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Relationship: 335

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Inhibition, VegfR2 leads to Reduction, Angiogenesis

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Disruption of VEGFR Signaling Leading to Developmental Defects adjacent High High Tom Knudsen (send email) Open for citation & comment EAGMST Under Review

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 KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

VEGF signals promote endothelial cell motility, filopodial extension and proliferation, and together with Notch signaling controls whether specific endothelial cells (ECs) become pioneering ‘EC-tip’ cells (non-proliferating) or trailing ‘EC-stalk’ cells (proliferating). VEGFR2 activation is the master switch that promotes motility and exploratory behaviors of leading EC-tip cells and a mitogenic effect on trailing EC-stalk cells [EIlken and Adams, 2010; Herbert and Stanier 2011; Blanco and Gerhardt, 2013]. An early step is EC-tip cell selection [Eilken and Adams, 2010]. Endothelial cells are normally suppressed in their tip cell behaviors by Notch-Delta signaling [Blanco and Gerhardt, 2013; Li et al. 2014]. This lateral inhibition is broken when VEGFR2 is activated by VEGF-A.  Delta-like 4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, is selectively expressed in response to VEGF-A induction. This down-regulates VEGFR-2 expression in prospective EC-stalk cells but promotes VEGFR2 expression in EC-tip cells, enabling them to extend filopodial processes along VEGF-A rich paths thus orienting the angiogenic sprout [Williams et al. 2006]. VEGF-A rich corridors are established during in vivo development by local VEGFA gradients and the distribution of soluble VEGFR-1, a so-called ‘decoy receptor’ sequestered and released during enzymatic remodeling of ECM, both serving to channel sprouting progression along VEGFA-rich corridors [Roberts et al. 2004; Chappell et al. 2009 and 2016].

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER.  For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility: The control of EC-tip cell dynamics is a central feature linking VEGFR-2 inhibition (MIE:305) to adverse angiogenic sprouting behaviors (AE:28) [Argraves et al. 2002; Williams et al. 2006; Eilken and Adams, 2010; Oladipupo et al. 2011; Venkatraman et al. 2016; Beloglazova et al. 2021].

Empirical Evidence: 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 in time-scale relationships for immature blood vessel formation and embryonic lethality in mutant mouse embryos heterozygous for the Vegfa-null allele [Ferrara et al. 1996; Carmeliet et al. 1996]. Targeted disruption of genes encoding VEGFR1 or VEGFR2 are also early embryonic lethal; however, the vascular phenotypes differ in either case. Whereas VEGFR1-mutant (Flt1-null) embryos display excessive endothelial cell growth and disorganization of the vascular network [Fong et al. 1995], VEGFR2-mutant (Flk1-null) embryos die from a lack blood vessel network formation [Shalaby et al. 1995]. The requirement of VEGFA signaling is relevant to KER:335 for angiogenesis not only during embryonic development but for the uterine cycle, pregnancy, wound healing, and tumorigenic vessel growth in the adult. The inferred ‘window of vulnerability’ for chemical teratogenesis involves key events during early postimplantation stages of human development.

Uncertainties and Inconsistencies: Many physiological states influence VEGF-A production (e.g., hypoxia, estrogen) and post-VEGFR2 signaling. For example, VEGFR2 signals may be influenced by crosstalk with VEGFR1 and VEGFR3, other receptor tyrosine kinases (FGFR, EGFR), G-protein coupled receptors (CXCRs and CCRs), and GPI-linked surface receptors (uPAR) [Kleinstreuer et al. 2011]. The ToxCast pVDC signature includes assays for many of these targets and shows that environmental chemicals perturbing VEGFR2 also affect molecular targets in other signaling system [Knudsen et al. 2016]. Crosstalk between VEGFR-2 and other pro-angiogenic receptor tyrosine kinase (RTK) activities such as PDGFR or FGFR is known. This crosstalk has been embraced in the search for clinically efficacious synergistic kinase anti-angiogenesis strategies in suppressing tumorigenic growth [Lin et al. 2018] but is an uncertainty for establishing a role for KER:335 in the disruption of blood vessel morphogenesis (KE:28). For example, the fungal metabolite Epoxyquinol B inhibits kinase activity across several RTKs including VEGFR and PDGFR and blocks VEGF-induced migration and tubulogenesis in human umbilical vein endothelial cells (HUVECs) [Kamiyama et al. 2008]. Anlotinib inhibits cell migration and microvessel formation in the rat aortic ring assay and chicken chorioallantoic membrane assay via the ERK signaling pathway in both species [Lin et al. 2018]. Derazantinib at 0.1 µM to 3 µM blocked intersegmental vessel (ISV) migration linked to VEGF, PDGF, or FGF pathways in zebrafish embryos [Kotini et al. 2020].

