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Iron-Ion Radiation Accelerates Atherosclerosis in Apolipoprotein E-Deficient MiceAuthor(s): Tao Yu, Brian W. Parks, Shaohua Yu, Roshni Srivastava, Kiran Gupta, Xing Wu, SamanKhaled, Polly Y. Chang, Janusz H. Kabarowski, and Dennis F. KucikSource: Radiation Research, 175(6):766-773. 2011.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR2482.1URL: http://www.bioone.org/doi/full/10.1667/RR2482.1

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Iron-Ion Radiation Accelerates Atherosclerosis in ApolipoproteinE-Deficient Mice

Tao Yu,a Brian W. Parks,b,1 Shaohua Yu,b Roshni Srivastava,b Kiran Gupta,a Xing Wu,a Saman Khaled,a

Polly Y. Chang,c Janusz H. Kabarowskib,2 and Dennis F. Kucika,d,e,2

Departments of a Pathology, b Microbiology and d Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama; d SRIInternational, Menlo Park, California; and e VA Medical Center, Birmingham, Alabama

Yu, T., Parks, B. W., Yu, S., Srivastava, R., Gupta, K., Wu,X., Khaled, S., Chang, P. Y., Kabarowski, J. H. and Kucik,D. F. Iron-Ion Radiation Accelerates Atherosclerosis inApolipoprotein E-Deficient Mice. Radiat. Res. 175, 766–773(2011).

Radiation exposure from a number of terrestrial sources isassociated with an increased risk for atherosclerosis. Recently,concern over whether exposure to cosmic radiation might pose asimilar risk for astronauts has increased. To address thisquestion, we examined the effect of 2 to 5 Gy iron ions (56Fe), aparticularly damaging component of cosmic radiation, targetedto specific arterial sites in male apolipoprotein E-deficient(apoE2/2) mice. Radiation accelerated the development ofatherosclerosis in irradiated portions of the aorta independentof any systemic effects on plasma lipid profiles or circulatingleukocytes. Further, radiation exposure resulted in a more rapidprogression of advanced aortic root lesions, characterized bylarger necrotic cores associated with greater numbers ofapoptotic macrophages and reduced lesional collagen comparedto sham-treated mice. Intima media thickening of the carotidarteries was also exacerbated. Exposure to 56Fe ions cantherefore accelerate the development of atherosclerotic lesionsand promote their progression to an advanced stage character-ized by compositional changes indicative of increased thrombo-genicity and instability. We conclude that the potentialconsequences of radiation exposure for astronauts on prolongeddeep-space missions are a major concern. Knowledge gainedfrom further studies with animal models should lead to a betterunderstanding of the pathophysiological effects of acceleratedion radiation to better estimate atherogenic risk and developappropriate countermeasures to mitigate its damaging ef-fects. g 2011 by Radiation Research Society

INTRODUCTION

Radiation exposure from a number of sources isassociated with an increased risk for developingatherosclerotic cardiovascular disease. For example,after having undergone radiation therapy for head andneck cancer, even relatively young patients who wouldotherwise be at very low risk have an increased incidenceof stroke (1). Similarly, major causes of death for atomicbomb survivors include myocardial infarction andstroke (2). For workers exposed at Chernobyl, the mostsignificant adverse effect was cardiovascular disease (3).Even radiation technologists working before 1950 (whenshielding was less rigorous) had an increased incidenceof myocardial infarction (4). An understanding of thesecardiovascular risks has come primarily from epidemi-ological data. Doubt remains about the causal nature ofthe association between radiation and atherosclerosis,however, due to a paucity of supporting animal and celldata.

Due to interest in deep-space travel to the Moon,asteroids and/or Mars, concern has grown over whetherexposure to cosmic radiation might pose a similar riskfor astronauts. Although radiation risks are wellestablished for terrestrial radiation sources, the risk ofdeep-space travel is difficult to predict, largely becauseso few humans have been exposed. On Earth, and evenin Earth orbit, the magnetic field provides protectionfrom the damaging effects of cosmic radiation. The onlytravel beyond the protection of the Earth’s magneticfield by humans so far has been the Moon missions, andthese exposures were relatively short. Thus so fewastronauts have been exposed to high levels of cosmicradiation that epidemiological data cannot be used topredict risk. Furthermore, there are no published studiesof the effects of cosmic radiation in animal models ofatherosclerosis.

