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Key Event Title
|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|
Key Event Description
Developmental angiogenesis most closely ties into the Gene Ontology term ‘Blood Vessel Morphogenesis’ (GO:0048514), defined as “The process in which the anatomical structures of blood vessels are generated and organized. The blood vessel is the vasculature carrying blood”. The molecular control of endothelial cell behaviors during blood vessel morphogenesis requires coordinated cell migration, proliferation, polarity, differentiation and cell-cell communication [Herbert and Stanier, 2011; Blanco and Gerhardt, 2013]. Among the genes linked to this process [Drake et al. 2007] are 660 genes presently curated in The Mouse Gene Ontology Browser (http://www.informatics.jax.org/vocab/gene_ontology/, last accessed November 30, 2021). Three subordinate annotations account for 593 (89.8%) of those genes: (i) vasculogenesis (96 genes, GO:0001570, defined as “The differentiation of endothelial cells from progenitor cells during blood vessel development, and the de novo formation of blood vessels and tubes”; (ii) angiogenesis (545 genes, GO:0001525, defined as “Blood vessel formation when new vessels emerge from the proliferation of pre-existing blood vessels”; and (iii) negative regulation of blood vessel morphogenesis (110 genes, GO:0016525, defined as “Any process that stops, prevents, or reduces the frequency, rate or extent of angiogenesis”. Vegfr2 alone mapped to both vasculogenesis and angiogenesis, consistent with its critical pro-angiogenic role. Vegfr1 alone mapped to negative regulation of blood vessel morphogenesis consistent with its role as an endogenous angiogenesis inhibitor.
The angiogenic state of a cell can be explained as a balance between pro- and anti-angiogenic signals. During vasculogenesis, endothelial progenitor cells (angioblasts) in the prevascular mesoderm undergo a mesenchymal-to-epithelial transition to assemble into nascent endothelial tubes. This is dependent on VEGF signaling as demonstrated by the lack of nascent tubules when the prevascular mesoderm from the early mouse embryo is treated with sFlt1 or VEGF antibodies [Argraves et al. 2002] and in vegfaa(-/-) zebrafish embryos lacking de novo assembly of angioblasts into major blood vessels (dorsal aorta, cardinal vein) [Jin et al. 2019]. The acquisition of arterial or venous fate during angioblast assembly occurs during vasculogenesis [Herbert and Stanier, 2011]. While VEGFA-signaling promotes arterial fate [Jin et al. 2019], it is not required by endothelial cells to maintain their organization as an endothelium and acquire arterial or venous fates [Argraves et al. 2002]. VEGFR1 plays a role in endothelial organization and prevents overgrowth but is not required for endothelial differentiation [Fong et al. 1995; Roberts et al. 2004]. The dynamics of endothelial sprouting from existing vasculature (angiogenesis) takes over from here. VEGF signaling induces filopodial extensions to sprout from extant endothelial cells at the site, forming an endothelial tip cell (EC-tip) as the critical VEGFR2-responsive event [Belair et al. 2016a and 2016b]. Together with lateral inhibition by Dll4-Notch signaling, the VEGF-Notch-Dll4 signaling system determines where the endothelium will sprout an EC-tip cell or stay behind as a proliferating EC-stalk cells [Williams et al. 2006; Oladipupo et al. 2011; Venkatraman et al. 2016]. Angiogenic sprouts migrate along VEGF corridors established by local signals and extracellular matrix interactions, lumenize to endothelial tubules, and form connections with other tubules [Herbert and Stanier, 2011]. This requires local suppression of cell motility, pruning of any overgrowth by apoptosis, and the formation of new cell-cell junctions [Eilkin and Adams, 2010]. VEGF primes the endothelium to respond to factors that promote EC-tip cells, tubulogenesis, cytoskeletal remodeling, basement membrane deposition, activation of focal adhesion, and pericyte recruitment and proliferation [Bowers et al. 2020]. VEGF priming requires VEGFR2, and the effect of VEGFR2 is selective to the priming response. Although the genetic signals and responses for vasculogenesis (de novo assembly of angioblasts) and angiogenesis (endothelial growth and sprouting) differ, MIE:305 is common to both processes embedded in KE:28.
