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Event: 305

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Inhibition, VegfR2

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Inhibition, VegfR2

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
somatic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
vascular endothelial growth factor receptor 2 binding vascular endothelial growth factor receptor 2 decreased
vascular endothelial growth factor receptor 2 binding vascular endothelial growth factor receptor 1 decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Developmental Vascular Toxicity MolecularInitiatingEvent Tom Knudsen (send email) Open for citation & comment EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
zebra fish Danio rerio Moderate NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus Low NCBI
human Homo sapiens Moderate NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

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Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

The VEGF-VEGFR system is an important molecular regulator of various processes linked to physiological and pathological blood vessel development, from early embryonic to adult stages. The central players in this system among vertebrate species are three vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3) and five VEGF family members that bind and activate these various receptors during vasculogenesis, angiogenesis and lymphogenesis [Shibuya, 2013]. A search of the PubMed literature for these Medical Subject Heading (MeSH) terms returned 41,371 entries 1989-2014 (December 4, 2014); 4982 of those entries had broad focus on ‘vascular’ and ‘development’ headings.

Vascular endothelial growth factor-A (VEGF-A), in particular the VEGF165 splice variant, plays a key role in the regulation of angiogenesis during early embryogenesis. This is evidenced by immature blood vessel formation and embryonic lethality in mutant mouse embryos heterozygous for the Vegfa-null allele [Ferrara et al. 1996; Carmellet et al. 1996]. VEGF-A is a soluble protein that acts directly on endothelial cells through two receptor tyrosine kinases: VEGFR1 (Flt-1) and VEGFR2 (KDR). The former is a decoy receptor that traps VEGF-A into corridors preventing interaction with the active receptor, VEGFR2. When liganded, VEGFR2 induces endothelial tip cell proliferation, survival, and vascular permeability. Various factors leading to a change in VEGF-A gradients will be considered.  This could include a change in the local production of VEGF-A, an increase in the decoy receptor (VEGFR1), or a drop in the expression or activity of VEGFR2. Chemical effects may commence at VEGF receptors (VEGFRs) by influencing local VEGF-A ligand production, ligand binding, receptor tyrosine kinase activity, or crosstalk with angiogenic chemokines, cytokines and growth factors. VEGF-A is locally produced in the vicinity of target endothelial cells. Hypoxia (and chemical hypoxia) increases VEGF-A production through the HIF-alpha transcription pathway. VEGF-A can be trapped in the extracellular milieu by binding to components in the extracellular matrix (ECM) or VEGFR1. Since VEGFR1 binds VEGF-A with 10-fold greater affinity than does VEGFR2, VEGF-A is liberated by ECM breakdown during morphogenetic remodeling of tissues or matrix metalloproteinase (MMP) production during chemical injury. These events can positively or negatively regulate the local bioavailability of VEGF-A to its cognate receptor.

Targeted disruption of VEGFR1 or VEGFR2 is early embryonic lethal; however, the vascular phenotypes differ in either case. VEGFR1-mutant (Flt1-null) embryos display excessive endothelial cell growth leading to disorganization of the vascular network [Fong et al. 1995] whereas VEGFR2-mutant (Flk1-null) embryos die from a lack blood vessel network formation [Shalaby et al. 1995]. This duality is relevant to the VEGFR disruption because receptor affinity for VEGF is ten-fold higher at VEGFR1 but kinase activity is ten-fold higher at VEGFR2 [Fischer et al. 2008; Shibuya, 2013]. As such, VEGFR2 promotes angiogenesis whereas VEGFR1 acts as a ligand-trap to prevent VEGF-A interaction with VEGFR2 [Hiratsuka et al. 1998]. However, VEGFR2 activation is considered the master switch of developmental angiogenesis.  In addition to upstream effects on VEGF-A production or trapping, the inhibition of VEGFR2 can be engaged by interference interference with VEGF-A binding to VEGFR2, inhibition of receptor tyrosine kinase activity, or reduced expression of the VEGFR2 protein. Given the complex nature of the VEGF-VEGFR system, disruption of VEGF2 has diverse origins.

