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SMFM Papers www.AJOG.org
aternal hypercholesterolemia leads to activationf endogenous cholesterol synthesis in the offspring
ima Goharkhay, MD; Esther H. Tamayo; Huaizhi Yin; Gary D.V. Hankins, MD;eorge R. Saade, MD; Monica Longo, MD, PhD
BJECTIVE: The purpose of this study was to determine the effect ofaternal hypercholesterolemia on hepatic cholesterol metabolism in
he offspring in a mouse model.
TUDY DESIGN: Male and female wild type and apoE�/�KO (knock-ut for the apoprotein E [apoE]) gene) mice were crossbred to ob-
ain all 4 possible genetic offspring types. The litters were main-ained on regular chow and sacrificed at 8 months of age. Liveramples were collected and the mRNA expression levels for SCAP,REBP-1a, SREBP-2, HMGCR, and LDLR determined using real-
percholesterolemic doi: 10.1016/j.ajog.2008.06.064
ESULTS: We found a significant activation of the transcriptional ac-ivity of genes involved in endogenous cholesterol synthesis, as wells LDLR, in the liver of adult mice born to hypercholesterolemic dams.
ONCLUSION: Reprogramming of hepatic cholesterol homeostasisay be the basis for an increased predisposition to hypercholesterol-
mia and atherosclerosis found in offspring of mice exposed to a highholesterol environment during early life.
ey words: atherosclerosis, fetal development, fetal programming,
ime RT-PCR. hypercholesterolemia, mouse modelite this article as: Goharkhay N, Tamayo EH, Yin H, et al. Maternal hypercholesterolemia leads to activation of endogenous cholesterol synthesis in the offspring.m J Obstet Gynecol 2008;199:273.e1-273.e6.
ardiovascular disease remains themain contributor to overall mor-
idity and mortality in most countries.therosclerosis secondary to hyperlipid-mia constitutes a major etiologic causeor cardiovascular disease.
Besides hereditary genetic factors, thenvironment in utero and during earlyostnatal life is evolving as a major con-ributor to the overall development ofndividuals.1-3 The basis of the impor-ance of environmental factors duringarly life is presumed to be the tremen-
dous cell turnover and tissue plasticitypresent during this period of differentia-tion and growth. The first recognition ofearly environmental influences on devel-opment was by David Barker and co-workers, who found an association be-tween undernutrition during fetaldevelopment with the development ofcardiovascular disease and other disor-ders later in life. More recently it is be-coming evident that many other factorsduring early development— besidesmalnutrition— can have important in-fluences on the long-term health ofindividuals.3
Although atherosclerosis was amongthe first conditions for which a role ofdevelopmental programming was de-scribed, the mechanisms underlying thisphenomenon have not been well stud-ied. Among human fetuses, Napoli andassociates demonstrated a decreased rateof regression of fatty streak lesions inchildren born to hypercholesterolemicmothers as compared to controls.4,5
They also described a correlation be-tween maternal and fetal cholesterol lev-els that persisted up to 26 weeks of ges-tation. The same group has furtherdemonstrated an increased rate ofplaque formation in litters born to hy-
model.6 These changes were preventablewhen the dams received treatment withvitamin E or cholestyramine duringpregnancy.
Apoprotein E (apoE) deficient miceare widely used in the study of athero-sclerosis. Animals that are homozy-gously apoE-deficient experience spon-taneous hypercholesterolemia when feda regular diet and develop atheroscle-rotic lesions that are similar to thosefound in humans.7,8
In a previous report, we described amurine model of developmental pro-gramming of atherosclerosis using off-spring of apoE deficient mice crossbredwith wild-type animals.9 We showed thatat 8 months of age heterozygous off-spring born to apoE deficient mothershad a near 3-fold increase in total choles-terol levels as compared to heterozygousmice born to wild-type dams. We alsofound a significantly increased rate ofatherosclerotic plaque formation in het-erozygous animals born to hypercholes-terolemic mothers. We concluded thatmaternal hypercholesterolemia leads toelevated cholesterol levels and the devel-opment of atherosclerosis in the off-spring in adulthood.
