Effects of Reduced Gcm1 Expression on Trophoblast Morphology, Fetoplacental Vascularity, and...

Sled John CP Kingdom and S Lee AdamsonShannon A Bainbridge Abhijeet Minhas Kathie J Whiteley Dawei Qu John G

Vascularity and Pregnancy Outcomes in Mice Expression on Trophoblast Morphology FetoplacentalGcm1Effects of Reduced

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Effects of Reduced Gcm1 Expression on TrophoblastMorphology Fetoplacental Vascularity and Pregnancy

Outcomes in MiceShannon A Bainbridge Abhijeet Minhas Kathie J Whiteley Dawei Qu John G Sled

John CP Kingdom S Lee Adamson

AbstractmdashPreeclampsia is a life-threatening disorder characterized by maternal gestational hypertension and proteinuriathat results from placental dysfunction Placental abnormalities include abnormal syncytiotrophoblast and a 50reduction in placental expression of the transcription factor Gcm1 In mice homozygous deletion of Gcm1 preventssyncytiotrophoblast differentiation and is embryonic lethal We used heterozygous Gcm1 mutants (Gcm1) to test thehypothesis that hypomorphic expression of placental Gcm1 causes defective syncytiotrophoblast differentiation andmaternal and placental phenotypes that resemble preeclampsia We mated wild-type female mice with Gcm1 fathersto obtain wild-type mothers carrying 50 Gcm1 conceptuses Gcm1 placentas had syncytiotrophoblastabnormalities including reduced gene expression of Gcm1-regulated SynB elevated expression of sFlt1 a thickenedinterhemal membrane separating maternal and fetal circulations and electron microscopic evidence in syncytiotrophoblast ofnecrosis and impaired maternal-fetal transfer Fetoplacental vascularity was quantified by histomorphometry and microcom-puted tomography imaging In Gcm1 it was 30 greater than wild-type littermates whereas placental vascularendothelial growth factor A (Vegfa) expression and fetal and placental weights did not differ Wild-type motherscarrying Gcm1 conceptuses developed late gestational hypertension (1182 versus 109607 mm Hg incontrols P005) We next correlated fetoplacental vascularity with placental Gcm1 expression in human controland pathological pregnancies and found that as in mice fetoplacental vascularity increased when GCM1 proteinexpression decreased (R2045 P005) These results support a role for reduced placental Gcm1 expressionas a causative factor in defective syncytiotrophoblast differentiation and maternal and placental phenotypes inpreeclampsia in humans (Hypertension 20125900-00) Online Data Supplement

Key Words syncytin placenta preeclampsia angiogenesis VEGFA placental growth factor sFlt1

Placental dysfunction is believed to be the major cause ofone of the most common and serious complications of

human pregnancy preeclampsia (PE)1 This potentially life-threatening hypertensive disorder adversely impacts 5 ofall pregnancies and has no known cure In PE growingevidence implicates abnormalities in syncytiotrophoblast(SynT) in the villous exchange region of the placenta1ndash6

SynTs are thin fetal-derived cells that lie between thematernal blood flowing through the villous exchange regionof the placenta and the fetal blood flowing through fetalvessels in the highly vascularized villi This surface isessential for fetomaternal communication and exchange andis formed through the differentiation of SynT from theunderlying villous cytotrophoblast cells under the control of

the transcription factor GCM1 (glial cells missing 1)7 Gcm1in the conceptus is almost exclusively expressed in a subset oftrophoblast in the placenta89 and is a critical regulator of theSynT cell type10

In the PE placenta Gcm1 expression is reduced by 504

as is expression of the immediate downstream target ofGcm111 Syn-156 That SynT is abnormal and potentiallycausative in PE is supported by abnormal levels of SynT-produced factors such as pregnancy-associated plasma pro-tein A human chorionic gonadotropin and inhibin in mater-nal blood during early pregnancy in women at high risk forthe later development of PE1213 When PE develops theabnormal syncytial surface is prothrombotic leading to in-farctions within the intervillous space14 and is excessively

Received October 19 2011 first decision November 11 2011 revision accepted December 21 2011From the Faculty of Health Sciences (SAB) University of Ottawa Ottawa Ontario Canada Samuel Lunenfeld Research Institute (SAB AM

KJW DQ JCPK SLA) Mount Sinai Hospital Toronto Ontario Canada Departments of Obstetrics and Gynaecology (SAB AM JCPKSLA) Physiology (SAB AM SLA) and Medical Biophysics (JGS) University of Toronto Toronto Ontario Canada Mouse Imaging Centre(JGS) Toronto Centre for Phenogenomics Hospital for Sick Children Toronto Ontario Canada

The online-only Data Supplement is available with this article at httphyperahajournalsorglookupsuppldoi101161HYPERTENSIONAHA111183939-DC1

Correspondence to Shannon A Bainbridge Interdisciplinary School of Health Sciences University of Ottawa Roger Guindon Hall Room 2058 451Smyth Rd Ottawa Ontario Canada K1H 8M5 E-mail shannonbainbridgeuottawaca

copy 2012 American Heart Association Inc

Hypertension is available at httphyperahajournalsorg DOI 101161HYPERTENSIONAHA111183939

1 at University of Toronto on February 12 2012httphyperahajournalsorgDownloaded from

shed as aponecrotic fragments into the maternal circulation13both are believed to be factors contributing to clinical signs ofdisease13

The availability of Gcm1 deletion mutants provides anopportunity to explore the function of Gcm1 in vivo Ho-mozygous deletion mutants show the critical importance ofGcm1 embryonic lethality results from absent SynT differ-entiation and failure to form the exchange region known asthe labyrinth in mice1516 Relative to placental differencesbetween species the placentas of humans and mice arestrongly similar1718 although differences exist In both spe-cies maternal blood perfuses an intervillous space lined byfetal-derived trophoblasts that encase the fetoplacental vas-culature within the exchange region In both species Gcm1 isexpressed in trophoblast cells differentiating into SynT1920

However whereas humans have a single SynT layer micehave 2 layers SynT-II is adjacent to the fetal vasculature andit is intimately connected to SynT-I via gap junctions andnutrient transporters2122 and unlike SynT-I only SynT-IIexpresses Gcm110 In humans the SynT surface is fullyexposed to maternal blood whereas in mice the SynT-Isurface is partially covered by a discontinuous layer ofsinusoidal trophoblast giant cells (sTGCs)23 Given the diffi-culty in establishing causal relationships in human studieshere we exploit similarities between humans and mice byusing heterozygous Gcm1 deletion mutants to determinewhether reduced Gcm1 expression in mice causes changes introphoblast morphology fetoplacental vascularity and preg-nancy outcomes that resemble PE in humans

Materials and MethodsMouse and human experiments were approved by institutionalreview committees For complete methods please see the online-onlyData Supplement

Mouse ModelCD1 females mated to males with heterozygous deletion of Gcm1(Gcm1)1516 carried 50 Gcm1 and 50 wild-type con-ceptuses Control pregnancies were CD1 females mated with CD1males At embryonic day 135 (E135) and E175 placentas weremicrodissected to remove decidual tissue and frozen for proteinassays or fixed for histology Others were microdissected to obtainlabyrinth-enriched samples for mRNA quantification by quantitativeRT-PCR (primers in Table S1 available in the online-only DataSupplement)

Human PatientsPlacental villous tissue and matched histological samples wereobtained from a BioBank (httpbiobanklunenfeldca) Sampleswere from pregnancies with PE intrauterine growth restriction(IUGR and without PE) and from gestation-matched pretermcontrols (Table) PE was diagnosed as new-onset gestational hyper-tension proteinuria and reversal of hypertension and proteinuria by12 weeks postpartum24 IUGR was diagnosed as fetal sex andgestation-adjusted birth weights less than the third centile25 andabsent end-diastolic blood flow velocity in umbilical artery Dopplerwaveforms

Histology and ImagingIn mice images from midline placental sections were quantified byhistomorphometry26 to obtain areas of placental components orevaluated by transmission electron microscopy Counts of Ki67positive cells in the labyrinth excluded sinusoidal trophoblast giantcells In humans the proportion of fetoplacental vasculature in villi

was quantified in hematoxylin-eosinndashstained sections Microcom-puted tomography was used to quantify fetoplacental arteries andarterioles 50 m in diameter as published27ndash29 Capillaries wereexamined in vascular corrosion casts as published1727

Maternal PhenotypesArterial pressure in awake mice was measured by tail-cuff plethys-mography urine protein by the Bradford method urine creatinine bythe Jaffe reaction and sFLT1 protein in maternal plasma by ELISA

Statistical AnalysisWild-type littermates from CD1Gcm1 matings were used ascontrols for Gcm1 conceptuses Statistical significance (P005)was determined using Wilcoxon sign-ranked test for quantitativeRT-PCR data 2-tailed Student t test (genotype effects) or 2-wayANOVA (genotype and gestation effects) followed by the Tukeypost hoc test Results show meanSE

