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haracterization of a murine model of fetal programming oftherosclerosis

ima Goharkhay, MD; Elena Sbrana, PhD; Phyllis K. Gamble; Esther H. Tamayo; Ancizar Betancourt, MSc; Karina Villarreal;ary D. V. Hankins, MD; George R. Saade, MD; Monica Longo, MD, PhD

BJECTIVE: The objective of the study was to investigate the effect ofetal programming on the development of atherosclerosis in the off-pring in a mouse model.

TUDY DESIGN: Male and female mice of the wild type and the knock-ut for the apoprotein E (apoE) gene were cross-bred to obtain all 4ossible genetic offspring types. The offspring were kept on regularhow and killed at 8 months of age. Levels of total cholesterol andriglycerides were determined. The aortic arch was examined for theresence and severity of atherosclerosis. Kidney and liver sections

mouse is a widely usedoi: 10.1016/j.ajog.2007.08.002

16.e1 American Journal of Obstetrics & Gynecology OCTOBER 2007

ESULTS: We found increased total cholesterol levels and incidence oftherosclerosis in offspring born to hypercholesterolemic mothers asompared with genomically similar animals born to wild-type mothers.hese animals also showed kidney and liver lesions consistent withhronic hypercholesterolemia.

ONCLUSIONS: There is a strong effect of fetal programming on theevelopment of atherosclerosis in the apoE mouse model.

ey words: atherosclerosis, fetal development, fetal programming,

ere analyzed for pathologic changes. hypercholesterolemia, mouse model

ite this article as: Goharkhay N, Sbrana E, Gamble PK, et al. Characterization of a murine model of fetal programming of atherosclerosis. Am J Obstetynecol 2007;197:416.e1-416.e5.

etal development is determinedby both inherited genetic factors

nd environmental influences duringhe prenatal period and early child-ood. The in utero environment maye an important contributor to theredisposition for chronic diseases inhe offspring. As hypothesized byarker and Osmond1 and Barker2

timuli or insults to the fetus duringritical periods of development lead tofetal programming” and producedaptive changes in the fetal anatomy,hysiology, and metabolism that have

ong-term consequences.

Cardiovascular disease is the num-ber 1 cause for morbidity and mortalityin industrialized as well as many devel-oping countries. Atherosclerosis sec-ondary to hyperlipidemia constitutesthe main etiologic cause for cardiovas-cular disease.

A role for developmental program-ming of atherosclerosis has been sug-gested by various investigators. A corre-lation between maternal and fetalcholesterol levels during the first 26weeks of gestation has been shown in hu-mans.3 Napoli et al4 further demon-strated decreased regression of fattystreak lesions in children born to hyper-cholesterolemic mothers as comparedwith controls. A rabbit model of fetalprogramming of atherosclerosis has alsobeen described.5 In this model, maternaltherapy during pregnancy with vitaminE and cholestyramine was shown to de-crease plaque formation in the adult off-spring to levels found in the litters bornto normocholesterolemic mothers. Theavailability of a murine model wouldgreatly facilitate the study of the effects offetal programming as well as its un-derlying mechanisms and possiblecountermeasures.

The apolipoprotein E (apoE) deficient

terized model to study atherosclerosissecondary to hypercholesterolemia.6-8

When fed a regular chow, homozygousapoE knockout animals display totalcholesterol levels that are 5 times higherthan those observed in wild-type ani-mals.6-8 In addition, feeding these ani-mals a high-fat diet leads to a further3-fold increase in circulating cholesterollevels. In contrast, heterozygous micepossess 1 functional copy of the apoEgene and do not show a significant in-crease in cholesterol levels.

The objective of the current study wasto investigate whether maternal hyper-cholesterolemia during pregnancy cor-relates with elevated cholesterol levelsand early development of atherosclerosisin the adult offspring in a mouse model.The secondary outcomes of interest wereto determine the effects of fetal program-ming on the severity of atherosclerosis,and the nature and extent of secondaryorgan damage in the liver and kidney ofoffspring born to hypercholesterolemicmothers.

