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Relationship: 36

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

Reduction, Angiogenesis leads to Impairment, Endothelial network

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Reduction, Angiogenesis

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Impairment, Endothelial network

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	Moderate	Cataia Ives (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 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

Blood vessel morphogenesis requires coordinated control of endothelial cell (EC) and supportive mural cells staged to develop interconnected networks required for a fully functional circulatory system. Formation of endothelial networks in vivo and in vitro are dependent on VEGF-Notch-Dll4 signaling that determines EC specification and sprouting outgrowth to form microvessels that lumenize for blood circulation. Cell motility, proliferation, differential cell adhesion) are indispensable for multicellular tubular networks to emerge in vivo or in vitro [Nguyen et al. 2017; Toimela et al. 2017; Pauty et al. 2018; van Duinen et al. 2019a and 2019b; Zurlinden et al. 2020]. In HUVEC cells, VEGFR2 activates phospholipase PLCβ3 generating a second messenger (inositol-3-phosphate) that promotes EC migration (CDC42 activation) and suppresses EC proliferation (cell cycle progression) [Bhattacharya et al. 2009]. The ephrins couple VEGF signaling to endothelial patterning [Patan, 2000]. Unlike VEGFR2 activation, EPH-class receptor tyrosine kinase activation requires direct contact between cells expressing a receptor (EPH) and complementary ligand (EFN). Ephrin-B4 expression (Efnb4) in the mouse embryo co-localizes with its Ephb2 receptor in developing arterial endothelial cells and with its Ephb4 receptor in prospective venous endothelial cells. This partitioning of prospective arterial and venous counterparts stimulates microvascular density [Wang et al. 1998]. A ToxCast signature for embryonic vascular disruption (pVDCs) built with bioactivity profiling data from functional assays on genes for developmental angiogenesis was 87% accurate when anchored to empirical observations on 38 chemicals summed across 10 in vitro platforms across endothelial network formation [Saili et al. 2019].

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: Endothelial network formation is dependent on proper regulation of angiogenic sprouting. Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic [Muller et al. 2002].

Empirical Evidence: Compounds that disrupt angiogenic sprouting behaviors [Belair et al. 2016] also disrupt endothelial tubular network formation [Nguyen et al. 2016]. Activation of VEGFA signaling expands the arterial cell population at the expense of venous cells during vasculogenesis of the axial vessels in zebrafish; Vegfa deficiency interferes with the pathfinding of intersegmental vessels (ISVs) and a loss of a cranial vasculature [Jin et al. 2017]. A zebrafish embryo vascular model in conjunction with a mouse endothelial cell model revealed a plethora of vascular perturbations including malformed ISVs, uncondensed caudal vein plexus, hemorrhages and cardiac edema [McCollum et al. 2017]. Ephrin-B4 expression (Efnb4) in the mouse embryo co-localizes with its Ephb2 receptor in developing arterial endothelial cells and with its Ephb4 receptor in prospective venous endothelial cells. This partitioning of prospective arterial and venous counterparts stimulates microvascular density [Wang et al. 1998].

Uncertainties and Inconsistencies: Downregulating the VEGF signaling pathway in early zebrafish embryos, while affecting the number of angioblasts, did not appear to affect their migratory behaviors [Jin et al. 2005]. These findings indicate that chemical effects on developmental angiogenesis may be cell-specific, stage-dependent, and regionally selective. The progression of chemical effects on blood vessel morphogenesis in vivo is complicated by uncertainties that reflect the recovery potential or natural selection of an exposed embryo. Improved molecular understanding is necessary to understand the complex variables for these effects.

