Concomitant Proliferation and Caspase-3 Mediated Apoptosis in Response to Low Shear Stress and Balloon Injury

Journal of Surgical Research 161, 146–155 (2010)doi:10.1016/j.jss.2008.11.001

Concomitant Proliferation and Caspase-3 Mediated Apoptosis

in Response to Low Shear Stress and Balloon Injury

Lisa R. P. Spiguel, M.D.,*,2 Amito Chandiwal, M.D.,‡,2 James E. Vosicky, C.V.T.,*Ralph R. Weichselbaum, M.D.,† and Christopher L. Skelly, M.D.*,1

*Section of Vascular Surgery, Department of Surgery; †Department of Radiation and Cellular Oncology,University of Chicago, Chicago, Illinois; and ‡Department of Medicine, Lincoln Medical and

Mental Health Center, Bronx, New York

Submitted for publication September 16, 2008

Background. Arterial remodeling occurs as a re-sponse to hemodynamic change and direct vessel wallinjury through the process of neointimal hyperplasia(NH). A concomitant response of vascular smooth mus-cle cell (VSMC) proliferation and apoptosis exists. Thepurpose of this study is to assess the cellular responseof vessels following exposure to low shear stress (t) andballoon injury in order to further elucidate the mecha-nisms underlying vascular injury. Our hypothesis isthat the combination of low t and balloon injury re-sults in NH approximating that seen in clinical arterialrestenosis, and that quantitative analysis of VSMC pro-liferation and apoptosis correlates with the associatedincrease in arterial remodeling.

Methods and Results. New Zealand White rabbitsunderwent surgery on the carotid artery creating lowt (n [11), balloon injury (n [ 11), combined low t andballoon injury (n [11), and sham (n [ 13) groups. Ex-periments were terminated at 1, 3, and 28 d. Day 1and 3 arteries were analyzed with immunohistochem-istry for apoptotic markers, terminal transferasedUTP nick end labeling (TUNEL), and activated cas-pase-3, and a cellular proliferation marker, accumu-lated proliferating cell nuclear antigen (PCNA), aswell as immunoblot analysis for activated caspase-3and PCNA at day 3. There was significantly greater ap-optosis in the combined group as compared with theother groups assessed by quantitative TUNEL and acti-vated caspase-3 levels at both days 1 and 3. Similarly,an increase in cellular proliferation assessed byPCNA expression, was significantly greater in the

1 To whom correspondence and reprint requests should be ad-dressed at Section of Vascular Surgery, Department of Surgery, Uni-versity of Chicago MC 5028, 5841 S. Maryland Ave., Chicago, IL60637. E-mail: [email protected].

2 These authors contributed equally to this study.

0022-4804/$36.00� 2010 Elsevier Inc. All rights reserved.

146

combined group as compared with the other groups. At28 d there was no difference in NH observed in the low t

(26 ± 3 mm) and balloon injury (51 ± 17 mm) groups. How-ever, significantly more NH was observed in the com-bined group (151 ± 35 mm) as compared with the othergroups.

Conclusions. An increase in VSMC apoptosis viaa caspase-3 dependent pathway is up-regulated by24 h in the face of combined low shear stress andballoon-induced vessel wall injury. Paradoxically,this increase in VSMC apoptosis is associated witha significant increase in neointimal thickening at28 d. The concomitant increase of both apoptosis andproliferation are indicative of a robust arterial remod-eling response. � 2010 Elsevier Inc. All rights reserved.

Key Words: neointimal hyperplasia; arterial remodel-ing; vascular injury; vascular smooth muscle cells;shear stress; balloon injury; apoptosis; hemodynamics.

