Key Event Title
Key Event Component
|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
|AOP Name||Role of event in AOP|
|Disruption of VEGFR Signaling Leading to Developmental Defects||MolecularInitiatingEvent|
Level of Biological Organization
|zebra fish||Danio rerio||Moderate||NCBI|
How This Key Event Works
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. The specific aspect of the MIE considered here would involve factors leading to a change in VEGF-A gradients. 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 MIE 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. As such, the central concept of the MIE for this is inhibition of VEGFR2 activity. In addition to upstream effects on VEGF-A production or trapping, the MIE 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, this MIE must consider the state of the system overall.
How It Is Measured or Detected
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.
The MIE for '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, this MIE 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 . 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 MIE 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 the MIE with the physiological initiating event; and evaluation of the half-maximal inhibitory concentration (AC50) from a concentration-response curve.
Evidence Supporting Taxonomic Applicability
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 this MIE, 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
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].
Evidence that this MIE 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 the MIE in the embryo.
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 D, Schwartz MP, Knudsen T and Murphy WL. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays. Acta Biomaterialia 2016. (in press).
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.
Knudsen TB, Kleinstreuer NC, Heinonen T (2014) Adverse Outcome Pathway for Embryonic Vascular Disruption: ToxCast HTS predictive model qualified by a validated human angiogenesis assay. Abstract presented at ToxCast Data Summit, manuscript in preparation.
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.
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 T, Kleinstreuer N, Toimela T, Sarkanen R, Heinonen T, Knudsen T and Padilla S (2014) Identification of chemical vascular disruptors during development using an integrative predictive toxicity model and zebrafish and in vitro functional angiogenesis assays. Abstract presented at ToxCast Data Summit, manuscript in preparation.
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 (2014) Immediate and long-term consequences of vascular toxicity during zebrafish development. Reproductive Toxicology 48:51-61.
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.