disruption of embryonic vascular development in predictive ... · review the problem of embryonic...

Disruption of Embryonic Vascular Development inPredictive Toxicology

Thomas B. Knudsen* and Nicole C. Kleinstreuer

Toxicity testing in the 21st century is moving toward using high-throughput screening assays to rapidly test thousands of chemicalsagainst hundreds of molecular targets and biological pathways, and toprovide mechanistic information on chemical effects in human cells andsmall model organisms. First-generation predictive models for prenataldevelopmental toxicity have revealed a complex web of biological proc-esses with many connections to vasculogenesis and angiogenesis. Thisreview examines disruption of embryonic vascular development as apotential adverse outcome pathway leading to developmental toxicity.We briefly review embryonic vascular development and important sig-nals for vascular development (local growth factors and cytokines suchas vascular endothelial growth factor-A and TGF-beta, components inthe plasminogen activator system, and chemotactic chemokines).Genetic studies have shown that perturbing these signals can lead tovarying degrees of adverse consequences, ranging from congenitalangiodysplasia to fetal malformations and embryolethality. The molecu-lar targets and cellular behaviors required for vascular development,stabilization and remodeling are amenable to in vitro evaluation. Evi-dence for chemical disruption of these processes is available for thalido-mide, estrogens, endothelins, dioxin, retinoids, cigarette smoke, andmetals among other compounds. Although not all compounds with de-velopmental toxicity show an in vitro vascular bioactivity signature,many ‘putative vascular disruptor compounds’ invoke adverse develop-mental consequences. As such, an adverse outcome pathway perspec-tive of embryonic vascular development can help identify useful infor-mation for assessing adverse outcomes relevant to risk assessment andefficient use of resources for validation. Birth Defects Research (Part C)93:312–323, 2011. VC 2012 Wiley Periodicals, Inc.

Key words: angiogenesis; vascular development; developmental toxic-ity; predictive toxicology; adverse outcome pathway

INTRODUCTIONA chemical’s capacity to disruptembryogenesis depends on manyfactors including inherent chemicalproperties, dose and time of expo-sure, genetic susceptibility of thespecies or individual, bioavailabil-

ity and biotransformation, andchemical interactions with biologi-cal systems. Standard practice forassessing disruptions in embryo-genesis involves testing pregnantlaboratory animals of two species,typically rats and rabbits, exposed

during the period of major organo-genesis and evaluated just beforeterm, while monitoring maternalstatus throughout pregnancy(Hurtt et al., 2003; Kimmel et al.2006; Carney et al., 2007). Underthis design, the major manifesta-tions of developmental toxicitymay be one or more of a numberof possible endpoints such asintrauterine death, fetal growthretardation, structural variations,and abnormalities. Although thecurrent testing paradigm may beeffective in assessing developmen-tal toxicity, it is low-throughput,slow and costly both in terms offinancial and animal resources.These restrictions result in a rela-tively low number of compoundsthat have sufficient in vivo data toassess the potential for adverseeffects on human development.Toxicity testing in the 21st

century is moving toward usinghigh-throughput screening (HTS)assays to rapidly test thousands ofchemicals against hundreds of mo-lecular targets and biological path-ways, and to provide mechanisticinformation on chemical effects inhuman cells and small modelorganisms (NRC 2007; Collinset al., 2008; Knudsen et al.,2011). For example, the US Envi-ronmental Protection Agency’sToxCast and ToxRefDB projectsare building large HTS in vitrodatasets coupled with a rich in

REVIE

W

VC 2012 Wiley Periodicals, Inc.

Birth Defects Research (Part C) 93:312–323 (2011)

Thomas B. Knudsen and Nicole C. Kleinstreuer are from the National Center for Computational Toxicology, Office of Researchand Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S.Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommenda-tion for use.

*Correspondence to: Thomas B. Knudsen, PhD, National Center for Computational Toxicology (B205-01), U.S. Environmental Protec-tion Agency, Research Triangle Park, NC 27711. E-mail: [email protected]

View this article online at (wileyonlinelibrary.com). DOI: 10.1002/bdrc.20223

vivo legacy database that providesa novel resource for buildingpredictive models of toxicity, withthe initial goal of prioritizing chem-icals for further testing (Dix et al.,2007; Judson et al., 2010). Theanchoring reference database(ToxRefDB) includes endpointsfrom traditional animal bioassaysin standardized fields for chronic/cancer studies in rats and mice(Martin et al., 2009a), multigener-ation reproductive studies in rats(Martin et al., 2009b), and prena-tal developmental toxicity studiesin pregnant rats and pregnant rab-bits (Knudsen et al., 2009). Thepublic dataset and relevant publi-cations can be accessed at http://actor.epa.gov/toxrefdb/faces/Home.jsp.The first phase of ToxCast pro-

filed the biological activities of 309unique chemicals using over 600HTS assays, including biochemicalassays (e.g., nuclear receptorbinding, enzyme inhibition), cell-based assays (e.g., cytotoxicityprofiles, reporter gene assays),complex culture systems (e.g.,embryonic stem cell differentia-tion, inflammatory/angiogenic sig-nals), and small animal models(e.g., zebrafish embryo develop-ment), as well as chemicalproperty information. The publicin vitro dataset and relevant pub-lications can be accessed athttp://actor.epa.gov/actor/faces/ToxCastDB/Home.jsp. First-gener-ation predictive models were builtanchoring ToxCast in vitro data toToxRefDB in vivo effects findingsfor chronic liver cancer in rodents(Judson et al., 2010; Shah et al.,2011), reproductive toxicity in rats(Martin et al., 2011), and prenataldevelopmental toxicity in rats andrabbits (Sipes et al., 2011). Thelatter predicts a complex scenarioof molecular targets in a web of bi-ological processes, many of whichare linked to blood vessel develop-ment.A functioning circulatory system