MATERIALS AND METHODSMature cycling female and male mice(4-6 weeks old) homozygous for dis-ruption of the apoE gene (apoE�/�KO,

rom the Division of Maternal-Fetaledicine, Department of Obstetrics andynecology, University of Texas Medicalranch, Galveston, TX.

resented at the 27th Annual Clinical Meetingf the Society for Maternal–Fetal Medicine,an Francisco, CA, Feb. 5-10, 2007.

eceived Mar. 12, 2007; revised May 8, 2007;ccepted Aug. 2, 2007.

eprints not available from the authors.

upported in part by the Department ofbstetrics and Gynecology, University ofexas Medical Branch, Galveston, TX.

002-9378/$32.002007 Mosby, Inc. All rights reserved.

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tock #002052) and their age-matchedild-type controls (apoE�/�WT, strain57BL/6J, stock #000664) were ob-

ained from the Jackson LaboratoryBar Harbor, ME). The mice wereoused separately in temperature- andumidity-controlled quarters withonstant 12-hour light–12-hour darkycles. They were provided with foodnd water ad libitum. All animals wereed regular chow, including the off-pring after weaning at 21 days of age.

aintenance and care was provided byertified personnel and veterinary staffccording to the guidelines of the In-titutional Animal Care and Use Com-

ittee at the University of Texas Med-cal Branch in Galveston.

Homozygous apoE deficient-knockoutapoE�/�KO) and wild type (apoE�/�WT)

FIGURE 1Cross-breeding pattern used to oboffspring

Heterozygous

poE+/+WT apoE-/-KO

n=7

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oharkhay. Murine model of fetal programming of atherosclerosis

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ice were cross-bred to obtain all 4 possi-le genetic variants of litters (Figure 1).he heterozygous offspring were classifieds apoE�/�mat or apoE�/�pat according tohe maternal or paternal origin of thenockout apoE gene, respectively. Ho-ozygous apoE�/�KO and apoE�/�WT lit-

ers were used as controls.The offspring from the 4 litter groupsere killed at 8 months of age using a

arbon dioxide chamber. Blood was ob-ained by cardiac puncture after openinghe thoracic and abdominal cavities. Se-um was collected and frozen at -80°Cntil analysis. Total cholesterol and tri-lyceride levels were determined using aolorimetric method with automatedquipment (Fusion 5.0, Ortho Clinicaliagnostics, Rochester, NY). The aortic

rch was dissected, cleaned from con-

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OCTOBER 2007 America

ective tissue, and fixed in a 10% buf-ered formalin solution. These speci-

ens were then stained with oil red Ond evaluated for presence and size oftherosclerotic plaques using a modifi-ation of the en face method with an im-ge analysis software package (ImageJ, v.37h, National Institutes of Health,ttp://rsb.info.nih.gov/ij/).9

Areas of aortic lesions that stained or-nge and red were classified as advancedlaques. The specimens containing areasf advanced plaques were considered asositive for the presence of atherosclero-is. We also measured areas that showedellow discoloration as compared withhe background. These areas were con-idered preatherosclerotic and catego-ized as early lesions.

Livers and kidneys were removed athe time the animals were killed andxed in 10% buffered formalin solution,repared as paraffin blocks, and stainedith hematoxylin– eosin. These samplesere reviewed by a clinical pathologist,ho was blinded to the sample origin. In

he specimens in which a pathologichange was noted, the severity wasraded using the following scheme:rade 0 � no pathologic change, grade 1

negligible or nonspecific change,rade 2 � mild change, grade 3 � mod-rate change, and grade 4 � severehange.

Statistical analyses were performedsing unpaired t test, �2 test, analysis ofariance, and Fisher’s protected leastignificant difference as appropriate.ll tests were 2 tailed, and a P value of

ess than .05 was considered statisti-ally significant. Data are expressed asean � SE.