The liver is a major site of regulation of
rom the Division of Maternal–Fetaledicine, Department of Obstetrics andynecology, University of Texas Medicalranch, Galveston, TX.
his research was presented in part at the 27thnnual Meeting of the Society for Maternal–etal Medicine, San Francisco, CA, Feb. 5-10,007, and in part at the 28th Annual Meeting ofhe Society in Dallas, TX, Jan. 28-Feb. 2, 2008.
eceived March 1, 2008; revised May 27,008; accepted June 21, 2008.
eprints not available from the authors.
his study was supported by grants03HD056087-01 (N.G.) and K12HD001269-9 (G.D.V.H.) from the National Institutes ofhild Health and Human Development.
002-9378/$34.002008 Mosby, Inc. All rights reserved.
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oth cholesterol uptake and de novoynthesis. Since our previous studyhowed significant dysregulation of cho-esterol metabolism in response to a hy-ercholesterolemic environment duringevelopment, we decided to study theholesterol metabolic pathways in theivers of affected versus control animals.
Endogenous cholesterol synthesis isrimarily under the control of the SCAP-REBP-HMGCR pathway.10,11 SCAPSREBP-cleavage activator protein) andREBP (sterol response element bindingrotein) are part of a cascade of proteinshat are activated in response to de-reased intracellular cholesterol levels.REBP in turn stimulates the expressionf lipogenic proteins, including HMGCR3-hydroxy-3-methylglutaryl-coenzymereductase), the enzyme that catalyzes the
ommitted step in the endogenous choles-erol synthetic pathway. SREBP exists as 3istinct isoforms, SREBP-1a, SREBP-1c,nd SREBP-2. It is believed that activationf isoforms SREBP-1a and SREBP-2 pref-rentially lead to increased endogenousholesterol synthesis. In contrast,REBP-1c overactivation does not in-rease cholesterol production in trans-enic animals. The LDL receptor (LDLR)s the main mediator for cholesterol uptakento the liver. LDLR expression is alsotimulated through SCAP and SREBPshen intracellular cholesterol levels are
FIGURE 1Crossbreeding pattern used to obta
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n=7
apoE-/-
apoE+/-pat aoharkhay. Maternal hypercholesterolemia leads to activatio
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73.e2 American Journal of Obstetrics & Gynecolo
In the present study, we aimed to de-ermine the effect of maternal hypercho-esterolemia on the transcriptional acti-ation of SCAP, SREBP-1a, SREBP-2,MGCR, and LDLR, and to study the
orrelations between the expression ofhese mediators.
ATERIALS AND METHODSature cycling female and male mice
4-6 weeks old) homozygous for dis-uption of the apoE gene (apoE�/�KO,train B6.129P2-Apoe, tm1Unc�/J,tock no. 002052) and their age-matchedild-type controls (apoE�/�WT, strain57BL/6J, stock no. 000664) were ob-
ained from The Jackson LaboratoryBar Harbor, ME). The animals wereoused separately in temperature andumidity controlled quarters with con-tant light:dark cycles of 12h:12h androvided with food and water ad libi-um. All mice (dams and litters) were
aintained on regular chow. Mainte-ance and care was provided by certifiedersonnel and veterinary staff accordingo the guidelines of the Institutional An-mal Care and Use Committee (IACUC)t the University of Texas Medicalranch in Galveston.Homozygous apoE deficient-knock-
ut mice (apoE�/�KO) and wild typeapoE�/�WT) were crossbred to obtainll 4 possible genetic variants of lit-
the 4 genetic variations of offspring
s
apoE+/+WT
=16
apoE+/+WT ap
n=15
E+/-mat apoE+/apoE+/apoE+/
endogenous cholesterol synthesis in the offspring. Am J Obste
ers (Figure 1). The heterozygous off- c
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pring were classified as apoE�/�mat orpoE�/�pat according to the maternal oraternal origin of the knockout apoEene, respectively. Litters from homozy-ous apoE�/�KO and apoE�/�WT weresed as controls.The offspring from the 4 litter