How It Is Measured or Detected
Methods to quantify angiogenesis are essential to management of neovascularization for disease progression, drug discovery, and assessing environmental chemicals. Diverse assays used to detect or measure the biological states represented in KE:28 broadly stated include: (i) in vitro measures from endothelial cell culture, pluripotent stem cells, automated high-throughput screening (HTS) platforms, high-content imaging of human endothelial cell reporter lines, and engineered microsystems; (ii) in vivo measures with endothelial reporter zebrafish lines, chick chorioallantoic membrane vascularization, and genetic mouse models; and (iii) in silico computational models for quantitative simulation and biological integration. Each has advantages and limitations for dissecting the biological complexity of blood vessel morphogenesis, which involves coordinated control of endothelial cell migration, proliferation, polarity, differentiation, and cell-cell communication [Herbert and Stanier, 2011; Irwin et al. 2014]. In vitro models to study activation of endothelial function and screen for angiogenesis inhibitors are optimized to detect effects such as EC- tip cell selection, sprout formation, EC-stalk cell proliferation, and ultimately vascular stabilization by support cells [Belair et al. 2016a].
Angiogenic sprouting: Pro-angiogenic signals such as VEGF promote endothelial motility, filopodia extension and proliferation, and, together with Notch signaling, controls whether specific endothelial cells become lead tip cells (EC-tip) or trailing stalk cells (EC-stalk) [Eilken and Adams, 2010]. During sprouting, a highly motile EC-tip cell migrates from the blood vessel and is trailed by proliferating EC-stalk cells that form the body of the nascent sprout. Chemotactic, haptotactic, and extracellular matrix (ECM) guide and support this migration; however, regulation converges ultimately on cytoskeletal remodeling in EC-tip cells that can be visualized with molecular probes and immunochemical reagents specific for actin (microfilaments) and tubulin (microtubules) [Lamalice et al. 2007]. Functional assays used to evaluate angiogenic sprouting must, however, incorporate natural (ECM) or synthetic (hydrogel) matrices to support growth factor-dependent endothelial cell proliferation, migration and VEGF-dependent invasive behaviors. Several traditional and newer methods have been used to meet that requirement.
Aortic explants: Aortic explants cultured from developing chick embryos or mouse/rat fetuses have been used as a source for evaluating drug/chemical effects on microvessel outgrowth [Baker et al. 2011; Beedie et al. 2015; Ellis-Hutchings et al. 2017; Kapoor et al. 2020; Katakia et al. 2020]. Microvascular streams from these explants are amenable to morphometric analysis of many sprouting behaviors, including cell migration, proliferation tube formation, branching, perivascular recruitment and remodeling. Sandwiching the explants in a 3D collagen matrix supplemented with optimal conditions for endothelial culture improves the spatial dimensionality of microvessel imaging [Kapoor et al. 2020]. An advantage of this platform is in its simplicity and capacity to monitor sprouting behaviors in explants sampled from different species, anatomical spaces, or stages of development [Katakia et al. 2020]. A disadvantage is that explants require animal resources in the first place.
Human cell-based in vitro tubulogenesis assay: Angiogenic sprouts convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions. In vitro assays for this assembly, commonly referred to as tubulogenesis, use human umbilical vein endothelial cells (HUVEC) co-cultured with fibroblasts [Bishop et al. 1999]. Routine cell culture methods support the organization of isolated HUVEC cells into endothelial networks that resemble a microvascular bed upon stimulation with VEGF. The standardized assay detects pro-angiogenic and anti-angiogenic activities that are tracked with with immunochemical biomarkers (eg, PECAM-1) and quantified by image analysis [Bishop et al. 1999]. Refinements improved the standardized assay to increase sensitivity (limits of detection and linearity of response), reliability (reproducibility and repeatability), and predictivity for human-relevant high-throughput testing [Sarkanen et al. 2010 and 2012; Huttala et al. 2015]. The improved platform was validated in a GLP laboratory following the OECD Guidance Document 34 for the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment [Toimela et al. 2017]. A vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into vascularized embryoid bodies has been described, where the microsystem cultured onto 3D-collagen gels recapitulates key features of in vivo sprouting including endothelial EC-tip cell selection, migration and proliferation, polarized guidance, tubulogenesis, and mural cell recruitment [Galaris et al. 2021].