The quality of particle radiation in the galactocosmicenvironment is very different from that of the conven-tional low-energy X or c rays commonly encountered onEarth. The profile of energy deposition and thus the

1 Brian W. Parks is currently is in the Department of Medicine atthe University of California, Los Angeles.

2 Addresses for correspondence: University of Alabama at Bir-mingham, 640A Kaul Building, 720 S. 20th Street, Birmingham, AL35294; e-mail: Kucik@uab.edu; University of Alabama at Birming-ham, 334 Bevil Biomedical Research Building, 845 19th Street South,Birmingham, AL 35294; e-mail: janusz@uab.edu.

RADIATION RESEARCH 175, 766–773 (2011)0033-7587/11 $15.00g 2011 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2482.1

766

radiation-induced effects resulting from exposure tothese highly charged and energetic (HZE) ions as theypass through the body are very different from those of Xrays or c rays. Depending on the initial energy andcharge of the HZE particles, the effect of such cosmicradiation on tissues can be very different and potentiallymore damaging to vascular tissue than the low-linearenergy transfer (LET) forms of radiation with which weare more familiar. Moreover, while avoidance ofradiation exposure, including the use of lead shielding,is a common strategy on Earth, this is not feasible formitigating exposure to cosmic radiation. Acceleratedions can interact with shielding to generate a number ofsecondary particles that may also have biological effects.Although it is possible to shield against accelerated ionsusing other materials, incorporation of such materialsinto spacesuits can present significant challenges.

The apolipoprotein E-deficient (apoE2/2) mouse is awidely used animal model of atherosclerosis thatspontaneously develops atherosclerotic lesions through-out the aortic tree in the absence of high-fat/cholesteroldiet feeding (5, 6). The apoE2/2 mouse has proven to beuseful for understanding pathological changes caused byterrestrial radiation (7, 8). However, the effect ofexposure to accelerated particle radiation such as 56Feions on atherogenesis has not been examined in ananimal model. To determine whether cosmic radiationhas the potential to increase risk for atherosclerosis, weexamined the development of atherosclerotic lesionsafter targeted exposure of 10-week-old male ApoE2/2

mice to 2 Gy and 5 Gy 56Fe ions. Analysis at 13 and40 weeks postirradiation indicated that the developmentof atherosclerosis was accelerated in irradiated arteriescompared both to nonirradiated portions of the aorta inthe same animal and to nonirradiated control apoE2/2

mice.

MATERIALS AND METHODS

Animal Handling and Radiation Treatment

Nine-week-old male apoE2/2 mice were shipped directly from TheJackson Laboratories, Bar Harbor, ME, to the Animal Facility atBrookhaven National Laboratory (BNL), where they were housed for1 week before irradiation for acclimation. Prior to irradiation, micewere anesthetized by intraperitoneal injection of ketamine andxylazine (150 mg/kg and 15 mg/kg body weight, respectively) andimmobilized in custom-built mouse holders. Mice were then exposedto targeted radiation with 0 Gy (sham-treated), 2 Gy or 5 Gy of600 MeV/u 56Fe ions at the NASA Space Radiation Laboratory(NSRL) at BNL. The LET of the iron-ion beam was ,190 keV/mm.Radiation was targeted exclusively to the upper aortic tree (includingthe aortic root, aortic arch and the carotid arteries) as shown inFig. 1A. Radiation dosimetry using ion chambers was conductedprior to exposure to ensure that the beam was properly calibrated andthat the dose distribution across the targeted zones was uniform withonly a minimal dose to the shielded portion of the animal. The micewere then shipped to the University of Alabama at Birmingham(UAB) within 1 week and maintained on a standard chow diet for theduration of the study. All animal handling and experimental

procedures were performed with approval from the Animal CareCommittees of UAB and BNL, following the guidelines provided bythe 1970 Animal Welfare Act and its amendments and the principlespromulgated by the National Research Council in its Guide for theCare and Use of Laboratory Animals (1996).

Shielding/Targeting of Radiation

Radiation was targeted by means of a collimator (provided byNSRL) consisting of a combination of lucite, aluminum andpolyethylene radiation shield (85 mm, 75 mm and 90 mm thick,respectively) through which holes had been machined to allowpassage of radiation in selected spots. This collimator was placed inthe beam line, and the mice were aligned so that only the areasindicated in Fig. 1A were exposed to the unshielded beam.

