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Key Event Title
Impairment, Endothelial network
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|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|
|AHR activation to ELS mortality, via VEGF||KeyEvent||Amani Farhat (send email)||Open for citation & comment||WPHA/WNT Endorsed|
Key Event Description
In embryological terms, the angiogenic cycle entails a stepwise progression of formation, maturation, and stabilization of the microvasculature [Hanahan, 1997; Drake et al. 2007; Chung and Ferrara 2011; Knudsen and Kleinstreuer, 2011; Coultas et al. 2005; Huang, 2020]. This level of impairment of blood vessel morphogenesis best maps to Gene Ontology (GO) annotations: GO:001885 for ‘endothelial cell development’, which is defined as “The progression of an endothelial cell over time, from its formation to the mature structure”; and/or GO:0045601 for ‘regulation of endothelial cell differentiation’, defined as “Any process that stops, prevents, or reduces the frequency, rate or extent of endothelial cell differentiation”. The numbers of curated genes associated with these categories in the MGI database (http://www.informatics.jax.org/vocab/gene_ontology/) are 75 genes and 44 genes, respectively, for a total of 97 genes altogether. In addition, pericyte-endothelial interactions are indispensable for maturation and stabilization via broader signaling pathways (eg, VEGFA, PDGFB, Notch-DLL4, AGPNT, Norrin, TGF-β) that have been characterized during patterning neovascularization [Azam et al. 2018; Huang, 2020]. Neovascular stabilization is an active process that requires specific cellular signaling, including pro-angiogenic pathways such as VEGF and FGF, angiopoietin-Tie2 for endothelial cell survival and junction stabilization, PDGF and TGF-β signaling that modify mural cell (pericytes, vascular smooth muscle cells) functions to fortify vessel integrity [Murakami, 2012]. Breakdown of these signaling systems results in pathological hyperpermeability and/or genetic vascular abnormalities such as vascular malformations, ultimately progressing to hemorrhage and edema. Vascular mural cells are recruited to the endothelial network by endothelial cell signals [Sinha and Santoro, 2018]. A number of anti-angiogenic compounds, including Vatalanib and Thalidomide, have been shown to impair neovascularization during developmental angiogenesis [Tran et al. 2007; 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]. In exposed zebrafish embryos, early effects of potential vascular disrupting chemicals (pVDCs) invoke changes to the anatomical development of intersegmental vessels from the dorsal aorta [Tran et al. 2007; Tal et al. 2014; McCollum et al. 2017]. Thalidomide, for example, has been shown to primarily disrupt immature vascular networks versus more mature vasculature in the embryo [Therapontos et al. 2009; Beedie et al. 2016a, 2016b, 2017]. Evidence for KE:110 in human studies is indirect, based on the association of malformations with altered vascular patterns and exposure to anti-angiogenic drugs in women of reproductive potential or during pregnancy [Husain et al. 2008; van Gelder et al. 2010; Gold et al. 2011; Ligi et al. 2014; Vargesson and Hootnick, 2017]. Key nodes in the ontogenetic regulation of angiogenesis have been investigated with human cell-based high-throughput assay (HTS) platforms in ToxCast to screen for pVDCs acting on the formation, maturation and/or stabilization of endothelial networks [Houck et al. 2009; Knudsen et al. 2011; Kleinstreuer et al. 2014; Saili et al. 2019; Zurlinden et al. 2020].
How It Is Measured or Detected
Microvascular structure: Endothelial network formation can be monitored quantitatively in vitro using different human cell-based angiogenesis assays that score endothelial cell migration, cell counts, tubule counts, tubule length, tubule area, tubule intensity, and node counts [Muller et al. 2002; Masckauchan et al. 2005; Sarkanen et al. 2010; Knudsen et al. 2016; Nguyen et al. 2017; Toimela et al. 2017; Saili et al. 2019; Zurlinden et al. 2020]. Cell types commonly employed are human umbilical endothelial cells (HUVECs) and more recently endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) through various differentiation and purification protocols [Belair et al. 2015 and 2016; Iwata et al. 2017; Bezenah et al. 2018; van Duinen et al. 2019 and 2020]. Synthetic hydrogels are shown to promote robust in vitro network formation by HUVEC or iPSC-ECs in response to angiogenic factors as superior sensitivity and reproducibility to detect pVDCs [Nguyen et al. 2017]. Although endothelial cell models of migration, proliferation, apoptosis, and tube formation are popular due to their simplicity and throughput, these assays lack the biological complexity of an in vivo system. Animal models, including the chick chorioallantoic membrane assay, corneal neovascularization assay, and 3D embedded matrices preserve biological complexity but are costly and low throughput [Tran et al. 2007]. Endothelial-specific transgenic zebrafish reporter embryos thus provide a test system that combines the biological complexity of in vivo models with automated high-throughput screening (HTS).