INTRODUCTION

Neointimal hyperplasia (NH) is an adaptive responseof blood vessels to both injury [1, 2] as well as lowflow hemodynamic states [3, 4]. The consequent arterialremodeling is multifaceted resulting in compensatoryluminal narrowing [5], through vascular smoothmuscle cell (VSMC) proliferation and migration [1, 2],in addition to VSMC apoptosis [6, 7]. It is the pathologicremodeling of NH that is considered to be a predomi-nant cause of arterial restenosis following percutaneousinterventions. The role of injury and hemodynamicfactors, such as decreased shear stress, increased walltension, or both on vascular remodeling have beenwell studied [8–10]. These stimuli lead to VSMC

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SPIGUEL ET AL.: CONCOMITANT PROLIFERATION AND CASPASE-3 MEDIATED APOPTOSIS 147

‘‘phenotypic modulation,’’ migration, and proliferation,in addition to extracellular matrix degradation [11–13], all of which are thought to be stimulated by locallyreleased cytokines derived from activated platelets,macrophages, white blood cells, and the VSMCs them-selves [14–18]. As more percutaneous interventionsare being performed clinically, it is important to explorethe role of decreased wall shear stress and balloon in-jury as it relates to arterial remodeling.

Apoptosis is a noninflammatory mechanism of celldeath that has been shown to play an important rolein both atherosclerosis as well as NH [19, 20]. Isneret al. demonstrated that up to 93% of human arterialrestenotic specimens contained foci of apoptosis, aswell as 43% of primary atherosclerotic lesions [19]. Inexperimental models, apoptosis has been shown to oc-cur early after balloon injury, and is thought to be a re-sult of the vessel’s homeostatic response to proliferativesignals when normal cell-matrix interactions are dis-rupted [21]. Although some studies demonstrate an in-crease in apoptosis following balloon angioplasty, otherstudies focus on the lack of apoptosis up-regulation incomparison with cell proliferation, indicating a nidusfor net intimal thickening [22].

Hemodynamics are of critical importance to clinicalsuccess [23]. Wall shear stress (t), a well-studied flowparameter, is attributable to the frictional force of theflowing blood on the endothelial cells and is known tobe a major regulator of arterial remodeling. Decreasingthe wall t by reducing the blood flow is shown to pro-duce a predictable neointimal response [9, 10, 24]. Sim-ilarly, endothelial denudation as occurs duringpercutaneous transluminal angioplasty augments thedevelopment of neointimal response [25–27]. Moreover,it is not uncommon to find low t following balloon injurydue to technical failure, as seen with embolization ofdownstream vessels, presence of persistent collateralcirculation, and constrictive remodeling in arteries[28, 29]. Although several animal models of NH usingeither balloon injury or low t have been studied [7, 30,31], the effect of low t following balloon injury thatcan occur clinically following angioplasty has yet to befully elucidated.

The purpose of this study was to examine the in vivo ef-fects of concomitant balloon injury and low t on the arte-rial remodeling response, inparticular to focus onsmoothmuscle cell viability as assessed by apoptosis and cellularproliferation. In a New Zealand White rabbit carotidmodel, the apoptotic and proliferative response of vascu-lar smooth muscle cells exposed to extremely low t, bal-loon injury, or both were examined. Apoptosis wasassessed via terminal transferase dUTP nick end label-ing (TUNEL) and activated caspase-3 immunostainingand immunoblotting. Cellular proliferation was assessedvia immunostaining and immunoblotting of accumu-

lated proliferating cell nuclear antigen (PCNA), a cellproliferation marker. Resultant neointimal thickeningwas assessed via histomorphometry.

METHODS

Animal Operations

Animals were cared for in accordance with the University of Chi-cago Institutional Animal Care and Use Committee (IACUC). MaleNew Zealand white rabbits (3 kg) were anesthetized with an intra-muscular injection of ketamine hydrochloride (40 mg/kg) and xyla-zine (5 mg/kg) augmented with isoflurane (1%–3%) titrated foreffect via endotracheal intubation. Antibiotic prophylaxis wasprovided with enrofloxacin 10 mg/kg intramuscularly daily for 3 d.Aspirin (1 mg/kg) and clopidogrel (25 mg/kg) dissolved in waterwere given as a loading dose 24 h prior to surgery and a maintenancedose (aspirin, 1 mg/kg and clopidogrel, 10 mg/kg) was continued un-til sacrifice at either 1, 3, or 28 d. Intra-arterial pressure was moni-tored through a 22-gauge cannula placed in the contralateral earartery.

