inhibition of vascular cell growth by x-ray irradiation: comparison with gamma radiation and...

PII S0360-3016(01)01526-7

BIOLOGY CONTRIBUTION

INHIBITION OF VASCULAR CELL GROWTH BY X-RAY IRRADIATION:COMPARISON WITH GAMMA RADIATION AND MECHANISM OF ACTION

NEAL A. SCOTT, M.D., PH.D.,* IAN R. CROCKER, M.D.,† QIQIN YIN, M.S.* DAN SORESCU, M.D.,*JOSIAH N. WILCOX, PH.D.,*‡

AND KATHY K. GRIENDLING, PH.D.*

Departments of *Medicine, Division of Cardiology and†Radiation Oncology, and‡Division of Hematology,Emory University, Atlanta, GA

Purpose: Catheter-based delivery of gamma and beta radiation effectively inhibits restenosis. Major disadvan-tages of these radioisotopes include continuous emission; excessive depth of penetration, creating safety hazards(gamma); and inadequate penetration, limiting effectiveness (beta). Low-voltage X-rays have a distinct potentialadvantage, because the source is active only when current is applied, and depth of penetration is voltagedependent. This study was performed to determine if low-voltage X-rays inhibit smooth muscle and adventitialcell growth in vitro and to determine the molecular mechanisms involved in this cellular response.Methods and Results: Vascular cells in culture were exposed to low-voltage X-ray radiation and analyzed fortheir subsequent ability to proliferate. X-ray irradiation caused a dose-dependent inhibition in proliferation,similar to the effect seen with equivalent doses of gamma radiation. The radiation-induced inhibition ofproliferation did not appear to be related to apoptosis, but rather to delayed progression through the cell cycle,because a 65% increase in the proportion of cells in S phase was seen 24–96 h after X-ray exposure comparedto control. Expression of p53, a cell cycle transcriptional activator, and p21, a cell cycle inhibitor, weresignificantly elevated after exposure to low-voltage X-rays, providing a potential mechanism for this delay.Conclusions: Low-voltage X-rays can effectively inhibit proliferation of vascular smooth muscle and adventitialcells. This inhibition is apparently due to a delay in progression through the cell cycle, which is mediated byincreases in the levels of cell cycle inhibitors. © 2001 Elsevier Science Inc.

X-ray irradiation, Restenosis, Vascular smooth muscle proliferation, Adventitia.

INTRODUCTION

Gamma and beta radiation have been shown to preventrestenosis after angioplasty in animal models and in clinicaltrials. Wiedermannet al. (1) and Waksmanet al. (2) dem-onstrated that intracoronary gamma radiation could preventrestenosis in a pig coronary overstretch injury model. Cath-eter systems using gamma radiation have been examined inclinical trials and have demonstrated efficacy in the preven-tion of restenosis (3, 4). Because of the depth of penetrationof the gamma energy, the staff members caring for thepatient during this procedure are at risk for radiation expo-sure. This practical issue led to the testing of beta isotopesas an alternative to gamma sources. Beta irradiation wassimilarly efficacious in animal models (5–7) and in humans(8) when equivalent amounts of radiation were deliveredacross the vessel wall to the adventitia. A major limitationof this alternative is that beta energy penetrates only severalmillimeters, making correct placement of the radiation cath-eter critical to the success of the procedure.

Because these limitations are difficult to overcome, alter-native sources of radiation may provide new therapeuticoptions. Since the early 1900s, X-rays have been shown toeffectively treat malignant and nonmalignant proliferativedisorders (9–12). A distinct advantage of X-rays is that thetissue penetration is proportional to the voltage. Low-volt-age X-rays (soft X-rays) have tissue penetration ranges of atleast 10 mm, depending on the energy and filtration method,and have demonstrated clinical utility in the treatment ofcutaneous proliferative disorders (12). An added advantageof X-rays is that they present a lower risk to the patient andstaff. A new catheter system is under development usinglow-energy X-rays as the source of radiation, but basicstudies are limited concerning the effect of this energysource on vascular cells (13).