Still other pathways may be relevant with regards to developmental angiogenesis. For example, the endothelial TIE2 receptor is essential for ISV outgrowth in zebrafish embryos [Li et al. 2014] and TGFβ1 signaling in the formation of tubular networks in human vascular endothelial cells (HUVECs) [Zhang et al. 2021]. VEGF-dependent cell migration in HUVECs is also facilitated by the urokinase-type plasminogen activator receptor (uPAR), a system linked to cell-ECM interactions and Notch components: Notch1 receptor and ligands (Dll1, Dll4, Jag1) in endothelial cells on one hand, and uPA, uPAR, TGFβ1, integrin β3, Jag1, Notch3 receptor in mural cells on the other hand [Beloglazova et al. 2021]. Both an increase on pro-angiogenic factors as well as a decrease in anti-angiogenic factors (Notch signaling) can have similar outcomes. Crosstalk in these heterogeneous systems point to cell-specific patterns of gene expression as a critical determinant of RTK expression and cell-type specificity. As such, quantitative linkages to VEGF signaling must consider the uncertainties from effects to other MIEs.

Quantitative Understanding of the Linkage: Studies with pharmacological VEGFR2 inhibitors have shown their concentration dependent effect on angiogenic sprouting. For example, the VEGFR2 antagonist Vatalanib (PTK787) suppressed zebrafish ISV outgrowth in a concentration-dependent manner that was characterized quantitatively at 72 hours post-fertilization (hpf) and became evident at the 0.07 µM concentration level [Tal et al. 2014]. An even lower concentration of Vatalanib (0.01 µM) inhibited angiogenic sprouting dynamics in a 3D microsystem of human endothelial cells derived from induced pluripotent stem cells (iPSC-ECs) [Belair et al. 2016b]. The response-response relationship for Vatalnib in zebrafish was maintained for dysmorphogenesis at 120 hpf (0.22 µM) and adult survival curves at 10 days (0.70 µM) [Tal et al. 2014]. While Vatalanib inhibits both VEGFR-2 and PDGFRβ, it is most selective for VEGFR-2 [Wood et al. 2000].

Shirinifard et al. [2013] examined angiogenic sprouting dynamics in zebrafish embryos exposed to high concentrations of arsenic (As). This resulted in a suppressed but chaotic pattern of ISV outgrowth. Quantitative mathematical models inferred increased exploratory filopodial behaviors of EC-tip cells accounting for the loss of directional sensing of during ISV outgrowth [Shirinifard et al. 2013]. The chaotic versus ordered EC-tip cell dynamics may be mechanistically linked to key modulatory factors that regulate the cytoskeletal cycle and/or cell-ECM biomechanics. Molecular pathways such as the Aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor-1 alpha (HIF-1α) that control genes in response to xenobiotic metabolism, hypoxia, and hypoglycemia have potential feedback roles. These pathways regulate genes in developmental angiogenesis. For example, functional inactivation of ARNT, the AhR nuclear translocator protein, results in critical embryonic vascular phenotypes in the yolk sac and branchial arches reminiscent of those observed in mouse embryos deficient in VEGF-signaling [Maltepe et al. 1997].

