Increased NAD(P)H Oxidase and Reactive Oxygen Speciesin Coronary Arteries After Balloon Injury

Yi Shi, Rodica Niculescu, Dian Wang, Sachin Patel, Kelly L. Davenpeck, Andrew Zalewski

Abstract—Reactive oxygen species (ROS), produced by cellular constituents of the arterial wall, provide a signalingmechanism involved in vascular remodeling. Because adventitial fibroblasts are actively involved in coronaryremodeling, we examined whether the changes in the redox state affect their phenotypic characteristics. To this end,superoxide anion production and NAD(P)H oxidase activity were measured in porcine coronary arteries in vivo, and theeffect of ROS generation on adventitial fibroblast proliferation was examined in vitro. Superoxide production (SOD- andTiron-inhibitable nitro blue tetrazolium [NBT] reduction) increased significantly within 24 hours after balloon-inducedinjury, with the product of NBT reduction present predominantly in adventitial fibroblasts. These changes wereNAD(P)H oxidase–dependent, because diphenyleneiodonium (DPI) abolished superoxide generation (P,0.001).Furthermore, the injury-induced superoxide production was associated with augmented NAD(P)H oxidase activity andupregulation of p47phox and p67phox in adventitial fibroblasts (immunohistochemistry). Serum stimulation of isolatedadventitial fibroblasts produced time-dependent increases in ROS production (peak 3 to 6 hours). The inhibition of ROSgeneration with NAD(P)H oxidase inhibitor (DPI) or the removal of ROS with antioxidants (Tiron, catalase) abrogatedproliferation of adventitial fibroblasts. These results indicate that vascular NAD(P)H oxidase plays a central role in theupregulation of oxidative stress after coronary injury, providing pivotal growth signals for coronary fibroblasts.(Arterioscler Thromb Vasc Biol. 2001;21:739-745.)

Key Words: reactive oxygen speciesn NAD(P)H oxidasen coronary remodelingn adventitial fibroblast

Oxidative stress has been implicated in several stepsleading to the development of vascular disease.1,2 Initial

observations focused on reactive oxygen species (ROS)derived from invading macrophages and their possible in-volvement in oxidative lipid modifications in the vessel wall.Subsequently, it has become apparent that ROS are alsoproduced in a controlled fashion by all vascular cells and thatthey act as “second messengers” regulating various cellularfunctions. Several extracellular signals, such as growth fac-tors or even physical stimuli, induce ROS and their deriva-tives in vascular smooth muscle cells (SMCs) and fibroblasts,activating the intracellular growth program.3–6 For example,superoxide anion (zO22) increases expression of extracellularsignal–regulated kinase 1/2 mitogen-activated protein kinase,whereas H2O2 activates p38 mitogen-activated protein kinaseand stress-activated proteins.7,8 Furthermore, H2O2 also stim-ulates early proto-oncogenes and redox-sensitive transcrip-tional factors (eg, NF-kB and AP-1).9

See page 722In vascular cells, the major enzymatic source of intracel-

lular ROS is NAD(P)H oxidase, which generateszO22 by

1-electron reduction of molecular oxygen.10–12 AlthoughNADPH oxidase is responsible for the burst ofzO2

2 in

phagocytic cells, the generation of ROS in vascular cellsdiffers from that in neutrophils. In the former, it occurs overa period of hours (rather than minutes), appears to be mostlyintracellular (rather than extracellular and intracellular), andmay involve the assembly of different enzymatic subunits ofNAD(P)H oxidase.13 Significant progress has been madetoward the identification of NAD(P)H oxidase subunits innormal vascular cells and in atherosclerotic lesions, includingboth membrane-associated (p22phox) and cytoplasmic (p67phox,p47phox, Rac1) components.6,14 –16 The activity of theNAD(P)H oxidase in vascular cells is modulated by extracel-lular signals known to influence vascular remodeling andlesion development (eg, thrombin and angiotensin II).6,15–17

Furthermore, gene polymorphism affecting at least one of thesubunits (p22phox) has been linked to the development ofatherosclerosis in humans.18,19

The regulation of the redox state appears to be heteroge-neous across the vessel wall. Higher expression of NAD(P)Hoxidase andzO2

2 production have been reported in normaladventitia than in the media.20,21 The importance of thisfinding initially remained unclear, because the activation ofmedial SMCs and lipid peroxidation occur in the proximity ofthe arterial lumen. Several studies from our laboratory andothers, however, have suggested active involvement of ad-

Received December 4, 2000; revision accepted January 8, 2001.From the Cardiovascular Research Center, Department of Medicine (Cardiology), Thomas Jefferson University, Philadelphia, Pa.Correspondence to Yi Shi, MD, Thomas Jefferson University, Cardiovascular Research Center, Division of Cardiology, Suite 403D, 1025 Walnut St,

Philadelphia, PA 19107. E-mail yi.shi@mail.tju.edu© 2001 American Heart Association, Inc.

