Upstream eventReduction, Angiogenesis
Impairment, Endothelial network
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Directness||Weight of Evidence||Quantitative Understanding|
|Disruption of VEGFR Signaling Leading to Developmental Defects||directly leads to||Strong||Moderate|
Life Stage Applicability
How Does This Key Event Relationship Work
Angiogenic sprouting dynamics drives the complexity and connectivity of endothelial networks in vivo and in vitro. This process is dependent on rates of endothelial tip cell migration and stalk cell proliferation, as well as differential cell adhesion.
Weight of Evidence
Endothelial network formation is dependent on proper regulation of angiogenic sprouting.
Empirical Support for Linkage
Compounds that disrupt angiogenic sprouting behaviors [Belair et al. 2016] also disrupt endothelial tubular network formation [Nguyen et al. 2016].
Uncertainties or Inconsistencies
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.
Evidence Supporting Taxonomic 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).