It has been clearly demonstrated that the major effect ofirradiation on malignancies is cell death (14, 15). In con-trast, when radiation is used in the prevention of restenosis,cellular necrosis is not a common or consistent finding (16,

Reprint requests to: Kathy K. Griendling, Ph.D., Emory Uni-versity, Division of Cardiology, 1639 Pierce Dr., 319 WMB,Atlanta, GA 30322. Tel: (404) 727-8386; Fax: (404) 727-3585;E-mail: [email protected]

This work was supported in part by a Grant-in-Aid from the

Georgia Chapter of the American Heart Association to N.A.S., andby NIH Grant HL57908.Acknowledgment—We thank Carolyn Morris for excellent secre-tarial assistance.

Accepted for publication 15 February 2001.

Int. J. Radiation Oncology Biol. Phys., Vol. 50, No. 2, pp. 485–493, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/01/$–see front matter

485

17). Furthermore, there is evidence on a cellular level thatneither gamma nor beta radiation induces apoptosis of vas-cular smooth muscle cells (VSMCs) (18, 19). Although it isclear that the proliferation normally seen 2–3 days aftercoronary injury is inhibited by intracoronary radiation (16),less is known about the molecular mechanism of growthinhibition. It has been shown that beta and gamma radiationinduce arrest in the G0/G1 phase of the cell cycle (18, 20),but the effect of radiation on specific cell cycle componentsis unclear. In the present study, we compared the effect ofX-ray and gamma irradiation on smooth muscle cell growthand investigated the effect of low-energy X-rays on theexpression of the cell cycle inhibitor p21 (cdkn 1a) and thetranscriptional activator p53 (Tp53).

METHODS AND MATERIALS

MaterialsPolyclonal antibodies to p21, Bax, and p53, and Bcl-2

monoclonal antibodies were from Santa Cruz (Santa Cruz,CA). Isoton II was from Fisher (Norcross, GA). ECL West-ern blotting detection reagent was from Amersham Pharma-cia (Piscataway, NJ), and propidium iodide was from FlukaAldrich (Milwaukee, WI). Human coronary artery smoothmuscle cells, their growth medium, and supplements werepurchased from Cell Systems (Kirkland, WA). All otherchemicals and reagents, including Dulbecco’s modified Ea-gle’s medium (DMEM) with 25 mmol/L Hepes and 4.5 g/Lglucose, were from Sigma (St. Louis, MO).

Cell cultureRat aortic smooth muscle cells (RASMs) were isolated

from male Sprague-Dawley rat thoracic aortas by enzymaticdigestion as described previously (21). Cells were grown inDMEM supplemented with 10% calf serum, 2 mmol/Lglutamine, 100 U/mL penicillin, and 100mg/mL streptomy-cin and were passaged twice a week by harvesting withtrypsin:ethylene diamine tetraacetic acid (EDTA) and seed-ing into 75 cm2 flasks. For experiments, cells betweenpassages 6 and 15 were plated into 6-well dishes in DMEMcontaining 10% calf serum at a concentration of 50,000cells/dish. After 4 h, media was replaced with DMEMsupplemented with 0.1% calf serum, and cells were madequiescent for 84 h.

Human coronary artery smooth muscle cells(HCASMCs) were purchased from Cell Systems. Cellswere grown in the HCASMC complete growth medium andwere passaged up to 5 times as described above for RASMs.Cells were seeded in 6-well dishes at a density of approx-imately 66,000 cells/dish. Before irradiation, cells weremade quiescent in complete growth medium without growthfactor for 72 h.

Porcine coronary artery adventitial cells were isolated byenzymatic dispersion as described previously for smoothmuscle (21). The adventitial cells are of fibroblast origin, asdetermined by initial negative staining fora-actin andmonolayer, rather than hill-and-valley type, growth. In cul-

ture, they gradually begin to expressa-actin, typical of themyofibroblast phenotype. Cell culture was performed in anidentical manner to RASMs, except that 10% fetal bovineserum was used in place of calf serum.

Radiation proceduresCells were exposed to low-voltage X-ray radiation with a

Philips RT250 machine. The energy used averaged 32 kVwith a 2-mm aluminum filter at 20 mA. The dose rate wasconstant (1.81 Gy/min), and doses were altered by changingthe exposure time. For the dose rate experiments, a Varian600CD linear accelerator generating 6-MV X-rays wasused. The dose rate was altered by changing parameterswithin the machine and by varying the distance between theX-ray source and the target. Solid water blocks were used toensure full dose at the surface of the plate.

Cells were exposed to gamma radiation at a dose rate of60 Csy per minute from a Theratron 780 -60Co teletherapyunit utilizing solid water blocks for full buildup.

