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Key Event Title
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Key Event Description
Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues [Hogan et al. 2004]. Many human diseases, including stroke, retinopathy, and cancer, are associated with the vascular biology, including endothelial cells and pericytes that establish the blood-brain barrier and control cerebrovascular exchanges [Bautch and James, 2009; Eichmann and Thomas, 2013; Saili et al. 2017]. Functionally, blood vessel morphogenesis is critical for providing oxygen, nutrients and molecular signals to developing tissues [Maltepe et al. 1997; Vargesson, 2003; Chung and Ferrara, 2011; Eshkar-Oren et al. 2015]. The developing vascular network is shaped into a hierarchical system of arteries and veins, through progressive effects on blood vessel arborization (microvasculature) and pruning (angio-adaptation) [Jin et al. 2017]. The former is morpho-regulatory whereas the reshaping is influenced by regional changes in blood flow and local metabolic demands [Tran et al. 2007]. Evidence supports the ability of physiological parameters such as oxygen and glucose concentrations to affect the expression of genes critical for developmental angiogenesis [Maltepe and Simon, 1998]. Growth in tissue mass during organogenesis is thought to lead to the formation of hypoxic/nutrient-deprived cells. The subsequent activation of sensors such as HIF-1 [Xia et al. 2009; Oladipupo et al. 2011; Li et al. 2018] and ARNT [Maltepe et al. 1997; Abbott and Buckalew, 2000] that rapidly trans-activate the expression of genes such as VEGF that drive angiogenesis.
While mammalian embryos become sensitive to hypoxia during early organogenesis, the small size of zebrafish embryos renders this species less vulnerable to hypoxia than vertebrate counterparts; however, the genetic control of microvascular development is conserved among vertebrate species as evidenced by hypoxia-responsive signaling (HIF-1) via local oxygen-sensing gradients in the zebrafish, chick and mouse embryo [Hogan et al. 2004; Liu et al. 2017; Gerri et al. 2017]. The neural tube, for example, provides vascular patterning signals that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation [Hogan et al. 2004]. This process is temporally and spatially associated with Vegfa expression as the neural tube signal through VEGFR-2. Mesodermal VEGFR-2 expression is localized to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube under the positive control of BMP4 signaling and negative control by Noggin, a BMP4 antagonist [Nimmagadda et al. 2005]. Reciprocal signaling between VEGF-induced endothelial cells and neuroprogenitor cells enhanced formation of the brain neurovascular unit [Vissapragada et al. 2014]. In transgenic zebrafish embryos, the VEGFR-2 antagonist, Vatalanib produced a direct concentration-dependent progression of impaired intersegmental vessel (ISV) outgrowth in early embryos, increased rates of malformed hatched larva, and reduced survival in juvenile cohorts [Tal et al. 2014]. These data show that disruption in the early embryo has a lasting impact on advanced life stages.
Another key cell sensing activity is the recruitment of macrophage (microglia?) cells that secrete pro-angiogenic cytokines and proteases, remodeling the extracellular matrix (ECM) and providing survival and guidance cues to endothelial cells [Gerri et al. 2017]. Macrophages play crucial roles at each step of the angiogenic cycle, from sprouting to maturation and remodelling of the vascular plexus through angiopoietin-TIE2 signaling [Du Cheyne et al. 2020], which is known to synergize with the VEGF-pathway during developmental angiogenesis [Li et al. 2014]. A seminal study showed that loss of immature blood vessels is the primary cause of Thalidomide-induced teratogenesis in the chick embryo, where anti-angiogenic but not anti-inflammatory analogues of Thalidomide induced limb reduction defects. Outgrowth and remodeling of more mature blood vessels delayed, whereas newly formed angiogenic vessels were lost prior to limb dysmorphogenesis and altered patterns of gene expression [Therapontos et al. 2009; Vargesson, 2015]. Vascular insufficiency is likely important in human embryos where the window of vulnerability to Thalidomide-induced phocomelia precedes full establishment of the adult arterial pattern by the 8th week of gestation [Hootnick et al. 2016; Hootnick et al. 2017; Vargesson and Hootnick, 2017].
As such, a chemical’s potential to disrupt vascular patterning and/or remodeling during organogenesis can have profound effects on many systems, including: early limb development [Beedie et al. 2016a, 2016b, 2017 and 2020]; neurovascular development [Hogan et al. 2004; Hallene et al. 2006; Bautch and James, 2009; Eichman and Thomas, 2013; Vissapragada et al. 2014; Fiorentino et al. 2016; Uwamori et al. 2017; Huang, 2020]; and utero-placental development [Abbott and Buckalew, 2000; Douglas et al. 2009; Rutland et al. 2009; Chen, 2014; Araujo et al. 2021].
How It Is Measured or Detected
Complex functional assays such as the rat aortic explant assay, rat whole embryo culture, and the zebrafish embryotoxicity along with transcriptomic signatures provide a tiered approach to evaluate HTS signatures and their taxonomic implications for conserved pathways to prioritize further in vivo testing studies [Ellis-Hutchings et al. 2017].