Analysis of Blood

Peripheral blood was obtained by retro-orbital puncture foranalysis of circulating leukocytes and plasma lipid profiles. Frequen-cies of peripheral blood T cells, B cells and myeloid cells weremeasured by flow cytometry on a BD FACSCalibur using anti-CD3,anti-B220 and anti-CD11b antibodies (BD Pharmingen, FranklinLakes, NJ), respectively. Plasma lipids were measured as describedpreviously (9).

Aorta En Face Lesion Quantification

Euthanized mice were perfused with phosphate-buffered saline(PBS) followed by formal sucrose (4% paraformaldehyde, 7.5%

sucrose, 10 mmol/liter sodium phosphate buffer, 2 mmol/liter EDTA,20 mmol/liter butylated hydroxytoluene). Aortas were cut open,pinned flat with 0.2-mm minutien pins, and incubated with Sudan IVstaining solution (0.5% Sudan IV, 35% ethanol, 50% acetone) for10 min. Aortas were destained with 80% ethanol and color imageswere acquired with a digital camera (Zeiss Axiocam) attached to aZeiss Stemi SV6 dissecting microscope. Lesion areas were outlined

FIG. 1. Targeted exposure to 56Fe ions accelerates the develop-ment of aortic atherosclerotic lesions in apoE2/2 mice. Panel A: Thewhite boxes over the image of the dissected mouse and en face aortapreparation delineate the portion of the aortic tree exposed toradiation. Panel B: Aortic lesion coverage in irradiated (IR area) andnonirradiated (non-IR area) portions of en face aorta specimens fromthe indicated apoE2/2 mice. n 5 10 in each group; mean ± SEM; *P, 0.05 by Mann-Whitney test. Upper panel: 13 weeks postirradiation,lower panel: 40 weeks postirradiation. Panel C: Radiation-targetedareas of aortas from representative apoE2/2 mice in panel B.

56Fe IONS ACCELERATE ATHEROSCLEROSIS 767

and measured using Carl Zeiss AxioVision Rel. 4.6 software. Aortaswere divided into areas that had been targeted by the iron-ionradiation beam and those that were not. The percentage of the aortathat was covered by plaques was then quantified.

Aortic Root Lesion Analysis

The upper haves of the hearts removed from mice were embeddedin OCT medium (Tissue-Tek), frozen and stored at 220uC. Uponcryosectioning, the orientations of the blocks were adjusted to makethree aortic valve leaflets appear at the same level. From the firstappearance of all three aortic valve leaflets, 48 sections (8 mm persection in thickness) were collected. Twenty-four sections per mousewere used for lesion quantification and 24 intervening sections wereused for further immunohistochemical and immunofluorescencestaining. Atherosclerotic lesions were stained with Oil red-O (Sigma)and lesion areas were measured morphometrically under a ZeissAxiostar Plus microscope using a 1-mm2 eyepiece grid (100 3

10,000 mm2) at 1003 magnification. For collagen component analysis,corresponding sections were stained with Masson’s trichrome(Newcomer Supply, Middleton, WI). Infiltration of macrophages inatherosclerotic lesions was detected by immunofluorescence staining(F4/80, Abcam, ab6640, 1:200).

Terminal deoxynucleotidyl transferase dUTP nick end-labeling(TUNEL) kits (Chemicon, Temecula, CA) were used for apoptosisanalysis according to the manufacturer’s instructions as describedpreviously (9, 10). The percentage TUNELz staining, macrophagez

staining and collagen content of aortic root lesions were measured inthree lesions from each of eight control (0 Gy) and eight irradiated(5 Gy) apoE2/2 mice (24 lesions total for each group).

Left Carotid Artery Intima Analysis

Left carotid arteries (3 cm in length) were harvested from the miceand dissected away from the surrounding tissue and excess fat,immersed in OCT and frozen. Twenty-four consecutive longitudinal8-mm sections were placed on four slides (six sections per slide). Forthe quantitative analysis of intima thickness, six sections per mousewere stained with hematoxylin and eosin (H&E, Sigma). Intimathicknesses were measured under 2003 magnification on an opticalmicroscope (Zeiss Axiostar Plus) with an eyepiece scale. The averagethickness of these measurements was calculated per mouse. Immu-nofluorescence staining was performed on the corresponding sectionto identify endothelial cells (anti-PECAM-1, BD Pharmingen 550274,1: 100), smooth muscle cells (anti-Actin Smooth Muscle, E2464, 1:200, Spring Bioscience Inc.), and cell nuclei (Hoechst 33258,Molecular Probes, H3569, 10 mg/ml).

