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Key Event Title
|Level of Biological Organization|
Key Event Components
|vascular endothelial growth factor receptor 2 binding||vascular endothelial growth factor receptor 2||decreased|
|vascular endothelial growth factor receptor 2 binding||vascular endothelial growth factor receptor 1||decreased|
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||MolecularInitiatingEvent||Tom Knudsen (send email)||Open for citation & comment||EAGMST Under Review|
Key Event Description
The VEGFR system is an important molecular regulator of physiological and pathological blood vessel development. The central players are vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3) and five VEGF ligands that bind and activate these receptors during vasculogenesis, angiogenesis and lymphogenesis [Shibuya, 2013]. The MIE:305 target, VEGFR2, belongs to Class IV transmembrane receptor tyrosine kinases (RTKs) that play critical roles in the origin and progression of many adverse outcomes linked to vascular biology. Direct evidence supporting its role in developmental angiogenesis comes from functional inactivation in mouse VEGFR knockout models. For example, a targeted mutation in flt-1 showed Vegfr1(-/-) embryos formed endothelial cells in both embryonic and extra-embryonic regions but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages [Fong et al. 1995]. Functional inactivation of flk-1 showed that Vegfr2(-/-) embryos died much earlier due to deficiencies in hematopoeisis and organized blood vessels [Shalaby et al. 1995]. It’s endogenous ligand, Vascular Endothelial Growth Factor-A (VEGF-A), in particular the VEGF165 splice variant, plays a key role in the regulation of angiogenesis during early embryogenesis. Mouse embryos heterozygous for the Vegf gene died from impaired angiogenesis and hematopoeisis in Vegf(+/-) heterozygotes during organogenesis [Ferrara et al. 1996]. Nullizygotes died earlier showing that progressive severity in a quantitative gene dose-dependent manner [Carmeliet et al. 1996]. VEGF-A is a soluble protein that acts directly on endothelial cells and their precursors through VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). The former is a decoy receptor that traps VEGF-A into corridors preventing interaction with the active receptor, VEGFR2 [Roberts et al. 2004]. Environmental stressors (drugs/chemicals) may perturb VEGFR-dependent angiogenesis [Belair et al. 1996a,b]. Multiple mechanisms are involved, including direct effects on VEGFR2 structure-function as well as VEGF-A bioavailability or binding kinetics [Gustafsdottir et al. 2008]. The duality is relevant to MIE:305 because receptor affinity for VEGF is ten-fold higher at VEGFR1, whereas kinase activity is ten-fold higher at VEGFR2 [Fischer et al. 2008; Shibuya, 2013]. As such, VEGFR2 promotes angiogenesis whereas VEGFR1 acts as a ligand-trap to prevent VEGF-A interaction with VEGFR2 [Hiratsuka et al. 1998]. In this AOP, decreased VEGFR2 binding is the quantitative basis for an effect of stressors on VEGFR2 activation of the ‘master switch’ in developmental angiogenesis.
How It Is Measured or Detected
A number of targeted and high-throughput assays are used to quantitively assess chemical effects leading to reduced VEGFR2 activity. Starting with VEGF availability as a preceding event, a cell-based reporter gene assay has screened approximately 73,000 compounds in a quantitative high-throughput screening (HTS) approach [Xia et al. 2009]. That assay measures cellular VEGF-secretion in an ME-180 cervical carcinoma HRE (hypoxia-response element) reporter cell line as a genetic response to hypoxia-induced Vegf expression. Proximity Ligation Assays (PLAs) have been used to evaluate small molecule inhibitors of VEGF-A165 binding to solubilized VEGFRs [Gustafsdottir et al. 2008]. PLAs are fit for the purpose of monitoring the kinetics of formation and inhibition of ligand–receptor complexes through different mechanisms of interference with VEGF-A165 or its cognate binding site. This allows quantitative evaluation of the potency of chemical inhibitors based on computing half-maximal inhibitory concentrations (IC50) in concentration-response curves. The inhibition of VEGF-A165 binding to VEGFR2 correlated well in these assays with results obtained by measuring receptor phosphorylation following exposure to molecular probes or pharmacological reagents specific to VEGF-VEGFR2 receptor capacity and kinase activity [Gustafsdottir et al. 2008]. HTS platforms have also been used to screen neary 1,000 compounds in the ToxCast/Tox21 chemical library for effects on human VEGFR2 bioactivity (https://comptox.epa.gov/dashboard/) [Kavlock et al. 2012; Judson et al. 2016; Richard et al. 2016; Thomas et al. 2018]. This biochemical (cell-free) assay is one of 331 enzymatic and receptor signaling assays under the ‘NovaScreen’ (ToxCast_NVS) platform [Knudsen et al. 2011; Sipes et al. 2013]. VEGFR2 enzymatic activity is measured as an electrophoretic shift in migration of a specific fluorescein-peptide substrate to the fluorescein-phosphopeptide upo 1-hour incubation with ATP. Concentration response to a test chemical is detected by a change in activity, which may be decreased or increased depending on the nature of a drug or chemical’s effect on VEGFR2 catalysis or autophosphorylation, respectively with automated curve-fits [Knudsen et al. 2011; Sipes et al. 2013]. Also, in ToxCast, a multiplex assay described under the ‘BioSeek’ (ToxCast_BSK) platform exists for VEGFR2 bioactivity in a cell-based co-culture system [Kleinstreuer et al. 2014]. This assay measures increased or decreased levels of VEGFR2-immunoreactive protein by ELISA in primary human umbilical vein cells (HUVEC) conditioned to simulate proinflammation with histamine and IL4. Concentration response to a test chemical is curve-fitted to indicate changes in VEGFR2 receptor density. This is one of 87 endpoints covering molecular functions relevant to toxic and therapeutic pathways generated in eight cell systems for 641 environmental chemicals and 135 reference pharmaceuticals and failed drugs [Kleinstreuer et al. 2014].
Domain of Applicability
There is strong phylogenetic conservation of VEGFR2 genes [Shibuya, 2002]. For example, the amino acid homology ranges from 79.9 - 96.1% for the critical autophosphorylation domain across species of fish, birds, rodents with humans. This suggests a conserved molecular basis to regulation of blood vessel development and implies broad taxonomic applicability to VEGFR2 inhibition. Direct evidence for this comes from the susceptibility of vascular development to pharmacological inhibitors of human VEGFR2 kinase activity. Vatalanib (PTK787), for example, is a potent inhibitor of human VEGFR2 kinase activity [Wood et al. 2002] and disrupted angiogenic vessel formation in early zebrafish embryos at submicromolar concentrations [Tal et al. 2014].
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