ResultsAbnormal Trophoblast DifferentiationGcm1 in mice is specifically expressed in the SynT-II layer ofthe labyrinth where it regulates expression of its downstreamtarget SynB10 In the Gcm1 labyrinth expression of Gcm1mRNA and protein was decreased by 50 relative towild-type littermates (Figure S1 in the online-only DataSupplement) Gcm1 deficiency resulted in a dysregulation oftrophoblast differentiation as shown by an 35 decrease inSynB mRNA expression (Figure S1A) an 2-fold increase incell proliferation within the labyrinth (Figure S2) and prom-inent ultrastructural abnormalities of the 2 syncytial layers(SynT-I and SynT-II) at E175 (Figure 1) There was evi-dence of necrosis230 in SynT-II including pale cytoplasmnumerous vacuoles and swollen and degenerating mitochon-dria in transmission electron microscopy images (Figure 1Athrough 1C) The Gcm1 SynT-I layer appeared to differ-entiate normally in that expression of its marker gene SynAwas not significantly affected (1002- and 1303-foldchange relative to wild-type littermates at E135 and E175respectively P005) Nevertheless the SynT-I layer exhib-ited ultrastructural abnormalities including increased elec-tron density and the presence of large fluid-filled vacuoles(Figure 1A and 1B) SynT-I and SynT-II appeared thicker in

Table Human Clinical Outcomes and Histomorphometry Data

VariablesPreterm Controls

(N5)PE

(N5)IUGR

(N5)

Gestational age atdelivery wk

30908 30512 30712

Range wk 28ndash33 28ndash35 28ndash34

Systolic BP mm Hg 1125 1855dagger 1387

Diastolic BP mm Hg 616 1094dagger 864

Fetal weight g 1772127 1364200 998179

Placental weight gDagger 27123 18931 16323

Fetal vasculature invilli area

11311 19112dagger 11114

BP indicates blood pressure PE preeclampsia IUGR intrauterine growthrestriction

P005 vs preterm controlsdaggerP005 vs preterm controls and vs IUGRDaggerData show placental weight after fixation meanSE

2 Hypertension March 2012


Gcm1 placentas compared with wild-type littermate con-trols at both E135 and E175 (Figure 1A and 1B) In additionloss of apical membrane integrity in focal areas of the sTGCs(in direct contact with maternal blood) was observed intransmission electron microscopy images of the Gcm1

placenta at E175 (Figure 1D)

Abnormal Placental Histomorphologyand VascularityAt E135 the Gcm1 labyrinth was significantly decreasedin cross-sectional area and there were significant increases inthe proportion of maternal blood spaces and in the thicknessof the interhemal membrane relative to wild-type littermates(Figure 2A through 2D) There was also an increase in thearea of clustered cuboidal cells within the Gcm1 labyrinth(Figure 2C) In contrast at E175 Gcm1 placentas had asignificantly higher proportion of fetal blood spaces in thelabyrinth compared with wild-type littermates (Figure 2G)

The latter is consistent with increased fetoplacental vascular-ity at E155 in Gcm1 placentas as shown by significantincreases in the total number and length of arterial segmentswith diameters 50 m by microcomputed tomography(Figure 3) and apparent increases in the density of capillariesin vascular casts and in histological sections (Figure S3Athrough S3C)

Intriguingly the mRNA and protein expression profile ofVEGFA a potent proangiogenic molecule thought to play animportant role in placental angiogenesis31 demonstrated de-creased expression across gestation within the Gcm1

placenta relative to that of wild-type littermates (Figure 4Aand 4B) A similar examination of the mRNA and proteinexpression of another proangiogenic molecule placentalgrowth factor (PGF) demonstrated a modest increase inexpression at E135 in the Gcm1 placenta and no differ-ence in expression by E175 (late gestation) compared withlittermate controls (Figure 4A and 4C)

Figure 1 Transmission electron micro-graphs of the interhemal membrane thatseparates the maternal and fetal bloodspaces in the labyrinth Representativeimages for the wild-type (Wt left) andGcm1 (right) labyrinths at embryonicday 175 are shown (from N4 Wt andN4 Gcm1) A Images showingmore numerous vacuoles in a darkersyncytiotrophoblast (SynT) I and palerSynT-II in Gcm1 placentas comparedwith Wt B Images in A with shading toshow cell layers C Image showingabnormal mitochondria in SynT-II ofGcm1 placenta (right) in contrast toWt (left) Arrows point to mitochondriaAdded lines demarcate layers D Exam-ple showing cellular debris from sinusoi-dal trophoblast giant cells (sTGCs) inadjacent maternal blood space in theGcm1 labyrinth (right) in contrast toWt (left) FBS indicates fetal bloodspace MBS maternal blood space Efetal endothelial cells Arrow mitochon-dria Scale bar2 m (A and B) 1 m(C and D)

Bainbridge et al Gcm1 and Pregnancy Outcomes in Mice 3


Fetal and Placental GrowthDespite pronounced abnormalities in trophoblast differentia-tion and placental morphology Gcm1 conceptuses grewnormally at E175 there were no significant differencesbetween fetal weights of Gcm1 (1201 g) versus wild-type littermates (1101 g) or in their placental weights(9215 versus 9010 mg respectively) There was noevidence for an effect of paternal genotype on litter size ornumber of resorptions

Clinical Signs of PE in MothersCD1 females mated with Gcm1 males carried litters inwhich 50 of conceptuses were Gcm1 These mothershad significantly higher mean arterial pressure at E175compared with CD1 females mated with CD1 males (1182versus 109607 mm Hg respectively Figure 5A) Theincrease in maternal pressure in late gestation was positivelycorrelated with the number of Gcm1 pups within the litter(R2054 P005 Figure 5B) CD1 mothers carrying mixedlitters did not have proteinuria in late gestation they excreted1805 mg of protein per milligram of creatinine versus2107 mg of protein per milligram of creatinine in controlsAs in human PE placentas32 the Gcm1 labyrinth demon-strated a 2-fold increase in sFlt1 mRNA expression relative tothe wild-type littermate controls (Figure 5C) However incontrast to human PE32 there was no significant increase inmaternal plasma sFLT1 protein (Figure 5D)

Correlation Between GCM1 and FetoplacentalVascularity in HumansHuman clinical outcomes for patients with PE with IUGRand for gestation-matched controls are shown in the TableThe proportion of fetal vascular space within the villoustissue of patients with PE was significantly elevated relativeto gestation-matched controls and IUGR placentas (Table)Across all of the groups the proportion of fetal vascular spacewithin villous tissue was negatively correlated with GCM1protein expression (R2045 P005 Figure 6)

DiscussionIn the current study hypomorphic expression of placentalGcm1 caused defective SynT differentiation and maternal andplacental phenotypes resembling PE in humans As antici-pated heterozygous Gcm1 gene deletion reduced placentalGcm1 expression by 50 a decrement similar to that inhuman PE placentas4 SynT-II cells exhibited prominentabnormalities including reduced expression of SynB andultrastructural evidence of necrosis30 Both SynT-I andSynT-II appeared thickened and ultrastructurally abnormalNevertheless Gcm1 fetuses were normally grown Simi-larly fetuses are normally grown in the majority of PEpregnancies33 despite evidence of hypomorphic Gcm1 ex-pression and SynT dysfunction1ndash6 An unexpected and strik-ing finding of the current study was increased fetoplacental

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Figure 2 Histomorphometry of Gcm1 placentas ( ) vs wild-type littermate controls (Wt N5 pregnancies) Gcm1 placentashad a smaller labyrinth (A and B) and increased maternal blood space (C) and cuboidal cell area (C) within the labyrinth as well as athicker interhemal membrane (D) at E135 By E175 there was no difference in labyrinth size in Gcm1 placentas (E and F) or inter-hemal membrane thickness (H) but fetal blood space was significantly increased (G) vs Wt Laby indicates labyrinth JZ junctionalzone CP chorionic plate FBS fetal blood space MBS maternal blood space sTGC sinusoidal trophoblast giant cells Cub cuboidalcells P005



vascularity in the Gcm1 mouse placentas We show asimilar increase in fetoplacental vascularity correlates withdecreased GCM1 protein expression in human placental villiFinally results show that conceptuses with hypomorphic

Gcm1 expression caused maternal hypertension in late ges-tation in otherwise healthy wild-type mothers

Hypomorphic Gcm1 expression caused abnormal differentia-tion of SynT-II cells as shown by a reduction in expression of

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Figure 3 Arterial vascularity in the labyrinth at embryonic day (E) 155 by microcomputed tomography (CT) A Images from a Gcm1

and wild-type littermate control (Wt) B Color-coded arterial tree to show the anatomic distribution of vessel diameters C and DQuantitative analysis showing significant overall increases in (C) the total number of vessel segments and (D) the total length of vesselsegments in arterial trees of Gcm1 placentas ( ) vs Wt () Increased vascularity was most prominent in the smallest diameterrange (ie 50ndash100 m) P005 N11 per genotype (2ndash3 conceptuses per genotype from 5 pregnancies)