becomes indispensible to themammalian embryo as its growthoutpaces the limits of oxygen dif-fusion at about 100–200 lm insize (3rd week of gestationin humans, 10th day of gestation

in rats, and 8th day of gestation inmouse). For this reason, we devel-oped a predictive model for ‘‘puta-tive vascular disruptor com-pounds’’ (pVDCs) based on Tox-Cast Phase I HTS data(Kleinstreuer et al., 2011). Angio-genic targets in inflammatory che-mokine signaling, the vascularendothelial growth factor (VEGF)pathway, and the plasminogen-activating system were stronglyperturbed by some environmentalcompounds, and we found positivecorrelations with developmentaleffects in ToxRefDB. Here, wereview the problem of embryonicvascular development and adversepregnancy outcomes potentiallylinked to vascular disruption byprenatal exposure to environmen-tal chemicals.

EMBRYONIC VASCULAR

DEVELOPMENT

The cardiovascular system is thefirst functional organ to develop inthe mammalian embryo (Maltepeand Celeste Simon et al., 1998;Walls et al. 2008; Chung and Fer-rara, 2011). During embryonicvascular development, endothelialcells (ECs) assemble into bloodvessel networks by two successiveprocesses: vasculogenesis andangiogenesis. The former refers tothe de novo formation of nascentvessels from EC precursors (angio-blasts) to a primary vascularplexus. This involves EC migration,proliferation, and assembly intoendothelial tubes. Angiogenesisrefers to the process by which pre-existing vessels are remodeled,new capillaries emerge and vesselstabilization and maturationoccurs.Vasculogenesis in the mouse

embryo commences around gesta-tion day (GD) 7.0. The first sign isthe appearance of Flk-1-positiveangioblasts migrating laterallyfrom the posterior primitive streak(Dumont et al. 1995). Thoseangioblasts entering the embryoproper aggregate to directly formmajor vessels, e.g., the dorsalaorta, posterior cardinal veins.Those entering extraembryonicmembranes (e.g., hemangio-

blasts) segregate into bloodislands, which are sites of hemato-poietic stem cell development, andthen aggregate into a polygonalmeshwork of homogeneously sizedcapillaries. Flk-1-null mutants failto develop vascular and hemato-poietic cells and these mutantembryos die around GD9 (Coultaset al., 2005). The primary vascularplexus forms a simple circulatoryloop in the early embryo consistingof the heart, dorsal aorta, sinusvenosus, and extra-embryonicvasculature prior. The vitelline(yolk sac) plexus connects by the5-7 somite stage (GD 8.5) and theumbilical (allantoic) plexus by the20-somite stage (GD 9.5).Virtually, all other blood vessels

in the embryo arise by the subse-quent sprouting of new capillariesfrom the primitive vascular plexus(angiogenesis). In the mouseembryo, the first angiogenicsprouting occurs in the dorsalaorta around the 8-somite stage.Navigation of sprouting vesselsbetween adjacent somites isguided by ephrinB2 (expressed inthe caudal-half of a somite) andephrin receptors expressed onECs, similar to axonal guidancecues (Walls et al., 2008).Angioblasts derived from para-

xial/somatic mesoderm migrate tovascularize, by angiogenesis, theneural tube, mesonephros, limb-buds, and somatic body wall(Ambler et al., 2001; Pudliszewskiand Pardanaud, 2005). Differenti-ated ECs subsequently support thedevelopment of organ rudiments.For example, the perineural vascu-lar plexus (PNVP), precursor to theblood-brain barrier, draws ECsfrom somatic mesoderm inresponse to VEGF. It surroundsthe neural tube between GD 8.5and GD 9.5. PNVP sprouting, thenvascularizes the neuroepitheliumaround GD10.0 (Walls et al.,2008). This is dependent onWnt7a and Wnt7b signals fromneuroepithelial cells acting on ECsto trigger angiogenesis and blood-brain barrier formation through acanonical Wnt signaling pathway(Stenman et al., 2008). Lack ofPNVP invasion due to loss of func-tion of the orphan G-protein

CONSEQUENCES OF EMBRYONIC VASCULAR DISRUPTION 313

Birth Defects Research (Part C) 93:312–323, (2011)

coupled receptor GP124 results inavascular neural tissue, neurode-generation and embryolethalitystarting around GD 15.5 (Kuhnertet al., 2010). In the limb-bud,angiogenic sprouts from the inter-somitic vessels invade limb mes-enchyme. Concurrently, somite-derived angioblasts undergo vas-culogenesis within the limb mes-enchyme to form a vascular plexus(Eshkar-Oren et al., 2009).

