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|
Level of Biological Organization
How this Key Event works
Developmental angiogenesis involves a complex interplay between the native extracellular matrix, vascular endothelial cells (EC), growth factors, and cytokines/chemokines [Knudsen and Kleinstreuer, 2011]. In vitro models are presently used to study EC function and screen for angiogenesis inhibitors based on effects on cell proliferation, sprouting behavior, and tubulogenesis. Sprouting is driven by matrix metalloproteinase (MMP) activity whereas tubulogenesis shows MMP-dependence only in three-dimensional (3D) contexts. Furthermore, the VEGF-dependence is unclear in some tubulogenesis assay platforms and this further limits comparisons of EC tubulogenesis to VEGF-dependent vascular formation in vivo. As such, EC sprouting models that recapitulate MMP-dependent and VEGF-dependent endothelial cell invasion provide a more physiologic context for modeling early stages of angiogenesis and can be used to evaluate the extracellular matrix (ECM) degradation and invasion characteristic of angiogenic sprouting in vivo [Belair 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?
Functional assays used to evaluate angiogenic sprouting utilize natural (ECM) and synthetic (hydrogel) matrices that support growth factor-dependent endothelial cell proliferation, migration and invasive behaviors. EC sprouting models that recapitulate matrix metalloproteinase-dependent and VEGF-dependent endothelial cell invasion provide a physiologic context for modeling early stages of angiogenesis. Endothelial cell migration is directed by chemotactic, haptotactic, and mechanotactic stimuli and degradation of the ECM to enable progression of the migrating cells. It requires the activation of several signaling pathways that converge on cytoskeletal remodeling and follows a molecular cascade in which the endothelial cells extend filopodial processes and progress forward [Lamalice et al. 2007]. Pro-angiogenic signals, such as VEGF-A, together with Notch signaling controls whether specific endothelial cells become leading 'tip cells' or trailing 'stalk cells'. Angiogenic sprouts then convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions [Eilken and Adams, 2010]. A unique method to encapsulate endothelial cells at a controlled cell density in hydrogel spheres surrounded by a synthetic ECM allows for quantitative analysis of EC sprouting for enhanced-throughput screening in a chemically-defined sprouting model [Belair et al. 2016]. Another approach to detecting effects on angiogenic sprouting dynamics is live-cell imaging in transgenic zebrafish embryos [Shirinfard et al. 2013].
Evidence Supporting Taxonomic Applicability
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 angiogenic sprouting in human endothelial cells [Belair et al. 2016] and trangenic zebrafish embryos [Tal et al. 2016].
Belair et al.  designed and characterized a chemically human angiogenesis pPSC-EC sprouting model that responded appropriately to several reference pharmacological angiogenesis inhibitors, including Vatalanib/PTK787, which suggests the functional role of VEGFR2. Several pVDCs from the ToxCast library also inhibited angiogenic sprouting in this assay. 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. Homology appeared first in the receptor tyrosine kinase signaling systems, followed in turn by the urokinase plasminogen activating (uPA) receptor (uPAR) system and chemokine/G-protein coupled receptor system.
Belair DG, Schwartz MP, Knudsen TB, Murphy WL. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays. Acta Biomaterialia 2016 (in press).
Knudsen TB, Kleinstreuer NC. Disruption of embryonic vascular development in predictive toxicology. Birth defects research Part C, Embryo today: reviews. 2011 Dec;93(4):312-23. PubMed PMID: 22271680.
Lamalice L, Le Boeuf F and Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007 Mar 30;100(6):782-94.
Eilken HM and Adams RH. Dynamics of endothelial cell behavior in sprouting angiogenesis. Curr Opin Cell Biol. 2010 Oct;22(5):617-25. doi: 10.1016/j.ceb.2010.08.010.
Shirinifard A, McCollum CW, Bondesson MB, Gustafsson JA, Glazier JA and Clendenon SG. 3D Quantitative analyses of angiogenic sprout growth dynamics Devel Dynam. 2013 242(5): 518-526.
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).