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Figure 4 Expression of proangiogenic factors at embryonic day (E) 135 and E175 A mRNA expression of Vegfa and Pgf in labyrinth-enriched Gcm1 samples normalized using 3 housekeeping genes and expressed as a fold change relative to wild-type littermatecontrols (Wt hashed line at 10) Vegfa mRNA was significantly reduced at E135 and E175 in Gcm1 whereas Pgf mRNA was sig-nificantly increased at E135 only B Placental VEGFA protein by ELISA was significantly decreased at both ages C Placental PGFprotein by ELISA was not significantly changed Number of pregnancies for each gestational age and genotype was N6 P005



the Gcm1-regulated fusogenic protein SynB and by prominentultrastructural abnormalities that directly paralleled the de-scribed focal necrosis abnormalities observed in the SynT layerin human PE3034 SynT-II is tightly integrated with the adjacentSynT-I layer SynT-I uniquely expresses Syncytin A (SynA)10

We found that SynA expression was not altered in Gcm1

placentas suggesting normal differentiation of SynT-I a non-Gcm1ndashexpressing cell type10 Similarly no change in SynA

expression was observed in homozygous Gcm1 knockout pla-centas10 Nevertheless morphology of the SynT-I layer wasmarkedly abnormal in Gcm1 placentas transmission electronmicroscopy imaging showed that it appeared thicker containedmore electron dense material and contained many large fluid-filled vacuoles SynT-I and SynT-II are intimately connected bygap junctions and ultimately function as a single syncytial layeracross which maternal-fetal transfer occurs2122 Thus it is likely

NP E135 E175-5

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Figure 5 Effect of Gcm1 conceptuses inCD1 mothers on maternal arterial pressureand placental and plasma sFlt1 levels AArterial pressure in CD1 mothers carryingmixed litters of wild-type (Wt) and Gcm1

conceptuses (f) was significantly higher atE175 than in CD1 mothers with Wt concep-tuses only () when expressed as a change() from the nonpregnant state (NP) BMaternal arterial pressure was positively cor-related with the number of Gcm1 con-ceptuses at E175 R2 value P005 CmRNA expression of the antiangiogenic fac-tor sFlt1 was higher in Gcm1 placentasexpressed relative to Wt littermate controlsat E175 (hashed line at 10) D Maternalplasma sFLT1 protein was not significantlydifferent whether CD1 mothers carried onlyWt fetuses () or mixed litters (f) at E175Number of pregnancies for each gestationalage and paternal genotype was N6 in A Cand D and N10 in B P005

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C PE IUGR C PE IUGR C PE IUGR C PE IUGR

Gcm1

BAct

A

BFigure 6 GCM1 protein expression and itscorrelation with villous fetoplacental vascu-larity in human placentas A Western blotshowing GCM1 protein expression in pla-centas from preterm control (C) pre-eclamptic (PE) and intrauterine growth-restricted (IUGR) pregnancies at delivery-Actin (BAct) was used as the loadingcontrol B There was a significant negativecorrelation between placental GCM1expression and the proportion of villoustissue composed of fetal vasculature in CPE and IUGR placentas (N5 per group)R2 value P005



that a primary limitation in transport across the abnormalSynT-II layer contributed to abnormal SynT-I morphology byimpeding the egress of protein and vacuoles from SynT-I

Abnormal SynT-II in Gcm1 placentas additionally in-fluenced the discontinuous sTGCs23 that overlie SynT-I in thematernal blood spaces of the labyrinth Apical membraneintegrity of sTGC was lost in localized areas and cytoplasmiccellular contents were visible within the adjacent maternalblood space This was unexpected given that the sTGCs areonly loosely connected to the underlying SynT-I layerthrough desmosomal attachments23 and are not in directcontact with SynT-II cells Nevertheless results suggest thathypomorphic Gcm1 expression in the PE placenta in humanpregnancy may play a similar causative role in promoting theshedding of placental debris into the maternal circulation Thisdebris is thought to be responsible at least in part for thewidespread maternal endothelial dysfunction and hypertensionin PE12 In mice sTGCs release hormones such as placentallactogen into the maternal circulation10 so abnormalities ob-served in this cell type in the current study may also altermaternal responses to pregnancy by an endocrine mechanism

A remarkable finding in the current study was that fetalgrowth was not compromised despite marked abnormalitiesin SynT-I SynT-II and sTGC cell morphology as well asaugmented interhemal membrane thickness It is possible thatnormal fetal growth was protected by increased fetoplacentalvascular density in the Gcm1 labyrinth Increased fetopla-cental vascularity may have been caused by feedback mech-anisms invoked by impaired transfer caused by the thickermalfunctioning fetal-maternal exchange barrier Howeverabnormal SynT-I in SynA knockout placentas also causesabnormal interhemal membrane morphology but in that caseresults in fetoplacental hypovascularity fetal growth restric-tion and fetal lethality by E13535 Furthermore Gcm1overexpression results in fetoplacental hypovascularity lead-ing to fetal growth restriction in late-gestation in mice36

Thus it is more likely that dysregulated SynT-II differentia-tion directly influences fetoplacental vascularization throughaltered signaling to the adjacent fetal endothelial and mesen-chymal cells How angiogenesis is augmented in the face ofdecreased mRNA and protein expression of the potent proan-giogenic factor VEGFA increased mRNA expression of theantiangiogenic sFlt1 and only modest increases in PGF atE135 is not known It is possible that alternate unidentifiedangiogenic mechanisms are activated within the Gcm1

placenta The same paradox among villous trophoblast pa-thology23036 high placental expression of the antiangiogenicsFlt132 and augmented villous angiogenesis (current study)occurs in PE where the majority of women give birth toinfants of normal weight33 Indeed the current results inGcm1 mice prompted the examination and discovery of asimilar negative correlation between placental vascularity andGCM1 protein expression in placental biopsies from normaland pathological pregnancies (PE and IUGR) Thus thismouse model provides an excellent opportunity to explorethis apparent incongruity and to advance our understanding ofthe influence of SynT on fetoplacental vascularization

Hypomorphic Gcm1 expression resulted in significantlyincreased maternal arterial pressure in late gestation although

how hypomorphic Gcm1 in SynT-II caused this change is notknown Mechanisms other than increased circulating sFLT1must be driving maternal hypertension because despiteincreased placental mRNA expression of sFlt1 in theGcm1 placenta sFLT1 protein in the maternal circulationwas not significantly elevated Maternal proteinuria was alsoabsent suggesting that renal function was unimpaired It ispossible that placental debris from sTGC entered the maternalcirculation and caused endothelial cell inflammation in-creased peripheral vascular resistance and hypertension (asin human PE12) or that abnormal endocrine signals from thedysfunctional sTGC are responsible The increase in arterialpressure in this mouse model was modest and this couldmean that alone the hypomorphic expression of Gcm1 in theplacenta of women with PE is insufficient to completelyexplain their hypertension This is consistent with evidencesuggesting that a combination of placental dysfunction andunderlying maternal predispositions to endothelial dysfunc-tion may be required for PE in women3738 Alternatively it isalso possible that relatively mild maternal effects wereobserved because not all of the conceptuses were Gcm1this cannot be achieved in a wild-type mother by naturalbreeding because Gcm1 deletion is homozygous lethalWild-type conceptuses may diminish maternal effects byeffectively diluting circulating factors released by Gcm1

placentas andor by releasing protective factors into thematernal circulation Indeed a positive correlation was ob-served when maternal arterial pressure was plotted against thenumber of Gcm1 conceptuses within the pregnancyInterestingly evidence supports a similar mechanism inhuman twin pregnancies complicated by IUGR where ahealthy co-twin appears to be protective PE affects 68 ofsingleton pregnancies complicated with IUGR versus 15 intwin pregnancies when only 1 twin has IUGR39 Thus results ofthe current study show that hypomorphic Gcm1 expression inSynT can elevate maternal arterial pressure in late-gestation inmice This means that hypomorphic placental Gcm1 expressionmay not just correlate with human PE but could also play acausative role in maternal hypertension one of the hallmarksigns of PE This conclusion is supported by the recent identi-fication of a fetal variant in the Gcm1 gene that is associated withgestational hypertension in human pregnancy40

PerspectivesHypomorphic placental Gcm1 expression caused dysregu-lated SynT-II differentiation abnormal morphology and dys-function of the interhemal membrane and augmented feto-placental vascularity and caused maternal hypertension inlate gestation In addition we showed in humans that there isa similar inverse correlation between fetoplacental vascular-ity of placental villi and Gcm1 expression These resultstherefore support a role for reduced placental Gcm1 expres-sion as a causative factor in defective SynT differentiationand maternal and placental phenotypes in PE in humans

