Activation, PPARα

117-81-7

BJQHLKABXJIVAM-UHFFFAOYNA-N

BJQHLKABXJIVAM-UHFFFAOYSA-N

Di(2-ethylhexyl) phthalate

1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester DEHP 1,2-Benzedicarboxylic acid, bis(2-ethyl-hexyl) ester 1,2-Benzenedicarboxylic acid bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid, 1,2-bis(2-ethylhexyl) ester 1,2-Benzenedicarboxylic acid,bis(2-ethylhexylester) Bis(2-ethylhexyl) 1,2-benzenedicarboxylate Bis(2-ethylhexyl) o-phthalate bis(2-ethylhexyl) phthalate Bis(2-ethylhexyl)phthalat Bis(2-ethylhexyl)phthalate Bisoflex 81 Bisoflex DOP Corflex 400 Di(2-ethylhexyl)phthalate Di(isooctyl) phthalate Di-2-ethylhexlphthalate Di-2-ethylhexyl phthalate DI-2-ETHYLHEXYL-PHTHALATE Diacizer DOP Diethylhexyl phthalate Dioctylphthalate DOF Ergoplast FDO Ergoplast FDO-S ETHYLHEXYL PHTHALATE Eviplast 80 Eviplast 81 Fleximel Flexol DOD Flexol DOP ftlalato de bis(2-etilhexilo) Garbeflex DOP-D 40 Good-rite GP 264 Hatco DOP Jayflex DOP Kodaflex DEHP Kodaflex DOP Monocizer DOP NSC 17069 Palatinol AH Palatinol AH-L Phtalate de Bis (Ethyle-2-Hexyle) Phtalate de bis(2-ethylhexyle) PHTHALATE, BIS(2-ETHYLHEXYL) Phthalic acid di(2-ethylhexyl) ester Phthalic acid, bis(2-ethylhexyl) ester PHTHALIC ACID, BIS(2-ETHYLHEXYL)ESTER PHTHALSAEURE-BIS-(2-AETHYLHEXYL)-ESTER Pittsburgh PX 138 Plasthall DOP Reomol D 79P Sansocizer DOP Sansocizer R 8000 Sconamoll DOP Staflex DOP Truflex DOP Vestinol AH Vinycizer 80 Vinycizer 80K Witcizer 312

DTXSID5020607

637-07-0

KNHUKKLJHYUCFP-UHFFFAOYSA-N

Clofibrate

ethyl-p-chlorophenoxyisobutyrate Propanoic acid, 2-(4-chlorophenoxy)-2-methyl-, ethyl ester 2-(p-Chlorophenoxy)-2-methylpropionic acid ethyl ester Abitrate Amotril Anparton Arteriosan Artevil Ateculon Ateriosan Atheropront Atromid S Atromidin Azionyl Bioscleran Cartagyl Claripex Claripex CPIB Clobren SF Clofibrat clofibrato Clofinit Ethyl (p-chlorophenoxy) isobutyrate Ethyl 2-(4-chlorophenoxy)-2-methylpropionate Ethyl 2-(4-chlorophenoxy)isobutyrate Ethyl 2-(p-chlorophenoxy)-2-methylpropionate Ethyl 2-(p-chlorophenoxy)isobutyrate Ethyl clofibrate Ethyl p-chlorophenoxyisobutyrate Ethyl α-(4-chlorophenoxy)isobutyrate Ethyl α-(4-chlorophenoxy)-α-methylpropionate Ethyl α-(p-chlorophenoxy)isobutyrate Ethyl α-(p-chlorophenoxy)-α-methylpropionate Hyclorate Lipavil Lipavlon Lipomid Liprinal Miscleron Misclerone Neo-Atromid Normolipol NSC 79389 p-Chlorophenoxyisobutyric acid ethyl ester Propionic acid, 2-(p-chlorophenoxy)-2-methyl-, ethyl ester Recolip Regelan Serotinex Sklerepmexe Sklerolip Skleromexe Sklero-Tablinene Ticlobran Xyduril