RESULTS

56Fe Ions Accelerate Aortic Atherosclerosis in apoE2/2

Mice

Male apoE2/2 mice fed a standard chow diet wereexposed to targeted radiation (2 Gy or 5 Gy of 600 MeV

56Fe ions) at 10 weeks of age. 56Fe ions were targetedexclusively to the upper aortic tree (including the aorticroot, aortic arch and carotid arteries) (Fig. 1A). Thisapproach simplified the interpretation of results becausethe direct effects of radiation on specific arterial sitescould be assessed in the absence of potentially con-founding complications arising from systemic radiationeffects on the bone marrow or other organs. This limitedfield of radiation exposure also spared most leukocytes.The apoE2/2 mice irradiated in this way were analyzed13 and 40 weeks postirradiation to determine whether56Fe-ion exposure had accelerated the development ofatherosclerosis and to evaluate advanced lesion compo-sition. Radiation had no significant effect (as determinedby a Mann-Whitney test) on the frequency of circulatingT cells, B cells and monocytes (Table 1) or on plasmalipid profiles (Table 2) 13 weeks and 40 weeks postirra-diation. Examination of atherosclerosis in lipid-staineden face aortic preparations from control (0 Gy) andirradiated (2 Gy and 5 Gy) apoE2/2 mice revealedsignificantly increased lesion areas in the radiation-targeted portion of the aortas (aortic arch) after 5 Gy(but not 2 Gy) in apoE2/2 mice 13 weeks postirradiation(average 54.3% increase) (Fig. 1B, C). However, lesionareas in the targeted portion of the aortas of control andirradiated apoE2/2 mice were comparable at 40 weekspostirradiation (Fig. 1B). The non-targeted portions ofaortas (thoracic and abdominal aorta) showed nosignificant differences in lesion area at any 56Fe-ion doseor time (Fig. 1B), demonstrating the absence of asystemic effect of targeted 56Fe-ion radiation on athero-sclerosis.

56Fe Ions Accelerate Atherosclerosis at the Aortic Root inapoE2/2 Mice

To determine whether 56Fe-ion radiation similarlyaccelerated atherosclerosis at other targeted arterialsites, we measured atherosclerotic lesion size at theaortic root. Radiation effects were significantly morepronounced at this site. A dose of 2 Gy 56Fe ionsresulted in an increase in average lesion size of 37.9%and 24.4% at 13 weeks and 40 weeks postirradiation,respectively (Fig. 2A). A dose of 5 Gy 56Fe ions resultedin an increase in average lesion size of 88.6% and 33.8%at 13 weeks and 40 weeks postirradiation, respectively

TABLE 1Percentages of CD3+ T Cells, B220+ B Cells and CD11b+ Myeloid Cells (monocytes and neutrophils) in the Peripheral

Blood of Control and 56Fe-Ion-Irradiated apoE2/2 Mice

Dose

13 weeks postirradiation 40 weeks postirradiation

CD3z B220z CD11bz CD3z B220z CD11bz

0 Gy 19.6 ± 1.1 57.3 ± 2.4 22.1 ± 0.9 16.8 ± 0.9 60.8 ± 2.1 21.5 ± 1.82 Gy 16.2 ± 0.8 57.1 ± 2.3 26.4 ± 2.6 18.5 ± 1.3 54.3 ± 2.7 25.6 ± 2.55 Gy 17.7 ± 1.0 53.7 ± 2.4 27.9 ± 2.7 18.9 ± 1.1 55.9 ± 2.1 24 ± 1.3

768 YU ET AL.

(Fig. 2A). Thus, unlike the aortic arch, in which only5 Gy 56Fe ions accelerated lesion development but hadno effect at 40 weeks postirradiation (Fig. 1B), bothdoses of 56Fe ions accelerated atherosclerosis at theaortic root and their pro-atherogenic effect was still seenup to 40 weeks postirradiation.

56Fe Ions Accelerate Carotid Intima Media Thickening inapoE2/2 Mice

Carotid intima media thickening is an independentpredictor of cardiovascular events in humans (11) and isa response to injury of the arterial wall characterized byproliferative expansion of intimal smooth muscle cells(SMCs) (12, 13). To determine whether targeted 56Fe-ionradiation elicited carotid intima media thickening, weexamined the left carotid arteries from control and 56Fe-ion-irradiated apoE2/2 mice by immunohistochemicaland immunofluorescence staining. The thickness of theintima media of the left carotid artery was significantlyincreased in apoE2/2 mice 13 weeks after irradiationwith 2 Gy and 5 Gy 56Fe ions (Fig. 3). However, carotidintimal thickness was comparable in control and

irradiated groups of apoE2/2 mice by 40 weeks postir-radiation (Fig. 3). Thus vascular injury caused by asingle targeted exposure to 56Fe ions was sufficient toaccelerate carotid intima media thickening in apoE2/2

mice.