AcknowledgmentsWe thank Dr Sascha Drewlo Dora Baczyk and Dr Monique Rennie fortechnical guidance Doug Holmyard for electron microscopy and thetissue donors and the Research Centre for Womenrsquos and Infantsrsquo Health



BioBank of Mount Sinai Hospital (httpbiobanklunenfeldca) for thehuman specimens used in this study

Sources of FundingThis work was supported by operating grants to SLA (CanadianInstitutes of Health Research MOP-12772 and MOP-93618) andJCPK (Canadian Institutes of Health Research MOP-64302) and aMount Sinai HospitalUniversity Health NetworkDepartment ofObstetrics and Gynaecology Research award to SAB We gratefullyacknowledge support to SAB as a Molly Towell Perinatal Fellowand a Canadian Institutes of Health Research postdoctoral fellow toJCPK as the Rose Torno Chair and to SLA as the Anne and MaxTanenbaum Chair at Mount Sinai Hospital

DisclosuresNone

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Science 20053081592ndash15942 Huppertz B Kingdom JC Apoptosis in the trophoblast role of apoptosis

in placental morphogenesis J Soc Gynecol Investig 200411353ndash3623 Huppertz B Kadyrov M Kingdom JC Apoptosis and its role in the

trophoblast Am J Obstet Gynecol 200619529ndash394 Chen CP Chen CY Yang YC Su TH Chen H Decreased placental

GCM1 (glial cells missing) gene expression in pre-eclampsia Placenta200425413ndash421

5 Langbein M Strick R Strissel PL Vogt N Parsch H Beckmann MWSchild RL Impaired cytotrophoblast cell-cell fusion is associated withreduced syncytin and increased apoptosis in patients with placental dys-function Mol Reprod Dev 200875175ndash183

6 Knerr I Beinder E Rascher W Syncytin a novel human endogenousretroviral gene in human placenta evidence for its dysregulation in pre-eclampsia and HELLP syndrome Am J Obstet Gynecol 2002186210ndash213

7 Baczyk D Drewlo S Proctor L Dunk C Lye S Kingdom J Glial cellmissing-1 transcription factor is required for the differentiation of thehuman trophoblast Cell Death Differ 200916719ndash727

8 NCBI GEO Profiles GCM1 in GDS3113 (expression profiling by arrayofvarioushumantissues)httpwwwncbinlmnihgovgeoprofilestermGDS3113[ACCN]gcm1 Accessed October 3 2011

9 Iwasaki Y Hosoya T Takebayashi H Ogawa Y Hotta Y Ikenaka K Thepotential to induce glial differentiation is conserved between Drosophila andmammalian glial cells missing genes Development 20031306027ndash6635

10 Simmons DG Natale DR Begay V Hughes M Leutz A Cross JC Earlypatterning of the chorion leads to the trilaminar trophoblast cell structurein the placental labyrinth Development 20081352083ndash2091

11 Lin C Lin M Chen H Biochemical characterization of the human placentaltranscription factor GCMa1 Biochem Cell Biol 200583188ndash195

12 Smith GC Stenhouse EJ Crossley JA Aitken DA Cameron AD ConnorJM Early pregnancy levels of pregnancy-associated plasma protein A andthe risk of intrauterine growth restriction premature birth preeclampsiaand stillbirth J Clin Endocrinol Metab 2002871762ndash1767

13 Dugoff L Hobbins JC Malone FD Vidaver J Sullivan L Canick JALambert-Messerlian GM Porter TF Luthy DA Comstock CH Saade GEddleman K Merkatz IR Craigo SD Timor-Tritsch IE Carr SR Wolfe HMDrsquoAlton ME FASTER Trial Research Consortium Quad screen as a pre-dictor of adverse pregnancy outcome Obstet Gynecol 2005106260ndash267

14 Sood R Kalloway S Mast AE Hillard CJ Weiler H Fetomaternal crosstalk in the placental vascular bed control of coagulation by trophoblastcells Blood 20061073173ndash3180

15 Anson-Cartwright L Dawson K Holmyard D Fisher SJ Lazzarini RACross JC The glial cells missing-1 protein is essential for branching mor-phogenesis in the chorioallantoic placenta Nat Genet 200025311ndash314

16 Schreiber J Riethmacher-Sonnenberg E Riethmacher D Tuerk EEEnderich J Bosl MR Wegner M Placental failure in mice lacking themammalian homolog of glial cells missing GCMa Mol Cell Biol 2000202466ndash2474

17 Adamson SL Lu Y Whiteley KJ Holmyard D Hemberger M Pfarrer CCross JC Interactions between trophoblast cells and the maternal andfetal circulation in the mouse placenta Dev Biol 2002250358ndash373

18 Georgiades P Ferguson-Smith AC Burton GJ Comparative develop-mental anatomy of the murine and human definitive placentae Placenta2002233ndash19

19 Baczyk D Satkunaratnam A Nait-Oumesmar B Huppertz B Cross JCKingdom JC Complex patterns of GCM1 mRNA and protein in villousand extravillous trophoblast cells of the human placenta Placenta 200425553ndash559

20 Basyuk E Cross JC Corbin J Nakayama H Hunter P Nait-OumesmarB Lazzarini RA Murine Gcm1 gene is expressed in a subset of placentaltrophoblast cells Dev Dyn 1999214303ndash311

21 Shin BC Suzuki T Matsuzaki T Tanaka S Kuraoka A Shibata YTakata K Immunolocalization of GLUT1 and connexin 26 in the ratplacenta Cell Tissue Res 199628583ndash89

22 Metz J Heinrich D Forssmann WG Gap junctions in hemodichorial andhemotrichorial placentae Cell Tissue Res 1976171305ndash315

23 Coan PM Ferguson-Smith AC Burton GJ Ultrastructural changes in theinterhaemal membrane and junctional zone of the murine chorioallantoicplacenta across gestation J Anat 2005207783ndash796

24 ACOG Practice Bulletin Diagnosis and management of preeclampsia andeclampsia Number 33 January 2002ndashAmerican College of Obstetriciansand Gynecologists Int J Gynaecol Obstet 20027767ndash75

25 Canadian Perinatal Surveillance System Health Canada Birth weightfor gestational age httpwwwphac-aspcgccarhs-ssgbwga-pnagpdfbwga-pnag_epdf Accessed August 2001

26 Coan PM Ferguson-Smith AC Burton GJ Developmental dynamics ofthe definitive mouse placenta assessed by stereology Biol Reprod 2004701806ndash1813

27 Whiteley KJ Pfarrer CD Adamson SL Vascular corrosion casting of theuteroplacental and fetoplacental vasculature in mice Methods Mol Med2006121371ndash392

28 Rennie MY Whiteley KJ Kulandavelu S Adamson SL Sled JG 3Dvisualisation and quantification of microcomputed tomography of lategestational changes in the arterial and venous feto-placental vasculatureof the mouse Placenta 200728833ndash840

29 Rennie MY Detmar J Whiteley KJ Yang J Jurisicova A Adamson SLSled JG Vessel tortuousity and reduced vascularization in the fetopla-cental arterial tree after maternal exposure to polycyclic aromatic hydro-carbons Am J Physiol Heart Circ Physiol 2011300H675ndashH684

30 Jones CJ Fox H An ultrastructural and ultrahistochemical study of thehuman placenta in maternal pre-eclampsia Placenta 1980161ndash76

31 Kaufmann P Mayhew TM Charnock-Jones DS Aspects of human feto-placental vasculogenesis and angiogenesis IIndashchanges during normalpregnancy Placenta 200425114ndash126

32 Tsatsaris V Goffin F Munaut C Brichant JF Pignon MR Noel A SchaapsJP Cabrol D Frankenne F Foidart JM Overexpression of the solublevascular endothelial growth factor receptor in preeclamptic patients patho-physiological consequences J Clin Endocrinol Metab 2003885555ndash5563

33 Xiong X Demianczuk NN Saunders LD Wang FL Fraser WD Impactof preeclampsia and gestational hypertension on birth weight by gesta-tional age Am J Epidemiol 2002155203ndash209

34 Huppertz B Kingdom J Caniggia I Desoye G Black S Korr H KaufmannP Hypoxia favours necrotic versus apoptotic shedding of placental syncy-tiotrophoblast into the maternal circulation Placenta 200324181ndash190

35 Dupressoir A Vernochet C Bawa O Harper F Pierron G Opolon PHeidmann T Syncytin-A knockout mice demonstrate the critical role inplacentation of a fusogenic endogenous retrovirus-derived envelopegene Proc Natl Acad Sci U S A 200910612127ndash12132

36 Kashif M Hellwig A Kolleker A Shahzad K Wang H Lang S Wolter J ThatiM Vinnikov I Bierhaus A Nawroth PP Isermann B p45NF-E2 repressesGcm1 in trophoblast cells to regulate syncytium formation placental vascular-ization and embryonic growth Development 20111382235ndash2247