roups were sacrificed at 8 months ofge using a carbon dioxide chamber.iver samples were collected, washed
n phosphate buffered saline, flash fro-en in liquid nitrogen, and stored at80°C. RNA extraction was per-
ormed using the RNAqueous kit (Am-ion, Austin, TX) according to theanufacturer’s instructions. Reverse
ranscription into cDNA was per-ormed using the MultiScribe RT sys-em (Applied Biosystems, Foster City,A) according to manufacturer’s in-
tructions. The cDNA was amplified onn ABI 7900 HT Fast Real Time PCR sys-em (Applied Biosystems) using mouse-pecific Taqman probe sets (all purchasedrom Applied Biosystems) for SCAPcatalog numbers Mm01250183_g1),REBP-1a (probe sequences availablepon request), SREBP-2 (catalog numberm01306294_m1), HMGCR (catalog
umber Mm01282497_g1), and LDLRcatalog number Mm00440169_m1) ac-ording to the manufacturer’s protocolnder the following conditions: 95°C (20econds) for 1 cycle followed by 40 cy-
omozygous
+/+WT apoE-/-KO apoE-/-KO
n=10
TTT apoE-/-KO
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econds). All samples were run in du-licate and were within the standardurve. Target gene expression was re-ated to the expression levels of 18S
RNA in each specific sample (catalogumber Hs99999901_s1; Applied Bio-ystems). All PCR reactions for eachample were performed simultaneouslyn the same plate.Statistical analyses were performed us-
ng one-way ANOVA, Fisher’s PLSD,nd linear regression analysis. All testsere 2-tailed and a P value of less than
05 was considered statistically signifi-ant. Data are expressed as mean � stan-ard error.
ESULTSverall, 48 animals were included in this
tudy: 15 apoE�/�WT (8 females, 7ales); 7 apoE�/�pat (3 females, 4ales); 16 apoE�/�mat (8 females, 8ales); and 10 apoE�/�KO (6 females, 4ales).We found differential expression of
he RNA message for SCAP between thestudy groups (P � .0001, ANOVA)
Figure 2, A). The highest level was en-ountered in the apoE�/�mat offspring,hich was significantly increased as
ompared to apoE�/�WT (P � .0001),poE�/�pat (P � .0001), and apoE�/�KO
nimals (P � .0088).The pattern of SREBP-1a message ex-
ression resembled that observed forCAP: The variation in mRNA levels wasverall statistically significant (P �
0001, ANOVA) (Figure 2, B). The high-st levels were found in apoE�/�mat off-pring. The difference in SRBEP-1a-spe-ific message expression was significantetween apoE�/�mat as compared withpoE�/�WT and apoE�/�pat groups (P �0001 and P � .0027, respectively).poE�/�KO animals showed intermedi-te levels of SREBP-1a mRNA, albeit theevels found were not statistically differ-nt from the apoE�/�WT and apoE�/�pat
roups.The expression of SREBP-2 mRNAas significantly different across the 4
tudy groups (P � .0016, ANOVA).poE�/�mat animals showed the high-st SREBP-2 expression, followed by
poE�/�KO offspring (Figure 2, C). TheFIGURE 2Mean relative mRNA expression levels and standard errors for SCAP (A),SREBP-1a (B), SREBP-2 (C), HMGCR (D), and LDLR (E) by study group
BSREBP-1a
apoE+/+WT apoE-/-KO apoE+/-mat apoE+/-pat
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abeling of columns by different letters denotes a significant difference (�0.05) between the meansn post-hoc analysis. The significance levels specifically are as follows: A, SCAP (P � .0001 byNOVA): P � .0001 for apoE�/�mat vs apoE�/�WT, P � .0001 for apoE�/�mat vs apoE�/�pat, P �
0088 for apoE�/�mat vs apoE�/�KO; B, SREBP-1a (P � .0001 by ANOVA): P � .0001 forpoE�/�mat vs apoE�/�WT, P � .0027 for apoE�/�mat vs apoE�/�pat; C, SREBP-2 (P � .0016 byNOVA): P � .0004 for apoE�/�mat vs apoE�/�WT, P � .0046 for apoE�/�mat vs apoE�/�pat; D,MGCR (P � .0109 by ANOVA): P � .0082 for apoE�/�mat vs apoE�/�WT, P � .0042 forpoE�/�mat vs apoE�/�pat; E, LDLR (P � .002 by ANOVA): P � .03 for apoE�/�mat vs apoE�/�WT.oharkhay. Maternal hypercholesterolemia leads to activation of endogenous cholesterol synthesis in the offspring. Am Jbstet Gynecol 2008.
