sites in the initiation of lipoprotein oxidationand impairment of vascular function in atherosclerotic pro-cesses remains unclear.We suggest that a cholesterol-enriched environment en-

hances vascular production of 02. This excess °2 can thenreact with *NO, reducing the vasoactive levels of NO anddiminishing the response to EC-dependent vasodilators. Inthis case, normal or even elevated levels of SOD may beinsufficient to effectively scavenge the excess 0j. This issuggested by the slightly higher endogenous levels of SODmeasured in Chol-fed animals. The hypercholesterolemicstate may lead to increased expression of vessel SOD (45, 46),but in our studies it was not significantly different fromcontrols. Impaired EC-dependent relaxation may then be dueto enhanced reaction of NO with O°-, yielding ONOO- (23).This reaction is rapid, with a rate constant of k = 6.7 x 109M'Ls-1, faster than both the SOD-catalyzed and spontane-ous dismutation of °- to H202 (47). At physiological pH,ONOO- is protonated to peroxynitrous acid (ONOOH),yielding nitrogen dioxide ( NO2) and a molecule with OH-likereactivity (23, 48). We have observed that ONOO- is a lesspotent activator of VSMC guanylate cyclase than *NO (un-published observations) and suggest that EC-dependent re-laxation is impaired in our model due to diminished rates ofcGMP formation. Alternatively, the impaired relaxation maybe the result of ONOO--derived OH, which stimulatesguanylate cyclase activity to a lesser extent than "NO (49).

Peroxynitrite exhibited potent oxidative effects on,3VLDL, the principal carrier of cholesterol in this model.When compared to 5 ,M Cu2+, a commonly used in vitrolipoprotein oxidant (50), ONOO- rapidly generated greaterquantities of lipid peroxidation products. Similar results havebeen observed using the syndominine, SIN-1, a compoundyielding "NO and O°, which react to form ONOO- (51). Thus,the product of NO and 0° reaction may play a critical rolein the initiation and extension of atherosclerotic lesions aswell as the altered vascular reactivity associated with thedisease.These studies also demonstrate that pH-sensitive lipo-

somes are effective vectors for the delivery of antioxidantenzymes to the wall of both normal and atheroscleroticvessels. Pharmacologic efficiency of Lip-SOD, as indicatedby enzymatic analysis and changes in vascular function,contrasts with the minimal effect of native SOD. It is notunexpected that native SOD did little to restore EC-dependent relaxation. The 32-kDa SOD is electrostaticallyrepelled from cell surfaces at pH 7.4 and is thus excludedfrom intracellular compartments, where significant extents ofboth the production and reactions of °- and NO will occur.

Medical Sciences: White et al.

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1048 Medical Sciences: White et al.

pH-sensitive liposomes facilitated delivery of SOD tointernal sites ofO2 production, thus lowering steady state °2concentration and limiting O2 reaction with NO to yieldONOO-. Once injected intravenously, liposomes gain directcontact with the blood vessel wall and become incorporatedvia endocytosis. In the acidic environment of the endosome(pH 5.0), the liposomal membrane undergoes a phase tran-sition (52), promoting liposome-endosome fusion and releaseof liposomal contents to the cytosol. An increase in SODcontent was demonstrated in both control and Chol-fedliposome-injected rabbits. Parallel immunocytochemicalquantitation of SOD distribution reveals that SOD gainedaccess not only to ECs but to VSMCs and the interstitium aswell. These locales are all critical sites of excess productionof oxidants associated with the development and mainte-nance of atherosclerosis. Thus, Lip-SOD delivery is partic-ularly effective in raising tissue levels of the enzyme. Poly-ethylene glycol-derivatized SOD (PEG-SOD) to enhancevascular endothelial antioxidant enzyme levels has beenpreviously used (53). However, higher doses of PEG-SOD(41,000 units kg-l d-1 vs. 1500 units-kg-1ld-l) were used toachieve similar results in an animal model of atherosclerosis(54).

In summary, our findings suggest that an imbalance invascular °2 and NO arises in response to a hyperlipidemicenvironment. Impaired vascular relaxation results in partfrom the reaction of NO with O°. The ONOO- formed as aproduct of this reaction can then participate in the pathogen-esis ofthe atherosclerotic lesion in two ways. First, impairedvascular reactivity results from the production of ONOO-,which acts as a relatively weak stimulus for guanylate cyclaseactivity in VSMCs. Second, ONOO- can initiate lipoproteinoxidation, which then contributes to the production of thefatty streak and subsequent plaque formation characteristicof the atherosclerotic lesion, and may also contribute toaltered EC-dependent vascular relaxation.

We wish to thank Drs. J. S. Beckman and M. Winn for theirtechnical expertise. This work was supported by National Institutesof Health Grants P01-HL 48676, R01-HL 41180, and T32-HL 07457and by grants from the Alabama Affiliate, American Heart Associ-ation. C.R.W. is the recipient of a Harriet P. Dustan fellowship fromthe Alabama Affiliate, American Heart Association; M.M.T. is therecipient of a Parker B. Francis Fellowship for Pulmonary Research.

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Proc. NatL Acad. Sci. USA 91 (1994)