Domain of Applicability: The de novo assembly of endothelial cells into the primitive capillary network in an early embryo (vasculogenesis) or a tubular network in vitro (tubulogenesis) are both driven by VEGF-A signaling. A critical effect on developmental angiogenesis aligns with the Gene Ontology (GO) term ‘negative regulation of blood vessel morphogenesis’ (GO:0016525), defined as “Any process that stops, prevents, or reduces the frequency, rate or extent of angiogenesis”. Differences exist among the 110 genes mapped to this annotation in the Mouse Gene Ontology Browser (http://www.informatics.jax.org/vocab/gene_ontology/, last accessed November 30, 2021). Although the genetic signals and responses may differ between vasculogenesis and angiogenesis [Drake et al. 2007; Knudsen and Kleinstreuer, 2011], disruption of the former process ultimately leads to a reduction in the latter during development and so both are in the DoA for this KER.

Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

The control of EC-tip cell dynamics is a central feature linking VEGFR-2 inhibition (MIE:305) to adverse angiogenic sprouting behaviors (AE:28) [Argraves et al. 2002; Williams et al. 2006; Eilken and Adams, 2010; Oladipupo et al. 2011; Venkatraman et al. 2016; Beloglazova et al. 2021].

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Many physiological states influence VEGF-A production (e.g., hypoxia, estrogen) and post-VEGFR2 signaling. For example, VEGFR2 signals may be influenced by crosstalk with VEGFR1 and VEGFR3, other receptor tyrosine kinases (FGFR, EGFR), G-protein coupled receptors (CXCRs and CCRs), and GPI-linked surface receptors (uPAR) [Kleinstreuer et al. 2011]. The ToxCast pVDC signature includes assays for many of these targets and shows that environmental chemicals perturbing VEGFR2 also affect molecular targets in other signaling system [Knudsen et al. 2016]. Crosstalk between VEGFR-2 and other pro-angiogenic receptor tyrosine kinase (RTK) activities such as PDGFR or FGFR is known. This crosstalk has been embraced in the search for clinically efficacious synergistic kinase anti-angiogenesis strategies in suppressing tumorigenic growth [Lin et al. 2018] but is an uncertainty for establishing a role for KER:335 in the disruption of blood vessel morphogenesis (KE:28). For example, the fungal metabolite Epoxyquinol B inhibits kinase activity across several RTKs including VEGFR and PDGFR and blocks VEGF-induced migration and tubulogenesis in human umbilical vein endothelial cells (HUVECs) [Kamiyama et al. 2008]. Anlotinib inhibits cell migration and microvessel formation in the rat aortic ring assay and chicken chorioallantoic membrane assay via the ERK signaling pathway in both species [Lin et al. 2018]. Derazantinib at 0.1 µM to 3 µM blocked intersegmental vessel (ISV) migration linked to VEGF, PDGF, or FGF pathways in zebrafish embryos [Kotini et al. 2020].

Still other pathways may be relevant with regards to developmental angiogenesis. For example, the endothelial TIE2 receptor is essential for ISV outgrowth in zebrafish embryos [Li et al. 2014] and TGFβ1 signaling in the formation of tubular networks in human vascular endothelial cells (HUVECs) [Zhang et al. 2021]. VEGF-dependent cell migration in HUVECs is also facilitated by the urokinase-type plasminogen activator receptor (uPAR), a system linked to cell-ECM interactions and Notch components: Notch1 receptor and ligands (Dll1, Dll4, Jag1) in endothelial cells on one hand, and uPA, uPAR, TGFβ1, integrin β3, Jag1, Notch3 receptor in mural cells on the other hand [Beloglazova et al. 2021]. Both an increase on pro-angiogenic factors as well as a decrease in anti-angiogenic factors (Notch signaling) can have similar outcomes. Crosstalk in these heterogeneous systems point to cell-specific patterns of gene expression as a critical determinant of RTK expression and cell-type specificity. As such, quantitative linkages to VEGF signaling must consider the uncertainties from effects to other MIEs.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Time-scale
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help
Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Domain of Applicability: The de novo assembly of endothelial cells into the primitive capillary network in an early embryo (vasculogenesis) or a tubular network in vitro (tubulogenesis) are both driven by VEGF-A signaling. A critical effect on developmental angiogenesis aligns with the Gene Ontology (GO) term ‘negative regulation of blood vessel morphogenesis’ (GO:0016525), defined as “Any process that stops, prevents, or reduces the frequency, rate or extent of angiogenesis”. Differences exist among the 110 genes mapped to this annotation in the Mouse Gene Ontology Browser (http://www.informatics.jax.org/vocab/gene_ontology/, last accessed November 30, 2021). Although the genetic signals and responses may differ between vasculogenesis and angiogenesis [Drake et al. 2007; Knudsen and Kleinstreuer, 2011], disruption of the former process ultimately leads to a reduction in the latter during development and so both are in the DoA for this KER.