Cell countsImmediately after exposure to radiation, media was re-

placed with fresh DMEM containing 10% calf serum. Me-dia was changed every other day, and cells were counted atthe indicated time. Cells were trypsinized and diluted 1:10with Isoton II solution and counted in a Coulter Counter(Coulter Electronics, Luton, Beds, England) with a lowerthreshold of 4 microns.

Fluorescence-activated cell sorting (FACS) analysisRASMs made quiescent as described above were subjected

to X-ray irradiation and stained with propidium iodide forFACS analysis of DNA (22). Cells were trypsinized andwashed with citrate buffer (250 mmol/L sucrose, 40 mmol/Ltrisodium citrate, and 5% v/v dimethyl sulfoxide [DMSO]), pH7.6, and resuspended in 1 mL of citrate buffer. Cells werecounted, and concentration was adjusted to 53 106 cells/mL.For the staining procedure, 13 106 cells were centrifuged at4003 g at room temperature. To the pellet, 1.8 mL of trypsinbuffer (3.4 mmol/L trisodium citrate, 0.1% v/v NP-40, 1.5mmol/L spermine tetrahydrochloride, 0.5 mmol/L Tris, and 30mg/mL trypsin) was added, mixed gently, and incubated for 10min. Trypsin inhibitor solution (1.5 mL) (3.4 mmol/L triso-dium citrate, 0.1% v/v NP-40, 1.5 mmol/L spermine tetrahy-drochloride, 0.5 mmol/L Tris, 0.5 mg/mL trypsin inhibitor and0.1 mg/mL ribonuclease A) was then added to the suspensionfor 10 min at room temperature, followed by the addition of 1.5mL of cold staining solution (3.4 mmol/L trisodium citrate,0.1% v/v NP-40, 4.8 mmol/L spermine tetrahydrochloride, 0.5mmol/L Tris, and 0.4 mg/mL propidium iodide) and incubatedin the dark on ice for 10 min. Propidium iodide–stained cellswere then subjected to FACS analysis using FACSort (BectonDickinson). The fluorescence distribution from 10,000 cellswas captured and analyzed using the CELLQuest program(Becton Dickinson). Analysis of the cell cycle phases wasperformed using ModFit LT for Macintosh Version 2 (VeritySoftware House, California) software.

486 I. J. Radiation Oncology● Biology ● Physics Volume 50, Number 2, 2001

Western analysisUntreated and irradiated cells were lysed with 500mL

ice-cold lysis buffer, pH 7.4 ([mmol/L] 50 HEPES, 5EDTA, 50 NaCl), 1% Triton X-100, protease inhibitors (10mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride,10 mg/mL leupeptin) and phosphatase inhibitors ([mmol/L]50 sodium fluoride, 1 sodium orthovanadate, 10 sodiumpyrophosphate, 0.001 microcystin). Solubilized proteinswere centrifuged at 14,0003 g in a microfuge (4°C) for 30min, and supernatant protein was quantified by the Bradfordassay. Proteins (25mg) were separated using 9–12% SDS-PAGE and transferred to nitrocellulose membranes. Mem-branes were blocked overnight at room temperature withphosphate-buffered saline containing 5% nonfat dry milkand 0.1% Tween 20. Blots were incubated with primaryrabbit polyclonal antibodies against p21, Bcl-2, Bax, or p53at 1:500. After incubation with secondary antibodies (horse-radish peroxidase–conjugated goat anti-rabbit antibody,1:2000), proteins were detected by enhanced chemilumines-cence. Band intensity was quantified by densitometry ofimmunoblots using NIH Image 1.61.

DNA ladderingGenomic DNA was isolated from both control and X-

ray–irradiated RASM cells. The cells were washed 23 with8 g/L NaCl, 0.2 g/L KCl, 3 g/L Tris, resuspended in 10mmol/L Tris, 1 mmol/L EDTA buffer, and lysed withextraction buffer containing 0.5% SDS. The viscous solu-tion was incubated with proteinase K for 3 h followed byphenol:chloroform purification. Genomic DNA was precip-itated with ethanol overnight at220°C and recovered withcentrifugation at 13,500 rpm for 30 min. DNA ladderingwas then analyzed by electrophoresis on a 1.5% agarose gel.

