This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Disruption of VEGFR Signaling Leading to Developmental Defects
Point of Contact
- Tom Knudsen
- Nancy Baker
|Author status||OECD status||OECD project||SAAOP status|
|Open for citation & comment||EAGMST Under Review||1.6||Included in OECD Work Plan|
This AOP was last modified on August 02, 2022 09:56
|Inhibition, VegfR2||January 17, 2022 10:05|
|Reduction, Angiogenesis||July 20, 2022 13:04|
|Impairment, Endothelial network||January 18, 2022 13:21|
|Insufficiency, Vascular||July 20, 2022 17:07|
|Increased, Developmental Defects||January 18, 2022 13:12|
|Inhibition, VegfR2 leads to Reduction, Angiogenesis||July 20, 2022 17:33|
|Reduction, Angiogenesis leads to Impairment, Endothelial network||January 18, 2022 12:54|
|Impairment, Endothelial network leads to Insufficiency, Vascular||January 18, 2022 13:05|
|Insufficiency, Vascular leads to Increased, Developmental Defects||January 18, 2022 13:07|
|Vatalanib||July 25, 2022 09:09|
|Sunitinib malate Sunitinib (INN)||July 25, 2022 09:19|
BACKGROUND: The cardiovascular system is the first functional organ system to develop in the vertebrate embryo, reflecting its critical role during normal development and pregnancy. Elucidating an AOP for embryonic vascular disruption must consider the stepwise events underlying blood vessel patterning. Vascular development commences in the early embryo with in situ formation of nascent vessels from angioblasts, leading to a primary capillary plexus (vasculogenesis). After the onset of blood circulation, the primary vascular pattern is further expanded as new vessels sprout from pre-existing vessels (angiogenesis). Both processes, vasculogenesis and angiogenesis, are regulated by genetic signals and environmental factors dependent on anatomical region, physiological state, and developmental stage of the embryo. The developing vascular network is further shaped into a hierarchical system of arteries and veins, through progressive effects on blood vessel arborization, branching, and pruning (angioadaptation). These latter influences include hemodynamic forces, regional changes in blood flow, local metabolic demands and growth factor signals. Disruptions in embryonic vascular patterning-adaptation may result in adverse pregnancy outcomes, including birth defects, angiodysplasias and cardiovascular disease, intrauterine growth restriction or prenatal death. Some chemicals may act as potential vascular disrupting compounds (pVDCs) altering the expression, activity or function of molecular signals regulating blood vessel development and remodeling. Critical pathways involve receptor tyrosine kinases (e.g., growth factor-signaling), G-protein coupled receptors (e.g., chemokine signaling), and GPI-anchored receptors (e.g. uPAR system).
DESCRIPTION: This AOP focuses on the regulation and disruption of vasculogenesis-angiogenesis during embryonic development via disruption of the VEGF-signaling pathway. VEGFA binding to its cognate receptor (VEGFR2) triggers angiogenic sprouting, growth and fusion during early development, and in flow-sensing adaptation of vascular development during later development. VEGFR2 inhibition, the postulated molecular initiating event (MIE) for this AOP, may be invoked by effects on VEGFA production, mobility, or receptor binding, and by effects on VEGFR2 cellular expression, molecular function or post-receptor signal transduction pathways. Downstream key events (KE) include altered cell fate and behavior of 'endothelial tip cells' (exploratory behavior, cell migration) and endothelial 'stalk cells' (cell proliferation, apoptosis). KE relationships (KERs) leading to vascular insufficiency then involve local interactions with other cell types (stromal cells, macrophages), the extracellular matrix (ECM) and micro-physiology (hemodynamics, metabolism). Adverse outcomes (AO) would ultimately vary by anatomical region, organ system, gestational stage and state of the embryo, fetus or placenta when an MIE is invoked.
RELEVANCE and APPLICATION: Angiogenesis and vascular disruption is a broad concept. The intended use of this AOP in a regulatory context is the predictive toxicology of developmental hazards, especially for integrating data from high-throughput screening (HTS) assays into cell agent-based models for predicting dysmorphogenesis. As part of an integrated assessment of toxicity, this AOP can identify useful information for assessing adverse outcomes relevant to risk assessment and efficient use of resources for validation through predictive models linking developmental toxicity to vascular disruption. AOP-based computer models that simulate vascular development can usher-in new virtual screening techniques to predict what might happen to a developing embryo when exposed to chemicals across different dose-time-stage scenarios, including the range of effects and how cellular injury propagates across development.