37 Rodie VA Freeman DJ Sattar N Greer IA Pre-eclampsia and cardio-vascular disease metabolic syndrome of pregnancy Atherosclerosis2004175189ndash202

38 Ness RB Roberts JM Heterogeneous causes constituting the singlesyndrome of preeclampsia a hypothesis and its implications Am J ObstetGynecol 19961751365ndash1370

39 Gouin K Pakenham S Barrett J Nevo O Incidence of hypertensivedisorders in pregnancies complicated by IUGR twins versus singletons[abstract] Am J Obstet Gynecol 2009S273

40 Wilson ML Brueggmann D Desmond DH Mandeville JE GoodwinTM Ingles SA A fetal variant in the GCM1 gene is associated withpregnancy induced hypertension in a predominantly hispanic populationInt J Mol Epidemiol Genet 20112196ndash206



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ONLINE SUPPLEMENT

EFFECTS OF REDUCED Gcm1 EXPRESSION ON TROPHOBLAST MORPHOLOGY

FETO-PLACENTAL VASCULARITY AND PREGNANCY OUTCOMES IN MICE

Shannon A Bainbridge 1234 Abhijeet Minhas234 Kathie J Whiteley 2 Dawei Qu 2 John G

Sled5 John CP Kingdom 23and S Lee Adamson 234

(1) Faculty of Health Sciences University of Ottawa Ottawa ON Canada (2) Samuel Lunenfeld Research Institute of Mount Sinai Hospital Toronto ON Canada (3) Department of Obstetrics and Gynaecology University of Toronto and (4) Department of Physiology University of Toronto (5) Department of Medical Biophysics University of Toronto and the Mouse Imaging Centre Toronto Centre for Phenogenomics Hospital for Sick Children Toronto Canada

Corresponding Author Shannon A Bainbridge PhD Interdisciplinary School of Health Sciences University of Ottawa Roger Guindon Hall Rm 2058 451 Smyth Rd Ottawa Ontario Tel (613) 562-5800 ext 8569 Fax (613) 562-5632 E-mail shannonbainbridgeuOttawaca


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MATERIALS AND METHODS

Mouse Model Experiments were approved by the Animal Care Committee of the Toronto Centre for

Phenogenomics (Toronto ON Canada) and were conducted in accord with guidelines established by the Canadian Council on Animal Care Virgin female CD1 mice (an outbred wild type strain Charles River Canada) between 6-10 weeks of age were mated with males with heterozygous deletion of the Gcm1 gene (Gcm1+-) which were maintained on a CD1 background (a gift from Dr J Cross University of Calgary 1) This breeding scheme yields pregnancies with ~50 offspring with heterozygous deletion of Gcm1 (Gcm1+-) and ~50 wildtype (Wt) offspring Wildtype littermates were used as intra-pregnancy controls Additional controls were obtained by mating virgin CD1 females (6-10 weeks) with CD1 males Noon of the day a vaginal plug was found was designated E05 Maternal weight blood pressure urine and maternal blood samples were collected prior to breeding and longitudinally across pregnancy At E135 and E175 a sub-set of mothers were euthanized by cervical dislocation and the uterus was removed into RNAse-free ice-cold PBS Each implantation site was individually opened and the corresponding fetus removed and weighed and a sample of fetus or yolk sac collected for genotyping Placentas were removed from the uterine wall and microdissected to separate them from their deciduas Placentas were either flash frozen in liquid nitrogen for protein analysis immersion fixed in 4 PFA for light microscopy or fixed in 2 glutaraldehyde (in 01M sodium cacodylate buffer) for transmission electron microscopy Some placentas were further microdissected to obtain labyrinth-enriched tissue These samples were processed in RNAlater (Ambion Applied Biosystems Canada) and stored at -80oC for mRNA analysis After genotyping fetuses samples for protein or mRNA analysis were pooled per pregnancy by genotype

Human patients Placental villous tissue (for GCM1 protein quantification) and matched histological

samples (for histomorphometry) were obtained from the specimen archive of the Research Centre for Womenrsquos and Infantsrsquo Health BioBank at Mount Sinai Hospital (httpbiobanklunenfeldca) The study was conducted in accordance and with approval from the Mount Sinai Hospital Research Ethics Board (Toronto ON) and patients had provided their informed consent Samples were collected from placentas from PE pregnancies (N=5) pregnancies with intra-uterine growth restriction without PE (IUGR N=5) and gestation matched idiopathic preterm rols (N=5) PE was diagnosed with new onset gestational hypertension proteinuria and reversal of hypertension and proteinuria by 12 weeks postpartum 2 Hypertension was defined as an increase of 30 mmHg systolic or 15 mmHg diastolic blood pressure compared to values obtained before 20 weeks gestation and an absolute blood pressure gt14090 mmHg Proteinuria was defined as gt300 mg24 h collection or gt2+ on voided or 1+ on catheterized random urine sample or a protein creatinine ratio gt03 IUGR was diagnosed by fetal sex and gestational age-adjusted birth-weight lt3rd centile 3 accompanied by absent end-diastolic blood flow velocity in umbilical artery Doppler waveforms

Gene Expression RNA was extracted from the labyrinth-enriched samples (N=6 pregnancies 3 pooled

samplesgenotypepregnancy) using the Trizol method with removal of any contaminants by means of DNase treatment (RNase-free DNase Set Qiagen) and RNeasy Mini-eltue cleanup kit


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(Qiagen) Samples were reverse transcribed to cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems) Expression of genes of interest were measured using qRT-PCR (Eppendorf Realplex2) with SYBER Green (iTaq SYBER Green Supermix with ROX Bio-Rad Laboratories) using 40 cycles of 95degC (15 sec) and 60degC (1 min) followed by melting curve analysis Primers were designed for murine Gcm1 Syncytin A (SynA) Syncytin B (SynB) vascular endothelial growth factor A (Vegfa) soluble fms-like tyrosine kinase 1 (sFlt1) placental growth factor (Pgf) along with three housekeeping genes Beta-actin (BAct) glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and TAT box binding protein (Tbp) All primer sequences (Table S1) were subjected to BLAST analysis to ensure specificity and were tested for efficiency Expression data was analyzed using the ΔΔ-Ct method with gene expression in the Gcm1+- placenta normalized to the geometric mean of the three housekeeping genes and expressed relative to the expression in the wildtype littermate sample

Protein Expression Frozen placental samples (N=6 pregnancies 3 pooled samplesgenotypepregnancy) were

ground into a fine powder using a mortar and pestle in the presence of liquid nitrogen Samples were then lysed and homogenized in the presence of RIPA buffer (Thermo Scientific Rockford IL) and protease inhibitors (Complete Mini EDTA-free Inhibitor Roche Applied Science Laval QC) and subjected to centrifugation Concentrations of VEGFA and PGF were measured in prepared lysates using commercially available ELISA kits (RampD Systems Burlington ON) as per manufacturersrsquo directions All results were normalized to total protein concentration in each sample as measured by Bradford Reagent GCM1 protein was measured by western blot analysis SDS- polyacrylamide gel electrophoresis was used to separate proteins Samples were then transferred to Immobilon-P membranes (Millipore Corp Bedford MA) After a 1-hour incubation in blocking solution consisting of 5 dry milk and 005 Tween 20 in PBS (PBS-T) the membranes were incubated overnight at 4degC with an anti-Gcm1 antibody for mice (sc-69406 1500 Santa Cruz Biotechnology Santa Cruz CA) or humans (OAAB04228 11000 Aviva Systems Biology San Diego CA) followed by three 15-minute washes in PBS-T The membranes were then incubated with secondary rabbit anti-goat IgG (31402 13000 Pierce Thermo Scientific) or goat anti-rabbit IgG (31460 13000 Pierce Thermo Scientific Ottawa ON) labeled with horseradish peroxidase After three additional 15-minute washes with PBS-T the blots were developed using a chemiluminescence HRP detection (Immun-Star Western C Chemiluminescence kit BioRad Laboratories Mississauga ON) and visualized using Versa-Doc Imaging System (BioRad Laboratories) Uniformity of loading of protein extracts was determined by probing with an anti-β-actin antibody (sc-1616 11000 Santa Cruz) Relative intensities of bands were determined by densitometry using Quantity One 1-D Analysis software (BioRad Laboratories)

Transmission Electron Microscopy Mouse placentas for transmission electron microscopy (EM) were fixed in 2

glutaraldehyde post fixed in 1 osmium tetroxide in 01M sodium cacodylate buffer dehydrated in a graded ethanol series followed by propylene oxide and embedded in Quetol-Spurr epoxy resin Ultra-thin sections (100 nm) were cut and counterstained with uranyl acetate and lead citrate and imaged by transmission EM (FEI CM100 Hillsboro OR) at magnifications between x2500 - x25000 Imaging focused on the interhaemal membrane of the placental labyrinth (ie the tissue layers separating the maternal and fetal blood spaces) with specific


