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Reduction, Angiogenesis leads to Impairment, Endothelial network
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||Moderate||Tom Knudsen (send email)||Open for citation & comment||EAGMST Under Review|
Life Stage Applicability
Key Event Relationship Description
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
Evidence Supporting this KER
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).
Endothelial network formation is dependent on proper regulation of angiogenic sprouting.
Compounds that disrupt angiogenic sprouting behaviors [Belair et al. 2016] also disrupt endothelial tubular network formation [Nguyen et al. 2016].
Uncertainties and Inconsistencies
Known modulating factors
Quantitative Understanding of the Linkage
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.
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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].
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).