Animal Models

For all of the animal groups, a 4 cm length of left common ca-rotid artery (CCA) was exposed, after surgical preparation,through an incision overlying the anterior border of sternocleido-mastoid muscle. Systemic anticoagulation was achieved with 200U/kg of heparin prior to proximal and distal arterial control. Forthe low t group (n ¼ 11), the left CCA was exposed as previouslydescribed and an arteriotomy was made just distal to the cranialthyroid branch. Arterial outflow was restricted by ligating the dis-tal CCA, distal to the patent cranial thyroid branch, over a 0.014guide wire, limiting arterial outflow through a narrowed distalCCA and cranial thyroid branch. For the balloon injury group (n¼ 11), the left CCA was exposed as described above and an arterio-tomy made just distal to the cranial thyroid branch. A 3F Fogartyballoon catheter (Edwards Life Sciences, Irvine, CA) was then in-serted retrograde into the CCA. The balloon catheter was inflatedwith 0.1 mL isotonic saline solution to a ratio of 1.1:1 (balloon toCCA diameter) and the inflated catheter was withdrawn in a step-wise fashion to the entry point with constant rotation. The previ-ous procedure was repeated a total of three times to create a 3to 4 cm area of balloon injury in the proximal CCA. For the com-bined low t and balloon injury group (n ¼ 11), the left CCA wasballoon injured first as previously described and then restrictedby ligating distal to the cranial thyroid branch over an 0.014guidewire as previously described with the low t group. The arte-riotomies of all injury models were repaired using interrupted 8-0 nonabsorbable prolene surgical suture (TE-145; Davis-Geck,Manatee, PR) under 4.53 loupe magnification. For the sham group(n ¼ 13), the left CCA was temporally exposed as for the othergroups and the incision was closed in a two layer fashion with 3-0 vicryl deep fascial and 4-0 maxon running subcuticular suturesimilar to the other groups. Anticoagulation was not reversed forany of the four groups.

After either 1, 3, or 28 d, the operated left CCA was re-exposed aspreviously described for harvest. The proximal CCA was cannulated,while the distal CCA and the cranial thyroid branch were clampedand the artery was perfusion fixed with 1.25% glutaraldehyde atthe last recorded mean arterial pressure for 10 min, followed byCCA excision. The proximal half of the harvested CCA was snap fro-zen in liquid nitrogen for later protein analysis, and the distal halfwas paraffin-embedded for later Weigert van Gieson staining, H &E staining, and immunostaining.

JOURNAL OF SURGICAL RESEARCH: VOL. 161, NO. 1, JUNE 1, 2010148

Flow and Pressure Monitoring

Blood flow and mean arterial pressures were each measured, beforeand after intervention and arteriotomy repair, as well as prior toartery harvest, using a transonic flow probe (2SB) and an ultrasonictransit-time flow meter during both the initial operation and duringthe harvest to ensure consistency and reproducibility of our model(HT207; Transonics Systems, Ithaca, NY) The readings were recordedwith a digital data acquisition system (Lab Master DMA; ScientificSolutions, Solon, OH). The length of the balloon-injured CCA andthe external diameter of the CCA were measured with digital calipers(CD-6; Mitutoyo Corp., Osaka, Japan).

Hemodynamic Assessment

Measured hemodynamic variables included mean arterial pressure(P) and blood flow (Q) while the wall shear stress (tw) was calculatedusing the Poiseuille formula:tw ¼ 4rQ/p ri

3(ri ¼ internal radius; r ¼viscosity of blood, assumed to be 0.03 poise). The internal radius mea-surement was calculated from immunohistochemical sections at day 1and day 28.

Immunohistochemistry

For immunohistochemistry, 5-mm sections of the paraffin-embed-ded CCAs were longitudinally cut at day 1 and day 3 from thesham, low t, balloon injury, combined low t and balloon injury groups.All paraffin sections were deparaffinized and rehydrated in a descend-ing alcohol series.