REGULATION OF

ANGIOGENESIS

Angiogenesis is typically quiescentin the adult, except for pathologicalsituations (e.g., wound healing,diabetic retinopathy, rheumatoidarthritis, cardiac ischemia, psoria-sis, tumor growth) and the femalereproductive system (e.g., ovula-tion, endometrial growth, implan-tation, placentation) (reviewed inChung and Ferrara, 2011; Herbertand Stanier 2011).Important signals for controlling

angiogenesis are VEGF-A(sprouting), Notch/Delta (specifi-cation) and TGF-beta/Angiopoietin(stabilization). VEGF-A releasefrom hypoxic cells is the mostcommon angiogenic switch, speci-fying a pioneering sproutingbehavior of EC ‘‘tip-cells.’’ Activa-tion of a G-protein signaling cas-cade (e.g., RhoA, Rac1, Cdc42) intip-cells triggers filopodial exten-sions that probe the surroundingenvironment and direct cell migra-tion along the VEGF-A gradient.The exploratory behavior and min-imal proliferation of a pioneeringtip-cell is similar to axonal growthcones, sensing and responding toguidance cues in the local micro-environment. Concurrently, VEGF-A signals suppress adjacent ECsfrom becoming tip-cells through aNotch/Dll4 negative feedbackloop. Tip-cells express DLL4 thatbinds Notch on adjacent ECs todown-regulate VEGFR2 expres-sion. These cells instead differenti-ate into ‘stalk-cells’ that proliferatebehind tip-cells to elongate thesprout (Potente et al., 2011).Stalk cells also release an alterna-tive splice of VEGFR1 (Flt-1) intothe extracellular matrix (ECM),

soluble VEGFR1 (sFlt-1), that actsas a ‘‘ligand sink" for VEGF-A dueto high-affinity binding (Kappaset al., 2008). VEGFR1 null mutantmice die on GD 8.5 – GD 9.0 dueto an overgrowth of ECs and vas-cular disorganization, consistentwith a negative role in angiogene-sis regulation (Hiratsuka et al.,2005).Chemotaxis and differential cell

adhesion are fundamental cellularbehaviors driving EC aggregation.ECs produce chemotactic chemo-kines such as CCL2 that attractmacrophage cells to nascent vas-cular junctions. Chemokines are asuperfamily of homologous hepa-rin-binding proteins, first de-scribed for their role in recruitingleukocytes to sites of inflamma-tion. During angiogenesis, macro-phage cells facilitate bridging ofadjacent tip-cells at vascular junc-tions (Fantin et al., 2010). CCL2through its G-protein coupledreceptor (CCR2) is the best-described chemokine mediator ofangiogenesis and appears to bedependent on membrane-typematrix metalloproteinase (MT1-MMP). Other chemokines areangiostatic, via inhibition of ECproliferation and tubulogenesis,such as CXCL10 and its receptorCXCR3B (Keeley et al., 2008). Inaddition to promoting anastamoticvessel connections, macrophagessecrete many enzymes, proteases,and cytokines that have direct andindirect effects on EC sprouting.Angiogenic sprouting is an inva-

sive behavior. As such, it isdependent on ECM interactionsand remodeling. Sprouting ECsexpress higher levels of severalmatrix metalloproteinases, includ-ing MT1-MMP, MMP-2, and MMP-9.The former (MT1-MMP) binds uro-kinase-type plasminogen activatorto facilitate EC migration, whereasMMP-2 binds a5b3-integrin to lib-erate or modify angiogenic growthfactors (van Hinsbergh et al.,2006). Macrophages release ser-ine proteases such as plasminogenactivator and metalloproteasessuch as collagenase that degradethe ECM and yield angiogenicstimuli. Members of the transform-ing growth factor beta (TGF-beta)

superfamily can act as indirectregulators of angiogenesis througheffects on ECM remodeling in addi-tion to their role in controlling ECproliferation. Macrophages arealso able to degrade heparan sul-phate and release ECM-boundfibroblast growth factor (FGF)expression of uPA. FGF, throughits FGFR1 receptor, may partici-pate in specification of angiogenicsprouting. Pro-angiogenic growthfactors (such as bFGF) that actthrough the AKT/PI3k pathway arenecessary for maintaining vasculardevelopment in the mouse embryoand yolk sac. This may be regu-lated by FoxO1 (Forkhead-type)family transcription factors thatare downstream targets of AKT/PI3K signaling (Furuyama et al.,2004).Structural support is brought to

the developing vessels by recruit-ment of mural cells (MCs) fromthe stroma (e.g., pericytes,smooth muscle cells) (Hanahanet al., 1997). ECs express plate-let-derived growth factor B(PDGFB) that binds heparin-sul-fate proteoglycan in the basementmembrane and pericellular matrix.This acts as a chemoattractant tomesenchymal cells expressing thePDGFRB receptor, triggeringrecruitment of MCs to the endo-thelium (Boyd et al., 2011). Inturn, Angiopoietin-1 released fromMCs sustains interaction betweenECs and MCs and stabilizes thevasculature through binding to itsTIE2 receptor. Sustained contactbetween ECs and MCs activatesthe TGF-beta pathway, whichinhibits EC proliferation and stimu-lates MC differentiation throughdownstream target genes such asplasminogen activator inhibitor(PAI-1) (Gaengel et al., 2009).MCs thus regulate blood vessel de-velopment at the maintenancestep, rendering capillary networksless susceptible to remodeling.Finally, the presence of blood

flow is known to be essential forremodeling of the primary vascu-lar plexus via hemodynamic fac-tors (hydrostatic pressures, fluidshear stress) (Lucitti et al., 2007).Without blood flow, differentiatingECs display defects in shape and

314 KNUDSEN AND KLEINSTREUER


cellular contacts resulting in lossof vessel integrity (May et al.,2004). This important concept isbeyond the scope of this articleand is reviewed elsewhere (leNoble et al., 2005).