Accelerated Progression of Advanced Aortic Root Lesionsin 56Fe-Ion-Irradiated apoE2/2 Mice

The pro-atherogenic effect of 56Fe ions in apoE2/2

mice was most robust at the aortic root compared toother arterial sites examined. Increases in lesion size atthe aortic root caused by a single targeted exposure to2 Gy or 5 Gy 56Fe ions were maintained up to 40 weekspostirradiation (Fig. 2A). We therefore investigated thecompositional features of these advanced lesions(40 weeks postirradiation), which are considered tocontribute to atherosclerotic plaque instability in hu-mans, and whether thrombogenicity could be exacer-bated by prior exposure to 56Fe ions. Such features are

TABLE 2Plasma Lipid Profiles in Control and Irradiated apoE2/2 mice

TC HDL-C LDL-C TG FFA

0 Gy 13 weeks (n 5 10) 344.4 ± 34.6 45.4 ± 3.4 308.1 ± 33.2 96.2 ± 15.2 15 ± 1.72 Gy 13 weeks (n 5 10) 355.5 ± 18.4 51.1 ± 4.2 304.4 ± 20.8 83 ± 8.1 17.1 ± 1.25 Gy 13 weeks (n 5 10) 311.8 ± 16.7 55.6 ± 3.8 256.2 ± 18.4 63.3 ± 14 13.9 ± 1.10 Gy 40 weeks (n 5 8) 577.5 ± 34.5 28.1 ± 1.4 549.4 ± 35.2 81.1 ± 14.4 21.6 ± 1.52 Gy 40 weeks (n 5 8) 495.6 ± 25.7 28.4 ± 3.1 467.3 ± 24.6 69.6 ± 11.9 17.3 ± 0.85 Gy 40 weeks (n 5 8) 559.3 ± 37.7 24.3 ± 1.5 535 ± 36.6 56.1 ± 3.6 21.4 ± 1.9

Notes. n: number of mice. TC: total cholesterol, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol,TG: triglycerides, FFA: free fatty acids.

FIG. 2. Targeted 56Fe-ion radiation accelerates atherosclerosis atthe aortic root in apoE2/2 mice. Panel A: Mean lesion areas persection in the aortic root of control and 56Fe-ion-irradiated apoE2/2

mice. n 5 10 in each group; mean ± SEM; *P , 0.05 by Mann-Whitney test. Panel B: Oil red-O-stained sections of the aortic rootfrom representative apoE2/2 mice in panel A.

FIG. 3. Targeted 56Fe-ion radiation accelerates carotid arteryintima media thickening in apoE2/2 mice. Panel A: Intima mediathickness of left carotid arteries in control and irradiated apoE2/2

mice 13 weeks and 40 weeks postirradiation (mean ± SEM).Thickness is significantly greater at 13 weeks after both 2 and 5 Gy(*P , 0.05 by Mann-Whitney test). Panel B: H&E-stainedlongitudinal carotid artery sections from representative control(0 Gy) and irradiated (5 Gy) apoE2/2 mice in panel A 13 weekspostirradiation. Panel C: Co-immunofluorescence staining of carotidartery sections from representative control (0 Gy) and irradiated(5 Gy) apoE2/2 mice in panel A 13 weeks postirradiation. Green:smooth muscle cells, red: endothelial cells, blue: Hoescht 33258z

nuclei. Note the significantly increased intimal smooth muscle celllayer in 56Fe-ion-irradiated apoE2/2 mouse.