Detection of apoptosis was performed through TUNEL assay usingthe VasoTACS in situ apoptosis detection kit (Trevigen, Inc., Gai-thersburg, MD). The sections were incubated with proteinase K (Tre-vigen, Inc., Gaithersburg, MD), washed with deionized water,incubated with 3% H2O2 for 5 min, and washed with deionized wateragain. TUNEL staining was performed per the manufacturer’sinstructions.

Detection of activated caspase-3, an end mediator of apoptosis, wasperformed using SignalStain IHC detection kit (Cell Signaling,Danvers, MA). Antigen unmasking was performed by immersingslides in 0.01 M sodium citrate buffer followed by peroxidase quench-ing to block endogenous peroxidase activity. To prevent nonspecificbinding, the sections were immersed in a 3% BSA blocking solutionfor 1 h at room temperature. For antibody staining, sections were firstincubated with a prediluted primary antibody at 4�C overnight,rinsed for 15 min with PBS, incubated with biotinylated secondaryantibody for 1 h at room temperature, and rinsed for a further 15min with PBS. The sections were then stained per the manufacturer’sprotocol and counterstained with hematoxylin.

Cell proliferation was assessed by staining for PCNA to look for theactively cycling cells within the media and intima. After antigen un-masking and peroxidase quenching as described above, the sectionswere incubated in a 3% BSA blocking solution for 1 h at room temper-ature to prevent nonspecific binding. Sections were then incubatedwith anti-PCNA mouse monoclonal IgG primary antibody (1:50 dilu-tion, Santa Cruz Biotechnology, Santa Cruz, CA) at 4�C overnight,then rinsed with PBS and incubated with a prediluted biotinylatedanti-mouse secondary antibody (DAKO, Carpinteria, CA) for 1 h atroom temperature followed by washing with PBS. The sections werethen incubated with a DABþchromagen substrate buffer (DAKO,Carpinteria, CA). The reaction was analyzed under a microscopeand the slides were rinsed when brown precipitate developed indicat-ing positive staining. The slides were counterstained with hematoxy-lin prior to coverslipping.

Western Blot

At day 3, after perfusion fixation with 1.25% glutaraldehyde, a por-tion of the left CCA was excised, snap frozen in liquid nitrogen, and

morselized via mortar and pestle with subsequent homogenizationon ice in lysis buffer containing 20 mM Hepes (pH 7.4), 2 mMEDTA, 1 mM DTT, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol,2 mM leupeptin, 400 mM PMSF, and 10 U/mL aprotinin (Sigma Chem-ical, St. Louis, MO). The homogenate was incubated for 30 min at 4�Cand then centrifuged at 10,000 3 g for 10 min at 4�C. The superna-tant was removed and protein concentration determined using theBradford method. Equal amounts of each protein extract (50 mg/lane) were heated at 95�C for 10 min in sample buffer (94 mM phos-phate buffer, pH 7.0, 1% SDS, 2 M urea, and 3% b-mercaptoethanol)and then separated onto 10% SDS-PAGE under reducing conditionsand transferred to nitrocellulose membrane (Millipore, Bedford,MA) in a Novex Western transfer apparatus (Invitrogen, Carlsbad,CA) per the manufacturer’s instructions. The blots were washedand incubated with anti-caspase-3 rabbit polyclonal primary anti-body (Calbiochem, San Diego, CA) and anti-rabbit secondary anti-body (Pierce Biotechnology, Rockford, IL) for detection of caspase-3.For detection of PCNA, HRP conjugated mouse monoclonal antibodyspecific for PCNA (DAKO) was used. GAPDH monoclonal antibody(Chemicon, Temecula, CA) was used as a loading control. The blotswere developed using an ECL Western blotting system (AmershamBiosciences; Piscataway, NJ) and densitometry analysis was per-formed using a KODAK Image Station 440 cf system (KODAK,Rochester, NY).