CONSEQUENCES OF

EMBRYONIC VASCULAR

DISRUPTION

Genetic studies have shown thatperturbing embryonic vascular de-velopment can have varyingdegrees of adverse consequences,ranging from benign vascular mal-formations (congenital angiodys-plasias) (Brouillard and Vikkula,2007) to embryolethality and con-genital defects (Maltepe and Cel-este Simon et al., 1998; Coultaset al., 2005). Vascular malforma-tions are thought to be due todefects in pathways linked to vas-cular development and angiogene-sis. Most are sporadic forms pre-senting with a single lesion,whereas multiple lesions areobserved in familial cases. Someof the latter include VEGF,PDGFRB, TGF-beta, TIE2, PI3K/PTEN, Ras, and EphB2 (Brouillardand Vikkula, 2007). Following theapproach of Drake et al. (2007),we searched the gene ontologyand mammalian phenotype (MP)browsers of the Mouse GenomeInformatics database (http://www.informatics.jax.org/) forterms affiliated with the disruptionof vascular development. Termsfor abnormal vasculogenesis[MP:0001622; 72 genotypes, 73annotations] and abnormal angio-genesis [MP:0000260; 610 geno-types, 894 annotations] were cap-tured into a table and then linkedto ToxCast assays. This list had 65target genes with bona fide rolesin vasculogenesis or angiogenesis,50 of which had evidence ofabnormal embryonic vascular de-velopment based on geneticmouse models. Table 1 shows asummary of the MP terms, targetgenes and associated ToxCastassays identified by this approach.Vascular development is also

sensitive to drug and chemicalexposures. One of the best known

inhibitors of blood vessel forma-tion is thalidomide. Introduced asa sedative and anti-nausea com-pound for pregnant women in1956, thalidomide was withdrawnfrom the market in 1961 after anepidemic of phocomelia and othermalformations affecting about10,000 children (D’Amato et al.,1994). This tragic episode clearlydemonstrates the potential foradverse pregnancy outcomes thatmay follow from environmentaldisruption of embryonic vasculardevelopment.

Thalidomide

D’Amato and coworkers (1994)provided the first demonstrationthat thalidomide is an angiogene-sis inhibitor. In that study, thalido-mide was found to inhibit VEGF-and bFGF-mediated angiogenesis.Micromolar concentrations of thali-domide disrupted EC differentia-tion in ‘‘embryoid bodies" frommurine embryonic stem cells(Sauer et al., 2000). Stephenset al. (2000) proposed direct inter-ference with IGF-I and FGF-2mediated transcription of integrinsubunits alpha-5, beta-3, respec-tively, due to intercalation of thali-domide or a planar metabolite intopolyG-rich (GGCGG) gene pro-moter regions of DNA leading todisruption of angiogenesis. Sev-eral metabolites of thalidomide(e.g., CPS49) were shown to havestrong anti-angiogenic propertiesin the low micromolar rangethrough inhibition of bFGF-inducedvascularization (Ng et al., 2003).CPS49 also disrupted the vascularnetwork in chick embryos throughexcessive cell death of immatureECs, affecting the limb-bud andother developing structures thathave a high-microvascular density(Therapontos et al., 2009; Varges-son, 2009). The thalidomide ana-log 5-hydroxy-2-(2,6-diisopropyl-phenyl)-1H-isoindole-1,3-dione(5HPP-33) was shown to havemore potent anti-angiogenic activ-ity than thalidomide (� 60% inhi-bition of tube formation in ahuman umbilical vein EC assayversus � 30% inhibition for thali-domide) (Noguchi et al., 2005).

A nonhuman primate model ofthalidomide embryopathy wasused to array the genomicresponse of the embryo to thalido-mide at 6 h post-exposure. Amongthe major changes were down-regulation of VEGF and up-regula-tion of PDGFRB expression, indi-cating changes in the angiogenicswitch (Ema et al., 2010). Otherstudies linked thalidomide-induceddown-regulation of VEGF receptorsto ceramide imbalance in thesphingosine-1-phosphate pathway(Yabu et al., 2005). Interestingly,thalidomide, through stimulatingPDGFB by EC tip cells, promotedMC stabilization in patients withHereditary Hemorrhagic Telangiec-tasia, a disease linked to a muta-tion in the TGF-beta pathway andloss of vessel wall integrity (Lebrinet al., 2010). These studies areconsistent with a central effect ofthalidomide compounds on mech-anisms of physiological angiogenicregulation. In support of thisnotion, Cereblon has been identi-fied recently as a candidate thali-domide-binding protein and asso-ciated inhibition of an E3 ubiquitinligase activity that is important forFGF8 expression during limb out-growth (Ito et al., 2010).