Statistical analysisResults are expressed as mean6 S.E. For p21 and p53,

statistical significance was assessed by Student’s unpairedone-tailedt test on untransformed data. For all other exper-iments, significance was assessed by analysis of variance onuntransformed data, followed by comparison of group av-erages by contrast analysis, using the SuperANOVA statis-tical program (Abacus Concepts, Berkeley, CA). Apvalue, 0.05 was considered to be statistically significant.

RESULTS

Effect of gamma irradiation on VSMC proliferationBecause gamma irradiation has been shown to effectively

inhibit restenosis in animal models and in humans (1–3, 23,24), we assessed its direct effect on RASM proliferation.Exposure of quiescent RASMs to 15 Gy gamma radiationsignificantly impaired the ability of RASMs to proliferate inresponse to a subsequent challenge with 10% serum (Fig.1A). After 4 days, the proliferation of irradiated cells was2.7 6 0.02-fold lower than the proliferation of untreatedcells (6.36 0.3 vs. 176 0.2-fold, respectively). This effectwas dose dependent, with an ED50 (dose at which 50% ofthe maximum response is achieved) of approximately 6.5Gy (Fig. 1B).

Effect of low-energy X-rays on vascular cell proliferationTo assess the effect of low-energy X-rays on growth, we

exposed subconfluent RASMs to varying doses of X-rays.As shown in Fig. 2A, after exposure to 15 Gy X-ray irra-diation, the proliferation of irradiated cells in response to10% serum was dramatically impaired compared with con-trol cells. Over a period of 5 days, cell number increased by

Fig. 1. Effects of gamma irradiation on cellular proliferation. (A) Survival curve: RASMs were plated at low density,made quiescent for 84 h, and exposed to 15 Gy gamma radiation (filled circle) or to sham treatment (open circle). Cellswere then immediately challenged with 10% serum and counted every day for 4 days. Each point is the mean6 SEMof triplicate determinations. Error bars are within the width of the symbol. *Indicatesp , 0.05 compared to same-daycontrol. (B) Dose response: Cells prepared as above were exposed to the indicated dose of radiation and counted at 4days. Each point is the mean6 SEM of triplicate determinations from a single experiment representative of three.*Indicatesp , 0.05 compared to dose 0.

487X-ray irradiation and VSMC growth● N. A. SCOTT et al.

only 4.4 6 0.5-fold in irradiated cells, while control cellsincreased by 9.86 0.5-fold (p , 0.001). Importantly, nocell loss was observed after X-ray treatment. The inhibitionof proliferation was dose dependent (Fig. 2B), with a thresh-old of 2.5 Gy, an ED50 of approximately 5 Gy, and amaximal effect at 15 Gy.

In a separate series of experiments, we tested the effect ofX-rays on growth of HCASMCs and porcine adventitialcells. The results were similar to those obtained withRASMs (Table 1), indicating that X-rays are as effective asgamma rays at inhibiting growth of both adventitial andsmooth muscle cells.

Effect of dose rate on RASM proliferationPrevious studies in a porcine coronary overstretch injury

model suggested that the response to radiation may varywith dose rate (25). To determine whether this effect waspresent in our model, we exposed RASMs to dose ratesbetween 1 and 28 Gy/min. There was no significant differ-ence in the inhibitory effect of X-ray radiation at any of thedose rates tested (Fig. 3).

Molecular mechanisms of growth inhibitionA reduced increase in cell number can result from an

increase in cell death (apoptosis) in the face of normal celldivision or a decrease in cell proliferation. To assess theeffect of X-ray irradiation on apoptosis, we measured DNAladdering as well as Bax and Bcl-2 expression. There wasno significant difference in DNA laddering at 4 or 7 daysafter irradiation (data not shown) or the Bcl-2/Bax ratiobetween untreated cells and cells exposed to 15 Gy of X-rayirradiation (Table 2). These data are consistent with previ-ous studies using beta and gamma radiation (18, 19).