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

A structure-activity relationship (SAR) analysis of a 73,000-compound library using an HIF-1a: VEGF secretion assay identified 350 actives for followup [Xia et al. 2009]. Proximity Ligation Assays have been used to identify small molecule inhibitors of VEGF-A binding to its receptors [Gustafsdottir et al. 2008]. This assay is: fit for the purpose of monitoring the formation and inhibition of VEGF-A–receptor complexes; defines chemical disruption of VEGF-A direct binding to its receptors (VEGFR1, VEGFR2); correlates well with results obtained by measuring receptor phosphorylation (VEGFR2); and allows evaluation of the half-maximal inhibitory concentration (AC50) from a concentration-response curve.

VEGFR2 inhibition can be detected by measures of capacity (receptor density, expression levels) and bioactivity (tyrosine kinase activity) utilizing molecular probes and pharmacological reagents. As part of a broader AOP framework for vascular developmental toxicity, VEGFR2 inhibition is anchored to genetic models having strong phenotypic evidence for adverse developmental outcomes. To organize what is known, an AngioKB knowledgebase was built from high-throughput PubMed queries utilizing Medical Subject Heading (MeSH) terms for vasculogenesis and angiogenesis in the embryo, fetus or development [Knudsen and Kleinstreuer, 2011]. These unstructured data were supplemented with information culled from the MGI Mammalian Phenotype Ontology (MPO) Browser (http://www.informatics.jax.org, accessed October 31, 2011) for 'abnormal vasculogenesis' (MP:0001622, aberrant process of the initial establishment of the vascular network; 78 annotations) and 'abnormal angiogenesis' (MP:0000260, aberrant process of blood vessel formation and the subsequent remodeling process - does not refer to the initial establishment of the vascular network; 892 annotations). The relevant genes were then mapped to the ToxCast assay portfolio [1]. This yielded an overlap of 25 different sectors for pathways in blood vessel development [Knudsen and Kleinstreuer 2011], including the VEGF-VEGFR system covering many diverse studies in the open literature. The predictive signature for vascular disruption (pVDCs) incorporates several assays that directly measure VEGFR2 capacity (BSK_4H_VEGFRII_down and BSK_4H_VEGFRII_up) and bioactivity (NVS_ENZ_hVEGFR1 and NVS_ENZ_hVEGFR2). Importantly, VEGFR2 is inactive when non-liganded so measures of VEGFR2 capacity in the BioSeek (BSK) human 4H primary endothelial cell assay can register an increase or decrease in capacity depending on whether the VEGFR2 inhibition is being detected by a change in VEGFR2 protein levels [Kleinstreuer et al. 2014]. Liganding VEGFR2 leads to receptor dimerization and autophosphorylation on tyrosine residues, which initiates signal transduction [Kendall et al. 1999]. This bioactivity is measured by a cell-free assay for human recombinant VEGFR2 that can register a concentration-dependent activation or inhibition [Knudsen et al. 2009; Sipes et al. 2011].

Analysis of VEGFR2 expression under different physiological and toxicological contexts can be determined by standard array-based assays to define regulation of the angiogenic transcriptome, as well as targeted non-array methods to characterize cell-specific profiles during in vivo and in vitro development [Dumont et al. 1995; Abbott et al. 2000; Drake et al. 2007; Murakami et al. 2011]. In addition, the ontogenetic profile of VEGFR1 and VEGFR2 expression can be mapped in reporter zebrafish embryos under specific control of Flk or Flt gene regulatory elements [Tal et al. 2014]. Zebrafish possess 72 orthologs for 70% of human genes and 86% of 1318 human drug targets. Transgenic zebrafish that express reporter gene in developing blood vessels are fit for purpose to: visualize blood vessel formation during early development; localize and quantitate regional effects of chemicals on a phenotypic readout of angiogenic vessel formation; assess the reproducibility of vascular disruption across species; and evaluate the half-maximal inhibitory concentration (AC50) from a concentration-response curve.