Estrogens

Hormonally controlled vascularchanges play a key role in endo-metrial development and blasto-cyst implantation. VEGF-A expres-sion is transactivated by theestrogen receptor (Mueller et al.,2000). Furthermore, VEGF is acentral regulator of uterine vascu-lature permeability and angiogen-esis during the peri-implantationperiod. Estradiol and ethynyl-es-tradiol induced proliferation ofhuman endometrial ECs from hys-terectomy patients (Bredhultet al., 2007). The weak environ-mental estrogen Bisphenol-A(BPA) induced VEGF-A levels inthe rat uterus (Long et al., 2001).Neonatal female pups exposed toBPA showed a decreased responseto VEGF and uterine EC prolifera-tion by estradiol, a possible conse-quence of developmental reprog-ramming that may affect fertility



TABLE

1.Overlap

Betw

een

ToxCastAssayTarg

ets

and

Abnorm

alVascula

rPhenoty

pesfrom

GeneticMouse

Models

ToxCast

genetarg

eta

MPannotatedterm

cToxCast

ass

aysd

AHR

Patentductusvenosu

s,abnorm

alvasc

ularre

gre

ssion

ATG_Ahr_

CIS

,NCGC_AhR,

BMPR2

Decrease

dangiogenesis

ATG_BRE_CIS

CASP8

Abnorm

alvitellinevasc

ulatu

remorp

hology

NVS_ENZ_hCASP8

CCL2

Decrease

dangiogenesis,

abnorm

alphysiologicalneovasc

ulariza

tion,ch

oro

idal

neovasc

ulariza

tion

BSK_3C_MCP1,BSK_4H_MCP1,

BSK_KF3

CT_MCP1,BSK_LP

S_MCP1,

BSK_SAg_MCP1,BSK_SM3C_MCP1

CEBPBa

Abnorm

alvasc

ulogenesis,

abse

ntorg

anizedvasc

ularnetw

ork

ATG_C_EBP_CIS

,ATG_CRE_CIS

CREB3a

Abnorm

alvasc

ulogenesis,

abse

ntorg

anizedvasc

ularnetw

ork

ATG_C_EBP_CIS

,ATG_CRE_CIS

CXCL1

0a

Abnorm


ulariza

tion

BSK_BE3C_IP

10,BSK_hDFC

GF_

IP10,

BSK_KF3

CT_IP

10

EDNRA

Abnorm

aldorsalaortamorp

hology,patentductusarteriosu

s,abnorm

albra

nch

ial

archartery

morp

hology,persistentrightdorsalaorta

NVS_GPCR_hETA

EphA2

Abnorm

alangiogenesis

NVS_ENZ_hEphA2

ETS1

Abnorm

alvasc

ularbra

nch

ingmorp

hogenesis

ATG_Ets_CIS

FGFR

1,FG

FR1a

Abnorm

albra

nch

ialarchartery

morp

hology,decrease

dangiogenesis

NVS_ENZ_hFG

FR1

FOXA2RE

Abnorm

aldorsalaortamorp

hology,abnorm

albra

nch

ialarchartery

morp

hology

ATG_Fo

xA2_CIS

FOX

OREa

Abse

ntorg

anizedvasc

ularnetw

ork

ATG_Fo

xO_CIS

GATA

REa

Abse

ntvitellinebloodvess

els

ATG_GATA_CIS

H2AFX

Abnorm


ulariza

tion,re

tinalneovasc

ulariza

tion

CLM

_PH2AX

HIF1a

Decrease

dangiogenesis,

abnorm

alangiogenesis,

abnorm

aldorsalaortamorp

hology,

abnorm

albra

nch

ialarchartery

morp

hology,abnorm

alvasc

ularre

gre

ssion

ATG_HIF1a_CIS

ICAM1

Dec

rease

dsu

scep

tibility

toinduce

dch

oroidaln

eova

sculariza

tion,dec

rease

dangiogen

esis

BSK_3C_IC

AM1,BSK_KF3

CT_IC

AM1

IFNG

Stim

REa

Corn

ealvasc

ulariza

tion

ATG_IS

RE_CIS

MAPK3

Decrease

dangiogenesis

NVS_ENZ_hMAPK3

MHC

Abnorm

alvasc

ularre

gre

ssion,abnorm

alvasc

ularbra

nch

ingmorp

hogenesis

MESC_MHC_Down,MESC_MHC_Up

MMP2

Decrease

dangiogenesis

NVS_ENZ_hMMP2

MMP7

Abnorm

alinduce

dre

tinalneovasc

ulariza

tion

NVS_ENZ_hMMP7

MMP9

Abnorm

alangiogenesis,

abnorm

alinduce

dre

tinalneovasc

ulariza

tion,abnorm

al

physiologicalneovasc

ulariza

tion,decrease

dangiogenesis

NVS_ENZ_hMMP9,BSK_KF3

CT_MMP9

NRF2

Abnorm

alanteriorca

rdinalvein

morp

hology,abnorm

alposteriorca

rdinalvein

morp

hology,decrease

dangiogenesis

ATG_NRF2

_ARE_CIS

p53

Increase

dangiogenesis

ATG_p53_CIS

,NCGC_p53

PAK4

Abnorm

alvitellinevasc

ularre

modelin

gNVS_ENZ_hPAK4

PAX6

Corn

ealvasc

ulariza

tion

ATG_Pax6_CIS

PI3

Ka

Abnorm

alangiogenesis,

abnorm

aldorsalaortamorp

hology,abnorm

