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USEPA - National Center for Computational Toxicology, Research Triangle Park NC, USA
LEAD: Tom Knudsen - email@example.com Nancy Baker (Lockheed-Martin) - firstname.lastname@example.org Richard Spencer (Lockheed-Martin) - email@example.com
USEPA - National Health and Environmental Effects Research Laboratory, Research Triangle Park NC, USA
Tamara Tal - firstname.lastname@example.org David Belair - email@example.com
Finnish Centre for Alternative Methods, University of Tampere, Tampere Finland
Tuula Heinonen - firstname.lastname@example.org
The Dow Chemical Company, Midland MI, USA
Rob Ellis-Hutchings - email@example.com
University of Houston, Houston TX, USA
Maria Bondesson - firstname.lastname@example.org
Indiana University, Bloomington IN, USA
James Glazier - email@example.com
University of Wisconsin, Madison WI, USA
William Daly - firstname.lastname@example.org
University of Aberdeen, Aberdeen, Scotland UK
Neil Vargesson – email@example.com
National Toxicology Program/NICEATM-ICCVAM, Research Triangle Park NC, USA
Nicole Kleinstreuer – firstname.lastname@example.org
Point of Contact
Tom Knudsen (email point of contact)
- Tom Knudsen
|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 December 02, 2016 11:34
|Inhibition, VegfR2||September 16, 2017 10:15|
|Reduction, Angiogenesis||September 16, 2017 10:15|
|Impairment, Endothelial network||February 13, 2018 15:56|
|Insufficiency, Vascular||September 16, 2017 10:15|
|Increased, Developmental Defects||November 30, 2016 11:30|
|Inhibition, VegfR2 leads to Reduction, Angiogenesis||November 30, 2016 11:40|
|Reduction, Angiogenesis leads to Impairment, Endothelial network||November 30, 2016 11:48|
|Impairment, Endothelial network leads to Insufficiency, Vascular||November 30, 2016 11:51|
|Insufficiency, Vascular leads to Increased, Developmental Defects||November 30, 2016 11:54|
|Vatalanib||November 29, 2016 18:42|
|Sunitinib malate Sunitinib (INN)||November 29, 2016 18:42|
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.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||305||Inhibition, VegfR2||Inhibition, VegfR2|
|2||KE||28||Reduction, Angiogenesis||Reduction, Angiogenesis|
|3||KE||110||Impairment, Endothelial network||Impairment, Endothelial network|
|4||KE||298||Insufficiency, Vascular||Insufficiency, Vascular|
|5||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
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.
Domain of Applicability
Toxicity testing in the 21st century is moving toward using high-throughput screening assays to rapidly test thousands of chemicals against hundreds of molecular targets and biological pathways, and to provide mechanistic information on chemical effects in human cells and small model organisms. First-generation predictive models for prenatal developmental toxicity have revealed a complex web of biological processes with many connections to vasculogenesis and angiogenesis. Disruption of embryonic vascular development as a potential adverse outcome pathway (AOP) framework leading to developmental toxicity. Further evidence comes from an analysis of pharma compounds to which women of reproductive age were exposed, leading to the implication of vascular disruption as one of six potential mechanisms of teratogenesis. We reviewed embryonic vascular development and important signals for vascular development (local growth factors and cytokines such as VEGF-A and TGF-beta, components in the plasminogen activator system, and chemotactic chemokines). Genetic studies have shown that perturbing these signals can lead to varying degrees of adverse consequences, ranging from congenital angiodysplasia to fetal malformations and embryolethality. The molecular targets and cellular behaviors required for vascular development, stabilization and remodeling are amenable to in vitro evaluation. Evidence for chemical disruption of these processes is available for thalidomide, estrogens, endothelins, dioxin, retinoids, cigarette smoke, and metals among other compounds. Although not all compounds with developmental toxicity show an in vitro vascular bioactivity signature, many 'putative vascular disruptor compounds' invoke adverse developmental consequences. As such, an adverse outcome pathway perspective of embryonic vascular development can help identify useful information for assessing adverse outcomes relevant to risk assessment and efficient use of resources for validation.