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

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ventitial fibroblasts in arterial repair.22–24 In particular, aftersevere coronary injury, these cells demonstrate preferentialproliferation and migration toward intima. This is not surpris-ing, because coronary SMCs display more advanced differ-entiation and a limited response to stimulation compared withnoncoronary SMCs.25,26 In view of our previous findings andthe established role of ROS in the regulation of cell prolifer-ation, we hypothesized that the increase in oxidative stressafter coronary injury involves adventitial fibroblasts. Theresults of this study demonstrated the upregulation ofNAD(P)H oxidase activity and ROS production in adventitialfibroblasts after coronary injury. In cell culture, ROS areimportant signals for growth response of coronary fibroblasts.We postulate that phenotypic responsiveness of coronaryfibroblasts to stimulation is mediated, in part, by NAD(P)Hoxidase–derived oxidative stress.

MethodsAnimal ModelDomestic crossbred female pigs (12 to 15 kg) were anesthetized andinstrumented as previously described.22,23 After the exposure of theright common carotid artery, heparin (5000 U) was administeredintravenously. Coronary ostia were cannulated under fluoroscopicguidance, and intracoronary nitroglycerin was given (100mg).Coronary injury was carried out in 2 coronary arteries in each animalwith an oversized balloon (balloon:artery ratio'1.3 to 1.5) inflated2 to 3 times for 30 seconds. The third artery was used as control. Theanimals were euthanatized with intravenous Euthasol (DelmarvaLaboratory) at times indicated in the text. All experiments werecarried out in accordance with institutional guidelines.

Measurement of zO22 Production

The production ofzO22 was measured by superoxide dismutase

(SOD)– or Tiron-inhibitable conversion of nitro blue tetrazolium(NBT) to formazan.20,21

zO22 Production in Coronary Arteries

Coronary arteries were dissected free from adipose tissue andmyocardium, then cut into'5-mm rings and placed in 24-wellplates. Tissues were balanced in phenol-free DMEM at 37°C in aCO2 incubator for 30 minutes with or without addition ofzO2

2 SOD(1000 U/mL) or Tiron (10 mmol/L). Freshly made NBT (100 mg/Lin phenol-free DMEM) was added to tissues with gentle rocking for3 hours. The reaction was terminated by addition of an equal volumeof 0.5N HCl, and tissues were rinsed twice with cold PBS. To extractformazan, tissues were pulverized in liquid nitrogen and dissolved in100% pyridine at 80°C for 30 minutes. After centrifugation, lightabsorbance was read in supernatants at 540 nm. The NBT reductionto formazan was calculated by the following formula: NBTreduction5A3V/(T3E3L), where A is absorbance, V is volume ofsolubilizing solution, T is time of incubation with NBT (minutes), Eis extinction coefficient50.72 mmol/mm, and L is length of lighttravel through the solution, 10 mm. Either SOD- or Tiron-inhibitableNBT reduction was calculated as a measure ofzO2

2 production (pmolz min21 z mg wet wt21). To determine pathways mediatingzO2

2

production, several inhibitors were used in the experiments, includ-ing diphenyleneiodonium (DPI, 100mmol/L), rotenone (50mmol/L),oxypurinol (300 mmol/L), Nv-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), and diethyldithiocarbamic acid (10 mmol/L).The n value represents the number of vascular rings obtained from$3 animals per experimental condition.