AOP Development Strategy
Functionalization of the ToxCast pVDC predictive signature
The ToxCast putative Vascular Disrupting Chemicals (pVDC) signature will be described here and parts will be incorporated into the relevant Key Events sections.
The sectors of the ToxPI are color-represented by features from ToxCast HTS assays indicated by the target of the assays, the characteristics as follows.
Vascular cell adhesion molecule 1 (VCAM1): the pVDC signature aggregates assays from the BioMAP Systems Predictive Toxicology panel [Houck et al., 2009, Kunkel et al., 2004] focusing here on chemical disruption of endothelial VCAM1 expression following stimulation by cytokines-growth factors. This assay endpoint is an in vitro surrogate for inflammatory cell recruitment per endothelial dysfunction and has been probed across five different cell systems: 4H (HUVECs stimulated with IL-4 + histamine); 3C (HUVECs stimulated with IL-1β + TNFα + IFNϒ); CASM3C (primary human coronary artery smooth muscle cells stimulated with IL-1β + TNFα + IFNϒ); LPS (HUVECs co-cultured with monocytes and stimulated with bacterial endotoxin); and hDFCGF (human dermal fibroblasts stimulated with IL-1β + TNFα + IFNϒ and EGF + bFGF + PDGF-BB)[Knudsen and Kleinstreuer, 2011, Kleinstreuer et al., 2014].
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.
Angiogenic growth factors: FGFs and VEGFs exert their effects on endothelial cell proliferation, migration, and differentiation via specific binding to receptor tyrosine kinases VEGFR and FGFR. The pVDC signature has features for liganding VEGFR1, VEGFR2, and VEGFR3 based on receptor kinase activity (RTK, inhibition or activation) from the NovaScreen biochemical profile [Sipes et al. 2013] and for down-regulation of VEGFR2 expression in the 4H BioMAP system (HUVECs stimulated with IL-4 + histamine, B). VEGFR1 is a non-signaling VEGF-A decoy receptor that can be cleaved from the cell surface; VEGFR2 is the most important VEGF-A receptor and a master switch for developmental angiogenesis; and VEGFR3 is a VEGF-C receptor up-regulated by Notch signals. The pVDC signature includes features for the basic helix-loop-helix transcription factors Aryl Hydrocarbon Receptor (AhR) and Hypoxia Inducible Factor-1 alpha (HIF1a) that are upstream regulators of VEGF gene expression during ischemia or hypoxia. HIF1a and AhR are measured in reporter assays (Attagene). In addition to HIF1a and AhR, the pVDC signature has features for the estrogen receptor alpha (ERa), also a trans-activator of VEGF expression. This included human ERa binding activity (NovaScreen), ERa reporter trans-activation (Attagene) and ERE (estrogen responsive element) reporter cis-activation (Attagene).
Angiogenic 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.
Endothelial cell migration and proliferation: the pVDC signature includes assays for human primary vascular cultures (endothelial and vascular smooth muscle cells). Assays for nuclear localization of beta-catenin (CTNB) are based on the principle that nuclear translocation activates pathways important for endothelial cell migration, proliferation and survival during capillary network formation in HUVEC cells [Muller et al. 2002; Masckauchan et al. 2005].