Image Analysis

For evaluating regions with positive TUNEL, caspase-3 and PCNAstaining, ImageJ, a processing and analysis software was used [32].In brief, digital images were acquired with a microscope equippedwith a 3.0 mega pixel digital camera (Olympus; Melville, NY). Theregions of interest were photographed in 8-bit grayscale at 34 mag-nification, the background normalized, and the density thresholdsset to 130 (minimum) and 255 (maximum). The image was then in-verted to give the positive staining as red on a black background.A region of 0.04 mm2 was selected to measure the positive area ineight equidistant regions on the section in both media and intima,and analysis was performed by using the ImageJ particle analysis al-gorithm in a blinded fashion. The percent positive area was calcu-lated by dividing the positive staining area by the total area (0.04mm2) of the region selected. For each region of interest, a spot checkwas performed by visually counting the cells under the microscope,and the consistency of results obtained with both methods was foundto exceed 95%.

Assessment of Morphology

For histomorphometry, 5-mm sections of the paraffin-embeddedCCA were longitudinally cut at day 28 from the sham, low t, ballooninjury, and combined low t and balloon injury groups, and stained us-ing the Weigert van Gieson stain. Sections of each specimen were an-alyzed histomorphometrically in a blinded fashion. Digital imageswere acquired with a microscope equipped with a 3.0 mega pixel dig-ital camera (Olympus, Melville, NY). Neointimal thickness, medialthickness, and luminal radius were digitally measured using ImageJsoftware, while the intima:media ratio and neointimal area werecalculated using the previously measured values.

Data Analysis

Data analysis was performed using standard software (MicrosoftExcel Data Analysis; Redmond, WA). Data is presented as mean 6

standard error of the mean (SEM). Comparison of means wasachieved using One-way analysis of variance (ANOVA) with Tukey’sHonestly Significant Differences test, as well as the Student t-test,with a significance assigned at P < 0.05.


RESULTS

Hemodynamic Assessment

Hemodynamic data are given in Tables 1 and 2. Asexpected, restriction of the CCA by ligating the distalCCA over a 0.014 guide wire in the low t group resultedin very low flow both at day 1 (2.90 6 0.72 mL/min) andday 28 (3.65 6 0.78 mL/min), in addition to shear stressat days 1 (0.64 6 0.16 dyne/cm2) and 28 (0.73 6 0.14dyne/cm2), compared with the sham (27.25 6 1.36 mL/min, 25.2 6 2.92 mL/min; 4.59 6 0.53 dyne/cm2, 6.656 2.07 dyne/cm2). The measurements in the combinedlow t and balloon injury group at day 1 and day 28(3.58 6 0.89 mL/min, 5.75 6 2.62 mL/min; 0.50 6 0.11dyne/cm2, 0.54 6 0.26 dyne/cm2) were statistically sim-ilar to the low t group, and significantly lower whencompared with both the sham group and the balloon in-jury group alone (17.00 6 1.26 mL/min, 32.58 6 2.72mL/min; 2.57 6 0.41 dyne/cm2, 6.49 6 1.47 dyne/cm2).

Assessment of Apoptosis

In situ detection of cell death using TUNEL assay re-vealed a significant increase in the percent positivearea at day 1 and day 3 for the combined low t and bal-loon injury group compared with the other threegroups. Although there was a significant difference inthe percent positive area of apoptosis between the lowt and balloon injury groups at day 1, there was no sig-nificant difference at day 3 (Fig.1 and Fig. 2A and B).