alanterior

card

inalvein

morp

hology

NVS_ENZ_hPI3

Ka

PLA

UDecrease

dsu

sceptibility

toinduce

dch

oro

idalneovasc

ulariza

tion

BSK_BE3C_uPA,BSK_KF3

CT_uPA

PLA

UR

Decrease

dangiogenesis

BSK_3C_uPAR,BSK_4H_uPAR,

BSK_BE3C_uPAR,BSK_SM3C_uPAR

PTEN

Increase

dangiogenesis

NVS_ENZ_hPTEN

PTGS1

Patentductusarteriosu

sNVS_ENZ_oCOX1



TABLE

1.Continued

ToxCast

genetarg

eta

MPannotatedterm

ToxCast

ass

ays

PTGS2

Patentductusarteriosu

s,abnorm

alinduce

dre

tinalneovasc

ulariza

tion

NVS_ENZ_oCOX2

PTPN11

Abnorm

alvitellinevasc

ularre

modelin

gNVS_ENZ_hPTPN11

PTPN12

Abnorm

alvasc

ularbra

nch

ingmorp

hogenesis

NVS_ENZ_hPTPN12

PTPRB

Abnorm

alvitellinevasc

ulatu

remorp

hology,abse

ntorg

anizedvasc

ularnetw

ork

,abnorm

alca

rdinalvein

morp

hology,abnorm

alvasc

ularre

gre

ssion

NVS_ENZ_hPTPRB

RARA

Abse

ntfetalductusarteriosu

s,persistentrightdorsalaorta,abnorm

albra

nch

ialarch

artery

morp

hology

ATG_RARa_TRANS,NVS_NR_hRARa

RARB

Abnorm

albra

nch

ialarchartery

morp


ATG_RARb_TRANS

RARG

Abnorm

albra

nch

ialarchartery

morp


ATG_RARg_TRANS

RXRA

Abnorm

alvasc

ulogenesis,

abnorm

albra

nch

ialarchartery

morp

hology

ATG_RXRa_TRANS

RXRB

Abnorm

albra

nch

ialarchartery

morp

hology

ATG_RXRb_TRANS

SERPIN

E1

Decrease

dangiogenesis

BSK_BE3C_PAI1

,BSK_hDFC

GF_

PAI1

TEK

Failu

reofvasc

ularbra

nch

ing,decrease

dangiogenesis,

abnorm

alAngiogenesis,

abnorm

alvasc

ularbra

nch

ingmorp

hogenesis

NVS_ENZ_hTie2

TGFb

,TGFb

aAbse


els,abnorm

alvasc

ularendoth

elia

lce

lldiffere

ntiation,

abnorm

alvitellinevasc

ulatu

remorp

hology,abnorm

alangiogenesis,

abnorm

al

dorsalaortamorp

hology,abnorm

albra

nch

ialarchartery

morp

hology

ATG_TGFb

_CIS

,BSK_BE3C_TGFb

1,

BSK_KF3

CT_TGFb

1

THBD

Abnorm

alvitellinevasc

ulatu

remorp

hology,abse


els

BSK_3C_Thro

mbomodulin

,BSK_SM3C_Thro

mbomodulin

VEGFR

1Abnorm


ulariza

tion,increase

dangiogenesis,

abnorm

aldorsal

aortamorp

hology,co

rnealvasc

ulariza

tion

NVS_ENZ_hVEGFR

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through impairment of VEGF-mediated uterine angiogenesis(Bosquiazzo et al., 2010). Gesta-tional BPA exposure led to a per-sistent increase in vascular area inthe postnatal mammary epithe-lium of female rats, and this effectwas preceded by a transitoryincrease in VEGF in the region(Durando et al., 2011). Severalnon-steroidal anti-estrogens(clomiphene, nafoxidine, tamoxi-fen), as well as pure estrogenreceptor antagonists inhibitedangiogenesis in the chick chorioal-lantoic (CAM) assay (Gagliardi andCollins, 1993). These findingsdemonstrate the clear potentialfor adverse developmental out-comes that may follow fromendocrine-mediated disruption ofangiogenesis.

Endothelins

Endothelins (ET-1, ET-2, ET-3)are vasoconstrictive peptides pro-duced by vascular ECs. Pharmaco-logical antagonists specific for thenative endothelin receptors ETAand ETB (SB-217242, SB-209670)given to pregnant rats or rabbitswere found to induce craniofacialand cardiovascular defects at rela-tively low dosages (Treinen et al.,1999). Another group reportedsimilar findings with a balancedETA-ETB antagonist (L-753,037)(Spence et al., 1999). In bothcases, the teratogenic effects ofendothelin receptor antagonistswere attributed to primary effectson altered neural crest migration.In stage 21, chick embryos ET-1infusion resulted in decreasedblood flow in the dorsal aorta andconstrictive effects on resistancearteries resulting in hemodynamicmediated changes in vascular de-velopment (Kajio and Nakazawa,1997).