183939-R1

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attention paid to the appearance and integrity of the 2 syncytial membranes (SynT-I and SynT-II) and the sinusoidal trophoblast giant cells (sTGC)

Immunohistochemistry and histomorphometry After genotyping 3 Wt and 3 Gcm1+- placentas per pregnancy (N=5 pregnancies for

each gestational age) were arbitrarily selected for placental histomorphometry Paraffin-embedded placental sections (5 microm) were de-paraffinized by sequential incubations in Xylene and decreasing ethanol baths (each for 2 x 3 min) Antigen retrieval was performed by heating the sections for 20 min followed by incubation in sodium citrate buffer (10 mM) Following washes in PBS endogenous peroxidase activity was quenched using 3 hydrogen peroxide and nonspecific binding was limited using Dako protein block (Dako Burlington ON) The sections

were treated with antibodies overnight at 4degC to detect trophoblast (anti-cytokeratin Z0622 11000 Dako) endothelial basement membranes (anti-CD34 MCA1825 1100 AbD Serotec Raleigh NC) or proliferating cells (anti-Ki67 RM-9106 1200 Thermo Fisher Scientific Fremont CA) A biotinylated anti-rat (712066153 1500 Research Diagnostics INC Flanders NJ) or anti-rabbit IgG (BA-1000 1200 Vector Laboratories Burlington ON) secondary antibody was added to the sections for 30 min at room temperature Additional processing of the sections for the detection of the trophoblast (cytokeratin) fetal vascular endothelial cells (CD34) or proliferating cells (Ki67) was performed according to the instructions provided with the Vectashield Elite ABC kit (Vector Laboratories) Colorimetric detection was achieved using diaminobenzidine as the peroxidase substrate

Histomorphometry of each placenta using newCAST stereological software (Visiopharm New York NY) 4 was performed on midline transverse sections immunohistochemically stained for cytokeratin Sections from 1-5 placentas per genotype (typically 3) were examined from 5 ICR x Gcm1+- pregnancies Placental tissue was outlined using the mask tool and a pre-defined grid superimposed Spongiotrophoblast labyrinth and chorionic plate were identified at grid intersection points at 20x magnification Proportions of each component were calculated as a percentage of total point counts Subsequently proportional counts specific to the components of the labyrinth (including fetal blood space maternal blood space sinusoidal trophoblast giant cells cuboidal cells and other) were performed at 40x on serial sections stained for CD34 to label the fetal endothelial cell basement membrane Proportions of each component were calculated as a percentage of total labyrinth point counts The grid sizes and coverage were sufficient to obtain a minimum of 250 total point counts per region of interest (in the placenta or labyrinth) per genotype per pregnancy Interhaemal membrane thickness was measured on the same midline sections stained for CD34 at 60x as previously described 4 A line grid with 50 coverage was used to obtain a minimum of 250 measurements per genotype per pregnancy The harmonic mean of the interhaemal membrane thickness was calculated per genotype per pregnancy taking into account 29 shrinkage factor as calculated based on measurements of red blood cell diameters in fresh blood smears vs those measured on the processed sections (data not shown) The total number of proliferating cells (Ki67 positive) per field of view was counted on midline sections stained with Ki67 This was performed using Image J freeware (National Institute of Health Bethesda MD) on captured images of 20 random fields of view per placenta (40x newCAST software) Sinusoidal trophoblast giant cells identified by large nuclear size were not counted to exclude cells undergoing DNA endoreduplication from the count of proliferating cells


183939-R1

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Transverse sections (5 microm) of human placental biopsies were stained with HampE Using a similar histomorphological approach described above newCAST analysis tools were used to mask the entire placental biopsy Counts of villous tissue and feto-placental vasculature were performed at 20x using a pre-defined point grid with 50 coverage to obtain a minimum of 250 total point counts per placental biopsy Proportions of feto-placental vasculature were calculated as a percentage of total villous tissue counts

Murine feto-placental vascularity Feto-placental vascularity was examined in Gcm1+- murine placentas using two imaging

approaches In one method the feto-placental vasculature was filled with X-ray opaque silicone rubber using our published methods and imaged by micro-computed tomography (micro-CT) 5-7 to visualize and quantify feto-placental arteries and arterioles ge 50 microm in diameter Automated vascular segmentation of micro-CT datasets as described previously 7 was used to determine the number of vessel segments and the total vessel length for given vessel diameters (all gt50 microm 50-100 microm 100-300 microm and all gt300 microm) Micro-CT analysis was performed on 2-3 conceptuses per genotype from 5 pregnancies (total of N = 11 per genotype) To visualize capillaries liquid plastic was infused into the feto-placental vasculature to prepare vascular corrosion casts using our previously published methods 5 8 Feto-placental vascular casts from two conceptuses per genotype from 3 pregnancies were imaged by scanning electron microscopy (FEI XL30 Hillsboro OR)

Maternal arterial pressure proteinuria and plasma sFlt Maternal heart rate and arterial blood pressure were measured in awake mice using an

automated tail cuff plethysmography system (BP-2000 Visitech Systems Apex NC) The tail-cuff system has previously been validated and accurately reflects mean carotid arterial blood pressure as measured by chronic arterial catheter in mice 9 Non-pregnant measurements were taken on 3 consecutive days prior to breeding and were averaged per female Following confirmation of a vaginal plug measurements were obtained longitudinally (N= 10 CD1 females mated with Gcm1+- males N=6 CD1 females mated with CD1 males) at E135 and at E175 (late gestation) All blood pressure measurements were obtained between 900 AM and 1100 AM

Proteinuria was assessed longitudinally in the same mice across gestation (non-pregnant E135 and E175) Urine samples were collect between 900 AM and 1100 AM and stored at -20degC until further processing Protein concentrations were measured in the urine samples (diluted 10-fold) using the Bradford Reagent and expressed relative to creatinine concentrations as measured by the Jaffeacute reaction At euthanasia maternal blood was collected from these same mice by cardiac puncture using heparin coated syringes Following centrifugation (1500 g for 15 minutes at room temperature) plasma was isolated and frozen at -80degC Concentrations of sFlt were measured in plasma samples (diluted 5-fold) using a commercially available ELISA kit (RampD Systems Burlington ON) as per manufacturerrsquos directions

Statistical analysis Results for wild type littermates did not significantly differ from those of wild type

conceptuses from pregnancy control breedings (ie CD1 x CD1 matings) by one-way ANOVA with Tukeyrsquos post-hoc analysis (not shown) Wild type littermates from CD1 x Gcm1+- matings were therefore used as controls for Gcm1+- conceptuses A Studentrsquos t-test was used for variables measured at one gestational age and a 2-way ANOVA when multiple gestational ages


183939-R1

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were studied Real time PCR data was analyzed using a Wilcoxon sign-ranked test Maternal blood pressure measurements were analyzed using a 2-way ANOVA using paternal genotype and gestational age as factors Linear regression analysis was performed on maternal arterial blood pressure at E175 in relation to number of heterozygous conceptuses per pregnancy (R2= correlation coefficient of linear regression) Linear regression analysis was also used to test the correlation between GCM1 protein expression and the proportion of fetal vasculature within villous tissue in the human placenta Statistical significance was set at plt005 All data are presented as mean plusmn standard error of the mean (SEM)

REFERENCES

1 Anson-Cartwright L Dawson K Holmyard D Fisher SJ Lazzarini RA Cross JC The

glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta Nat Genet 200025311-314

2 ACOG practice bulletin Diagnosis and management of preeclampsia and eclampsia Number 33 January 2002 American College of Obstetricians and Gynecologists Int J Gynaecol Obstet 20027767-75

3 Canadian Perinatal Surveillance System Health Canada Birth Weight for Gestational Age Available at httpwwwphac-aspcgccarhs-ssgbwga-pnagpdfbwga-pnag_epdf

4 Coan PM Ferguson-Smith AC Burton GJ Developmental dynamics of the definitive mouse placenta assessed by stereology Biol Reprod 2004701806-1813

5 Whiteley KJ Pfarrer CD Adamson SL Vascular corrosion casting of the uteroplacental and fetoplacental vasculature in mice Methods Mol Med 2006121371-392

6 Rennie MY Whiteley KJ Kulandavelu S Adamson SL Sled JG 3D visualisation and quantification of microcomputed tomography of late gestational changes in the arterial and venous feto-placental vasculature of the mouse Placenta 200728833-840

7 Rennie MY Detmar J Whiteley KJ Yang J Jurisicova A Adamson SL Sled JG Vessel tortuousity and reduced vascularization in the fetoplacental arterial tree after maternal exposure to polycyclic aromatic hydrocarbons Am J Physiol Heart Circ Physiol 2011300H675-H684

8 Adamson SL Lu Y Whiteley KJ Holmyard D Hemberger M Pfarrer C Cross JC Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta Dev Biol 2002250358-373