Zebrafish reporter assays: Blood flow begins in the zebrafish embryo at ∼24 h postfertilization. Shortly after this, the angiogenic vessels that perfuse the trunk of the embryo (intersegmental vessels) sprout from the vasculogenic vessels [Tran et al. 2007]. These effects can be visualized in automated, quantitative screening assays using transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the vascular endothelial growth factor receptor (VEGFR) Vegfr2 promoter that restricts reporter gene expression to developing blood vessels. Phenotypic readouts have been used to screen and validate anti-angiogenic compounds [Tran et al. 2007; Yano et al. 2012; Yozzo et al. 2013; Tal et al. 2014; McCollum et al. 2017]. Live-cell imaging has been used to quantitatively detect the trajectory dynamics of vascular patterning [Clendenon et al. 2013; Shirinfard et al. 2013] and confocal cell imaging has been used to develop a quantitative assay capable of detecting relatively subtle changes (~8%) in relative to controls during chemical exposure [Tal et al. 2017].
ToxCast: A 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 the ToxCast HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay, rat whole embryo culture, and zebrafish embryotoxicity [Saili et al. 2019]. Both compounds disrupted angiogenesis and embryogenesis in the functional assays, with differences in potency and adverse effects. 5HPP-33 was embryolethal, whereas TNP-470 produced caudal defects at low concentrations [Ellis-Hutchings et al. 2017]. Anti-angiogenic modes of action are known for 5HPP-33, which blocks tubulin polymerization inhibition [Yeh et al. 2000; Inatsuki et al. 2005; Kizaki et al. 2008; Rashid et al. 2015); and TNP-470, a methionine aminopeptidase II (MetAP2) inhibition, through non-canonical Wnt inhibition of 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, either directly or via effects on placental morphology [Rutland et al. 2009].
Computational models: Critical pathways for developmental angiogenesis and potential disruptions have critical signal-response systems embedded in three types of receptors that play key roles in a number of morphoregulatory processes: receptor tyrosine kinases (e.g., growth factor-signaling), G-protein coupled receptors (e.g., chemokine signaling), and GPI-anchored receptors (e.g., uPAR system). Computational approaches have been used to predict vascular insufficiency for potential vascular disrupting chemicals (pVDCs) that are developmental toxicants or non-toxicants [Kleinstreuer et al. 2011; Knudsen and Kleinstreuer, 2011]. This has been applied to the ToxCast inventory to rank order over a thousand chemicals for validation testing [McCollum et al. 2017; Tal et al. 2017; Saili et al. 2019; Zurlinden et al. 2020].
Domain of Applicability
Complex functional assays such as the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) along with transcriptomic signatures provide a tiered approach to evaluate HTS signatures and their taxonomic implications for conserved pathways to prioritize further in vivo testing studies [Ellis-Hutchings et al. 2017].
Chung AS, Ferrara N. Developmental and pathological angiogenesis. Annual review of cell and developmental biology. 2011;27:563-84. PubMed PMID: 21756109.
Clendenon SG, Sankaran DG, Shirinifard A, McColluma CW, Bondesson MB, Gustafssona JA and Glazier JA. Arsenic exposure inhibits angiogenesis in zebrafish via downregulation of both VEGFA and VEGFR2. Microscopy and Microanalysis. 2013 19(S2): 778-779.
Ellis-Hutchings RG, Settivari RS, McCoy AT, Kleinstreuer N, Franzosa J, Knudsen TB and Carney EW. Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences. Reprod Toxicol. 2017; 70: 82-96. PMID:28527947.
Eshkar-Oren I, Krief S, Ferrara N, Elliott AM, Zelzer E. Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism. Development (Cambridge, England). 2015 Feb 15;142(4):672-80. PubMed PMID: 25617432.
Jin SW, Beis D, Mitchell T, Chen JN,Stainier DY. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development. 2005 132: 5199-209.
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.
Maltepe E, Schmidt JV, Baunoch D, Bradfield CA, Simon MC. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature. 1997 Mar 27;386(6623):403-7. PubMed PMID: 9121557.
McCollum CW, Conde-Vancells J, 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 analysis in zebrafish embryos and mouse embryonic endothelial cells. Reprod Toxicol. 2017; 70: 60-69. PMID:27838387.
Saili KS, Franzosa JA, Baker NC, Ellis-Hutchings RG, Settivari RS, Carney EW, Spencer R, Zurlinden TJ, Kleinstreuer NC, Li S, Xia M and Knudsen TB. Systems Modeling of Developmental Vascular Toxicity. Curr Opin Toxicol. 2019; 15(1): 55-63. PMID:32030360.
Shirinifard A, McCollum CW, Bondesson MB, Gustafsson JA, Glazier JA and Clendenon SG. 3D Quantitative analyses of angiogenic sprout growth dynamics Devel Dynam. 2013 242(5): 518-526.
Tal T, Kilty C, Smith A, LaLone C, Kennedy B, Tennant A, McCollum CW, Bondesson M, Knudsen T, Padilla S and Kleinstreuer N. Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity. Reprod Toxicol. 2017; 70: 70-81. PMID:28007540.
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.
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.
Tran TC, Sneed B, Haider J, Blavo D, White A, Aiyejorun T, et al. Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish. Cancer research. 2007;67: 11386-92.
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.
Vargesson N. Thalidomide-induced teratogenesis: history and mechanisms. Birth defects Research Part C, Embryo today: reviews. 2015 Jun;105(2):140-56. PubMed PMID: 26043938.
Yozzo KL, Isales GM, Raftery TD,Volz DC. High-content screening assay for identification of chemicals impacting cardiovascular function in zebrafish embryos. Environmental science & technology. 2013;47: 11302-10.