Since apoptosis did not appear to be a major mechanismof growth inhibition, and radiation has been shown to causeboth G0/G1 arrest and G2 arrest (26), we used FACS anal-ysis to determine the cell cycle distribution of both un-treated and irradiated cells. At 24 h after addition of 10%serum, a greater proportion of irradiated cells were retainedin G0/G1 compared to the control population (71.46 0.9%vs. 57.26 0.5%, p , 0.0002). By 48 h, we found unex-pectedly that there was a 656 2% increase in the proportionof cells retained in S phase in X-ray–irradiated cells (34.66

Fig. 2. Effects of low-energy X-rays on cellular proliferation. (A) Survival curve: RASMs were plated at low density,made quiescent for 84 h, and exposed to 15 Gy X-ray radiation (filled circle) or to sham treatment (open circle). Cellswere then immediately challenged with 10% serum and counted every day for 5 days. Each point is the mean6 SEMof triplicate determinations from a single experiment representative of 3 identical experiments. Error bars not shown arewithin the width of the symbol. *Indicatesp , 0.05 compared to same-day control. (B) Dose response: Cells preparedas above were exposed to the indicated dose of radiation and counted at 5 days. Each point is the mean6 SEM of 3independent experiments performed in triplicate. *Indicatesp , 0.05 compared to dose 0.

Table 1. Effect of X-ray irradiation on cell growth in vascular cells

Cell type

Cell number, day 0 (3105 cells/dish) Cell number, day 4* (3105 cells/dish)

Control X-ray (15 Gy) Control X-ray (15 Gy)

Rat aortic SMC 0.856 0.04 0.816 0.07 8.36 0.2 3.66 0.4Human coronary artery SMC 0.666 0.05 0.666 0.05 11.46 1.1 2.06 0.1Porcine adventitial fibroblasts 1.16 0.1 1.16 0.1 4.96 0.3 2.96 0.1

* Cells were made quiescent and exposed to 15-Gy X-ray irradiation. Cells were counted immediately (Day 0), or the medium wasreplaced with medium containing 10% calf serum (or complete growth medium for HCASMCs), and cells were counted at Day 4, or, inthe case of the more slowly growing adventitial cells, Day 9. Data are mean6 SEM for triplicate determinations.

Abbreviations:SMC 5 smooth muscle cell.


3.4% vs. 20.96 1.7%, p , 0.03), and a correspondingdecrease in the number of cells in G0/G1 (Fig. 4). By 96 hafter treatment, the proportion of X-ray–treated cells inG2/M was enhanced compared to control (10.76 0.7% vs.6.2 6 0.4%,p , 0.03). Together with the observation thatcell number was significantly reduced in X-ray–irradiatedcells, these data suggest that the major effect of X-rays wasto delay progression through each phase of the cell cycle.

To clarify the potential mechanisms of this delay in cellcycle progression, we investigated the effect of X-ray radi-ation on p53 protein levels in VSMCs. p53 is a transcrip-tional activator that increases the expression of the cellcycle inhibitor p21 (27) and ultimately arrests the cells atvarious points in the cell cycle through p21-dependent andp21-independent mechanisms (28). Preliminary experi-ments showed that 24 h after X-ray treatment, p21 levelswere increased threefold, while p53 levels were unchanged(data not shown). As expected, p53 protein levels were

increased 4 and 7 days after X-ray exposure as comparedwith untreated cells (Fig. 5). By 7 days, there was a 360.1-fold increase in p53 levels in X-ray–treated cells com-pared to untreated controls. To determine whether p53 ac-tivation was associated with an upregulation of p21, weinvestigated the effect of X-ray radiation on p21 proteinlevels in VSMCs. As shown in Fig. 6, exposure of VSMCsto 15 Gy irradiation caused a 66 0.25-fold increase in p21protein expression after 4 days, and p21 levels remainedelevated for at least 7 days.

DISCUSSION

A major impetus for this study was the anticipated de-velopment of an intravascular catheter system that utilizeslow-energy X-rays to prevent restenosis. Low-energy X-rays have distinct advantages over both gamma and betaisotopes that have already proven effective in animal mod-els of restenosis and clinical studies. Since X-rays are de-pendent on electrical current and voltage, radiation is pro-duced on demand as opposed to continuously emittingradioisotopes. The penetration of X-rays can be accuratelycontrolled with changes in voltage. In comparison, the pen-etration of isotopes is solely a function of their atomiccomposition. These studies were performed to compare theeffects of equivalent energies of low-energy X-rays andgamma rays on vascular cell growth and indicate that low-energy X-rays have similar effects on the inhibition ofvascular cell proliferation.