Maturation and stabilization: Chemical effects may be detected by HTS assays for phenotypic profiling in endothelial co-culture systems based on specific biomarker protein readouts [Kleinstreuer et al. 2014]. The ToxCast portfolio includes eight human cell-based systems for screening chemicals that disrupt physiologically important cell-cell signaling pathways, including vascular biology. The endpoints measured can be closely linked to in vivo outcomes. Local signals may act through several receptor modalities, including receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and glycosyl phosphatidyl-inositol (GPI)-anchored receptors as part of a ToxCast in vitro signature for profiling potential vascular disrupting compounds (pVDCs) [Knudsen and Kleinstreuer, 2011; Kleinstreuer et al. 2013; Tal et al. 2017; Saili et al. 2019].
Assessing weight of evidence with a ToxCast pVDC predictive signature assays for KE:110:
ToxCast HTS predictions for 38 potential pVDCs and non-pVDCs were tested across ten in vitro platforms from laboratories addressing different aspects of the vasculogenic/angiogenic cycle. Three tubulogenesis platforms used traditional HUVECs [Sarkanen et al. 2010; Toimela et al. 2017]; 3D endothelial sprouting and network assays used endothelial cells derived from human induced pluripotent stem cells (iPSCs) [Belair et al. 2016b; Nguyen et al. 2017; Zurlinden et al. 2020]; microvessel outgrowth in rat fetal aortic explants [Ellis-Hutchings et al. 2017] and transgenic endothelial reporter zebrafish lines [Tal et al. 2017; McCollum et al. 2017] rounded out the panel. While no single study confirmed all of the pVDC predictions, the combined vascular disrupting effects across all studies aligned well with the in silico predictions (87% accuracy; positive predictive value of 93%, negative predictive value of 73%) [Saili et al. 2019]. ToxCast assay features input to the prediction model were detected as follows.
Vascular cell adhesion molecule 1 (VCAM1): the pVDC signature aggregates assays from the BioMAP Systems Predictive Toxicology panel [Kunkel et al., 2004; Houck et al., 2009] 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 [Knudsen et al. 2011; Kleinstreuer et al. 2013; Tal et al. 2017; Saili et al. 2019; Zurlinden et al. 2020].
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 outgrowth: 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
Endothelial networks are necessary components of normal development. Direct evidence comes from the observation of severe dysmorphogenesis and embryolethality in genetic mouse models lacking a functional VEGF signaling pathway [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]. These alterations may follow impairment of the primitive capillary network in the early embryo and extraembryonic membranes (vasculogenesis) or its subsequent expansion and patterning of the embryonic and placental vasculature (angiogenesis). Several anti-angiogenic compounds are known to impair these stages of vascular development across multiple vertebrate species (e.g., zebrafish, frog, chick, mouse, rat) [Tran et al. 2007; 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]. Vascular patterning is known to be sensitive event in human pregnancy as well [Husain et al. 2008; van Gelder et al. 2010; Gold et al. 2011; Vargesson and Hootnick, 2017]. Anatomically, the stabilization and has varied themes for arterial, venous, and lymphatic channels [Beedie et al. 2017; Tal et al. 2017]. These events are mediated by angiogenic factors through receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and glycosyl phosphatidyl-inositol (GPI)-anchored receptors, and later vascular flow-mediated signals [Drake et al. 2007; Knudsen and Kleinstreuer, 2011]. These provide assayable targets for high-throughput screening (HTS) assays, and an open source of data screening hundreds of chemicals for impairment to the angiogenic cycle [Tran et al. 2007; Houck et al. 2009; Kleinstreuer et al. 2011; Knudsen et al. 2011 and 2013; Kleinstreuer et al. 2014; Tal et al. 2014 and 2017; McCollum et al. 2017; Saili et al. 2019; Zurlinden et al. 2020].
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