Engineered microtissues: To better recapitulate angiogenesis in vivo, in vitro assays for drug and chemical screening must adopt physiological relevant culture conditions with robustness and scalability. Human endothelial lines have been derived from induced pluripotent stem cells (iPSC-EC) and cultured in engineered platforms that mimic the 3D microenvironment [Belair et al. 2015]. They formed VEGF-dependent 3D perfusable vascular networks when co-cultured with fibroblasts and aligned with flow in microfluidic devices [Belair et al. 2015]. Encapsulating endothelial cells at controlled densities in hydrogel microspheres surrounded by a synthetic ECM [Belair et al. 2016a] or VEGF-binding peptides [Belair et al. 2016b] can be used to evaluate the activation by ECM and ECM-sequestered VEGF and other angiogenic factors. Synthetic hydrogels proved advantageous over Matrigel for consistency in screening for drug/chemical effects [Nguyen et al. 2017]. Applying an array of individually addressable microfluidic circuits to differentiating EC-tip cells in a 3D collagen enables continuous exposure to VEGF-165 and other test agents for optimizing conditions for directional sprouting, microvascular anastomosis, and vessel maturation [van Duinen et al. 2019]. The 3D micro-perfusion angiogenesis assay showed similar performance between primary endothelial cells and iPSC-ECs with regards to sprouting behaviors (eg, EC-tip cell formation, directional sprouting, and lumenization) as well as VEGF gradient-driven angiogenic sprouting [van Duinen et al. 2020]. The role of VEGF-priming has been precisely defined for serum-free 3D microvessel formation using a cocktail of growth factors needed in combination [Bowers et al. 2020]. VEGF failed to support this process under serum-free conditions but an 8-hour pretreatment with VEGF-165 led to marked increases in the endothelial cell response to angiogenic factors.
Computational models: These aspects of angiogenic sprouting have been modelled in silico mathematically or computationally, probing deeply into the molecular control of tip/stalk switching dynamics linked to the VEGF-Notch-DLL4 signaling [Venkataraman et al. 2016], uncovering the critical determinants of EC-tip and EC-stalk differentiation that influence the morphology of sprout progression [Palm et al. 2016], establishing canonical growth trajectories in normal and chemical-disrupted zebrafish embryos [Shirinifard et al. 2013], and simulating cell-cell interactions in a self-organizing computer model of tubulogenesis for predictive toxicology [Kleinstreuer et al. 2013].
Domain of Applicability
ToxCast high-throughput screening (HTS) data for 25 assays mapping to targets in embryonic vascular disruption signature [Knudsen and Kleinstreuer, 2011] were used to rank-order 1060 chemicals for their potential to disrupt vascular development. The predictivity of this signature is being evaluated in various angiogenesis assays, including angiogenic sprouting in human endothelial cells [Belair et al. 2016] and trangenic zebrafish embryos [Tal et al. 2016].
Belair et al.  designed and characterized a chemically human angiogenesis pPSC-EC sprouting model that responded appropriately to several reference pharmacological angiogenesis inhibitors, including Vatalanib/PTK787, which suggests the functional role of VEGFR2. Several pVDCs from the ToxCast library also inhibited angiogenic sprouting in this assay. Because gene sequence similarity of the ToxCast pVDC signature is comprised of proteins that primarily map to human in vitro and biochemical assays, the U.S. EPA SeqAPASS tool was used to assess the degree of conservation of signature targets between zebrafish and human, as well as other commonly used model organisms in human health and environmental toxicology research [Tal et al. 2017]. This approach revealed that key nodes in the ontogenetic regulation of angiogenesis have evolved across diverse species. Homology appeared first in the receptor tyrosine kinase signaling systems, followed in turn by the urokinase plasminogen activating (uPA) receptor (uPAR) system and chemokine/G-protein coupled receptor system.
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