Aryl hydrocarbon receptorpathway

Hypoxia potentially inducesmany genes in the embryo, includ-ing VEGF through hypoxia-induci-ble factor (HIF-1a) (Semenza,2006). HIF-1a also interactsdirectly with the aryl hydrocarbon

receptor and its dimerization part-ner (ARNT), which are required forvascular development, and ARNT-deficiency in the mouse is embryo-lethal by GD 10.0 due to aberrantplacental vascularization. Thisadverse effect in ARNT-nullmutants correlated with decreasedVEGF (the primary angiogenic sig-nal related to hypoxia) andVEGFR2 expression in the cho-rioallantoic placenta on GD 9.5,whereas their expression in theyolk sac and embryo appearedunaltered (Abbott and Buckalew,2000). Exposure of pregnant ratsto 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin), which binds arylhydrocarbon receptor, decreasedthe placental expression of TIE2,reduced the size of maternal bloodsinusoids, and caused the con-striction of fetal capillaries in theplacenta (Ishimura et al., 2006).

Retinoids

Retinoids are potent angiogene-sis inhibitors. All-trans retinoicacid suppressed microvasculatureformation in the chick CAM assaywhereas the anti-retinoid, Ro41-5253, had the opposite effect. Inmurine embryonic stem cell-derived vascular progenitor cells,retinoids down-regulated TIE2 sig-naling. This predicts an interfer-ence with EC-MC interactions thatcould impair vascular stability invivo (Suzuki et al., 2004).

Smoking

Smoking has been shown toimpair angiogenesis during preg-nancy, either via side-stream ormainstream patterns of exposure.Both kinds of exposure inhibitedangiogenesis in the chick CAMassay, decreasing vascular area,vessel diameter, and branching.The active compounds were identi-fied as pyrazines (Melkonian et al.,2002; 2003). Anti-angiogenicproperties of the pyrazines couldbe a contributing factor in the lowbirth weight found in infantsexposed to tobacco smoke duringpregnancy (Rogers and Abbott,2003).

Metals

The hypoxic response can beinvoked by some environmentalmetals (e.g., Co, Cu, Ni) that maystimulate angiogenesis. Dietarycopper deficiency in mice wasshown to produce vascular abnor-malities in the yolk sac of earlyembryos (reduced vascular den-sity, vessel wall thinning, andblebbing). Monitoring Cu-deficientembryos in culture revealed a cor-relation with dysmorphogenesisand general up-regulation ofVEGF, VEGFR1, VEGFR2, ANG1,and TIE2 genes (Yang et al.,2006).

SCREENING FOR VASCULAR

DISRUPTORS

The multitude of steps, targets,and cellular behaviors required forvascular development, stabiliza-tion and remodeling render thissystem amenable to in vitroscreening with techniques such asthe Flt-1-EGFP transgenic zebra-fish embryos, chick CAM assay, rataortic explant assay, human vas-cular endothelial cell culture (seeSarkanen et al., 2011), and HTSof critical vascular biomarkers (Dixet al., 2007; Kleinstreuer et al.,2011).Unlike mammalian embryos,

zebrafish embryos are smallenough that they can receiveenough oxygen by passive diffu-sion for them to survive and de-velop for several days in the com-plete absence of blood circulation(Cha and Weinstein, 2007). Thismakes Flt-1-EGFP transgenic fishan attractive screening platform toidentify compounds with effects onembryonic vascular development(McCollum et al., 2011). Retinalvascularization was assessed intransgenic fish exposed to a 200-chemical small molecular library.This screen identified five com-pounds that disrupted vascular de-velopment specifically in the eye:Enalapril maleate (an acetylcholin-esterase inhibitor), pyrogallin,albendazole, and mebendazole(microtubule inhibitors), and zear-alenone (an estrogenic mycotoxin)(Kitambi et al., 2009).



Using a human vascular EC cul-ture-fibroblast co-culture assay toassess vascular tubule formation,Sarkanen et al. (2011) evaluatedsix anti-angiogenic reference com-pounds: Levamisole, acetyl sali-cylic acid, thalidomide, erlotinib,anti-VEGF, and 2-methoxyestra-diol. In vitro tubulogenesis pre-dicted the known angiogenic orangiostatic effects of these com-pounds observed in vivo confirm-ing the predictive value of theassay (Sarkanen et al., 2011).VEGF secretion has been used asbiomarker for HTS to screen largerlibraries of chemicals. Of 1353

unique compounds screened in aHIF-1a reporter cell line, five com-pounds were identified thatinduced VEGF-secretion (Xia et al.,2009).We used EPA’s 309-chemical

ToxCast library to build a predic-tive model for pVDCs based on invitro HTS data (Kleinstreuer et al.,2011). Model features includedassays for VEGF receptors, pro-and anti-angiogenic chemokines,and the plasminogen activatingsystem. Not all ToxCast chemicalswith evident developmental toxic-ity showed an in vitro vascularbioactivity signature; however,

most with a vascular bioactivitysignature had prenatal develop-mental effects in vivo. An intrigu-ing preliminary finding was thedistinctly different correlative pat-terns observed for chemicals witheffects in prenatal rabbit versusrat studies, despite derivation ofin vitro signatures based onhuman cells and cell-free bio-chemical targets. This impliespotentially differential contribu-tions of developmental toxicitypathways between these species.A follow-up analysis was under-taken with thalidomide and 5HPP-33, the potent anti-angiogenic