References

List of the literature that was cited for this KER description. More help

Argraves, W. S., Larue, A. C., Fleming, P. A. et al. (2002). 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 225, 298-304. doi:10.1002/dvdy.10162. PMID:12412012

Belair, D. G., Schwartz, M. P., Knudsen, T. et al. (2016). Human ipsc-derived endothelial cell sprouting assay in synthetic hydrogel arrays. Acta biomaterialia 39, 44554-44554. doi:10.1016/j.actbio.2016.05.020. PMID:27181878

Beloglazova, I., Stepanova, V., Zubkova, E. et al. (2021). Mesenchymal stromal cells enhance self-assembly of a huvec tubular network through upa-upar/vegfr2/integrin/notch crosstalk. Biochimica et biophysica acta. Molecular cell research 1869, 119157-119157. doi:10.1016/j.bbamcr.2021.119157. PMID:34619163

Blanco, R. and Gerhardt, H. (2013). Vegf and notch in tip and stalk cell selection. Cold Spring Harbor Perpect Med 3, a006569-a006569. doi:10.1101/cshperspect.a006569. PMID:23085847

Carmeliet, P., Ferreira, V., Breier, G. et al. (1996). Abnormal blood vessel development and lethality in embryos lacking a single vegf allele. Nature 380, 435-439. doi:10.1038/380435a0. PMID:8602241

Chappell, J. C., Taylor, S. M., Ferrara, N. et al. (2009). Local guidance of emerging vessel sprouts requires soluble flt-1. Developmental cell 17, 377-386. doi:10.1016/j.devcel.2009.07.011. PMID:19758562

Chappell, J. C., Cluceru, J. G., Nesmith, J. E. et al. (2016). Flt-1 (vegfr-1) coordinates discrete stages of blood vessel formation. Cardiovascular research 111, 84-93. doi:10.1093/cvr/cvw091. PMID:27142980

Drake, C. J., Fleming, P. A. and Argraves, W. S. (2007). The genetics of vasculogenesis. Novartis Foundation symposium 283, 61-71; discussion 71. doi:10.1002/9780470319413.ch6. PMID:18300414

Eilken, H. M. and Adams, R. H. (2010). Dynamics of endothelial cell behavior in sprouting angiogenesis. Current opinion in cell biology 22, 617-625. doi:10.1016/j.ceb.2010.08.010. PMID:20817428

Ferrara, N., Carver-Moore, K., Chen, H. et al. (1996). Heterozygous embryonic lethality induced by targeted inactivation of the vegf gene. Nature 380, 439-442. doi:10.1038/380439a0. PMID:8602242

Fong, G. H., Rossant, J., Gertsenstein, M. et al. (1995). Role of the flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66-70. doi:10.1038/376066a0. PMID:7596436

Herbert, S. P. and Stainier, D. Y. (2011). Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nature reviews. Molecular cell biology 12, 551-564. doi:10.1038/nrm3176. PMID:21860391

Kamiyama, H., Kakeya, H., Usui, T. et al. (2008). Epoxyquinol b shows antiangiogenic and antitumor effects by inhibiting vegfr2, egfr, fgfr, and pdgfr. Oncology research 17, 44521-44521. doi:10.3727/096504008784046063. PMID:18488711