ESULTSverall, 51 offspring were included in

he study: 12 apoE�/�WT (7 females, 5ales); 7 apoE�/�pat (3 females, 4ales); 18 apoE�/�mat (8 females, 10ales); and 14 apoE�/�KO (7 females, 7ales).Cholesterol levels were higher in both

poE�/�mat (289 � 47 mg/dL) andpoE�/�KO (396 � 62 mg/dL) offsprings compared with apoE�/�pat (105 � 8

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L) offspring (P � .0021; Figure 2). Cir-ulating cholesterol levels in thepoE�/�mat group of offspring were sig-ificantly higher than in the apoE�/�WT

P � .037) and apoE�/�pat offspringice (P � .027). No significant variation

etween sexes was noted, except in thepoE�/�WT group in which the male off-pring had higher total cholesterol levelshan females (136 � 10 vs 89 � 9 mg/dL,� .038).No significant difference in the triglyc-

ride levels were found among the studyroups (172 � 42 mg/dL in thepoE�/�WT, 148 � 14 mg/dL in thepoE�/�pat, 152 � 14 mg/dL in thepoE�/�mat, and 135 � 13 mg/dL in thepoE�/�KO offspring). Male offspringad higher levels of triglycerides than fe-ales in the apoE�/�KO (167 � 16 vs 997 mg/dL, P � .037) but not in the

ther groups of animals (data nothown).

There was a significant difference inhe incidence of atherosclerosis (ad-anced lesions) among the groups:8.6% in apoE�/�KO, 72.2% inpoE�/�mat, 28.6% in apoE�/�pat, and.1% in apoE�/�WT (P � .0001). Theffspring sex did not influence the rate oflaque formation (data not shown). Fig-re 3 depicts an example of moderately

arge atherosclerotic plaques found inhe aortic arch of an apoE�/�mat mousefter oil red O staining.

As for the extent of atherosclerosis, theatio of the surface area of the advanced

FIGURE 3Portion of aortic arch of anapoE�/�mat mouse depictingatherosclerotic lesions

oharkhay. Murine model of fetal programming oftherosclerosis. Am J Obstet Gynecol 2007

laques to the total surface of the aortic

16.e3 American Journal of Obstetrics & Gynecol

rch was significantly different amonghe offspring groups: 0.16 � 0.16% inpoE�/�WT, 0.22 � 0.16% inpoE�/�pat, 2.99 � 0.89% inpoE�/�mat, and 5.10 � 1.39% inpoE�/�KO (P � .0031). We further an-lyzed the total surface area covered byhe plaque combining the early and ad-anced lesions within each group. Theean total area of atherosclerotic

hanges in the apoE�/�WT offspring was.96 � 1.45% vs 7.57 � 2.65% for thepoE�/�pat, 15.3 � 2.07% for thepoE�/�mat, and 28.9 � 4.98% forpoE�/�KO groups (P � .0001). Sex didot account for any significant difference

n advanced or combined area of athero-clerosis (data not shown).

We found an association between cho-esterol levels and the presence of athero-clerosis. Animals showing atheroscle-otic plaques had mean cholesterol levelsf 385 � 41 mg/dL as compared with 104

4.9 mg/dL in those with no evidence of

FIGURE 4Examples of kidney lesions observoffspring mice

eft and insert, glomerulosclerosis with Kimmubular casts.oharkhay. Murine model of fetal programming of atherosclerosis

TABLE 1Total cholesterol levels (milligramspresence or absence of advancedGroup Plaque

apoE�/�WT 145...................................................................................................................

apoE�/�pat 117 � 15...................................................................................................................

apoE�/�mat 307 � 65...................................................................................................................

apoE�/�KO 484 � 54

ogy OCTOBER 2007

uch lesions (P � .0001). This relation-hip also held true when analyzing eachubgroup individually, as shown in Ta-le 1.The histological examination of the kid-

eys did not show any pathologic changesn either apoE�/�WT or apoE�/�pat off-pring. The predominant lesions found inhe kidneys of apoE�/�mat and apoE�/�KO

nimals were glomerulosclerosis withimmelstiel-Wilson–type lesions as well asroteinaceous tubular casts (Figure 4).uch changes were found in 50% ofpoE�/�mat and 69.2% of apoE�/�KO off-pring mice. The distribution of the sever-ty of the anomalies found is depicted inable 2.In the liver of the animals examined,

he predominant pathologic change wasultifocal hepatocyte necroapoptosis

Figure 5). Although we did not encoun-er such lesions in apoE�/�WT mice, theyere present in 28.6% of apoE�/�pat,5.5% of apoE�/�mat and 76.9% of

in apoE�/�mat and apoE�/�KO

stiel-Wilson–type lesion; right, proteinaceous

J Obstet Gynecol 2007

er deciliter, means � SE) byques within each study group

No plaque P value

97 � 10 n/a..................................................................................................................

