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Inhibition, VegfR2 leads to Reduction, Angiogenesis
Key Event Relationship Overview
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||Cataia Ives (send email)||Open for citation & comment||EAGMST Under Review|
Life Stage Applicability
Key Event Relationship Description
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
Evidence Supporting this KER
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.  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.
VEGFR2 is the most important VEGF-A receptor and is the 'master switch' for angiogenic sprouting [Herbert and Stanier 2011].
Uncertainties and Inconsistencies
Many physiological signals 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 some other signaling system [Knudsen et al. 2016]. As such, quantitative linkages to VEGF signaling must consider the uncertainties from effects to other MIEs.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Studies have demonstrated a quantitative relationship between VEGFR2 signaling and angiogenic sprouting dynamics in human endothelial cells [Belair et al. 2016] and zebrafish embryos [Shirinifard et al. 2013].
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).
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.
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.
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.
Herbert SP and Stainier DY. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol. 2011 Aug 23;12(9):551-564. doi: 10.1038/nrm3176.
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.
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.
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.