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Screening for Angiogenic Inhibitors in Zebrafish to Evaluate A Predictive Model for Developmental Vascular Toxicity

Tamara Tal, Claire Kilty, Andrew Smith, Carlie LaLone, Brendán Kennedy, AlanTennant, Catherine W. McCollum, Maria Bondesson, Thomas Knudsen, Stephanie Padilla, and Nicole Kleinstreuer.
Reproductive Toxicology (2017) DOI: https://doi.org/10.1016/j.reprotox.2016.12.004 PMID: 28007540



Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals for their potential to disrupt vascular development. Thirty-seven compounds were selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP) zebrafish embryos to identify chemicals that impair developmental angiogenesis. We hypothesized that zebrafish angiogenesis toxicity data would correlate with human cell-based and cell-free in vitro HTS ToxCast data. Univariate statistical associations used to filter HTS data based on correlations with zebrafish angiogenic inhibition in vivo revealed 132 total significant associations, 33 of which were already captured in the pVDC signature, and 689 non-significant assay associations. Correlated assays were enriched in cytokine and extracellular matrix pathways. Taken together, the findings indicate the utility of zebrafish assays to evaluate an HTS-based predictive toxicity signature and also provide an experimental basis for expansion of the pVDC signature with novel HTS assays.


Figure 1. An AOP for embryonic vascular disruption.

A predictive toxicity model was generated to group chemicals by their in vitro bioactivity profile and look for signatures that correlate with in vivo toxicity. (A) An AOP for embryonic vascular disruption was constructed by identifying initial molecular targets that are linked to developmental angiogenesis and coarsely map to 124/821 human in vitro ToxCast assays. ToxCast assays (124) mapping to 30 molecules are included in the ToxPi for putative vascular disrupting compounds (pVDCs). (B) The signature was used to rank order 1060 ToxCast chemicals and a 37 member chemical test set was selected. CASP8 (Caspase 8); CCL2 (chemokine (C-C motif) ligand 2); CXCL9.10 (C-X-C motif chemokine 9 and 10); EPHA1.A2.B1 (Ephrin receptor type A1, A2, and B2); ERa (Estrogen receptor alpha); FGFR (Fibroblast growth factor receptor); HIF1a (Hypoxia inducible factor 1 alpha); IL1a.6.8 (Interleukin 1a, 6, and 8); MMP1.2.9 (Matrix metalloproteinase 1, 2, and 9); NFkB (Nuclear factor kappa B); PAI1 (Plasminogen activator 1); PTEN (Phosphatase and tensin homolog); PTPN11.12 (Protein tyrosine phosphatase non-receptor type 11 and 12); PTPRB (Protein tyrosine phosphatase receptor type B); TBXA2 (Thromboxane A2): THBD (Thrombomodulin); Tie2 (TEK tyrosine kinase); TNFa (Tumor necrosis factor alpha); TGFb (Transforming growth factor beta); uPA (Urokinase-type plasminogen activator); uPAR (Urokinase receptor); VEGFR1.2.3 (Vascular endothelial growth factor receptor 1, 2, and 3); VCAM1 (vascular cell adhesion protein 1); VC_Prolif (Vascular cell proliferation).

Figure 2. Sequence similarity of the vascular toxicity predictive signature across model organisms.

SeqAPASS was used to compare amino acid sequence conservation across common model organisms (relative to human) for each of the 30 molecules represented in the predictive signature. (A) ToxPi key with each protein represented by a single slice. (B) Percent similarity comparisons of selected key functional domains are shown. See Supplemental Table S4 and Supplemental Fig. S2 for percent similarity comparisons across the entire primary amino acid sequences.

Figure 3. Developmental angiogenesis toxicity assay.

Dechorionated Tg(kdr1:EGFP) zebrafish embryos were exposed to 37 test chemicals from 26 to 72 hpf and imaged at 72 hpf. (A) Study design. (B) Representative negative and positive control images. (C) Hits were rescreened and representative images are shown here and in Supplemental Fig. S3 (n = 2). Eye (E), cranial (C), caudal vein (CV), inter segmental vessels (ISV), sub intestinal vessels (SIV), and yolk (Y) vessels are noted.

Figure 4. Hyaloid vessel assay.

Tg(fli1:EGFP) zebrafish embryos were exposed to 37 test chemicals from 48 to 120 hpf. GFP positive hyaloid primary branches covering the back of the lens were imaged and quantified on an Olympus SZX16 fluorescence microscope (n = 3 with 5 larvae per well per concentration) (Supplemental Fig. S4). (A) Representative negative and positive control (Su) images are shown. Primary hyaloid vessel (HV) branches (white arrows) emerge from the optic disk (asterisk). (B) Representative images and (C) HV vessel quantification for haloperidol, 1-hydroxypyrene, and bisphenol A are shown.

Figure 5. Evaluation of the pVDC signature using zebrafish angiogenesis toxicity data.

ToxCast in vitro HTS assay data consisted of AC50 values for 1060 chemicals across 821 assays. To identify HTS assays that are correlated with angiogenic inhibition in zebrafish, ToxCast data were compared to chemicals that produce angiogenic inhibition in any zebrafish assay (EPA and UCD data in addition to data on 161 ToxCast Phase 1 chemicals screened by M. Bondesson Lab at UH; Supplemental Tables S6 and S7) via univariate analysis. (A–B) Univariate schematic reveals that 132 ToxCast assays are correlated with angiogenic inhibition in zebrafish, 33 of which were already captured in the pVDC signature. (C) Signature assays by AOP pathway/quadrant are shown. ToxCast assays for CCL2 (red), IL1a.6.8 (pink), vascular cell proliferation (dark pink), VEGFR2 (blue) Ephrins (purple), and PTPN (light purple) are highlighted among all correlated assays. Thirty of the 33 correlative pVDC signature assays mapped to chemokine and ECM pathways. Univariate associations identified were mined for relationships that may be informative of angiogenic inhibition in vivo but are not contained in the pVDC signature. (D) Assay platform for all ToxCast assays significantly associated with angiogenic inhibition in one or more zebrafish assays. (E) Newly identified ToxCast assays positively correlated with developmental angiogenic inhibition in zebrafish that relate xbp1-mediated flow are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


Table 1. Human query proteins and select functional domains for sequence similarity evaluation.

Human query proteins and select functional domains for sequence similarity evaluation across species. aType of Domain Hit defined by the NCBI CDD: Specific hits (S) meet or exceed a domain-specific e-value threshold and represent a very high confidence that the query sequence belongs to the same protein family as the sequences used to create the domain model; non-specific hits (N) meet or exceed the RPS-BLAST (reverse-position specific-Basic Local Alignment Search Tool) threshold for statistical significance (default E-value cutoff of 0.01). National Center for Biotechnology Information (NCBI); Conserved Domain Database (CDD); amino acid (aa).

Table 2. Chemical hits that disrupt vessel development in zebrafish.

pVDC scores, hit calls, and the LOELs (Lowest Observable Effect Level) for angiogenic-specific toxicity are shown. Environmental Protection Agency (EPA); University College Dublin (UCD); putative Vascular Disrupting Compound (pVDC).

Supplemental Materials

Supplementary data