Quantitative Understanding of the Linkage: A ToxCast signature for potential Vascular Disrupting Chemical (pVDC) [Knudsen and Kleinstreuer, 2011; Kleinstreuer et al. 2013] has been tested for predictivity [Saili et al. 2019]. The pVDC signature included biochemical features for three receptor systems prominent in developmental angiogenesis (receptor tyrosine kinases for growth factor signals; the urokinase-type plasminogen activator (uPA) system that functions in VEGFR2-induced changes to focal adhesion and extracellular matrix (ECM) degradation during sprout progression; and G-protein coupled receptors (GPRCs) for angiogenic cytokines and chemokines) [Knudsen et al. 2011; Sipes et al. 2013; Kleinstreuer et al. 2014] (see image below). The battery of assays represented 21 ToxPi slices (see below) for a ToxPi [Marvel et al. 2018] based profile of Aop43 in sectors for G-protein coupled receptors (red-orange), receptor tyrosine kinases (blue-purple), and uPAR system (green-yellow) [Knudsen and Kleinstreuer, 2011; Kleinstreuer et al. 2013]. 38 ToxCast chemicals were selected for targeted testing by different laboratories having expert-qualified in vitro assays that are sensitive to, or specific for, different stages of the angiogenesis cycle (e.g., activation, sprouting, migration, tubulogenesis, vascular patterns). The ToxPi prediction was 87% accurate when in vitro observations were summed across all 10 platforms [Saili et al. 2019]. This shows the value of Aop43 in combining HTS data from ToxCast with biological knowledge of the angiogenesis cycle derived from curated knowledge from genetic mouse models – in this case for developmental angiogenesis, that establishes a course of predictivity from sprouting to patterning [Saili et al. 2019]. The U.S. EPA SeqAPASS tool revealed how the genetic signature may have evolved phylogenetically [Tal et al. 2017].

Response-response Relationship: Consequences of Vatalnib exposure to early zebrafish embryos was maintained for inhibition of ISV sprouting progression (0.07 µM) at 72 hours post-fertilization (hpf), dysmorphogenesis at 120 hpf (0.22 µM), and adult survival at 10 days (0.70 µM) [Tal et al. 2014]. The progression of critical concentrations through development and adult stages may be explained by recovery or natural selection processes.

Known modulating factors: The importance of canonical and non-canonical Wnt signaling in embryonic development and tissue homeostasis is widely known for its ability to influence cell movement, ECM degradation and paracrine signaling [Sedgwick et al. 2016]. Differences in Wnt signaling could, for example, contribute to the differential recovery processes in the embryo across space and time.

Domain of Applicability: Morphology of endothelial networks with regards to their completeness and complexity is a feature dependent on cell-cell signaling within the endothelial network as well as their microenvironment with regards to the ECM and other cell types. A critical effect on developmental angiogenesis aligns with the Gene Ontology (GO) term GO:001885 ‘endothelial cell development’, which is defined as “The progression of an endothelial cell over time, from its formation to the mature structure” and/or GO:0045601, ‘regulation of endothelial cell differentiation’, defined as “Any process that stops, prevents, or reduces the frequency, rate or extent of endothelial cell differentiation”. Differences exist among the 119 genes mapped to this annotation in the Mouse Gene Ontology Browser (http://www.informatics.jax.org/vocab/gene_ontology/, last accessed November 30, 2021).

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

Endothelial network formation is dependent on proper regulation of angiogenic sprouting.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Compounds that disrupt angiogenic sprouting behaviors [Belair et al. 2016] also disrupt endothelial tubular network formation [Nguyen et al. 2016].

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

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

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Linking the ToxCast assays from the putative Vascular Disrupting Chemical (pVDC) signature to sprouting:

The ephrins (EFNA1 and EFNB2 in particular) couple VEGF signaling to angiogenic sprouting during early development of the embryonic vasculature (vasculogenesis, angiogenesis). The ToxCast pVDC signature included features for EPH-receptor tyrosine kinase biochemical activity (increased or decreased) for receptors EPHA1, EPHA2, EPHB1 and EPHB2 via their cognate cell membrane-anchored ligands (EFNAs). In contrast to the ephrin system, a number of chemicals had activity on diverse assays for urokinase-type plasminogen activator (uPA). That system, consisting of uPA (4 features) and its GPI-anchored receptor, uPAR (8 features) - both assayed in the BioMAP System [Kleinstreuer et al. 2014], functions in VEGFR2-induced changes to focal adhesion and extracellular matrix (ECM) degradation at the leading edge of endothelial cells during angiogenic sprouting. Binding of uPA to uPAR results in serine-protease conversion of plasminogen to plasmin that initiates a proteolytic cascade leading to degradation of the basement membrane and angiogenic sprouting. The uPA proteolytic cascade is suppressed by the serine protease inhibitor, endothelial plasminogen activator inhibitor type 1 (PAI1). The PAI1/uPA/uPAR assays report chemical effects on the system (up or down) across diverse cellular platforms: 4H, 3C, CASM3C, and hDFCGF noted above; BE3C (human bronchial epithelial cells stimulated with IL-1β + TNFα + IFNϒ); and KF3T (human keratinocytes + fibroblasts stimulated with IL-1β + TNFα + IFNϒ + TGF-β). The pVDC signature has features for thrombomodulin (THBD) and the thromboxane A2 (TBXA2) receptor that participate in the regulation of endothelial migration during angiogenic sprouting. THBD is a type I transmembrane glycoprotein that mediates regulator of uPA/uPAR and TBXA2 is an angiogenic eicosanoid generated by endothelial cyclooxygenase-2 (COX-2) following VEGF- or bFGF stimulation. THBD protein expression was monitored in the 3C and CASM3C BioMAP systems (up, down) and TBXA2 was assayed for ligand binding in the NovaScreen platform.