To assess the location of injury-inducedzO22 production, the

injured coronary arteries were incubated with NBT and processed tovisualize formazan deposits. Briefly, coronary rings were fixed in10% formalin and embedded in paraffin. They were sectioned into6-mm-thick sections and deparaffinized by heating at 65°C for 1hour. To avoid solubilization of NBT in tissue, the sections wererinsed with Clear-Rite 3 solution (Richard-Allan Scientific) andcounterstained with nuclear fast red.

zO22 Production in Isolated Adventitial Fibroblasts

Adventitial fibroblasts (passages 2 through 6) were plated in 6-wellplates at 100 000 cells/well in 10% FBS. At 2 days later, when cellswere'80% confluent, they were arrested in 0.5% FBS for the next48 hours. After that, they were stimulated with 10% FBS for 1 to 24hours, followed by incubation with NBT (0.5 mg/mL in phenol-freeDMEM) for 1 hour. After brief washing, cells were trypsinized andcell pellets were dissolved in 100% pyridine. The light absorbancewas measured at 540 nm and the NBT reduction to formazan wascalculated as described above and corrected by cell number. Valueswere derived from 6 to 9 wells from 3 separate experiments.

Measurement of NAD(P)H Oxidase Activity inCoronary ArteriesNAD(P)H oxidase activity was measured by SOD-inhibitable cyto-chrome c reduction using NADH or NADPH as substrate.17 Tomeasure NAD(P)H oxidase activity in injured coronary arteries, thearteries were harvested at 2 days after injury, and the injuredsegments (including the adventitia and media) were dissected freefrom adipose tissue and myocardium. The noninstrumented coronaryarteries were used as control. After the removal of endothelial cells,tissues were minced in 10 volumes of ice-cold Tris-sucrose buffer(pH 7.1) containing Tris base 10 mmol/L, sucrose 340 mmol/L,PMSF 1 mmol/L, EDTA 1 mmol/L, leupeptin 10mg/mL, aprotinin10 mg/mL, and pepstatin 10mg/mL. Then the tissue homogenateswere sonicated for 20 seconds on ice, followed by extraction for 30minutes. After centrifugation at 15 000gfor 10 minutes, an aliquot(20 mL) of supernatant (50 to 150mg of protein) was added to thereaction buffer (980mL) containing cytochromec (78 mmol/L),NADH, or NADPH (100 mmol/L), with or without SOD (1000U/mL). The samples were then incubated at 37°C for 1 hour, and theabsorbance at 550 nm was measured. There was no measurableactivity in absence of NADH. A buffer blank was measured in eachassay, and SOD-inhibitable cytochromec reduction in buffer blankwas subtracted from each sample. The activity of NAD(P)H oxidasewas calculated as SOD-inhibitable cytochromec reduction andexpressed aszO2

2 in pmol z mg21 z min21.

Measurement of SOD Activity inCoronary ArteriesSOD activity in vascular tissues was measured by SOD-dependentinhibition of cytochromec reduction catalyzed by xanthine/xanthineoxidase.27 To assess SOD activity in uninjured coronary arteries,coronary adventitia and media were dissected after the removal ofendothelium. SOD activity after coronary injury was measured inarterial segments including the adventitia and media. The tissueswere minced and homogenized in 10 volumes of 50 mmol/Lpotassium phosphate (pH 7.4) containing 0.3 mol/L KBr and acocktail of protease inhibitors (0.5 mmol/L PMSF, 90mg/mLaprotinin, 10mg/mL pepstatin, 10mg/mL leupeptin). After sonica-tion for 20 seconds, the homogenates were extracted at 4°C for 30minutes, followed by centrifugation at 15 000g for 10 minutes. Thesupernatants were added to the reaction mixture consisting of0.1 mmol/L EDTA, 0.090 mmol/L xanthine, and 0.018 mmol/Lcytochromec (pH 7.4). SOD activity was assessed by monitoring theinhibition of xanthine oxidase–mediated cytochromec reduction,with the absorbance measured at 550 nm over 3 minutes.

ImmunohistochemistryThe Vectastain Elite ABC system (Vector Laboratories) was used forimmunohistochemistry as previously described.22,28 Sections weredeparaffinized, incubated with 0.6% H2O2 in methanol for 30minutes, and blocked with 5% horse or rabbit serum. After beingwashed in PBS, sections were incubated with primary antibodies for1 hour at room temperature in a moisture chamber. The followingprimary antibodies were used: polyclonal antibodies against p47phox

and p67phox (1:200, Santa Cruz), monoclonal antibody recognizingSM myosin heavy chain (SM-MHC, 1:800, Sigma), and porcinemacrophages (1:10, ATCC HB 142). Then slides were washed andincubated with biotinylated secondary horse anti-mouse or rabbitanti-goat antibodies (1:2000, Vector Laboratories) for 1 hour. Theywere visualized with DAB substrate (Vector Laboratories) followed

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by counterstain with Gill’s hematoxylin (Sigma Diagnostics). Neg-ative controls were carried out with nonimmune serum instead ofprimary antibody.