Kleinstreuer, N. C., Judson, R. S., Reif, D. M. et al. (2011). Environmental impact on vascular development predicted by high-throughput screening. Environmental health perspectives 119, 1596-1603. doi:10.1289/ehp.1103412. PMID:21788198

Knudsen, T. B., Martin, M. T., Kavlock, R. J. et al. (2009). Profiling the activity of environmental chemicals in prenatal developmental toxicity studies using the u.S. Epa's toxrefdb. Reproductive toxicology (Elmsford, N.Y.) 28, 209-219. doi:10.1016/j.reprotox.2009.03.016. PMID:19446433

Knudsen, T. B. and Kleinstreuer, N. C. (2011). Disruption of embryonic vascular development in predictive toxicology. Birth defects research. Part C, Embryo today : reviews 93, 312-323. doi:10.1002/bdrc.20223. PMID:22271680

Kotini, M. P., Bachmann, F., Spickermann, J. et al. (2020). Probing the effects of the fgfr-inhibitor derazantinib on vascular development in zebrafish embryos. Pharmaceuticals (Basel, Switzerland) 14, doi:10.3390/ph14010025. PMID:33396726

Li, W., Chen, J., Deng, M. et al. (2014). The zebrafish tie2 signaling controls tip cell behaviors and acts synergistically with vegf pathway in developmental angiogenesis. Acta biochimica et biophysica Sinica 46, 641-646. doi:10.1093/abbs/gmu055. PMID:25001479

Lin, B., Song, X., Yang, D. et al. (2018). Anlotinib inhibits angiogenesis via suppressing the activation of vegfr2, pdgfrß and fgfr1. Gene 654, 77-86. doi:10.1016/j.gene.2018.02.026. PMID:29454091

Maltepe, E., Schmidt, J. V., Baunoch, D. et al. (1997). Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein arnt. Nature 386, 403-407. doi:10.1038/386403a0. PMID:9121557

Oladipupo, S., Hu, S., Kovalski, J. et al. (2011). Vegf is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting. Proceedings of the National Academy of Sciences of the United States of America 108, 13264-13269. doi:10.1073/pnas.1101321108. PMID:21784979

Shalaby, F., Rossant, J., Yamaguchi, T. P. et al. (1995). Failure of blood-island formation and vasculogenesis in flk-1-deficient mice. Nature 376, 62-66. doi:10.1038/376062a0. PMID:7596435

Shirinifard, A., McCollum, C. W., Bolin, M. B. et al. (2013). 3d quantitative analyses of angiogenic sprout growth dynamics. Developmental dynamics : an official publication of the American Association of Anatomists 242, 518-526. doi:10.1002/dvdy.23946. PMID:23417958

Tal, T. L., McCollum, C. W., Harris, P. S. et al. (2014). Immediate and long-term consequences of vascular toxicity during zebrafish development. Reproductive toxicology (Elmsford, N.Y.) 48, 51-61. doi:10.1016/j.reprotox.2014.05.014. PMID:24907688

Venkatraman, L., Regan, E. R. and Bentley, K. (2016). Time to decide? Dynamical analysis predicts partial tip/stalk patterning states arise during angiogenesis. PloS one 11, e0166489-e0166489. doi:10.1371/journal.pone.0166489. PMID:27846305

Williams, C. K., Li, J. L., Murga, M. et al. (2006). Up-regulation of the notch ligand delta-like 4 inhibits vegf-induced endothelial cell function. Blood 107, 931-939. doi:10.1182/blood-2005-03-1000. PMID:16219802

Wood, J. M., Bold, G., Buchdunger, E. et al. (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 research 60, 2178-2189. PMID:10786682

Zhang, Y., Liu, J., Zou, T. et al. (2021). Dpscs treated by tgf-ß1 regulate angiogenic sprouting of three-dimensionally co-cultured huvecs and dpscs through vegf-ang-tie2 signaling. Stem cell research & therapy 12, 281-281. doi:10.1186/s13287-021-02349-y. PMID:33971955