99 � 9 .35..................................................................................................................

117 � 10 .065..................................................................................................................

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poE�/�KO offspring mice. Most ofhese lesions were of low severity (Ta-le 3).

OMMENTe found that, despite being genomi-

ally similar, heterozygous offspring thateveloped in a hypercholesterolemic en-ironment had higher cholesterol levelsnd were more predisposed to athero-clerosis as compared with heterozygousffspring growing in a normal maternalnvironment. In accordance with previ-us reports of normal triglyceride levels

n apoE knockout animals, the triglycer-de levels were not different among theroups of offspring. Although the varia-ion in cholesterol levels and atheroscle-osis between homozygous wild-typend homozygous knockout animals isxpected, the discrepancy found be-ween the heterozygous apoE�/�pat andpoE�/�mat groups cannot be explainedn the basis of genetic differences.ence, this finding is likely attributable

o the influence of the altered intrauter-

FIGURE 5Examples of liver lesions observed

eft, hepatocellular necrosis; right, acidophilicoharkhay. Murine model of fetal programming of atherosclerosis

TABLE 2Severity distribution of histopathogroupStudy group Grade 0, % Grade 1, %

apoE�/�WT 100 —...................................................................................................................

apoE�/�pat 100 —...................................................................................................................

apoE�/�mat 50 22.2...................................................................................................................

apoE�/�KO 30.8 61.5...................................................................................................................

Total number of samples available for examination of kidney

ne environment because of the maternalypercholesterolemia present during de-elopment in the apoE�/�mat andpoE�/�KO but not in the apoE�/�pat

nd apoE�/�WT offspring.Our findings strongly support a role

or developmental programming of hy-erlipidemia and atherosclerosis. Thenderlying mechanism for this phenom-non is currently unknown. Palinski etl3,4 and Napoli et al5 have shown an as-ociation between maternal and fetalholesterol levels in humans and a rever-al of the effect of fetal programminghen treating high cholesterol levels inregnant rabbits. More recently, Yo-hida and Wada10 provided evidence forransplacental transfer of cholesterol in

ice during early and late gestation.One may hypothesize that exposure of

he fetus to elevated cholesterol levelsuring early development permanentlylters cholesterol homeostasis. The va-idity of this hypothesis, as well as its pos-ible cellular and molecular basis, needso be further studied. An alternative hy-

the animals examined

ies (arrows).J Obstet Gynecol 2007

ic kidney changes in each litter

Grade 2, % Grade 3, % Grade 4, %

— — —..................................................................................................................

— — —..................................................................................................................

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OCTOBER 2007 America

othesis may relate the changes in cho-esterol levels to possible differences inenetic imprinting between apoE�/�pat

nd apoE�/�mat offspring.A previous study on the apoE model of

therosclerosis did not show a compara-le effect of fetal programming.11 We be-

ieve the discrepancy in the outcome isainly caused by 2 differences in study

esign. In the report by Madsen et al,11

he offspring were maintained on antherogenic Western (high-fat) diet.his resulted in elevated cholesterol lev-ls in all study groups, much higher thanhose found in our investigation. Thether difference lies in the age at whichhe animals were killed. We chose 8

onths of age in contrast to 6 months inhe study by Madsen et al.11 This morelosely corresponds to middle-aged hu-ans, a time when variations in the pre-

isposition to the development of ath-rosclerosis are more likely to becomeanifest.The role of developmental program-ing is highlighted by the consistent ef-

ect of the uterine milieu across the var-ous observations in our current study,anging from the effect on cholesterolevels, the incidence, and the severity oftherosclerotic lesions, to the secondaryrgan damages seen in the kidney andhe liver. This model seems to offer andequate tool to study the effects of fetalrogramming on the development oftherosclerotic disease later in life.

One of the limitations of the presenttudy was the lack of data on cholesterolevels in the pregnant dams. Because allnimals were maintained on the sameiet, one would expect the cholesterol

evels in the mothers to be similar tohose in the offspring of the same genetic

akeup. This, of course, will have to beonfirmed in our future studies. Anothermportant question is the degree tohich high cholesterol levels in mother

each the fetal compartment in utero.his topic has partially been addressed inrevious studies by Palinski et al5 andoshida and Wada,10 as mentioned ear-

ier. We plan direct measurement of lipidevels in fetuses or neonates to more spe-ifically address this question.