Cell Proliferation AssayFibroblasts were isolated from the adventitia of porcine coronaryarteries as described.26 The cells (passages 2 to 6) were plated intriplicate at 10 000 cells/well in 24-well plates in DMEM supple-mented with 10% FBS. At 24 hours later, cells were arrested inDMEM containing 0.5% FBS for 48 hours. They were then stimu-lated with 10% FBS for 3 days with or without addition of indicatedinhibitors. Cells were trypsinized at 72 hours after stimulation andcounted in a Coulter counter. Values were derived from 3 wells pertreatment, and the experiments were repeated$3 times on separateoccasions.

Statistical AnalysesData were expressed as mean6SD. The statistical significance regard-ing multigroup comparisons was determined by ANOVA with Bonfer-roni correction. A value ofP,0.05 was considered significant.

ResultsOxidative Stress in Normal and InjuredCoronary Arteries

Normal Coronary ArteriesNormal coronary adventitia exhibited higher basalzO2

2 gen-eration (4.461.2 pmolz mg21 z min21 SOD-inhibitable NBTreduction) than did the media (0.460.5 pmolz mg21 z min21,n56, P,0.01). As shown in Figure 1, the preincubation ofcoronary rings with DPI [NAD(P)H oxidase inhibitor] abol-ished zO2

2 production in coronary adventitia, whereas rote-none (mitochondrial dehydrogenase inhibitor) and oxypurinol(xanthine oxidase inhibitor) had no effect. The difference inbasalzO2

2 generation between coronary adventitia and mediais probably due to heterogeneous distribution of endogenousSOD, inasmuch as adventitia exhibited lower SOD activitythan did media (10167 versus 16669 U/g,P,0.001, n55).Subsequently, SOD inhibitor (diethyldithiocarbamic acid)augmented morezO2

2 generation in the media (adventitia19.662.5 and media 36.368.2 pmolz mg21 z min21, P,0.01versus no diethyldithiocarbamic acid).

Injured Coronary ArteriesBecause coronary injury induces a short-lived adventitial cellproliferation, the change in oxidative stress during this timeperiod was examined. To this end, the SOD activity andzO2

2

generation were measured in the entire coronary segments,because precise separation of the adventitia from media is nottechnically feasible at early time points after injury. SODactivity showed no difference between control and injuredcoronary segments (not shown).zO2

2 generation, as measuredby SOD- and Tiron-inhibitable NBT reduction, increasedsignificantly within 1 day after injury, and it remainedelevated for$10 days (Figure 2). Higher values of Tiron-inhibitable NBT reduction were probably due to bettercellular permeability of Tiron than SOD. To ascertain the site

Figure 1. Superoxide anion (zO22) generation in coronary adven-

titia and media in control (uninjured) arteries. The adventitiashowed a higher basal level of zO2

2 production than the media(SOD-inhibitable NBT reduction). An NAD(P)H oxidase inhibitor,DPI, inhibited zO2

2 generation in coronary adventitia, whereasoxypurinol (OXY) and rotenone (ROT) showed no inhibitoryeffects. The lack of SOD-inhibitable NBT reduction in boiledadventitia further confirmed an enzymatic source of zO2

2 pro-duction. *P,0.001 vs coronary media; †P,0.001 vs coronaryadventitia without treatment (n56 to 9 vascular rings per bar).

Figure 2. The time course of zO22 production after coronary

injury. The zO22 production (SOD- and Tiron-inhibitable NBT

reduction) was measured in transmural segments of uninjuredand injured vessels. The increase in zO2

2 generation wasobserved within 1 day after injury. *P,0.05 and †P,0.01 vsuninjured coronary arteries. Numbers represent the number ofvascular rings.

Figure 3. Localization of superoxide in injured coronary arteries.Injured coronary arteries (2 days) were incubated with NBT for 3hours and processed for NBT histology. A, Cross section ofinjured coronary artery with apparent site of injury. The areas ofthe adventitia (a) and media (m) outlined by rectangular boxesare depicted at higher magnification. B, Intracellular deposits offormazan (blue) were present in adventitial cells in the center ofinjury. C, Coronary medial SMCs showed no intracellularformazan deposits despite medial dissection. Magnification: A340, B and C 3410.