A key mediator of apoptosis, caspase-3, was signifi-cantly greater in the combined low t and balloon injurygroup compared with the other three groups at day 1and day 3 on immunostaining. Unlike the TUNEL as-say apoptosis data at day 1, there was no difference inthe percent positive areas of apoptosis between thelow t and balloon injury groups at day 1. (Fig. 1 andFig.2 A,B) These findings may result from potentialareas of cell necrosis that may be stained with TUNELassay. At day 3, similar to the TUNEL assay apoptosisdata, there was no difference in caspase-3 percent

TABL

Carotid Artery Hemodyn

ModelBlood flow day 0

(mL/min)

Sham (n ¼ 4) 31.50 6 2.87Low t (n ¼ 4) 3.30 6 0.45*,yBalloon injury (n ¼ 4) 29.87 6 3.18z

Low t and balloon injury (n ¼ 4) 2.91 6 0.69*

Note. Expressed as mean 6 SEM.*P < 0.05 compared with Sham Model.yP < 0.05 compared with Balloon Injury Model.zP < 0.05 compared with Combined Low t and Balloon Injury Model.

positive area between low t and balloon injury groups,and both continued to be significantly greater thanthe sham group (Figs.1 and 2A and B). The Westernblot densitometry further supports these results forcaspase-3, demonstrating a 3- to 4-fold increase in cas-pase-3 in the combined low t and balloon injury groupcompared with the other three groups at day 3(Fig. 3A and B).

Assessment of Proliferation

Cell proliferation as assessed by percent positive areaof PCNA was also significantly greater in the combinedlow t and balloon injury group compared with the otherthree groups at day 1 and day 3 (Fig.1 and Fig. 2A andB). Although at day 1, the PCNA percent positive areaof the sham group compared with the low t and ballooninjury groups is higher, this lacked statistical signifi-cance (Fig. 1B). At day 3, these results differed, demon-strating an increase in cell proliferation with all of theinjury groups compared with the sham group, withthe combined low t and balloon injury group creatingstatistically the greatest cell proliferation than all theother three groups.(Fig. 2B) Supportively, our Westernblot analysis at day 3 from the combined low t and bal-loon injury group revealed more than a 2.5-fold increasein the PCNA expression at day 3 compared with eitherthe low t or the balloon injury group alone. There wasno PCNA expression in the sham group (Fig.4A and B).

Carotid Artery Morphology at 28 Days

Representative samples from all of the different co-horts; sham (n ¼ 6) (28 d carotid artery morphologydata from four of the six sham animals was previouslypublished [33]), low t (n ¼ 4), balloon injury (n ¼ 4),and combined low t and balloon injury (n ¼ 4), areshown in Fig. 5, and numeric data given in Table 3.All arteries remained patent at sacrifice at 28 d. Allthree injury models, low t (26 6 3 mm), balloon injury(51 6 17 mm), and combined low t and balloon injury

E 1

amics at Harvest (Day 1)

Blood flow at harvestday 1 (mL/min)

Shear stress at harvestday 1 (dyne/cm2)

27.25 6 1.36 4.59 6 0.532.90 6 0.72*,y 0.64 6 0.16*,y

17.00 6 1.26*,z 2.57 6 0.41z

3.58 6 0.89* 0.50 6 0.11*

TABLE 2

Carotid Artery Hemodynamics at Harvest (Day 28)

ModelBlood flow day 0

(mL/min)Blood flow at harvest

day 28 (mL/min)Shear stress at harvest

day 28 (dyne/cm2)

Sham (n ¼ 4) 31.3 6 3.06 25.2 6 2.92 6.65 6 2.07Low t (n ¼ 4) 3.98 6 0.85*,y 3.65 6 0.78*,y 0.73 6 0.14*,yBalloon injury (n ¼ 4) 23.75 6 1.11z 32.58 6 2.72z 6.49 6 1.47z

Low t and balloon injury (n ¼ 4) 3.59 6 0.25* 5.75 6 2.62* 0.54 6 0.26*



(151 6 35 mm), resulted in a significant increase in theneointimal thickness compared with the sham group(0 mm). When comparing the NH observed in the low tgroup with the balloon injury group, there was no signif-icant difference. However, a significantly greater NH

FIG. 1. (A) In situ detection of apoptosis using TUNEL assay and camunostaining. Sham, low t, balloon injury, and combined low t and ballnification 340). (B) The percent positive area by TUNEL assay, caspase-

was observed with the combination of low t and ballooninjury compared with all of the other three groups. Sim-ilarly, a significantly greater intima:media ratio wasobserved for all injury models when compared withthe sham group, with statistically the greatest ratio

spase-3 immunostaining, as well as cell proliferation using PCNA im-oon injury at day 1. The arrow indicates positively stained cells. (Mag-3, and PCNA immunostaining for the different injury models at day 1.

