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Relationship: 1036

Title

The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”. More help

Insufficiency, Vascular leads to Increased, Developmental Defects

Upstream event

Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Insufficiency, Vascular

Downstream event

Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Increased, Developmental Defects

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Disruption of VEGFR Signaling Leading to Developmental Defects	non-adjacent	High	Moderate	Tom Knudsen (send email)	Open for citation & comment	EAGMST Under Review

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

Blood vessels in a developing embryo change to accommodate rapid growth, morphogenesis and differentiation. The importance of development and maintenance of the vasculature is evident in the association between developmental defects and vascular insufficiency, particularly arterial dysgenesis, derived by experimental teratogenesis and inferred in clinical teratology [Vargesson and Hootnick, 2017]. Several known anti-angiogenic compounds have been shown to cause dose-dependent developmental defects in various animal models (e.g., zebrafish, frog, chick, mouse, rat) [Therapontos et al. 2009; Jang et al. 2009; Rutland et al. 2009; Tal et al. 2014; Vargesson, 2015; Beedie et al. 2016; Ellis-Hutchings et al. 2017; Kotini et al. 2020]. Human studies of malformations showed a correlation with genetic and/or environmental factors that target vascular development [Husain et al. 2008; Gold et al. 2011]. Broad analysis of medicinal compounds to which women of reproductive age were exposed identified ‘vascular disruption’ as one of six potential mechanisms of teratogenesis [van Gelder et al. 2010].

Evidence Supporting this KER

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help

Biological Plausibility: A failure of correct vessel patterning, vessel occlusion in the embryo, or placental defects limiting maternal-fetal nutrition could result in tissue damage to an embryo invoking malformations and other developmental defects at critical periods of development. This perhaps best known for limb reduction defects (e.g., phocomelia) following thalidomide exposure during early limb development, when the critical response coincides with nascent vascular patterning prior to innervation [Therapontos et al. 2009]. At this stage, the early limb-bud receives its blood supply from a single axial artery at which time the undifferentiated mesenchyme is perfused by a simple capillary network. Susceptibility to thalidomide-induced dysmorphogenesis declines as the vascular pattern transitions to a more complex and definitive system of maturing vessels and emergence of the skeletal elements [Vargesson and Hootnick, 2017].

Biological Plausibility

Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information). More help

Empirical Evidence

In this section authors are encouraged to cite specific evidence that supports the idea that a change in the upstream KE (KEupstream) will lead to, or is associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. Given the likelihood that new empirical support will be developed over time, particularly as various AOPs are tested and applied, it is most practical to provide empirical support in the form of bulleted lists or tables that include a short description of the nature of the empirical support along with the corresponding reference(s).In particular, it is useful to cite evidence showing that stressors that perturb KEupstream also perturb KEdownstream. Because this section of the KER description cites evidence from specific studies, it is also helpful to provide as much detail about the toxicological and biological context in which the measurements were made, as is feasible, including the stressor(s) tested, the effective doses at each KE, etc. While the KER itself is not intended to be stressor-specific, those details can aid the overall assessment of the individual AOPs that include that KER. These details also help inform the question of consistency of supporting data, consistency across different biological contexts for which the KER is relevant, and the applicability domain of the KER. However, authors are cautioned that this evidence should focus on data that only relate KEupstream to KEdownstream, and should avoid reference to other KEs, KERs and AOPs as much as possible in order to maintain modularity of the KER. Please see pages 40-41 of the User Handbook for more information. More help

Empirical Evidence: Two lines of evidence support this KER for developmental vascular toxicity: (i) spatial correlation between altered vascular patterning and dysmorphogenesis; and (ii) concentration-dependent developmental toxicity with known anti-angiogenic compounds. Therapontos et al. [2009] determined that loss of immature blood vessels was the primary cause of thalidomide-induced teratogenesis in the chick limb, an effect phenocopied by anti-angiogenic but not anti-inflammatory metabolites/analogues of thalidomide. The thalidomide analog CPS49 suppressed chick limb-bud outgrowth only when the vasculature was at an immature stage of development; CPS49 did not suppress limb development post-innervation [Mahony et al. 2018]. Eight mechanistically diverse angiogenesis inhibitors (sunitinib, sorafenib, TNP-470, axitinib, pazopanib, vandetanib, everolimus, CPS49) suppressed vascularization and invoked dysmorphogenesis in a concentration-dependent manner in both the chick limb-bud and zebrafish embryo models [Beedie et al. 2016]. Vatalanib, a selective VEGFR2 antagonist, suppressed vascular development in zebrafish embryos at 0.07 µM leading to vascular insufficiency by 72 hours post-fertilization (hpf), foreshadowing dysmorphogenesis at 0.22 µM by 120 hpf reduced survival of 10-day adults at 0.70 µM [Tal et al. 2014]. A tiered study evaluated two anti-angiogenic agents, 5HPP-33, a synthetic Thalidomide analog [Noguchi et al. 2005] and TNP-470, a synthetic Fumagillan analog [Ingber et al. 1990] across several complex in vitro functional assays: rat aortic explant assay, rat whole embryo culture, and zebrafish embryotoxicity [Ellis-Hutchngs et al. 2017]. Both compounds disrupted angiogenesis and embryogenesis but with modal differences: 5HPP-33 was embryolethal, and TNP-470 dysmorphic. The former blocks tubulin polymerization [Yeh et al. 2000; Inatsuki et al. 2005; Kizaki et al. 2008; Rashid et al. 2015] and the latter is a methionine aminopeptidase II inhibitor that suppresses non-canonical Wnt signals for endothelial proliferation [Ingber et al. 1990]. Transcriptomic profiles of exposed embryos pathways unique to each and in common to both, strongest being the TP53 pathway [Saili et al. 2019]. In mouse, TNP-470 reduced fetal intraocular microvasculature and induced microphthalmia [Rutland et al. 2009], which is a TP53-dependent phenotype [Wubah et al. 1996].