Previous studies have shown that 5–8 Gy gamma irradi-ation effectively inhibits proliferation of VSMCs by 50%(19). Our results confirm this observation and show that

Fig. 3. Effect of dose rate on cellular proliferation. RASMs wereplated at low density, made quiescent for 84 h, and exposed to theindicated dose rates of X-ray radiation. Cells were then immedi-ately challenged with 10% serum and counted 5 days later. Eachpoint is the mean6 SEM of triplicate determinations from a singleexperiment representative of 2 identical experiments. Error barsnot shown are within the width of the symbol.

Table 2. Effect of X-ray irradiation on Bcl-2 and Baxexpression in RASMs

Time after radiation exposure

2 h 24 h 7 days

Bcl-2 1.36 0.2 0.96 0.1 2.26 0.1Bax 1.16 0.1 1.16 0.2 1.46 0.2Bcl-2/Bax ratio 1.2 0.8 1.6

Cells were made quiescent, exposed to 15-Gy X-ray irradiation,and harvested at the indicated times for analysis of Bcl-2 or Baxexpression by Western blot. Blots were quantified by densitome-try, and protein levels are expressed as fold-change over nonirra-diated cells harvested at the same time. Data are means6 SEM fortwo experiments.

Abbreviations:RASMs 5 rat aortic smooth muscle cells.

Fig. 4. Cell cycle analysis of X-ray–irradiated cells. RASMs pre-pared as described in the legend to Fig. 1 were sham treated (openbars) or exposed to 15 Gy X-ray irradiation (hatched bars). Cellswere prepared for cell cycle analysis as described in “Methods.”Each bar represents the mean6 SEM of 3 independent experi-ments and indicates the percentage of cells in each phase of the cellcycle. *Indicatesp , 0.05 compared to control.


short exposure to X-rays was equally effective at inhibitingVSMC growth (Fig. 1 and Table 1). The ED50 for X-rayirradiation was about 5 Gy, with a maximal effect observedby 7.5–10 Gy (Fig. 2). Our studies do not confirm an effectof dose rate on the extent of inhibition of cell proliferation(Fig. 3).

Radiation can induce apoptosis, necrosis, and inhibitionof normal cell cycle progression in various cell types (26).Apoptosis seems to be a consequence of radiation in radi-osensitive cells or in tissues in which apoptosis is a normaloccurrence (26). However, the response of cells to radiationhas been shown to be cell cycle dependent. Cells in S or G2

phase (e.g., tumor cells) are more sensitive to mutagenicdoses of radiation and arrest in G2 and undergo apoptosissecondary to p53 upregulation (29). Cells in G0/G1 (quies-cent) are either delayed in their progression into S phase orarrest in G1 when exposed to either X-ray or gamma radi-

ation (18, 29). Others have shown that radiation of VSMCswith either beta or gamma emitters fails to increase apopto-sis (18, 19), and the present results indicate a similar lack ofeffect of X-rays on apoptosis in these cells. It is possible thatthe response to radiation may be cell specific and dependenton the duration and amount of radiation exposure. Irradia-tion of tumor cells usually occurs over prolonged timeswhen the cells are actively proliferating (S/G2/M). In con-trast, in studies with vascular cells, the cells are semiquies-cent (G0/G1 phase) when they are exposed to radiation.

The ability of beta particles and gamma radiation toattenuate VSMC proliferation has been attributed to inhibi-tion of cell cycle progression (18, 30). Farehet al. (18)showed that prolonged exposure of cells (24–72 h) to betaparticles induced arrest in G1. In contrast, we observed thatat 24 h, a greater proportion of irradiated cells were retainedin G0/G1 compared to the control population. By 48 h, a

Fig. 5. Effect of X-ray irradiation on the expression of p53 protein. Cells prepared as described in the legend to Fig. 1were exposed to 15 Gy X-ray radiation or sham treatment and harvested after 4 or 7 days. Upper panel showsrepresentative Western blotted for p53. Lower panel shows mean6 SEM of densitometric data derived from 3independent experiments. Open bars, control; hatched bars, X-ray treatment. *Indicatesp , 0.05 compared to control.