SEPTEMBER 2008 American Journal of Obstetrics & Gynecology 273.e3
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FIGURE 3Correlations between mRNA expression levels of mediators of cholesterol metabolism
G H
D E F
SCAP
AP SCAP
A B C
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-1
0
1
2
3
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
2
3
4
1
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-1
0
2
3
4
1
0.0 0.5 1.0 1.5 2.0 2.5
-1
0
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1
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-1
0
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-1
0
1
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4
r=0.724, P <.001 r=0.644, r=0.691,
r=0.846, r=0.783,
SREBP-1a SREBP-2
r=0.586, r=0.635, r=0.836,
SREBP-1a SREBP-2 SCAP
SCAP SCAP SCAP
P <.001 P <.001
P <.001 P <.001 P <.001
P <.001 P <.001
, SCAP and SREBP-1a, B, SCAP and SREBP-2, C, SCAP and HMGCR, D, SREBP-1a and HMGCR, E, SREBP-2 and HMGCR, F, SCAP and LDLR,, SREBP-1a and LDLR, and H, SREBP-2 and LDLR. Abnormal activation of hepatic endogenous cholesterol synthesis may be the basis ofevelopmental programming of atherosclerosis in the apoE mouse model.oharkhay. Maternal hypercholesterolemia leads to activation of endogenous cholesterol synthesis in the offspring. Am J Obstet Gynecol 2008.
73.e4 American Journal of Obstetrics & Gynecology SEPTEMBER 2008
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ifferences in SREBP-2 message levelsere statistically significantly differentetween apoE�/�mat and apoE�/�WT
roups (P � .0004), as well betweenpoE�/�mat and apoE�/�pat animalsP � .0046).
HMGCR mRNA expression levels var-ed significantly by study group (P �0109, ANOVA) (Figure 2, D). Theuantity of HMGCR message was high-st in the apoE�/�mat group as com-ared with apoE�/�WT (P � .0082),poE�/�pat (P � .0042), and apoE�/�KO
ffspring (P � .0295). In contrast to thether gene products described so far,he expression of HMGCR in thepoE�/�KO animals did not show a trendowards higher levels than those inpoE�/�WT or apoE�/�pat animals.
We found evidence for differential ex-ression of LDLR among the 4 studyroups (P � .002, ANOVA). LDLRRNA levels were significantly higher in
poE�/�mat versus apoE�/�WT (P �0004) and apoE�/�pat (P � .0085) off-pring (Figure 2, E). apoE�/�KO ani-
als displayed higher HMGCR expres-ion levels than either apoE�/�WT orpoE�/�pat offspring, but the differencenly reached statistical significance inomparison to wild-type (apoE�/�WT)ice (P � .03).We compared the levels of mRNA ex-
ression for SCAP, SREBP-1a, SREBP-2,MGCR, and LDLR in each group by
ender, but did not find any significantifference for any of the studied generoducts (data not shown).Linear regression analyses were per-
ormed to correlate the expression levelsf the mRNA for SCAP, SREBP-1a,REBP-2, HMGCR, and LDLR. Weound that the expression of all theseene products significantly correlatedith each other. The regression plots and
egression coefficients found are de-icted in Figure 3.
