Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Disruption of VEGFR Signaling Leading to Developmental Defects||KE||Moderate|
|Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty||KE||Moderate|
Level of Biological Organization
How this Key Event works
In embryological terms the angiogenic cycle entails a stepwise progression of de novo blood vessel morphogenesis (vasculogenesis), maturation and expansion (angiogenesis), and remodeling [Hanahan, 1997; Chung and Ferrara 2011; Coultas et al. 2005]. These events commence as angioblasts migrate, proliferate, and assemble into a tubular network. With maturation, the endothelial tubules co-opt local stromal cells as pericytes and smooth muscle. Local signals acting on receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and glycosyl phosphatidyl-inositol (GPI)-anchored receptors, and later vascular flow-mediated signals. The process of endothelial assembly into a tubular network may be disrupted by environmental agents [Sarkanen et al. 2010; Bondesson et al. 2016; Knudsen et al. 2016; Nguyen et al. 2016; Tal et al. 2016].
How it is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
Endothelial tubule formation (tubulogenesis) can be monitored both qualitatively and quantitatively in vitro using different human cell-based angiogenesis assays that score endothelial cell migration and the degree of tubular network formation, including cell counts, tubule counts, tubule length, tubule area, tubule intensity, and node counts [Muller et al. 2002; Masckauchan et al. 2005; Sarkanen et al. 2010; Knudsen et al. 2016; Nguyen et al. 2016]. Standard practice for reproducible in vitro tubule formation uses endothelial cells co-cultured with stromal cells [Bishop et al. 1999]. Cell types commonly employed are human umbilical endothelial cells (HUVECs) or more recently induced pluripotent stem cells (iPSCs) derived to endothelial cells through various differentiation and purification protocols. The assay is run in agonist or antagonist modes to detect chemical enhancement or suppression of tubulogenesis. Synthetic hydrogels are shown to promote robust in vitro network formation by HUVEC or iPSC-ECs as well as their utilization to detect putative vascular disruptive compounds [Nguyen et al. 2016]. Endothelial networks formed on synthetic hydrogels showed superior sensitivity and reproducibility when compared to endothelial networks formed on Matrigel.
Evidence Supporting Taxonomic Applicability
Blood vessel development utilizes highly conserved molecular pathways that are active across vertebrate species. Anatomically, however, the molecular toolbox for vasculogenesis/angiogenesis has varied themes for arterial, venous, and lymphatic channels, as well as across different organs and species [Tal et al. 2016]. ToxCast high-throughput screening (HTS) data for 25 assays mapping to targets in embryonic vascular disruption signature [Knudsen and Kleinstreuer, 2011] were used to rank-order 1060 chemicals for their potential to disrupt vascular development. The predictivity of this signature is being evaluated in various angiogenesis assays, including tubulogenesis in endothelial cells from zebrafish, chick, mouse and human species [Tal et al. 2016; Vargesson et al. 2003; Knudsen et al. 2016; McCollum et al. 2016; Nguyen et al. 2016]. As an example, 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 pVDCs inhibited endothelial tubulogenesis in an yolk-sac-derived endothelial cell line [McCollum et al. 2016]. The VEGF pathway was implicated across mouse-zebrafish species. Because gene sequence similarity of the ToxCast pVDC signature is comprised of proteins that primarily map to human in vitro and biochemical assays, the U.S. EPA SeqAPASS tool was used to assess the degree of conservation of signature targets between zebrafish and human, as well as other commonly used model organisms in human health and environmental toxicology research [Tal et al. 2016]. This approach revealed that key nodes in the ontogenetic regulation of angiogenesis have evolved across diverse species.
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Chung AS, Ferrara N. Developmental and pathological angiogenesis. Annual review of cell and developmental biology. 2011;27:563-84. PubMed PMID: 21756109.
Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development. Nature. 2005 Dec 15;438(7070):937-45. PubMed PMID: 16355211.
Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 1997 Jul 4;277(5322):48-50. PubMed PMID: 9229772.
Knudsen TB, Kleinstreuer NC. Disruption of embryonic vascular development in predictive toxicology. Birth defects research Part C, Embryo today : reviews. 2011;93(4):312-23.
Knudsen TB, Tomela T, Sarkanen R, Spencer RS, Baker NC, Franzosa J, Tal T, Kleinstreuer NC, Cai B, Berg EL and Heinonen. ToxCast Model-Based Prediction of Human Vascular Disruption: Statistical Correlation to Endothelial Tubulogenesis Assays and Computer Simulation with Agent-Based Models. 2016 (in preparation).
Masckauchan TN, Shawber CJ, Funahashi Y, Li CM, Kitajewski J. Wnt/beta-catenin signaling induces proliferation, survival and interleukin-8 in human endothelial cells. Angiogenesis. 2005;8(1):43-51. PubMed PMID: 16132617.
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).
Muller T, Bain G, Wang X, Papkoff J. Regulation of epithelial cell migration and tumor formation by beta-catenin signaling. Experimental cell research. 2002 Oct 15;280(1):119-33. PubMed PMID: 12372345.
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).
Vargesson N. Vascularization of the developing chick limb bud: role of the TGFβ signalling pathway. J Anat. 2016 Jan, 202(1): 93-103. PMCID: PMC1571066.