Uncertainties and Inconsistencies

In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook). More help

Uncertainties and Inconsistencies: The cellular basis of tissue damage linked to vascular insufficiency is not well and represents a gap in understanding. During limb development, programmed cell death (PCD) contributes to separation of the digits. The onset of PCD is preceded by a genetically programmed increase of vascular density that directly determines with the extent of PCD and oxygen-dependent generation of reactive oxygen species (ROS) [Eshkar-Oren et al. 2015]. While many human and animal phenotypes associate with genetic signals and responses that control circulatory development, the causal relationship between vascular insufficiency and dysmorphogenesis is less understood due to various modes of tissue damage that may follow insufficient blood support (e.g., slow or weak heartbeat, poor vascularization, vessel occlusion, or reperfusion injury).

Quantitative Understanding of the Linkage

The quantitative understanding section of the KER description is intended to capture information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. Empirical support addresses whether data between the two KEs are consistent with the patterns that are expected if the upstream event is causing the downstream event to occur, while the quantitative understanding section helps to define the precision with which the state of the downstream KE can be predicted from knowledge of the state of the upstream KE. These quantitative relationships may be defined in terms of correlations, response-response relationships, dose-dependent transitions or points of departure (i.e., a threshold of change in KEupstream needed to elicit a change in KEdownstream), etc. They may take the form of simple mathematical equations or sophisticated biologically-based computational models that consider other modulating factors such as compensatory responses, or interactions with other biological or environmental variables. Regardless of form, the idea is to briefly describe what is known regarding the quantitative relationship between the KEs and cite appropriate literature that defines those relationships and/or provides support for them.Data that confer quantitative understanding of a KER are not necessarily mutually exclusive from those addressing other weight of evidence considerations. In that respect, the quantitative understanding section of the KER description is not intended to be redundant with the other WoE sections. Rather, it is intended to aid application of the AOP by allowing a reader to rapidly identify the relationships that would support a quantitative prediction of the probability or magnitude of change in KEdownstream based on a known state of KEupstream. For transparency, the toxicological and biological context in which the quantitative relationships were defined should be indicated within the description. However, the ultimate goal is to identify quantitative relationships that generalize across the entire applicability domain of the two KEs being linked via the KER. More help

Concentration-dependent linkages reported for at least 9 anti-angiogenic compounds in chick limb and/or zebrafish embryos with regards to both vascular suppression and dysmorphogenesis [Tal et al. 2014; Beedie et al. 2016]. The general response on endothelial cells preceded effects on morphogenesis. Potential modulating factors include species susceptibility and stage dependency. Developmental buffering (canalization) systems may support resilience to exposure via angio-adaptative recovery mechanisms that are spatially and temporally differentiated.

Response-response Relationship

This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture. More help

Time-scale

This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help

Known modulating factors

This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help

Known Feedforward/Feedback loops influencing this KER

This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook). More help

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

Wilson's Principles of Teratology (circa 1977) support the taxonomic applicability of teratogenesis. According to these long-standing Wilson's principles, the first on "Susceptibility to Teratogenesis Depends on the Genotype of the Conceptus and a Manner in which this Interacts with Adverse Environmental Factors". This principle has four main tenets: (i) species differences account for the fact that certain species respond to particular teratogens where others do not, or at least not to the same extent (e.g., humans and other primates are vulnerable to thalidomide induced phocomelia whereas rodents are not); (ii) strain and individual differences account for the fact that some lineages of the same species with different genetic backgrounds can differ in teratogenic susceptibility; (iii) gene-environment interplay results in different patterns of abnormalities between organisms with the same genome raised in different environments, and between organisms with different genomes raised in the same environment; and (iv) multifactorial causation accounts for the complex interactions involving more than one gene and/or more than one environmental factor.

References

List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help

Gold NB, Westgate MN, Holmes LB. Anatomic and etiological classification of congenital limb deficiencies. American journal of medical genetics Part A. 2011 Jun;155A(6):1225-35. PubMed PMID: 21557466.

Husain T, Langlois PH, Sever LE, Gambello MJ. Descriptive epidemiologic features shared by birth defects thought to be related to vascular disruption in Texas, 1996-2002. Birth defects research Part A, Clinical and molecular teratology. 2008 Jun;82(6):435-40. PubMed PMID: 18383510.

Kleinstreuer NC, Judson RS, Reif DM, Sipes NS, Singh AV, Chandler KJ, et al. Environmental impact on vascular development predicted by high-throughput screening. Environmental health perspectives. 2011 Nov;119(11):1596-603. PubMed PMID: 21788198. Pubmed Central PMCID: PMC3226499.

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.

Therapontos C, Erskine L, Gardner ER, Figg WD, Vargesson N. Thalidomide induces limb defects by preventing angiogenic outgrowth during early limb formation. Proceedings of the National Academy of Sciences of the United States of America. 2009 May 26;106(21):8573-8. PubMed PMID: 19433787. Pubmed Central PMCID: 2688998.

van Gelder MM, van Rooij IA, Miller RK, Zielhuis GA, de Jong-van den Berg LT, Roeleveld N. Teratogenic mechanisms of medical drugs. Human reproduction update. 2010 Jul-Aug;16(4):378-94. PubMed PMID: 20061329.