56Fe IONS ACCELERATE ATHEROSCLEROSIS 769

consistent with a more rapid progression of atheroscle-rotic lesions. Aortic root sections were stained withMasson’s trichrome to examine lesion morphology andto quantify lesion-associated collagen, a key protectivecomponent of atherosclerotic plaques that contributes totheir stability and resistance to rupture (14). In addition,lesion sections were subjected to TUNEL staining toquantify apoptotic cells. Enlarged necrotic cores wereobserved in lesions from apoE2/2 mice irradiated with56Fe ions compared to their nonirradiated counterparts(Fig. 4A) that were associated with significantly in-creased lesional apoptotic (TUNELz) macrophagestaining (average 3.8-fold/284.3% increase in the per-centage TUNELz lesion area) (Fig. 4B and C). This wasnot simply due to an increased number of lesionalmacrophages in the irradiated apoE2/2 mice (Fig. 4D).Lesion areas occupied by collagen were significantlyreduced in the apoE2/2 mice irradiated with 5 Gy(average 23.3% reduction in percentage lesion collagen)(Fig. 4A and E), although this may be primarily due tothe larger necrotic cores that are devoid of collagendeposition. Taken together, these observations indicatethat the ability of 56Fe-ion radiation to accelerateatherogenesis in apoE2/2 mice results in a moreadvanced lesion phenotype compared to that in theirnonirradiated age-matched counterparts that is charac-terized by larger necrotic cores (resulting from morelesional macrophage death) and reduced lesional colla-gen. Considering that the necrotic core is a major sourceof thrombogenic material released from rupturedunstable atherosclerotic plaques (15), this suggests that56Fe-ion radiation exposure can accelerate the develop-ment of advanced thrombogenic and unstable lesions.These findings have direct implications regardingsusceptibility to stroke and myocardial infarction.

DISCUSSION

In this study, acceleration of atherogenesis by 56Feions was demonstrated in apoE2/2 mice. A single dose of56Fe ions resulted in consequences at a distant time.Notably, atherosclerotic lesions were increased in size atthe aortic arch 13 weeks postirradiation with 5 Gy 56Feions but not with 2 Gy or at 40 weeks postirradiation ateither dose (Fig. 1B), demonstrating a dose-specific andtime-dependent acceleration of atherosclerosis at thissite. Intima media thickening of the carotid arteries, anindependent predictor of stroke and myocardial infarc-tion in humans (11), was also accelerated by 56Fe-ionradiation in apoE2/2 mice (Fig. 3), suggesting involve-ment of components in the arterial wall in the biologicalresponse to 56Fe-ion-induced injury. However, carotidintima thickness was comparable in control andirradiated groups of apoE2/2 mice by 40 weeks postir-radiation (Fig. 3). Lesion size at the aortic root, on theother hand, was significantly increased by both doses of

56Fe ions and at both 13 and 40 weeks postirradiation(Fig. 2), demonstrating a significantly more robust pro-atherogenic effect of 56Fe ions at this aortic site. Thecomposition of aortic root lesions in irradiated apoE2/2

mice 40 weeks postirradiation was indicative of a moreadvanced stage characterized by larger necrotic coresassociated with a greater incidence of macrophage deathwithin lesions (Fig. 4). Furthermore, reduced collagencontent of these advanced lesions was observed,consistent with a report of the effects of X rays oncarotid lesions in apoE2/2 mice (7). Potentially fatalclinical manifestations of atherosclerosis such as strokeand myocardial infarction can be caused by the ruptureof advanced atherosclerotic plaques and subsequentrelease of their thrombogenic contents into the circula-tion (16). Because lesion-associated apoptosis andcollagen content are major deterministic factors ofplaque thrombogenicity and stability (15), these obser-vations in apoE2/2 mice indicate that exposure to asingle dose of 56Fe ions not only can increase the rate atwhich atherosclerotic lesions develop but also canaccelerate their progression to an advanced stage witha composition typically associated with plaque instabil-ity and increased thrombogenicity.

In this study, radiation was targeted to the aortic archand carotids, avoiding most other organs. In particular,irradiation of bone marrow and most circulatingleukocytes was avoided. This did not result in significantdifferences in the frequencies of circulating T cells, Bcells or monocytes/neutrophils in irradiated and controlmice (Table 1). Moreover, lipid profiles were similarregardless of the radiation history (Table 2). Further-more, C-reactive protein, a systemic marker of inflam-mation, was not significantly increased in irradiatedmice regardless of their degree of atherosclerosis (4.7 ±

0.2 ng/ml in 5-Gy-irradiated apoE2/2 mice 13 weekspostirradiation compared to 5.2 ± 0.2 ng/ml in controlage-matched apoE2/2 mice). The absence of systemiceffects of radiation targeted to major arteries suggests adirect local pro-atherogenic effect specifically due to 56Feions on the arteries themselves. Our results are alsostrongly supported by the fact that atherosclerosis atnon-targeted aortic sites was unaffected (Fig. 1B).