9 Wong AYH Kulandavelu S Whiteley KJ Qu D Langille BL Adamson SL Maternal cardiovascular changes during pregnancy and postpartum in mice Am J Physiol Heart Circ Physiol 2002282H918-H925


183939-R1

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SUPPLEMENTAL FIGURE LEGENDS

FIGURE S1 Gcm1 and SynB expression in the placental labyrinth at E135 and E175 in Gcm1+- (Het) compared to wild type littermate controls (Wt) (A) mRNA expression of Gcm1 and its downstream target SynB was significantly decreased in Het vs Wt (hashed line at 10) (B) Western blot showing lower protein expression of GCM1 in Het vs Wt β-actin (BAct) was used as the loading control p lt 005 Number of pregnancies for each gestational age and genotype was N = 6 in (A) N = 2 in (B)

FIGURE S2 Cell proliferation in the labyrinth at E175 (A) Ki67 positive cells in 20x view of the labyrinth of Gcm1+- and (B) wild type littermate controls (Wt) as shown by brown nuclear stain (eg nuclei marked by arrows in 60x image in right panel of A) (C) The number of Ki67 positive cells per 20x field of view was significantly greater in Gcm1+- vs Wt Number of pregnancies examined per genotype was N=5 plt005

FIGURE S3 Images illustrating increased fetal capillary density in the labyrinth at E175 in Gcm1+- vs wild type littermate controls (Wt) (A B) Scanning electron micrographs of vascular corrosion casts of Wt and Gcm1+- feto-placental vasculature showing increased capillary density in Gcm1+- placentas (B) Higher magnification view of (A) Representative images from casts of two conceptuses per genotype from 3 pregnancies are shown (C) Increased fetal capillaries in Gcm1+- placentas compared Wt CD34 stains the basement membrane of endothelium brown Representative images from 3 conceptuses per genotype from 5 pregnancies Scale bars in (A) are 500 microm and in (BC) they are 60 microm


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SUPPLEMENTAL TABLE

Table S1 Primers for qRT-PCR

Gene of interest Forward Primer (5 3) Sequence Reverse Primer (5 3) Sequence

Gcm1 NM_008103 TGA AGC TTA TTC CCT GCC GA TGA TCA TGC TCG CCT TTG G

SynA NM_001013751 TGC TGG TTT TCA GGC CTT GT ATC TTG TCC ACG TGT CCT TCG

SynB NM_173420 AAC CTG AGA GCC TAC AGC CA TAA GGA GGA GGG GAA AAG GA

VegfA NM_001025250 GAG CAG AAG TCC CAT GAA GTG TGT CCA CCA GGG TCT CAA TC

sFlt D88690 AGA AGA CTC GGG CAC CTA TG GCA GTG CTC ACC TCT AAC GA

PGF NM_008827 GTG GAA GTG GTG CCT TTC AAC CAG CAG CCA CTA CAG CGA CT

BAct NM_007393 TCG TGC GTG ACA TCA AAG AGA GAA CCG CTC GTT GCC AAT A

Gapdh NM_008084 AGG AGT AAG AAA CCC TGG ACC AGG CCC CTC CTG TTA TTA TGG

Tbp NM_013684 CGG ACA ACT GCG TTG ATT TTC AGC CCA ACT TCT GCA CAA CTC





Effects of Reduced Gcm1 Expression on TrophoblastMorphology Fetoplacental Vascularity and Pregnancy

Outcomes in MiceShannon A Bainbridge Abhijeet Minhas Kathie J Whiteley Dawei Qu John G Sled

John CP Kingdom S Lee Adamson

AbstractmdashPreeclampsia is a life-threatening disorder characterized by maternal gestational hypertension and proteinuriathat results from placental dysfunction Placental abnormalities include abnormal syncytiotrophoblast and a 50reduction in placental expression of the transcription factor Gcm1 In mice homozygous deletion of Gcm1 preventssyncytiotrophoblast differentiation and is embryonic lethal We used heterozygous Gcm1 mutants (Gcm1) to test thehypothesis that hypomorphic expression of placental Gcm1 causes defective syncytiotrophoblast differentiation andmaternal and placental phenotypes that resemble preeclampsia We mated wild-type female mice with Gcm1 fathersto obtain wild-type mothers carrying 50 Gcm1 conceptuses Gcm1 placentas had syncytiotrophoblastabnormalities including reduced gene expression of Gcm1-regulated SynB elevated expression of sFlt1 a thickenedinterhemal membrane separating maternal and fetal circulations and electron microscopic evidence in syncytiotrophoblast ofnecrosis and impaired maternal-fetal transfer Fetoplacental vascularity was quantified by histomorphometry and microcom-puted tomography imaging In Gcm1 it was 30 greater than wild-type littermates whereas placental vascularendothelial growth factor A (Vegfa) expression and fetal and placental weights did not differ Wild-type motherscarrying Gcm1 conceptuses developed late gestational hypertension (1182 versus 109607 mm Hg incontrols P005) We next correlated fetoplacental vascularity with placental Gcm1 expression in human controland pathological pregnancies and found that as in mice fetoplacental vascularity increased when GCM1 proteinexpression decreased (R2045 P005) These results support a role for reduced placental Gcm1 expressionas a causative factor in defective syncytiotrophoblast differentiation and maternal and placental phenotypes inpreeclampsia in humans (Hypertension 20125900-00) Online Data Supplement

Key Words syncytin placenta preeclampsia angiogenesis VEGFA placental growth factor sFlt1

Placental dysfunction is believed to be the major cause ofone of the most common and serious complications of

human pregnancy preeclampsia (PE)1 This potentially life-threatening hypertensive disorder adversely impacts 5 ofall pregnancies and has no known cure In PE growingevidence implicates abnormalities in syncytiotrophoblast(SynT) in the villous exchange region of the placenta1ndash6

SynTs are thin fetal-derived cells that lie between thematernal blood flowing through the villous exchange regionof the placenta and the fetal blood flowing through fetalvessels in the highly vascularized villi This surface isessential for fetomaternal communication and exchange andis formed through the differentiation of SynT from theunderlying villous cytotrophoblast cells under the control of

the transcription factor GCM1 (glial cells missing 1)7 Gcm1in the conceptus is almost exclusively expressed in a subset oftrophoblast in the placenta89 and is a critical regulator of theSynT cell type10

In the PE placenta Gcm1 expression is reduced by 504

as is expression of the immediate downstream target ofGcm111 Syn-156 That SynT is abnormal and potentiallycausative in PE is supported by abnormal levels of SynT-produced factors such as pregnancy-associated plasma pro-tein A human chorionic gonadotropin and inhibin in mater-nal blood during early pregnancy in women at high risk forthe later development of PE1213 When PE develops theabnormal syncytial surface is prothrombotic leading to in-farctions within the intervillous space14 and is excessively

Received October 19 2011 first decision November 11 2011 revision accepted December 21 2011From the Faculty of Health Sciences (SAB) University of Ottawa Ottawa Ontario Canada Samuel Lunenfeld Research Institute (SAB AM

KJW DQ JCPK SLA) Mount Sinai Hospital Toronto Ontario Canada Departments of Obstetrics and Gynaecology (SAB AM JCPKSLA) Physiology (SAB AM SLA) and Medical Biophysics (JGS) University of Toronto Toronto Ontario Canada Mouse Imaging Centre(JGS) Toronto Centre for Phenogenomics Hospital for Sick Children Toronto Ontario Canada

The online-only Data Supplement is available with this article at httphyperahajournalsorglookupsuppldoi101161HYPERTENSIONAHA111183939-DC1

Correspondence to Shannon A Bainbridge Interdisciplinary School of Health Sciences University of Ottawa Roger Guindon Hall Room 2058 451Smyth Rd Ottawa Ontario Canada K1H 8M5 E-mail shannonbainbridgeuottawaca

copy 2012 American Heart Association Inc

Hypertension is available at httphyperahajournalsorg DOI 101161HYPERTENSIONAHA111183939

1 at University of Toronto on February 12 2012httphyperahajournalsorgDownloaded from














(N5)PE

(N5)IUGR

(N5)


30908 30512 30712




Fetal weight g 1772127 1364200 998179



11311 19112dagger 11114


















0

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20

30

40

50

60

FBS

MBS

sTGC

Cub

Other

0

1

2

3

4

5

6

7

8

Thic

knes

s (micro

m)