Downstream consequences to signal transduction can be measured by specific targets of VEGFR2 tyrosine kinase activity. Various examples of bioassays that measure the growth of blood vessels and the effects of specific inhibitors include in vitro assays of endothelial cell migration and proliferation. Some assays test human endothelial cells in primary culture models (e.g., HUVEC), stem-cell derived systems that are capable of de novo assembly into capillary networks, or genetically engineered mouse and zebrafish embryo, and computational models [Mueller et al. 2000; Dorrell et al. 2002; Xia et al. 2009; Chapell et al. 2013; Kleinstreuer et al. 2013; Shirinifard et al. 2013]. These assays and models are: fit for the purpose of defining optimal VEGF-A levels for angiogenesis; screening large inventories of small molecules for VEGF-A secretion over a range of chemical concentrations and low oxygen tension; linkage of VEGFR2 inhibition with the physiological initiating event; and evaluation of the half-maximal inhibitory concentration (AC50) from a concentration-response curve.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

There is strong phylogenetic conservation of VEGFR2 genes [Shibuya, 2002]. For example, the amino acid homology ranges from 79.9 - 96.1% for the critical autophosphorylation domain across species of fish, birds, rodents with humans. This suggests a conserved molecular basis to regulation of blood vessel development and implies broad taxonomic applicability to VEGFR2 inhibition. Direct evidence for this comes from the susceptibility of vascular development to pharmacological inhibitors of human VEGFR2 kinase activity. Vatalanib (PTK787), for example, is a potent inhibitor of human VEGFR2 kinase activity [Wood et al. 2002] and disrupted angiogenic vessel formation in early zebrafish embryos at submicromolar concentrations [Tal et al. 2014].

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

Chemical effects on VEGF-A binding to VEGFR2 has been demonstrated for 6 different inhibitors using recombinant VEGF-A(165) [Gustafsdottir et al. 2008]. Among the inhibitors were DNA/RNA aptamers, neutralizing antibodies directed against VEGF-A or VEGFR2, recombinant competitive protein, and a low molecular weight synthetic molecule. A pharmacological panel of small molecule inhibitors of VEGFR inhibitors is known, having varied activities on VEGFR2 and other members of the same receptor tyrosine kinase family as the VEGF receptors, including the platelet-derived growth factor receptor β (PDGFR-β). These compounds include Vatalanib (VEGFR2/PDGFRβ/c-kit inhibitor), Sunitinib (VEGFR1/VEGFR2/PDGFR inhibitor), and Semaxinib (VEGFR2 inhibitor).

Vatalanib, also known by the code name PTK787, is a potent vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor that inhibits VEGFR2/KDR and VEGFR1/Flt-1 with the half maximal inhibition concentration IC50 values of 0.037 μM and 0.077 μM, respectively [Wood et al. 2000]. It also inhibits to a lesser degree PDGFR-β. Liganding VEGFR2 leads to receptor dimerization and autophosphorylation on tyrosine residues, which initiates signal transduction [Kendall et al. 1999]. Using a double antibody chemiluminescence assay, PTK787 was shown to block VEGF-induced auto-phosphorylation of VEGFR2 with an IC50 of 0.017 μM in human endothelial cells (HUVECs) and concentration-dependent suppression of endothelial migration and tumorigenic formation of microvessels [Wood et al. 2000].

Vatalanib

Evidence that this VEGFR2 inhibition can be chemically initiated with impacts on embryogenesis, transgenic TG(flk1:GFP) zebrafish embryos were used to visualize and quantify blood vessel formation [Tal et al. 2014]. The embryos were exposed to Vatalanib at concentrations ranging from 0.07-1.25 uM during the period from 24- to 72 hours post fertilization (hpf). An evaluation of blood vessel development and developmental toxicity showed clear evidence for concentration-dependent disruption, and a comparison of the VEGFR2 inhibitor (PTK787) with an EGFR inhibitor (AG1478) showed regional specificity for adverse effects on vascular patterning and gross morphology. This specificity provides evidence for chemical initiation of VEGFR2 inhibition in the embryo.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Argraves WS, Larue AC, Fleming PA, Drake CJ. VEGF signaling is required for the assembly but not the maintenance of embryonic blood vessels. Developmental dynamics : an official publication of the American Association of Anatomists. 2002;225(3):298-304.