Figure 1. Proposed adverse outcome pathway (AOP) for embryonic vascular disruption. The model is built incorporating ToxCastHTS data into the AOP schema presented in Ankley et al. 2010. Anchor 1 (red boxes on the left side) address chemical properties ofthe toxicant and nature of macromolecular interactions. MIEs may include receptor/ligand interactions (weak interactions), enzymeinhibition or activation, DNA binding (reactive chemistry, alkylation, intercalation), protein oxidation (ROS/RNS), and so forth.Anchor 2 (red boxes on the right) refer to relevant organism responses and community-level population responses. The middle col-umns address cellular and organ responses. The color wheel indicates ToxPi sectors for chemical prioritization (shown here withoutdata applied). The 25 assays shown are those which had evidence of abnormal embryonic vascular development based on geneticmouse models (from Table 1) and mapped to previously-identified critical pathways (hypoxia/growth factor signaling, chemokinenetworks, ECM interactions and vessel remodeling/stabilization). They are color-coded as such in the ToxPi schema.



analogue. Results were consistentwith the pVDC signature predic-tions 5HPP-33 ranking as amongthe most active compounds testedin this screen (Kleinstreuer et al.,2011).These predictions implicate the

embryonic vasculature as a targetfor environmental chemicals actingas pVDCs and illuminates potentialspecies differences in sensitivevascular developmental pathways.One of the key ideas in the NRCreport is of a pathway of toxicity,which is a cellular response path-way that, when sufficiently per-turbed, is expected to result in anadverse health effect (NRC,2007). This definition focuses onan organism’s cellular response tochemicals that could be measuredand modeled from in vitro expo-

sure. A broader term, ‘‘adverseoutcome pathway’’ (AOP) was pro-posed (Ankley et al., 2010) tomore explicitly translate this infor-mation into endpoints meaningfulto risk assessment (e.g., develop-ment, reproduction, survival).An AOP delineates the docu-

mented, plausible, and testableprocesses by which a chemicalinduces molecular perturbationsand the associated biologicalresponses that describe how themolecular perturbations causeeffects at the subcellular, cellular,tissue, organ, whole animal, andpopulation levels of observation.This concept requires an anchor toa molecular initiating event (MIE)and an adverse outcome with sig-nificance to risk assessment(Ankley et al., 2010). We present

a preliminary AOP for embryonicvascular disruption based on thatschema (Fig. 1).Figure 1 includes a toxicity pri-

oritization index (ToxPi; Reif et al.,2010) schema consisting of the 25assay targets associated withabnormal embryonic vascular de-velopment based on geneticmouse models from the MPBrowser (see Table 1) andmapped to previously-identifiedcritical pathways (hypoxia/growthfactor signaling, chemokine net-works, ECM interactions and ves-sel remodeling/stabilization) basedon the gene ontology browserfrom the mouse genome infor-matics database. The sectors arecolor-coded by the respectivepathway that each target infers,although genes such as TGF-beta

Figure 2. AOP for embryonic vascular disruption predicted for an environmental chemical (Maneb). The model is built incorporatingToxCast HTS data into the schema from Figure 1, applying the HTS data for Maneb. ToxPi scores for Maneb relative to the 309chemicals in ToxCast Phase I are indicated in the ToxPi to right.



have widespread involvementacross many pathways.A case example is shown in Fig-

ure 2 for a proposed AOP andToxPi for Maneb, the highest scor-ing ToxCast Phase I chemical bythis ranking scheme. Maneb pro-duced AC50 values (defined asmicromolar concentration with50% activity on a particular invitro assay) on CCL2, CXCL10,IL1a, TNFa, MMP9, uPAR, uPA,PAI-1, FGFR1, VEGFR1, VEGFR2,VEGFR3, TIE2, and VCAM-1 inmultiple ToxCast assay platforms.In ToxRefDB prenatal guidelinestudies in rat, Maneb resulted infetal weight reduction, skeletalmalformations, and embryolethal-ity. The proposed AOP card forManeb demonstrates how thesetargets are plausible MIEs thatcould lead to an adverse effect onembryonic vascular developmentand, in turn consequences to pre-natal development. The caseexample shown in Figure 2 isclearly focused on in vitro hazardidentification. Bringing this in vitroinformation together with informa-tion on chemical exposure, toxico-kinetics, and interactions withother AOPs could help guide theefficient use of resources for ex-perimental validation and riskassessment.

CONCLUSIONS

First-generation predictive modelsfor prenatal developmental toxic-ity have revealed a complex webof biological processes with manyconnections to important signalsfor vascular development (localgrowth factors and cytokines suchas VEGF-A and TGF-beta, compo-nents in the plasminogen activatorsystem, and chemotactic chemo-kines). Evidence for chemical dis-ruption of these processes is accu-mulating for a number of mecha-nistically diverse compounds.Although not all environmentalchemicals with developmental tox-icity show an in vitro vascular bio-activity signature, many pVDCsinvoke adverse developmentalconsequences. For a subset ofthese chemicals, the disruption ofblood vessel development may be

part of an AOP that can be appliedto predictive toxicology and mech-anistic modeling of prenatal devel-opmental toxicity. As such, anAOP perspective of embryonicvascular development can helpidentify useful information forassessing adverse outcomes rele-vant to risk assessment and effi-cient use of resources for valida-tion.

ACKNOWLEDGMENTS

This work was performed underEPA’s Chemical Safety for Sustain-ability Research Program (CSS)Project 2.2.2. The authors aregrateful to Dr. Nancy C. Baker ofLockheed Martin, contractor to theNational Center for ComputationalToxicology, for assistance with thelarge scale text-mining operations.

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