Angiogenic cytokines and chemokines: the pVDC signature aggregates features for LPS-induced TNFα protein expression (see BioMAP descriptor above), nuclear factor-kappa B (NFkB) mediated reporter gene activation (Attagene; cis- configuration), and caspase 8 enzymatic activity (NovaScreen; inhibition or activation). TNFα is a proinflammatory cytokine that can promote angiogenesis indirectly through NFkB-mediated expression of angiogenic growth factors, or inhibit angiogenesis by direct effects on endothelial proliferation and survival. The pVDC signature also aggregates features for signaling activity of the pro-angiogenic cytokines interleukin-1 alpha (IL1a, a macrophage-derived activator of TNFα) and interleukin 6 (IL6). These cytokines act through the G-protein coupled receptors (GPCRs) IL1R and IL6R, respectively. CXCL8 (chemokine (C-X-C motif) ligand 8), formerly known as interleukin 8 (IL8), is angiogenic through its cognate GPCRs (CXCR1, CXCR2). In contrast to CXCL8, the chemokines CXCL9 (alias MIG, monokine induced by IFNϒ) and CXCL10 (alias IP10, interferon-inducible cytokine IP-10) are considered anti-angiogenic through their cognate receptor, CXCR3.

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

Blood vessel development utilizes highly conserved molecular pathways that are active across vertebrate species. A zebrafish embryo vascular model in conjunction with a mouse endothelial cell model identified 28 potential vascular disruptor compounds (pVDCs) from ToxCast. These exposures invoked a plethora of vascular perturbations in the zebrafish embryo, including malformed intersegmental vessels, uncondensed caudal vein plexus, hemorrhages and cardiac edema; 22 of the also inhibited endothelial endothelial tubulogenesis in an yolk-sac-derived endothelial cell line [McCollum et al. 2016]. The U.S. EPA SeqAPASS tool revealed that key nodes in the ontogenetic regulation of angiogenesis have evolved across diverse species [Tal et al. 2016].

References

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

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).

Kleinstreuer NC, Yang J, Berg EL, Knudsen TB, Richard AM, Martin MT, et al. Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms. Nat Biotechnol. 2014 Jun;32(6):583-91. PubMed PMID: 24837663.

McCollum CW, Vancells JC, Hans C, Vazquez-Chantada M, Kleinstreuer N, Tal T, Knudsen T, Shah SS, Merchant FA, Finnell RH, Gustafsson JA, Cabrera R and Bondesson M. Identification of vascular disruptor compounds by a tiered analysis in zebrafish embryos and mouse embryonic endothelial cells. 2016 (in preparation).

Nguyen EH, Daly WT, Le NNT, Belair DG, Schwartz MP, Lebakken CS, Ananiev GE, Saghiri A, Knudsen TB, Sheibani N and Murphy WL. Identification of a synthetic alternative to matrigel for the screening of anti-angiogenic compounds. 2016 (in preparation).

Tal T, Kilty C, Smith A, LaLone C, Kennedy B, Tennant A, McCollum C, Bondesson M, Knudsen T, Padilla S and Kleinstreuer N. Screening for chemical vascular disruptors in zebrafish to evaluate a predictive model for developmental vascular toxicity. Reprod Toxicol (submitted).