Vascular stabilization: The signature has features for transforming growth factor-beta 1 (TGF-b), which regulates vascular morphogenesis and integrity, and for Tie2 - a receptor tyrosine kinase activated by the angiopoietins (ANG1, ANG2) that function stabilize nascent vasculature. The pVDC signature has features for the anti-angiogenic phosphatases PTEN (phosphatase and tensin homolog), PTPN11 (tyrosine-protein phosphatase non-receptor type 11) and PTPN12, and endothelial-specific receptor tyrosine protein phosphatase beta (PTPRB). Matrix metalloproteinases (MMPs) 1/2/9 aggregate features on biochemical activity and cellular function of zinc-dependent endopeptidases MMP1, MMP2 and MMP9 that facilitate angiogenesis through ECM degradation by activated endothelial cells.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||305||Inhibition, VegfR2||Inhibition, VegfR2|
|KE||28||Reduction, Angiogenesis||Reduction, Angiogenesis|
|KE||110||Impairment, Endothelial network||Impairment, Endothelial network|
|KE||298||Insufficiency, Vascular||Insufficiency, Vascular|
|AO||1001||Increased, Developmental Defects||Increased, Developmental Defects|
Relationships Between Two Key Events (Including MIEs and AOs)
|Inhibition, VegfR2 leads to Reduction, Angiogenesis||adjacent||High||High|
|Reduction, Angiogenesis leads to Impairment, Endothelial network||adjacent||High||Moderate|
|Impairment, Endothelial network leads to Insufficiency, Vascular||non-adjacent||Moderate||Low|
|Insufficiency, Vascular leads to Increased, Developmental Defects||non-adjacent||High||Moderate|
Life Stage Applicability
|Conception to < Fetal||High|
Overall Assessment of the AOP
Domain of Applicability
The cardiovascular system is the first organ system to function in the vertebrate embryo, reflecting its critical role during organogenesis [Chan et al. 2002; Jin et al. 2005; Walls et al. 2008]. Blood vessel development commences in the early (sexually undifferentiated) embryo with de novo assembly of angioblasts into a primary capillary plexus (vasculogenesis). With the onset of blood circulation, the primary vascular pattern is further expanded as new vessels sprout from pre-existing vessels (angiogenesis). Both processes, vasculogenesis and angiogenesis, are developmentally regulated by genetic signals and environmental factors dependent on anatomical region, physiological state, and gestational age of the embryo-fetus [Shalaby et al. 1995; Patan, 2000; Jin et al. 2005; Knudsen and Kleinstreuer, 2011; Eberlein et al. 2021]. Disruption of embryonic vascular development is a potential framework for adverse outcome pathways (AOPs) in developmental toxicity [Knudsen and Kleinstreuer, 2011; Kleinstreuer et al. 2013; Saili et al. 2019; Zurlinden et al. 2020]. Developmental angiogenesis is supported by evidence in genetic models of abnormal vascularization leading to severe developmental phenotypes [Fong et al. 1995; Shalaby et al. 1995; Carmeliet et al. 1996; Maltepe et al. 1997; Abbott and Buckalew, 2000; Chan et al. 2002; Coultas et al. 2005; van den Akker et al. 2007; Eberlein et al. 2021]. This may include cell signals and responses driving formation of the primitive capillary network in the early embryo and extraembryonic membranes (vasculogenesis), the subsequent expansion and patterning of the embryonic and placental vasculature (angiogenesis), and its further stabilization, specialization, and remodeling during growth, organogenesis and differentiation. Additional evidence comes from dysmorphogenesis induced with known anti-angiogenic compounds across multiple vertebrate species (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] and human studies of malformations correlated with genetic and/or environmental factors that target vascular development [Husain et al. 2008; Gold et al. 2011; Vargesson and Hootnick, 2017]. An analysis of pharma 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]. This AOP is focused on disruption of ‘developmental angiogenesis’ from the perspective of dysmorphogenesis leading to severe developmental defects. Although uterine-decidual vascularization is critically important for healthy pregnancy outcomes, the emphasis here is the direct role on anatomical development of the embryo proper.