Another subject that needs to be ad-

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SMFM Meeting Papers www.AJOG.org

4

ndings may be due to a postnatal effecturing the neonatal period. Such an effectould have to be centered on breastfeed-

ng because all litters were kept on the samehow after 21 days of age. Although we hy-othesize that this contribution is not sig-ificant, cross-fostering studies will specif-

cally allow targeting this topic.Assuming that the major effect of de-

elopmental programming occurs dur-ng gestation, it will be important to de-ne the “critical window period” duringhich the fetus is most susceptible tooxious stimuli. Manipulation of mater-al cholesterol metabolism during dif-

erent time periods of pregnancy, withse of diets or pharmacologic agents,ay give further clues as to the impor-

ance of timing in fetal programming.ransfer of embryos in between low-risknd high-risk mothers can be utilized to

TABLE 3Severity distribution of histopathogroupStudy group Grade 0, % Grade 1, %

apoE�/�WT 100 —...................................................................................................................

apoE�/�pat 71.4 28.6...................................................................................................................

apoE�/�mat 44.4 44.4...................................................................................................................

apoE�/�KO 23.1 61.5...................................................................................................................

Total number of samples available for examination of liver h

tudy other aspects of fetal programming 5

16.e5 American Journal of Obstetrics & Gynecol

f atherosclerosis, including possiblepigenetic mechanisms leading to thebserved effects.The primary event leading to the in-

reased development of atherotic lesionsn developmentally programmed off-pring appears to be an elevation in cir-ulating cholesterol levels. It remainsnclear how fetal programming causes aysregulation of cholesterol metabolism

n the affected offspring. Further studiesre clearly needed to evaluate the effectf the fetal environment on cholesterolegulatory pathways. f

EFERENCES. Barker DJ, Osmond C. Infant mortality, child-ood nutrition, and ischaemic heart disease inngland and Wales. Lancet 1986;1:1077-81.. Barker DJ. In utero programming of cardio-ascular disease. Theriogenology 2000;53:

ic liver changes in each litter

Grade 2, % Grade 3, % Grade 4, %

— — —..................................................................................................................

— — —..................................................................................................................

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ogy OCTOBER 2007

. Napoli C, D’Armiento FP, Mancini FP, et al.atty streak formation occurs in human fetalortas and is greatly enhanced by maternal hy-ercholesterolemia. Intimal accumulation of lowensity lipoprotein and its oxidation precedeonocyte recruitment into early atherosclerotic

esions. J Clin Invest 1997;100:2680-90.. Napoli C, Glass CK, Witztum JL, Deutsch, D’Armiento FP, Palinski W. Influence ofaternal hypercholesterolaemia during preg-

ancy on progression of early atheroscleroticesions in childhood: Fate of Early Lesions inhildren (FELIC) study. Lancet 1999;354:234-41.. Palinski W, D’Armiento FP, Witztum JL, et al.aternal hypercholesterolemia and treatmenturing pregnancy influence the long-term pro-ression of atherosclerosis in offspring of rab-its. Circ Res 2001;89:991-6.. Reardon CA, Getz GS. Mouse models of ath-rosclerosis. Curr Opin Lipidol 2001;12:67-73.. Jawien J, Nastalek P, Korbut R. Mouse mod-ls of experimental atherosclerosis. J Physiolharmacol 2004;55:503-17.. Wouters K, Shiri-Sverdlov R, van Gorp PJ,an Bilsen M, Hofker MH. Understanding hy-erlipidemia and atherosclerosis: lessons fromenetically modified apoe and ldlr mice. Clinhem Lab Med 2005;43:470-9.. Nunnari JJ, Zand T, Joris I, Majno G. Quan-itation of oil red O staining of the aorta in hy-ercholesterolemic rats. Exp Mol Pathol989;51:1-8.0. Yoshida S, Wada Y. Transfer of maternalholesterol to embryo and fetus in pregnantice. J Lipid Res 2005;46:2168-74.1. Madsen C, Dagnaes-Hansen F, Moller J,alk E. Hypercholesterolemia in pregnant miceoes not affect atherosclerosis in adult off-

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pring. Atherosclerosis 2003;168:221-8.