Belair DG, Schwartz MP, Knudsen T and Murphy WL. Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays. Acta Biomater. 2016; 39: 12-24. PMID:27181878.

Bhattacharya R1, Kwon J, Li X, Wang E, Patra S, Bida JP, Bajzer Z, Claesson-Welsh L and Mukhopadhyay D (2009) Distinct role of PLCbeta3 in VEGF-mediated directional migration and vascular sprouting. J Cell Sci. 122: 1025-1034.

Carmellet P, Ferreira V, Breier G, Pollefeyt S, Kleckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawlling J, Moons L, Collen D, Resau W, Nagy A (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380: 435–439.

Chan J, Bayliss PE, Wood JM, Roberts TM (2002) Dissection of angiogenic signaling in zebrafish using a chemical genetic approach. Cancer Cell. 1: 257-267. (Note: this paper describes the use of Vatalanib to inhibit VEGFR; PTK787 is a synonym for Vatalanib.)

Chappell JC, Taylor SM, Ferrara N, Bautch VL. Local guidance of emerging vessel sprouts requires soluble Flt-1. Developmental cell. 2009;17(3):377-86.

Chen DB, Zheng J. Regulation of placental angiogenesis. Microcirculation (New York, NY : 1994). 2014;21(1):15-25. Douglas NC, Tang H, Gomez R, Pytowski B, Hicklin DJ, Sauer CM, et al. Vascular endothelial growth factor receptor 2 (VEGFR-2) functions to promote uterine decidual angiogenesis during early pregnancy in the mouse. Endocrinology. 2009;150(8):3845-54.

Dorrell MI, Aguilar E, Friedlander M. Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Investigative ophthalmology & visual science. 2002;43(11):3500-10.

Eichmann A, Thomas JL. Molecular parallels between neural and vascular development. Cold Spring Harbor perspectives in medicine. 2013;3(1):a006551. Habeck H, Odenthal J, Walderich B, Maischein H, Schulte-Merker S. Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis. Current biology : CB. 2002;12(16):1405-12.

Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 380: 439–442.

Fischer et. al (2008) Nat Rev Cancer 8: 942–956.

Fong GH, Rossant J, Gertsenstein M, Breitman ML (1995) Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376: 66-70.

Gustafsdottir SM, Wennstrom S, Fredriksson S, Schallmeiner E, Hamilton AD, Sebti SM and Landegren U (2008) Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors. Clinical Chem 54: 1218-1225.

Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M (1998) Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci USA 95: 9349-9354.

Hogan KA, Ambler CA, Chapman DL, Bautch VL. The neural tube patterns vessels developmentally using the VEGF signaling pathway. Development (Cambridge, England). 2004;131(7):1503-13.

Jang GH, Park IS, Lee SH, Huh TL, Lee YM. Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling. Biochemical and biophysical research communications. 2009;382(3):486-91.

Kendall RL, Rutledge RZ, Mao X, Tebben AJ, Hungate RW and Thomas KA (1999) J Biol Chem 274: 6453-6460.

Kleinstreuer N, Dix D, Rountree M, Baker N, Sipes N, Reif D, Spencer R and Knudsen T (2013) A computational model predicting disruption of blood vessel development. PLoS Comp Biol 9(4): 1-20. e1002996.

Kleinstreuer NC, Judson RS, Reif DM, Sipes NS, Singh AV, Chandler KJ, DeWoskin R, Dix D, Kavlock R and Knudsen TB (2011) Environmental impact on vascular development predicted by high-throughput screening. Environmental Hlth Persp 119: 1596-1603.

Kleinstreuer N, Yang J, Berg E, Knudsen T, Richard A, Martin M, Reif D, Judson R, Polokoff M, Kavlock R, Dix D and Houck K (2014) Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms. Nature Biotech 32: 583-591.