FIG. 2. (A) In situ detection of apoptosis using TUNEL assay and caspase-3 immunostaining, as well as cell proliferation using PCNAimmunostaining. Sham, low t, balloon injury, and combined low t and balloon injury at day 3. The arrow indicates positively stained cells.(Magnification 340). (B) The percent positive area by TUNEL assay, caspase-3, and PCNA immunostaining for the different injury modelsat day 3.


observed with the combined low t and balloon injurygroup. The resultant neointimal area was also signifi-cantly greater in all of the injury models comparedwith the sham. Although there was a dramatic trendtowards increased neointimal area for the combinedlow t and balloon injury compared with both the low tand balloon injury groups alone, there was no statisticalsignificance other than when compared with the shamgroup.

DISCUSSION

Balloon-induced direct vessel wall injury and lowflow hemodynamic states lead to neointimal hyperpla-

sia (NH) as a result of induced arterial remodeling.Resultant NH plagues the success of catheter-basedtherapies for arterial occlusive disease, with intermedi-ate-term patency rates varying between 50% and 90%[34, 35]. Although studies demonstrate the effect of bal-loon injury as well as low flow hemodynamic statesalone on NH [6], our study evaluates the effect of lowflow hemodynamic states, assessed via shear stress,and balloon injury on NH, as well as the effect of com-bined low shear stress and balloon injury on NH—a phe-nomenon that frequents the clinical realm.

In this study, we evaluated the effect of low shearstress and balloon injury on vascular smooth musclecell proliferation and apoptosis underlying arterial

FIG. 3. (A) Western blot analysis for caspase-3 expression for thedifferent injury models at day 3. Lane 1 represents the positive con-trol, lane 2, sham group, lane 3, low t group, lane 4, balloon injurygroup, and lane 5, combined low t and balloon injury group. (B) Thedensitometry data for caspase-3 expression at day 3 for the differentinjury models. FIG. 4. (A) Western blot analysis for PCNA expression at day 3.

Lane 1 represents the positive control, lane 2, sham group, lane 3,low t group, lane 4, balloon injury group, and lane 5, combined lowt and balloon injury group. (B) The densitometry data for PCNAexpression at day 3 for the different injury models.


remodeling following injury. Evidence suggests thatthere is an integral role of programmed cell death invessel remodeling and neointimal lesion development[19]. Animal models have shown resultant smooth mus-cle cell apoptosis following low flow hemodynamicstates [6], as well as balloon injury [21]. In the presentstudy, we studied both the apoptotic and cellular prolif-erative response in a combined low shear stress and bal-loon injury animal model. Our model focused not onlyon the occurrence of cell proliferation and apoptosis,but also the onset of cellular response to injury.

Our results revealed an early onset of both cellularproliferation and apoptosis by 24 h, which continuedfor at least 72 h after injury. The resultant cellular pro-liferation and apoptosis was statistically greater follow-ing combined low shear stress and balloon injury thanlow shear stress, balloon injury, or uninjured arteriesalone, concluding that arterial exposure to the combi-nation of low shear stress and balloon injury createsa synergistic response to both smooth muscle cell prolif-eration and apoptosis. This early onset of apoptosis issupported by other investigators evaluating apoptosisfollowing balloon injury alone in rat carotid arteries[21]. However, in our study, we not only demonstratedan early onset of apoptosis following arteries exposedto balloon injury alone as well as low shear stress alone,

but the largest apoptotic response to vessels followingexposure to concurrent balloon injury and low shearstress states.