OMMENTe have found evidence of activation of
he cholesterol lipogenic pathway in theiver of animals born to hypercholester-lemic mothers. This activation is appar-nt across all the major components of
he SCAP-SREBP-HMGCR pathway. wxposure to a high cholesterol environ-ent during early development appears
o reprogram the set point for choles-erol homeostasis in the liver of affectednimals. This mechanism could explainhe hypercholesterolemia observed indult offspring born to hypercholester-lemic dams.SCAP is the most upstream compo-
ent studied in the present investigation.CAP activity itself is affected by otheregulatory proteins, most notably In-ig-1 and Insig-2.11,12 Whether Insig ex-ression is altered in response to devel-pmental programming events needs toe determined.Another question that remains to be
nswered is the mechanism that leads tohe resetting of cholesterol homeostasisnd the abnormal overactivation ofCAP in the presence of high cholesterolevels. Epigenetic mechanisms, such asene methylation events, are likely in-olved in this process. This needs to beurther studied.
Although there is some evidence forransplacental transfer of cholesterolrom the mother to the fetus in mice andn humans, we do not know for certainhether offspring of hypercholester-lemic mothers are directly exposed to aigh-cholesterol environment.5,13 It isonceivable that the initial insult in theetus may be an indirect event such as ahange in the feto-placental environ-ent as a result of endocrine and meta-
olic changes in the placenta in responseo maternal hyperlipidemia.
In a recent follow-up to our originaltudy we have shown a paradoxic de-rease in cholesterol levels in 21-day-oldpoE�/�mat and apoE�/�KO offspringersus apoE�/�WT and apoE�/�pat con-rols.14 In contrast to 8-month-old off-pring, preliminary studies in the 21-ay-old offspring fail to show evidence
or an activation of hepatic cholesterolynthesis in apoE�/�mat animals. It ismportant to note that in our studies allffspring remain with and breastfeedrom the natural dams up to 3 weeks ofge. The lack of any evidence of a mal-daptive response at this age may be hy-othesized to be a reflection of a contin-ation of the feto-placental environment
hile breastfeeding from a hypercholes- PSEPTEMBER 2008 Americ
erolemic dam. Studies using cross-fos-ering of animals and/or embryo transferxperiments will be utilized in the futureo determine the critical time periods forhe observed programming effects.
A limitation of the present study is theack of protein and enzyme activity mea-urements. While this aspect is the sub-ect of ongoing studies, we believe thathe significant and consistent activationf the entire SCAP-SREBP-HMGCRathway (and of LDLR) in the apoE�/�mat
roup underscores the validity of our hy-othesis based on the current data.In summary, our observations provide
plausible mechanism that may underliehe significant effects of the early devel-pmental environment on the predispo-ition to develop hypercholesterolemiand atherosclerosis in the apoE mouseodel. Further studies are needed to ad-
ress the exact details of the mechanismseading to the dysregulation of choles-erol synthesis in response to a hyper-holesterolemic environment duringarly life. f
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esions. J Clin Invest 1997;100:2680-90.. Napoli C, Glass CK, Witztum JL, Deutsch R,’Armiento FP, Palinski W. Influence of mater-al hypercholesterolaemia during pregnancy onrogression of early atherosclerotic lesions inhildhood: Fate of Early Lesions in Children (FE-IC) study. Lancet 1999;354:1234-41.. 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;12:167-73.. Jawien J, Nastalek P, Korbut R. Mouse mod-ls of experimental atherosclerosis. J Physiol
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. Goharkhay N, Sbrana E, Gamble PK, et al.haracterization of a murine model of fetal pro-ramming of atherosclerosis. Am J Obstet Gy-ecol 2007;197:416.e1-5.0. Horton JD, Goldstein JL, Brown MS.REBPs: Activators of the complete program ofholesterol and fatty acid synthesis in the liver.
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cad Sci USA 2007;104:6519-26. sgy SEPTEMBER 2008
3. Yoshida S, Wada Y. Transfer of maternalholesterol to embryo and fetus in pregnantice. J Lipid Res 2005;216874.4. Goharkhay N, Mateus J, Egbert K, et al. Lackf hypercholesterolemia and of activation of cho-
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