greater proportion of X-ray–treated than nonirradiated cellswere retained in S phase (the next stage of progression). At96 h, the proportion of cells in G2/M was greater in X-ray–treated cells than in nonirradiated cells. Thus, there was nospecific arrest in any phase of the cell cycle. Together withthe observation that cell number was significantly reducedin X-ray–irradiated cells (Fig. 2), these data suggest that themajor effect of X-rays was to delay progression througheach phase of the cell cycle. In other words, it takes longerfor each cell to progress through the cell cycle when it hasbeen exposed to X-ray irradiation, leading to apparentgrowth inhibition. This might have biologic significance inthe setting of coronary angioplasty or stenting. Within 2–3days after angioplasty, there is abundant proliferation ofcells in the adventitia (31). X-rays, by virtue of their abilityto delay, but not prevent, adventitial and smooth muscleproliferation, may allow cells to minimally proliferate toensure normalization of the arterial wall stress while at thesame time blocking the pathologic remodeling process. Fur-

ther studies are required to assess the efficacy of X-raytreatment in prevention of restenosisin vivo.

X-ray irradiation of RASMs dramatically upregulatedboth p53 and p21, similar to results reported for gammairradiation (30). p53 transcriptionally activates p21, whichin turn binds to the cyclin-dependent kinases mediatingentry into S phase, thus inhibiting their activity (32). More-over, p21 binds to proliferating cell nuclear antigen andprevents its interaction with DNA polymerase-d, blockingDNA synthesis and S phase progression without affectingDNA repair activity (33). p53 is activated in response toDNA damage, a well-known effect of radiation (34), so it ispossible that the increase in p53 is a consequence ratherthan a cause of growth inhibition. However, cells defectivein p53 do not undergo G1 arrest in response to gammairradiation (35), and this p53-dependent G1 stasis requiresinduction of p21 (27). There are also several potentialp21-independent mechanisms that may be responsible forthe p53-induced delay of cell cycle progression in smooth

Fig. 6. Effect of X-ray irradiation on the expression of p21 protein. Cells prepared as described in the legend to Fig. 1were exposed to 15 Gy X-ray radiation or sham treatment and harvested after 4 or 7 days. Upper panel showsrepresentative Western blotted for p21. Lower panel shows mean6 SEM of densitometric data derived from 3independent experiments. Open bars, control; hatched bars, X-ray treatment. *Indicatesp , 0.05 compared to control.


muscle cells. p53 binds and negatively regulates the pro-moter of cyclin A, which is essential for activation ofcyclin-dependent kinase-2 and progression of the cells be-yond G1/S checkpoint and through S phase (28). Further-more, p53 may upregulate GADD45 (growth arrested andDNA damage), which in turn inhibits proliferating cellnuclear antigen and blocks DNA synthesis and therefore Sphase progression (36). Thus, our results suggest that X-rays may cause a delay in cell cycle progression of VSMCsat least in part by inducing the expression of p53 and p21.

Intravascular radiation with catheters utilizing gamma orbeta isotopes has clinical efficacy as follows: the primaryprevention of restenosis after balloon angioplasty and stent-ing in native coronary arteries (4, 8), prevention of therecurrence of restenosis (3, 37), and prevention of restenosisin peripheral vessels (38). In the swine coronary injurymodel, a single dose of radiation (8–30 Gy) administeredimmediately after the interventional procedure results in adose-dependent decrease in neointimal formation (1, 2, 23).One problem with beta radiation is a short penetration depthin the vessel wall (39), limiting its usefulness in attenuating

the proliferation and remodeling that begins in the adven-titial layer (40). Low-energy X-rays are more graduallyattenuated (they can penetrate at least 10 mm depending onvoltage and filtration), suggesting that they may more ef-fectively influence the contribution of the adventitia torestenosis. In our study, X-rays were effective in slow-ing the proliferation of adventitial fibroblastsin vitro, rein-forcing the possibility that prevention of adventitial cellactivation is one of the potential benefits of low-energyX-rays.

In summary, these studies demonstrate that similar levelsof growth inhibition in VSMCs and adventitial cells can beobtained with equivalent physical doses of low-energy X-ray and gamma irradiation. The mechanism of inhibition ofVSMC proliferation by low-energy radiation involves theinduction of endogenous cell cycle inhibitors (p21 and itstranscriptional activator p53), leading to a delay in cell cycleprogression and not to radiation-induced cell killing. Radi-ation with low-energy X-rays may thus be a viable and safealternative to treatment of restenosis after angioplasty withgamma or beta radiation.

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inhibition of vascular cell growth by x-ray irradiation: comparison with gamma radiation and...

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