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Event: 28

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Reduction, Angiogenesis

Short name

The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. More help

Cell term
stromal cell

Organ term

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help

Process	Object	Action
angiogenesis		decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Developmental Vascular Toxicity	KeyEvent	Tom Knudsen (send email)	Open for citation & comment	EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Developmental angiogenesis involves a complex interplay between the native extracellular matrix, vascular endothelial cells (EC), growth factors, and cytokines/chemokines [Knudsen and Kleinstreuer, 2011].

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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].

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].

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information. More help

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. [2016] 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. 2017]. 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.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Belair DG, Schwartz MP, Knudsen T and Murphy WL. Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays. Acta Biomater. 2016; 39: 12-24. PMID:27181878.

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 CW, Bondesson M, Knudsen T, Padilla S and Kleinstreuer N. Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity. Reprod Toxicol. 2017; 70: 70-81. PMID:28007540.