E175


E135

A B C D

E F G H







0

500

1000

1500

2000

2500

ge 50

Num

ber

0

250

500

750

1000

1250

1500

1750

2000

50-100

0

50

100

150

200

250

300

100-3000

2

4

6

8

10

12

14

16

gt300

0

100

200

300

400

500

ge 50

mm

0

50

100

150

200

250

300

350

400

50-1000

20

40

60

80

100

100-3000

1

2

3

4

5

6

gt300

Wt Gcm1+-






A

B

C

D



Vegf Pgf Vegf Pgf00

05

10

15

20

25

30

E175

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

E135

VEGF protein

0

5

10

15

20

25

30

Gcm1+-Wt

E135 E175

Gcm1+-Wt

pg V

EGF

mg

prot

ein

PGF protein

0

200

400

600

800

1000

1200

pg P

GF

mg

prot

ein

Gcm1+-Wt

E135 E175

Gcm1+-Wt

p=008

A B C








NP E135 E175-5

0

5

10


Gestational Age

∆ A

rter

ial p

ress

ure

(mm

Hg)

0 1 2 3 4 5 6 7 8 9 10 11 12 1350

70

90

110

130

150

R2 =05373


Arte

rial p

ress

ure

(mm

Hg)

sFlt100

05

10

15

20

25

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

Plas

ma

sFLT

1 (n

gm

l)


10

20

30

40

50

A C

B D



0 5 10 150

5

10

15

20

25

R2=045IUGR


prop

ortio

n of

vill

ous

tissu

eco

mpr

ised

of f

etal

vas

cula

ture

()

PE

PE3769

C3809

IUGR2729

C2014

CT


Gcm1

BAct

A


















DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE





























(N5)PE

(N5)IUGR

(N5)


30908 30512 30712




Fetal weight g 1772127 1364200 998179



11311 19112dagger 11114


















0

1

2

3

4

5

6

7

8

Total Laby

mm

2

0

10

20

30

40

50

60

70

LabyJZ CP

0

10

20

30

40

50

60

FBS

MBS

sTGC

Cub

Other

0

1

2

3

4

5

6

7

8

Thic

knes

s (micro

m)

0

1

2

3

4

5

6

7

8

Total Laby

mm

2

0

10

20

30

40

50

60

70

LabyJZ CP0

10

20

30

40

50

60

FBS

MBS

sTGC

Cub

Other

0

1

2

3

4

5

6

7

8

Thic

knes

s (micro

m)

E175


E135

A B C D

E F G H







0

500

1000

1500

2000

2500

ge 50

Num

ber

0

250

500

750

1000

1250

1500

1750

2000

50-100

0

50

100

150

200

250

300

100-3000

2

4

6

8

10

12

14

16

gt300

0

100

200

300

400

500

ge 50

mm

0

50

100

150

200

250

300

350

400

50-1000

20

40

60

80

100

100-3000

1

2

3

4

5

6

gt300

Wt Gcm1+-






A

B

C

D



Vegf Pgf Vegf Pgf00

05

10

15

20

25

30

E175

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

E135

VEGF protein

0

5

10

15

20

25

30

Gcm1+-Wt

E135 E175

Gcm1+-Wt

pg V

EGF

mg

prot

ein

PGF protein

0

200

400

600

800

1000

1200

pg P

GF

mg

prot

ein

Gcm1+-Wt

E135 E175

Gcm1+-Wt

p=008

A B C








NP E135 E175-5

0

5

10


Gestational Age

∆ A

rter

ial p

ress

ure

(mm

Hg)

0 1 2 3 4 5 6 7 8 9 10 11 12 1350

70

90

110

130

150

R2 =05373


Arte

rial p

ress

ure

(mm

Hg)

sFlt100

05

10

15

20

25

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

Plas

ma

sFLT

1 (n

gm

l)


10

20

30

40

50

A C

B D



0 5 10 150

5

10

15

20

25

R2=045IUGR


prop

ortio

n of

vill

ous

tissu

eco

mpr

ised

of f

etal

vas

cula

ture

()

PE

PE3769

C3809

IUGR2729

C2014

CT


Gcm1

BAct

A


















DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE





























0

1

2

3

4

5

6

7

8

Total Laby

mm

2

0

10

20

30

40

50

60

70

LabyJZ CP

0

10

20

30

40

50

60

FBS

MBS

sTGC

Cub

Other

0

1

2

3

4

5

6

7

8

Thic

knes

s (micro

m)

0

1

2

3

4

5

6

7

8

Total Laby

mm

2

0

10

20

30

40

50

60

70

LabyJZ CP0

10

20

30

40

50

60

FBS

MBS

sTGC

Cub

Other

0

1

2

3

4

5

6

7

8

Thic

knes

s (micro

m)

E175


E135

A B C D

E F G H







0

500

1000

1500

2000

2500

ge 50

Num

ber

0

250

500

750

1000

1250

1500

1750

2000

50-100

0

50

100

150

200

250

300

100-3000

2

4

6

8

10

12

14

16

gt300

0

100

200

300

400

500

ge 50

mm

0

50

100

150

200

250

300

350

400

50-1000

20

40

60

80

100

100-3000

1

2

3

4

5

6

gt300

Wt Gcm1+-






A

B

C

D



Vegf Pgf Vegf Pgf00

05

10

15

20

25

30

E175

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

E135

VEGF protein

0

5

10

15

20

25

30

Gcm1+-Wt

E135 E175

Gcm1+-Wt

pg V

EGF

mg

prot

ein

PGF protein

0

200

400

600

800

1000

1200

pg P

GF

mg

prot

ein

Gcm1+-Wt

E135 E175

Gcm1+-Wt

p=008

A B C








NP E135 E175-5

0

5

10


Gestational Age

∆ A

rter

ial p

ress

ure

(mm

Hg)

0 1 2 3 4 5 6 7 8 9 10 11 12 1350

70

90

110

130

150

R2 =05373


Arte

rial p

ress

ure

(mm

Hg)

sFlt100

05

10

15

20

25

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

Plas

ma

sFLT

1 (n

gm

l)


10

20

30

40

50

A C

B D



0 5 10 150

5

10

15

20

25

R2=045IUGR


prop

ortio

n of

vill

ous

tissu

eco

mpr

ised

of f

etal

vas

cula

ture

()

PE

PE3769

C3809

IUGR2729

C2014

CT


Gcm1

BAct

A


















DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE



















0

500

1000

1500

2000

2500

ge 50

Num

ber

0

250

500

750

1000

1250

1500

1750

2000

50-100

0

50

100

150

200

250

300

100-3000

2

4

6

8

10

12

14

16

gt300

0

100

200

300

400

500

ge 50

mm

0

50

100

150

200

250

300

350

400

50-1000

20

40

60

80

100

100-3000

1

2

3

4

5

6

gt300

Wt Gcm1+-






A

B

C

D



Vegf Pgf Vegf Pgf00

05

10

15

20

25

30

E175

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

E135

VEGF protein

0

5

10

15

20

25

30

Gcm1+-Wt

E135 E175

Gcm1+-Wt

pg V

EGF

mg

prot

ein

PGF protein

0

200

400

600

800

1000

1200

pg P

GF

mg

prot

ein

Gcm1+-Wt

E135 E175

Gcm1+-Wt

p=008

A B C








NP E135 E175-5

0

5

10


Gestational Age

∆ A

rter

ial p

ress

ure

(mm

Hg)

0 1 2 3 4 5 6 7 8 9 10 11 12 1350

70

90

110

130

150

R2 =05373


Arte

rial p

ress

ure

(mm

Hg)

sFlt100

05

10

15

20

25

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

Plas

ma

sFLT

1 (n

gm

l)


10

20

30

40

50

A C

B D



0 5 10 150

5

10

15

20

25

R2=045IUGR


prop

ortio

n of

vill

ous

tissu

eco

mpr

ised

of f

etal

vas

cula

ture

()

PE

PE3769

C3809

IUGR2729

C2014

CT


Gcm1

BAct

A


















DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE




















NP E135 E175-5

0

5

10


Gestational Age

∆ A

rter

ial p

ress

ure

(mm

Hg)

0 1 2 3 4 5 6 7 8 9 10 11 12 1350

70

90

110

130

150

R2 =05373


Arte

rial p

ress

ure

(mm

Hg)

sFlt100

05

10

15

20

25

mRN

A (fo

ld c

hang

e re

lativ

e to

Wt)

Plas

ma

sFLT

1 (n

gm

l)


10

20

30

40

50

A C

B D



0 5 10 150

5

10

15

20

25

R2=045IUGR


prop

ortio

n of

vill

ous

tissu

eco

mpr

ised

of f

etal

vas

cula

ture

()

PE

PE3769

C3809

IUGR2729

C2014

CT


Gcm1

BAct

A


















DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE






























DisclosuresNone












































183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

1

ONLINE SUPPLEMENT








183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

2









183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

3







183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

4







183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

5










183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

6


REFERENCES












183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

7






183939-R1

8

SUPPLEMENTAL TABLE
















183939-R1

8

SUPPLEMENTAL TABLE
















Effects of Reduced Gcm1 Expression on Trophoblast Morphology, Fetoplacental Vascularity, and...

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Transcript of Effects of Reduced Gcm1 Expression on Trophoblast Morphology, Fetoplacental Vascularity, and...