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Event: 305

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Inhibition, VegfR2

Short name

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Inhibition, VegfR2

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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.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. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Cell term
somatic cell

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). 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 signaling 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. Further information on Event Components and Biological Context may be viewed on the attached pdf. 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

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AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Developmental Vascular Toxicity	MolecularInitiatingEvent	Undefined (send email)	Open for citation & comment	WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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

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Sex Applicability

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Key Event Description

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The VEGFR system is an important molecular regulator of physiological and pathological blood vessel development. The central players are vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3) and five VEGF ligands that bind and activate these receptors during vasculogenesis, angiogenesis and lymphogenesis [Shibuya, 2013]. The MIE:305 target, VEGFR2, belongs to Class IV transmembrane receptor tyrosine kinases (RTKs) that play critical roles in the origin and progression of many adverse outcomes linked to vascular biology. Direct evidence supporting its role in developmental angiogenesis comes from functional inactivation in mouse VEGFR knockout models. For example, a targeted mutation in flt-1 showed Vegfr1(-/-) embryos formed endothelial cells in both embryonic and extra-embryonic regions but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages [Fong et al. 1995]. Functional inactivation of flk-1 showed that Vegfr2(-/-) embryos died much earlier due to deficiencies in hematopoeisis and organized blood vessels [Shalaby et al. 1995]. It’s endogenous ligand, 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. Mouse embryos heterozygous for the Vegf gene died from impaired angiogenesis and hematopoeisis in Vegf(+/-) heterozygotes during organogenesis [Ferrara et al. 1996]. Nullizygotes died earlier showing that progressive severity in a quantitative gene dose-dependent manner [Carmeliet et al. 1996]. VEGF-A is a soluble protein that acts directly on endothelial cells and their precursors through VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). The former is a decoy receptor that traps VEGF-A into corridors preventing interaction with the active receptor, VEGFR2 [Roberts et al. 2004]. Environmental stressors (drugs/chemicals) may perturb VEGFR-dependent angiogenesis [Belair et al. 1996a,b]. Multiple mechanisms are involved, including direct effects on VEGFR2 structure-function as well as VEGF-A bioavailability or binding kinetics [Gustafsdottir et al. 2008]. The duality is relevant to MIE:305 because receptor affinity for VEGF is ten-fold higher at VEGFR1, whereas 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]. In this AOP, decreased VEGFR2 binding is the quantitative basis for an effect of stressors on VEGFR2 activation of the ‘master switch’ in developmental angiogenesis.

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

A number of targeted and high-throughput assays are used to quantitively assess chemical effects leading to reduced VEGFR2 activity. Starting with VEGF availability as a preceding event, a cell-based reporter gene assay has screened approximately 73,000 compounds in a quantitative high-throughput screening (HTS) approach [Xia et al. 2009]. That assay measures cellular VEGF-secretion in an ME-180 cervical carcinoma HRE (hypoxia-response element) reporter cell line as a genetic response to hypoxia-induced Vegf expression. Proximity Ligation Assays (PLAs) have been used to evaluate small molecule inhibitors of VEGF-A₁₆₅ binding to solubilized VEGFRs [Gustafsdottir et al. 2008]. PLAs are fit for the purpose of monitoring the kinetics of formation and inhibition of ligand–receptor complexes through different mechanisms of interference with VEGF-A₁₆₅ or its cognate binding site. This allows quantitative evaluation of the potency of chemical inhibitors based on computing half-maximal inhibitory concentrations (IC50) in concentration-response curves. The inhibition of VEGF-A₁₆₅ binding to VEGFR2 correlated well in these assays with results obtained by measuring receptor phosphorylation following exposure to molecular probes or pharmacological reagents specific to VEGF-VEGFR2 receptor capacity and kinase activity [Gustafsdottir et al. 2008]. HTS platforms have also been used to screen neary 1,000 compounds in the ToxCast/Tox21 chemical library for effects on human VEGFR2 bioactivity (https://comptox.epa.gov/dashboard/) [Kavlock et al. 2012; Judson et al. 2016; Richard et al. 2016; Thomas et al. 2018]. This biochemical (cell-free) assay is one of 331 enzymatic and receptor signaling assays under the ‘NovaScreen’ (ToxCast_NVS) platform [Knudsen et al. 2011; Sipes et al. 2013]. VEGFR2 enzymatic activity is measured as an electrophoretic shift in migration of a specific fluorescein-peptide substrate to the fluorescein-phosphopeptide upo 1-hour incubation with ATP. Concentration response to a test chemical is detected by a change in activity, which may be decreased or increased depending on the nature of a drug or chemical’s effect on VEGFR2 catalysis or autophosphorylation, respectively with automated curve-fits [Knudsen et al. 2011; Sipes et al. 2013]. Also, in ToxCast, a multiplex assay described under the ‘BioSeek’ (ToxCast_BSK) platform exists for VEGFR2 bioactivity in a cell-based co-culture system [Kleinstreuer et al. 2014]. This assay measures increased or decreased levels of VEGFR2-immunoreactive protein by ELISA in primary human umbilical vein cells (HUVEC) conditioned to simulate proinflammation with histamine and IL4. Concentration response to a test chemical is curve-fitted to indicate changes in VEGFR2 receptor density. This is one of 87 endpoints covering molecular functions relevant to toxic and therapeutic pathways generated in eight cell systems for 641 environmental chemicals and 135 reference pharmaceuticals and failed drugs [Kleinstreuer et al. 2014].

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). 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].

References

List of the literature that was cited for this KE description. 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.