There were both site-specific and dose-dependenteffects of 56Fe ions on atherosclerosis in apoE2/2 mice,with the aortic root being the most sensitive siteexamined with respect to both the dose of radiationand the longevity of its effects. The site-specific effect of56Fe ions may be related to the differential predilectionof different vascular sites to the development ofatherosclerotic lesions. Site-specific effects of experi-mental manipulation on atherosclerosis are not uncom-mon in atherogenic mouse models (17, 18) and may bedue to the considerable variability in local hemodynamicforces at different arterial sites that exert distinct effectson the expression of genes encoding pro-atherogenic

770 YU ET AL.

factors in endothelial cells (19). These factors influenceprocesses that are central to the pathogenesis ofatherosclerosis such as intimal permeability to lipopro-teins, endothelial adhesivity and inflammatory cellrecruitment (19). Site-specific influences on these pro-cesses are reflected by the high predilection of arterialsites exposed to disturbed laminar blood flow (particu-larly the aortic root) to development of atheroscleroticlesions (6). The higher sensitivity of the aortic root to56Fe ions could therefore be due to the greater magnitudeof pre-existing pro-atherogenic stimuli inherent at thissite in apoE2/2 mice that may have exacerbated theeffects of radiation-induced vascular injury.

Unlike other targeted portions of the aorta, a dose of2 Gy 56Fe ions was sufficient to accelerate atherosclerosisat the aortic root (Fig. 2A). This dose is much lowerthan the 14 Gy of X rays shown to produce a similareffect 13 weeks after irradiation of male apoE2/2 mice ina previous study (7), although lower doses were nottested and can have effects in other experimentalsystems. For example, a single 8-Gy dose of X raysincreased the total number of carotid lesions in femaleapoE2/2 mice at 30 weeks after irradiation (the lowestdose tested) (8). The fact that 56Fe ions can produceeffects at lower doses than X rays is not surprising, since

particle radiation can be particularly damaging. Inradiobiology, the relative biological effect (RBE) isdefined as the ratio of the absorbed dose of a radiationin question to the absorbed dose of a reference radiation(usually c rays) required to produce the same biologicaleffect in a particular tissue (20). Based on this definition,the RBE of approximately 7 for 56Fe-ion-inducedatherosclerosis [compared to ref. (7)] is consistent withthose associated with other effects (21). However, theRBE can vary depending on the doses used and thebiological effect studied. For example, the RBE for600 MeV 56Fe ions has been reported to be as high as 27for the induction of some tumors (22). Nevertheless,caution must be exercised in extrapolating this dose–response relationship to humans, because the doses maynot scale between the mouse model and humans forseveral reasons. Mice are relatively resistant to athero-genesis and normally do not develop atherosclerosis asthey age. Thus mouse models require either geneticmanipulation or extreme diets to induce atherosclerosis.Even for X rays, detectable changes in atherosclerosis inapoE2/2 mice have only been demonstrated using 8 to14 Gy (although lower doses were not tested in thosestudies) (7, 8), but it is known from epidemiologicalstudies that radiation can be a risk factor for humans at

FIG. 4. Larger necrotic cores associated with increased macrophage apoptosis and reduced collagen contentin advanced atherosclerotic lesions of apoE2/2 mice exposed to 5 Gy 56Fe ions. Panel A: Masson’s trichromestaining of collagen deposition (blue) in aortic root lesions of representative control (0 Gy) and irradiated (5 Gy)apoE2/2 mice 40 weeks postirradiation. Necrotic cores are indicated by arrows. Panel B: Co-immunofluores-cence staining of lesional macrophages (MØ) and apoptosis (TUNEL) in aortic root sections fromrepresentative control (0 Gy) and irradiated (5 Gy) apoE2/2 mice 40 weeks postirradiation. Panel C:Quantification of percentage lesional TUNELz (apoptotic) cells in aortic root sections from control (0 Gy) andirradiated (5 Gy) apoE2/2 mice 40 weeks postirradiation. *P , 0.05 by Mann-Whitney test. Panel D: Meanpercentage lesional macrophage (F4/80z) content in aortic root sections of control (0 Gy) and irradiated (5 Gy)apoE2/2 mice. Panel E: Mean percentage lesion area occupied by collagen in aortic root sections from control(0 Gy) and irradiated (5 Gy) apoE2/2 mice 40 weeks postirradiation. *P , 0.05.