To further evaluate the up-regulated apoptotic re-sponse, we selectively isolated caspase-3, a criticalmember of the caspase/interleukin-1b-converting en-zyme protease family, which is an end mediator of pro-grammed cell death [36, 37]. Through immunostainingand Western blot analysis, we showed that the apopto-tic response to vessel injury is caspase-3 mediated withconcomitant low shear stress and balloon injury, pro-ducing the greatest response.

Coexistent to the apoptotic response was a robustproliferative response as shown by early PCNA activa-tion by 24 h. This proliferative response is a well knownresponse in NH and has been well studied [11]. Kamenzet al. investigated both the proliferative and apoptoticresponse following balloon angioplasty in rabbit carotidarteries, showing an overall greater incidence in cellu-lar proliferation in comparison with cellular apoptosis,leading to overall net NH at 28 d [22]. They alluded tothe existence of a delicate balance between cell

FIG. 5. Rabbit CCA exposed to sham (A,a), low t (B,b), balloon injury (C,c), and combined low t and balloon injury (D,d) harvested after 4 wkand stained with Weigert-van Gieson stain. / represents Internal Elastic Lamina while 4 indicates NH. Note profound NH in the combinedlow t and balloon injury group (A, B, C, D, 34; a, b, c, d, 340 magnification).


proliferation and apoptosis within the smooth musclecells of the vessel wall, and that small shifts lead to ei-ther net cell accumulation or cell death [22].

NH is a complex physiologic response with multiplephysiologic variables [38], and the balloon injury-lowflow model has helped elucidate many of its mediators[31, 39]. With this in mind, we limited the study tothe assessment of concomitant proliferation and apo-ptosis. In our study, we demonstrated an up-regulationof both cellular proliferation and apoptosis with almostevery injury model compared with our control rabbits,with the combination of low shear stress and balloon in-jury leading to the greatest up-regulation. We believethis up-regulation of both apoptosis and cellular prolif-eration indicates not necessarily a balance, but a wellcoordinated response of cellular demise and cell sur-vival underlying arterial remodeling, since the greatestNH in our study resulted from the combined low shear

TABL

Carotid Artery Morp

Model Neointimal thickness (mm) M

Sham (n ¼ 6) 0Low t (n ¼ 4) 26 6 3*,zBalloon injury (n ¼ 4) 51 6 17*,zLow t and balloon injury (n ¼ 4) 151 6 35*


stress and balloon injury, which also produced both thegreatest cellular proliferation and apoptosis. One puta-tive explanation for the considerable increase in NH de-spite increased apoptosis in the combined low shearstress and balloon injury model may be that the apopto-tic cells induce a release of growth factors and cytokineswhich, by a paracrine effect, induce ERK1/2 pathwaysleading to increased smooth muscle cell proliferation,thereby leading to increased NH. Certainly, the NHresponse has been shown to be mediated through mito-gen activated protein kinase signaling pathway [40],modulation by the cytoskeleton due to changes in thephysical flow parameters [41], and by inflammatorymediators and platelets [42]. Although it is difficult toascertain whether the cellular events of apoptosislead to cellular proliferation, or vice versa, it reasonableto conclude that the combination of both low shearstress and balloon injury lead to a significant increase

E 3

hometry at 28 Days

edial thickness (mm) Intima:media Neointimal area (mm2)

149 6 13 0 0165 6 14z 0.16 6 0.02*,z 0.12 6 0.01*,y120 6 17 0.45 6 0.17*,z 0.67 6 0.18*107 6 15* 1.41 6 0.19* 2.05 6 0.81*


in both cell proliferation and cell apoptosis, which inturn results in a synergistic increase NH approximat-ing that seen in clinical restenosis.

ACKNOWLEDGMENTS

CLS was supported by grant K-08 HL091053-01A1 from NIH/NHLBI and by an American College of Surgeons Faculty ResearchFellowship. The contents are solely the responsibility of the authorsand do not necessarily represent the official views of the NHLBI orNIH.

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Concomitant Proliferation and Caspase-3 Mediated Apoptosis in Response to Low Shear Stress and Balloon Injury