Essentiality of the Key Events
No experimental evidence
Contradictory experimental evidence
MIE: Inhibition, VegfR2
KE1: Reduction, Angiogenesis
KE2: Impairment, Endothelial network
KE3: Insufficiency, Vascular
AO: Increased, Developmental Defects
The Vascular Endothelial Growth Factor (VEGF) pathway is a critical regulatory system for assembly of embryonic blood vessels [Fong et al. 1995; Shalaby et al. 1995; Carmeliet et al. 1996; Ferrara, et al. 1996; Argraves et al. 2002; Hogan et al. 2004; Roberts et al. 2004; Chung and Ferrara, 2011; Shibuya, 2013; Chapell et al. 2016; Jin et al. 2017; Queisser et al. 2021]. The VEGF growth factors belong to the platelet-derived growth factor supergene family. VEGF-A, the major regulator for angiogenesis, binds receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR2 (KDR/Flk-1) to regulate endothelial cell proliferation, survival, migration, tubular morphogenesis, and sprouting [Hogan et al. 2004; Douglas et al. 2009; Shibuya, 2013]. This pro-angiogenic effect is locally fashioned as VEGF gradients where the soluble VEGFR1 (sFlt-1) is released from the cell surface as an endogenous VEGF inhibitor that sets up VEGF-A corridors in the developing embryo [Roberts et al. 2004; Chappell et al. 2016]. Genetic studies have shown that perturbing the VEGF signaling system can invoke varying degrees of adverse consequences, ranging from congenital angiodysplasia to fetal malformations and embryolethality [Fong et al. 1995; Ferrara et al. 1996; Eshkar-Oren et al. 2015; Jin et al. 2017]. Filopodial sprouting of the endothelial cell tip (EC-tip) is the critical VEGFR2 responsive cell type specifically in this AOP [Belair et al. 2016a and 2016b]; however, other relevant cell types include: angioblasts (AB) as direct precursors to primary endothelial cells; endothelial ‘stalk’ cells (EC-stalk) proliferate in the wake of an angiogenic sprout; macrophage/microglial cells (MCs) release cytokines, chemokines, and growth factors; and stromal cells (SCs) are recruited to the nascent vascular wall for vessel stabilization. As such, the VEGF gradient/response system influences a multicellular dimension determined by cellular patterns of VEGF expression and processing (eg, MCs, SCs) and biochemical corridors set up by the extracellular matrix and the VEGFR1 decoy receptor (eg, EC-stalk). Evidence supporting an AOP for chemical disruption is available for thalidomide, estrogens, endothelins, dioxin, retinoids, cigarette smoke, and metals among other compounds [Kleinstreuer et al. 2011; Knudsen and Kleinstreuer, 2011; Shirinifard et al. 2013; Tal et al. 2014 and 2017; McCollum et al. 2017; Toimela et al. 2017; Mahony et al. 2018; Saili et al. 2019; Zurlinden et al. 2020]. Although not all compounds with developmental toxicity show an in vitro vascular bioactivity signature, many 'putative vascular disruptor compounds' (pVDCs) invoke adverse developmental consequences [Kleinstreuer et al. 2011 and 2013]. The molecular and cellular biology of human vascular development, stabilization and remodeling is amenable to in vitro assays with human cells [Bishop et al. 1999; Sarkanen et al. 2010; Kleinstreuer et al. 2014; Belair et al. 2016a and 2016b; Nguyen et al. 2017; Toimela et al. 2017; Pauty et al. 2018; van Duinen et al. 2019a and 2019b; Zurlinden et al. 2020], pluripotent stem cells induced to endothelial differentiation [Belair et al. 2015; Sinha and Santoro, 2018; Li et al. 2018; Galaris et al. 2021], and endothelial-specific reporter zebrafish [Tran et al. 2007; Shirinifard et al. 2013; Tal et al. 2014 and 2017; Beedie et al. 2017; McCollum et al. 2017]. An integrated portfolio of assays is thus available to cover many aspects of the angiogenic cycle and its ramifications during neurovascular development [Bautch and James, 2009; Eichman and Thomas, 2013; Saili et al. 2017; Uwamori et al. 2017; van Duinen et al. 2019; and Zurlinden et al. 2020]. Evidence is also available to support the essentiality of this AOP outside the embryo proper, such as uterine angiogenesis [Douglas et al. 2009; Araujo et al. 2021], placentation [Abbott and Bucklew, 2000; Chen and Zheng, 2014], and human pregnancies complicated by preeclampsia and small-for-gestational age infants [Andraweera et al. 2012].
Weight of evidence for the MIE and AO are strong; the intermediate KEs have in some cases strong evidence but in other cases weaker evidence, due to the lack of quantitative information. the KERs are biologically plausible. Several manuscripts have been published recently that bolster weight of evidence [Belair et al. 2016; Nguyen et al. 2017; Tal et al. 2017; McCollum et al. 2017; Ellis-Hutchings et al. 2017; Saili et al. 2019; Zurlinden et al. 2020].
Known Modulating Factors
Considerations for Potential Applications of the AOP (optional)
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