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of zO22 generation in injured vessels, reduced NBT

(formazan) was identified in cross sections. Figure 3 demon-strates preferential adventitial localization of intracellulardeposits of formazan in injured segments. As in uninjuredvessels, NAD(P)H oxidase inhibitor (DPI) almost entirelyabolished the production ofzO2

2 after coronary injury (n54per time point,P,0.001 versus no treatment). Althoughdynamic changes in inducible nitric oxide synthase expres-sion during coronary repair could contribute to oxidativestress, its inhibitor, L-NAME, showed no effect (Table 1).

NAD(P)H Oxidase Activity and Expressionof Subunits

NAD(P)H Oxidase ActivityTo ascertain that NAD(P)H oxidase is the major pathwayresponsible for oxidative stress after coronary injury, NADH/NADPH oxidase activity was measured by SOD-inhibitablecytochromec reduction using NADH or NADPH as sub-strates. At baseline, coronary arteries exhibited similar levelsof NADH and NADPH oxidase activity. At 2 days aftercoronary injury, NADH oxidase activity was significantlyaugmented in the injured and adjacent segments (Table 2),whereas NADPH oxidase activity showed no changes aftercoronary injury.

Expression of p47phox and p67phox

To localize NAD(P)H oxidase in injured coronary arteries,expression of p47phox and p67phox [cytoplasmic subunits ofNAD(P)H oxidase] were examined by immunohistochemis-try. They were low in normal coronary arteries (not shown)but showed a marked increase in adventitial cells after injury.The expression began at 1 day and peaked at 2 days afterinjury. Positive cells were of fibroblastic origin, because they

lacked SM differentiation markers (SM-MHC,a-SM actin,desmin, and caldesmon), and only infrequent cells (,5%)were positive for macrophage immunoreactivity (Figure 4).

Role of NAD(P)H Oxidase–Derived ROSProduction in Coronary Fibroblast Proliferation

Serum-Induced Superoxide Production inAdventitial FibroblastsTo assess the functional importance of increased oxidativestress in coronary adventitia, thezO2

2 production was exam-ined in serum-stimulated adventitial fibroblasts. In responseto serum stimulation, adventitial fibroblasts demonstrated atime-dependent increase inzO2

2 production, reaching maxi-mum levels at 3 to 6 hours (Figure 5). As expected, either theinhibition of NAD(P)H oxidase with DPI (10mmol/L) ordismutation ofzO2

2 with exogenous SOD (500 U/mL) pro-duced significant reduction inzO2

2 production (Figure 6),whereas L-NAME, rotenone, and oxypurinol showed noeffects (not shown).

Serum-Induced Superoxide Generation and AdventitialFibroblast ProliferationTo assess whether altering ROS generation could modulateadventitial fibroblast proliferation in vitro, growth inhibitionof serum-stimulated cells was determined either by inhibiting

TABLE 1. NAD(P)H Oxidase–Dependent Superoxide Generationin Injured Coronary Arteries

Time AfterInjury, d

Superoxide Generation

Control DPI L-NAME

2 14.867 0.560.9* 12.969.4

10 13.862.7 0.260.3* 11.566.1

Superoxide anion was measured as SOD-inhibitable NBT reduction(pmol z mg21 z min21). Vascular rings derived from injured coronary arterieswere pretreated without or with DPI (100 mmol/L) or L-NAME (1 mmol/L) for 30minutes. NAD(P)H oxidase inhibitor DPI significantly inhibited superoxidegeneration, whereas iNOS inhibitor L-NAME showed no effects.

*P,0.001 vs control, n54 per time point.

TABLE 2. NAD(P)H Oxidase Activity in InjuredCoronary Arteries

NADH NADPH

Uninjured (n56) 7.765.4 10.368.2

Injured (n510) 27.064.4*† 19.867.1

Adjacent (n55) 17.162.3* 16.965.9

NADH oxidase activity was measured in uninjured and injured coronarysegments at 2 days after injury. Injury significantly augmented NADH oxidaseactivity (zO2

2pmol z mg21 z min21), whereas NADPH oxidase activity showed nomajor changes compared with uninjured coronary arteries. Numbers inparenthesis represent the number of vessels.

*P,0.01 vs uninjured coronary arteries.†P,0.01 vs adjacent coronary arteries.