56Fe IONS ACCELERATE ATHEROSCLEROSIS 771

doses as low as 1 Gy (3, 4). Therefore, it is possible thateven lower doses of 56Fe ions might result in significanteffects on atherosclerosis in other models, at othervascular sites, or by other assays.

The doses used in this study were higher than thoseastronauts are expected to encounter on a trip to Mars.In this study, mice were exposed to a single dose ofparticle radiation. Astronauts in space are likely toexperience low but protracted exposures to HZE-particle radiation. It has been estimated that astronautswould absorb a cumulative dose of 0.42 Gy of HZE-particle radiation during a 1000-day Mars mission (23).Such an unrelenting, low-dose-rate exposure may bemore effective than a single acute exposure at promotingthe vascular inflammation that contributes to athero-sclerosis, however (24). Moreover, while the mice in thisstudy were exposed to only 56Fe ions, astronauts will beexposed to a mixture of accelerated ions and high-energyphotons. Since both X rays and accelerated ionsexacerbate atherosclerosis, it is likely that all compo-nents of radiation in the galactocosmic radiationspectrum will contribute to disease. While it is impos-sible to estimate fatal risk from this model, since deathfrom cardiovascular disease is rare for mice even withadvanced atherosclerosis, the maximum acceptable riskof death for astronauts is typically considered to be 3%(23).

The effects of 56Fe ions on atherogenesis wereexamined at 13 and 40 weeks after irradiation. Thesetimes were chosen so that the effect of 56Fe ions on therate of development of atherosclerosis could be mea-sured in addition to determining whether it resulted inlong-term deleterious consequences for the progressionof advanced lesions (as observed at the aortic root)(Fig. 4). Furthermore, these times allowed us to directlycompare the effects of 56Fe ions on atherosclerosis inapoE2/2 mice to those of X rays reported in a previousstudy in which a dose of 14 Gy X rays, which is similarto that used for therapeutic radiation, accelerated thedevelopment of atherosclerotic lesions (7). Although it ispossible that the effects of 56Fe ions on atherosclerosismay be more pronounced at other times, it was notpossible to irradiate a greater number of mice to assaymultiple times due to the limited amount of beam timeavailable at BNL. Nevertheless, this study provides thefirst demonstration of pro-atherogenic effects elicited byan accelerated ion such as would be encountered indeep-space travel. Other types of radiation such as c raysor X rays consist of photons and are different fromaccelerated ions such as 56Fe or protons in the way thatthey deposit energy in tissue. Energy deposited in tissueby photons decreases exponentially with distance fromthe source, while accelerated ions deposit most of theirenergy close to the end of their path. While damage fromc rays and X rays is caused primarily by the photonitself, accelerated ions such as 56Fe can generate

secondary particles as they interact with a target.Damage from these secondary particles can be a majorcontributor to the radiation effect. Despite these andother differences, both types of radiation have beenshown to exacerbate atherosclerosis. Our data demon-strate that a pro-atherogenic effect of cosmic radiationshould be considered a potential major concern for thoseinvolved in deep-space missions and suggest thatstringent therapeutic management of traditional athero-sclerosis risk factors may be a prudent countermeasurein these individuals. This has implications not only forastronauts but also for those undergoing therapeuticirradiation, especially new modalities that use accelerat-ed ions. For example, protons are increasingly beingused for radiotherapy due to the ability to moreprecisely target the radiation beam to the tumor (33).Similarly, therapeutic radiation protocols using acceler-ated carbon ions are being developed in Japan andEurope (34). While there are clear advantages to thesenew treatments, the long-term risk is unknown. Knowl-edge gained from studies with animal models such as thiswill therefore lead to a better understanding of thepathophysiological effects of accelerated ions to betterestimate risk and develop appropriate strategies tomitigate its damaging effects. Current efforts by ourgroup include determining how 56Fe-ion radiationaffects aortic endothelial cell adhesiveness and expres-sion of cell adhesion molecules and chemotactic factorsinvolved in atherogenesis.

ACKNOWLEDGMENTS

The authors especially thank Drs. Adam Rusek and Peter Guidaand the entire BNL/NSRL support crew for their able assistance inconducting these radiation studies. This work was supported by theNational Space Biomedical Research Institute through NASA NCC9-58.

Received: October 25, 2010; accepted: January 26, 2011; publishedonline: April 5, 2011

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56Fe IONS ACCELERATE ATHEROSCLEROSIS 773