Knudsen TB, Houck K, Sipes NS, Judson RS, Singh AV, Weissman A, Kleinstreuer NC, Mortensen H, Reif D, Setzer RW, Martin MT, Richard A, Dix DJ, and Kavlock RJ (2011) Activity profiles of 320 ToxCast™ chemicals evaluated Across 292 biochemical targets. Toxicology 282: 1-15

Knudsen TB, Kleinstreuer NC (2011). Disruption of embryonic vascular development in predictive toxicology. Birth defects research Part C 93: 312-323.

Ligi I, Simoncini S, Tellier E, Grandvuillemin I, Marcelli M, Bikfalvi A, et al. Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2014;27(3):233-8.

Liu H, Yang Q, Radhakrishnan K, Whitfield DE, Everhart CL, Parsons-Wingerter P, et al. Role of VEGF and tissue hypoxia in patterning of neural and vascular cells recruited to the embryonic heart. Developmental dynamics : an official publication of the American Association of Anatomists. 2009;238(11):2760-9.

Nimmagadda S, Geetha Loganathan P, Huang R, Scaal M, Schmidt C, Christ B. BMP4 and noggin control embryonic blood vessel formation by antagonistic regulation of VEGFR-2 (Quek1) expression. Developmental biology. 2005;280(1):100-10.

Roberts DM, Kearney JB, Johnson JH, Rosenberg MP, Kumar R, Bautch VL. The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation. The American journal of pathology. 2004;164(5):1531-5.

Saili KS, Franzosa JA, Baker NC, Ellis-Hutchings RG, Settivari RS, Carney EW, Spencer R, Zurlinden TJ, Kleinstreuer NC, Li S, Xia M and Knudsen TB. Systems Modeling of Developmental Vascular Toxicity. Curr Opin Toxicol. 2019; 15(1): 55-63. PMID:32030360.

Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376: 62-66.

Shibuya M (2013) Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases. J Biochem. 153: 13-19.

Shirinifard A, McCollum CW, Bolin MB, Gustafsson JA, Glazier JA, Clendenon SG. 3D quantitative analyses of angiogenic sprout growth dynamics. Developmental dynamics : an official publication of the American Association of Anatomists. 2013;242(5):518-26.

Sipes NS, Martin MT, Kothiya P, Reif DM, Judson R, Richard A, Houck KA, Dix DJ, Kavlock RJ and Knudsen TB (2013) Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays Chem Res Toxicol 26: 878-895.

Stankunas K, Ma GK, Kuhnert FJ, Kuo CJ, Chang CP. VEGF signaling has distinct spatiotemporal roles during heart valve development. Developmental biology. 2010;347(2):325-36.

Tal TL, McCollum CW, Harris PS, Olin J, Kleinstreuer N, Wood CE, Hans C, Shah S, Merchant FA, Bondesson M, Knudsen TB, Padilla S and Hemmer MJ. Immediate and long-term consequences of vascular toxicity during zebrafish development. Reprod Toxicol. 2014; 48: 51-61. PMID:24907688.

van den Akker NM, Molin DG, Peters PP, Maas S, Wisse LJ, van Brempt R, et al. Tetralogy of fallot and alterations in vascular endothelial growth factor-A signaling and notch signaling in mouse embryos solely expressing the VEGF120 isoform. Circulation research. 2007;100(6):842-9.

Williams CK, Li JL, Murga M, Harris AL, Tosato G. Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function. Blood. 2006;107(3):931-9.

Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, Persohn E, Rösel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, Schirner M, Thierauch KH, Schneider MR, Drevs J, Martiny-Baron G, Totzke F (2000) PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 60: 2178-2189.

Xia M, Bi K, Huang R, Cho MH, Sakamuru S, Miller SC, et al. Identification of small molecule compounds that inhibit the HIF-1 signaling pathway. Molecular cancer. 2009;8:117.

Yabu T, Tomimoto H, Taguchi Y, Yamaoka S, Igarashi Y, Okazaki T. Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate. Blood. 2005;106(1):125-34.