Essentiality of the Key Events
We devised an AOP framework for embryonic vascular disruption based on evidence from the open literature and public databases [Knudsen and Kleinstreuer, 2011]. Cellular behaviors linked to molecular targets in angiogenesis, and to some extent vasculogenesis, are well-described in the literature. Critical cell types include: angioblasts (AB) as direct precursors to primary endothelial cells; endothelial ‘tip’ cells (EC-tip) mediate angiogenic sprouting; endothelial ‘stalk’ cells (EC-stalk) proliferate in the wake of an angiogenic sprout; macrophage cells (MCs) release cytokines, chemokines, and growth factors; and stromal cells (SCs) are recruited to the nascent vascular wall for vessel stabilization. As such, cellular consequences vary spatiotemporally and may be defined by the VEGF-gradient and VEGF-response. Setting up VEGF gradients is a multicellular phenomenon, determined by VEGF expression and processing (eg, MCs, SCs) and biochemical corridors set up by the extracellular matrix and the VEGFR1 decoy receptor (eg, EC-stalk). EC-tip is the critical VEGFR2-responsive cell type displaying exploratory and migratory behavior during angiogenesis. Arsenic, for example, was shown to disrupt these behaviors [Shirinifard et al., 2013]. The impact of chemicals on distinct cellular behaviors or the signaling networks can be studied in vitro utilizing pluripotent stem cells, endothelial tubulogenesis assays, and aortic explants [Sarkanen et al. 2011;Kleinstreuer et al. 2013; Tal et al. 2014]. Downstream consequences of vascular disruption can be tracked in more integrated embryonic systems including rodent whole embryo culture and transgenic endothelial zebrafish reporter lines (e.g., VEGF/Rs). Disruption of vasculogenesis or angiogenesis can adversely impact the embryo in many ways, leading to intrauterine growth retardation (IUGR), skeletal malformations, functional deficits and neonatal death. Blood vessel formation is necessary for the uterine cycle and placentation and, therefore, can affect female fertility leading to implantation failure, pregnancy loss, preeclampsia and preterm labor. This AOP will focus on embryonic development where there is sufficient information on the necessity of vascular development for normal development (e.g., appendicular and axial structures) as well as a direct link between in utero vascular disruption and limb and other defects in humans [Husain et al. 2008; Gold et al. 2011].
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 submitted or are in preparation that will help bolster weight of evidence [Belair et al. 2016; Nguyen et al. 2016; Tal et al. 2016; McCollum et al. 2016; Ellis-Hutchings et al. 2016; Franzosa et al. 2016; Knudsen et al. 2016].
Considerations for Potential Applications of the AOP (optional)
Ellis-Hutchings RG, Settivari RS, McCoy AT, Kleinstreuer N, Franzosa J, Knudsen TB and Carney EW. Embryonic vascular disruption: linking high throughput signaling signatures with functional consequences. 2016 (in preparation).
Franzosa JA, Settivari RS, Ellis-Hutchings RG, Kleinstreuer NC, Houck KA, Carney EW and Knudsen TB. RNA-Seq analysis of the functional-link between vascular disruption and adverse developmental consequences. 2016 (in preparation).
Gold NB, Westgate MN, Holmes LB. Anatomic and etiological classification of congenital limb deficiencies. American journal of medical genetics Part A. 2011;155A(6):1225-35.
Houck KA, Dix DJ, Judson RS, Kavlock RJ, Yang J, Berg EL. Profiling bioactivity of the ToxCast chemical library using BioMAP primary human cell systems. Journal of biomolecular screening. 2009 Oct;14(9):1054-66. PubMed PMID: 19773588.
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;82(6):435-40.
Kleinstreuer NC, Yang J, Berg EL, Knudsen TB, Richard AM, Martin MT, et al. Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms. Nat Biotechnol. 2014 Jun;32(6):583-91. PubMed PMID: 24837663.
Kleinstreuer NC, Dix D, Rountree M, Baker NC, Sipes NS, Reif D, et al. A computational model predicting disruption of blood vessel development. PLoS computational biology. 2013;9(4):e1002996.
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;119(11):1596-603.
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).
Kunkel EJ, Plavec I, Nguyen D, Melrose J, Rosler ES, Kao LT, et al. Rapid structure-activity and selectivity analysis of kinase inhibitors by BioMAP analysis in complex human primary cell-based models. Assay and drug development technologies. 2004 Aug;2(4):431-41. PubMed PMID: 15357924.
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).
Sarkanen JR, Mannerstrom M, Vuorenpaa H, Uotila J, Ylikomi T, Heinonen T. Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators. Frontiers in pharmacology. 2010;1:147.
Shirinifard A, McCollum CW, Bolin MB, Gustafsson JA, Glazier JA, Clendenon SG. 3D quantitative analyses of angiogenic sprout growth dynamics. Developmental dynamics : an official publication of the American Association of Anatomists. 2013;242(5):518-26.
Sipes NS, Martin MT, Kothiya P, Reif DM, Judson RS, Richard AM, et al. Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays. Chem Res Toxicol. 2013 Jun 17;26(6):878-95. PubMed PMID: 23611293. Pubmed Central PMCID: 3685188.
Tal TL, McCollum CW, Harris PS, Olin J, Kleinstreuer N, Wood CE, Hans C, Shah S, Merchant FA, Bondesson M, Knudsen TB, Padilla S and Hemmer MJ. Immediate and long-term consequences of vascular toxicity during zebrafish development. Reproductive Toxicology. 2014;48:51-61.
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).