Figure 4. Localization of NAD(P)H oxidase subunits in injuredcoronary arteries. A, Cross section of injured coronary arterystained for SM-MHC. At 2 days after injury, coronary medialSMCs exhibited a strong SM-MHC immunoreactivity, whereasadventitial cells were negative. The areas of the adventitia (a)and media (m) outlined by rectangular boxes are depicted athigher magnification. B, Injured adventitia contained mostly rep-licating fibroblasts with only infrequent macrophages. C, Themajority of activated adventitial cells exhibited increased immu-noreactivity for p47phox. D, Coronary media showed no increasein p47phox immunoreactivity. E, Likewise, p67phox is increased inadventitial cells, but not in the media (not shown). F, Negativecontrol (NC; primary antibody was omitted). Magnification: A340, B through E 3410.

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the generation of ROS (DPI) or facilitating their removal(zO2

2: Tiron, SOD; and H2O2: catalase). The inhibitor ofNAD(P)H oxidase (DPI) significantly inhibited fibroblastgrowth in a concentration-dependent manner (Figure 7,P,0.001). In contrast, L-NAME and oxypurinol produced nosignificant effects (not shown), consistent with the lack ofinhibition of ROS generation by these inhibitors. The removalof either zO2

2 with Tiron or H2O2 with catalase inhibitedfibroblast proliferation. In contrast, dismutation ofzO2

2 toH2O2 after SOD did not prevent serum-induced cellreplication.

DiscussionThe major findings of this study were that (1) coronaryadventitia is an important source of increased oxidative stressafter endoluminal coronary injury, (2) the NAD(P)H oxidaseis the major pathway for ROS generation in injured coronary

arteries and stimulated adventitial fibroblasts, and (3) ROSare involved in the regulation of growth response of coronaryadventitial fibroblasts.

Oxidative stress is known to increase after various forms ofvascular insult.6,29,30 Although the presence of NAD(P)Hoxidase has been shown in normal adventitia,16,20,21its role incellular proliferation during arterial repair has not previouslybeen elucidated. In noncoronary vasculature, there is a rapiddecrease in glutathione level, an indirect marker of the redoxstate, after mechanical injury.30 Others have reported theinduction of p47phox, thus implicating NAD(P)H oxidase andROS generation in initial SMC proliferation.6 Likewise,p22phox expression and oxidative stress are increased in aorticmedial SMCs after angiotensin II infusion.31 Unique charac-teristics of coronary SMCs, however, raise the question ofwhether similar events occur during coronary repair.25,26

Earlier studies showed an increase inzO22 production at 2

weeks after coronary injury, although the presence of theneointima, containing cells of adventitial and medial origin,blood-borne cells, and regenerating endothelial cells, did notallow for the identification of its source.29 To characterize themechanism of oxidative stress and its role in cellular prolif-eration, the present study focused on earlier stages of coro-nary response to injury, with cellular constituents still remain-ing at their sites of origin. Predominant increases in ROSgeneration and vascular NAD(P)H oxidase (p47phox andp67phox subunits) were evident in the adventitia (Figures 3 and4). In contrast, coronary media exhibited higher levels ofSOD and subsequently lower oxidative stress. It remains to bedetermined whether the degree of SMC differentiation, whichdiffers among vascular beds, contributes to regional differ-ences in the activation of NAD(P)H oxidase and ROSgeneration after injury. The inhibition of NAD(P)H oxidasewith DPI or the removal of ROS (zO22 and H2O2 with Tironor H2O2 with catalase) abrogated serum-induced growthresponse of isolated coronary fibroblasts in vitro (Figure 7).Not surprisingly, dismutation ofzO2

2 to H2O2 after SOD was

Figure 5. Serum-induced zO22 generation in coronary fibro-

blasts. Adventitial fibroblasts were plated in 6-well plate at100 000 cells/well in 10% FBS for 2 days. The cells weregrowth-arrested with 0.5% FBS for 48 hours, followed by stimu-lation with 10% FBS. The NBT was added to cells for 1 hour,and intracellular accumulation of formazan was measured.Adventitial fibroblasts demonstrated a time-dependent zO2

2 pro-duction peaking at 6 hours after stimulation (n59 per timepoint). *P,0.001 vs no stimulation.

Figure 6. Inhibition of serum-induced zO22 generation in coro-

nary fibroblasts. Adventitial fibroblasts were pretreated with DPI(10 mmol/L) or SOD (500 U/mL) for 30 minutes, followed bystimulation with 10% FBS for 3 hours. The cells were then incu-bated with NBT for 1 hour, and the inhibition of formazan accu-mulation was measured. Both DPI and SOD showed significantinhibition of zO2

2 production (n54 per treatment). *P,0.001 vs10% FBS.

Figure 7. ROS and adventitial fibroblast proliferation. Adventitialfibroblasts (10 000 cells/well) were arrested with 0.5% FBS for48 hours and then stimulated with 10% FBS in presence of vari-ous inhibitors. Cell growth was examined at 3 days after treat-ment by cell counting. The inhibitor of NAD(P)H oxidase (DPI),the scavenger of zO2

2 (Tiron), or the removal of H2O2 (catalase)significantly inhibited fibroblast growth in a concentration-dependent manner. In contrast, dismutation of zO2

2 to H2O2 withSOD showed no significant inhibition of fibroblast proliferation.*P,0.05 and †P,0.01 vs 10% FBS alone (n53 per bar). Theexperiments were repeated 3 times, yielding similar results.

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ineffective in preventing cell replication, pointing to theessential role of H2O2 in the regulation of vascular cellgrowth.3 The above findings suggested the involvement ofROS in a rapid proliferation of adventitial fibroblasts aftercoronary injury in vivo.22–24 The relatively slow and pro-longed ROS production in adventitial fibroblasts (Figure 5)was similar to that in noncoronary SMCs but quite distinctfrom the faster and greater response previously seen inphagocytes.32 Preferential utilization of NADH as substratefor NAD(P)H oxidase in injured coronary arteries contrastswith the observations by others that aortic adventitial fibro-blasts primarily generatezO2

2 in response to NADPH.12,21

Several experimental conditions (eg, cell origin and type ofstimulation), as well as assay methods (eg, cytochromecreduction versus lucigenin assay) may be responsible forthese differences.33

The increase in oxidative stress stimulates cell growth, butROS can also cause cellular death (reviewed by Griendlingand Harrison).34 It is likely that these opposite results arerelated to the level and the type of ROS (zO2

2 versusH2O2).35,36 Much less is known, however, regarding theconsequences of oxidative stress in vascular cells with abroad range of differentiation. When terminally differentiatedcardiomyocytes and interstitial fibroblasts were exposed toH2O2, apoptosis was induced in the former and proliferationin the latter.37 Although endoluminal injury in a porcinemodel did not significantly enhance intracellular ROS gener-ation in coronary media, extracellular oxidative stress mayimpact SMC survival. In chronic intimal lesions, inflamma-tory cells, particularly active in the generation of oxidativestress, have been shown to contribute to SMC apoptosis.38

The loss of differentiated coronary SMCs may lead to adecrease of a protective barrier of the intact media, resultingin the expansion of less differentiated fibroblasts and thedevelopment of intimal lesions. Our results support the notionthat ROS production may serve as an attractive target fortherapeutic interventions. Nevertheless, several questions re-main unresolved, including the choice of antioxidants, be-cause negative clinical results with vitamin E.39 In contrast, 2independent clinical studies suggested a reduction in coro-nary restenosis in patients pretreated with the antioxidantprobucol before angioplasty.40,41 The recently publishedHeart Outcomes Prevention Evaluation (HOPE) trial alsoprovided evidence for the reduction of cardiovascular mor-tality after chronic administration of the ACE inhibitorramipril.42 These results are particularly notable becauseNAD(P)H oxidase activity is regulated by angiotensinII.10,15,21Undoubtedly, better understanding of the regulationof NAD(P)H oxidase in different vascular cells may providefurther insights into the pathogenesis of coronary arterydisease and aid the development of therapeutic interventions.

In conclusion, this study demonstrated an increase inNAD(P)H oxidase–derivedzO2

2 production in coronary ad-ventitial fibroblasts after balloon injury. The inhibition ofNAD(P)H oxidase and the attenuation of ROS productionabrogated proliferative responses of adventitial fibroblasts.The results imply that ROS serve as pivotal signals forgrowth response of coronary fibroblasts.

AcknowledgmentThis study was supported by National Institutes of Health grantsHL-44150 and HL-60672.

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Shi et al Oxidative Stress 745

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Yi Shi, Rodica Niculescu, Dian Wang, Sachin Patel, Kelly L. Davenpeck and Andrew ZalewskiBalloon Injury

Increased NAD(P)H Oxidase and Reactive Oxygen Species in Coronary Arteries After

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