DTXSID3020336

3771-19-5

XJGBDJOMWKAZJS-UHFFFAOYNA-N

XJGBDJOMWKAZJS-UHFFFAOYSA-N

Nafenopin

DTXSID8020911

52214-84-3

KPSRODZRAIWAKH-UHFFFAOYNA-N

KPSRODZRAIWAKH-UHFFFAOYSA-N

Ciprofibrate

DTXSID8020331

25812-30-0

HEMJJKBWTPKOJG-UHFFFAOYSA-N

Gemfibrozil

Pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl- 2,2-Dimethyl-5-(2,5-xylyloxy)valeric acid 5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanoic acid Decrelip gemfibrozilo Gevilon Lopizid Trialmin 900 Valeric acid, 2,2-dimethyl-5-(2,5-xylyloxy)-

DTXSID0020652

41859-67-0

IIBYAHWJQTYFKB-UHFFFAOYSA-N

Bezafibrate

Propanoic acid, 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methyl- Befizal Benzofibrate Bezafibrat bezafibrato Bezalip Bezatol Difaterol

DTXSID3029869

49562-28-9

YMTINGFKWWXKFG-UHFFFAOYSA-N

Fenofibrate

Propanoic acid, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-, 1-methylethyl ester 2-[4-(4-Chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester Ankebin Clorofibrate Elasterin Fenobrate Fenofibrat fenofibrato Fenogal Fenotard Isopropyl 2-[p-(p-chlorobenzoyl)phenoxy]-2-methylpropionate Lipanthyl Lipantil Lipicard Lipidil Lipidil Supra Lipirex Lipoclar Lipofene Liposit MeltDose Nolipax NSC 281319 Procetofen Procetofene Procetoken Protolipan Secalip

DTXSID2029874

79902-63-9

RYMZZMVNJRMUDD-HGQWONQESA-N

Simvastatin

Butanoic acid, 2,2-dimethyl-, (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyl ester (+)-Simvastatin Apo-Simvastatin Bestatin 20 Butanoic acid, 2,2-dimethyl-, 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester, [1S-[1α,3α,7β,8β(2S*,4S*),8aβ]]- Cholestat Co-Simvastatin Kolestevan L 644128-000U Lipinorm Liponorm Lipovas Lodales Modutrol Nor-Vastina Novo-Simvastatin Pms-simvastatin Simastin 20 Simovil Simvastatin lactone Simvotin Sinvacor Sinvascor Sivastin Starstat 20 Synvinolin Valemia Velostatin

DTXSID0023581

PR:000013056

PR peroxisome proliferator-activated receptor alpha

PR:000017284

PR vascular endothelial growth factor A

GO:0035357

GO peroxisome proliferator activated receptor signaling pathway

GO:0010467

GO gene expression

MP:0008469

MP abnormal protein level

GO:0001525

GO angiogenesis

MP:0008762

MP embryonic lethality

D009026

MESH mortality

WIKI increased

WIKI decreased

Di(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Mono(2-ethylhexyl) phthalate

2016-11-29T18:42:26

Stressor:205 pirinixic acid (WY-14,643)

2020-12-19T09:06:20

Clofibrate

2016-11-29T18:42:27

Nafenopin

2016-11-29T18:42:27

ciprofibrate

2016-11-29T18:42:27

Gemfibrozil Fibrate drug

2016-11-29T18:42:27

2020-03-31T10:24:40

PERFLUOROOCTANOIC ACID

2016-11-29T18:42:27

Bezafibrate

2016-11-29T18:42:27

Fenofibrate

2016-11-29T18:42:27

Simvastatin

2020-05-06T09:41:35

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

2017-02-09T14:32:32

10116

NCBI rat

10090

NCBI mouse

WCS_9606

common toxicological species human

WCS_9031

common ecological species chicken

WikiUser_17

mammals

WCS_93934

common ecological species Japanese quail

WCS_7955

common ecological species zebrafish

WCS_8355

common ecological species Xenopus laevis

WikiUser_28

Vertebrates

Activation, PPARα

Molecular

Gene expression occurs in a coordinated fashion (Judson et al., 2012). The many observations of altered gene expression following binding of ligand to PPARα led to systematic investigations of the genomic signature that corresponds to PPARα activation (Tamura et al., 2006; Kupershmidt et al., 2010; Rosen et al., 2017; Rooney et al., 2018; Corton et al., 2020; Hill et al., 2020; Lewis et al., 2020). Specific gene with increased expression following PPARα activation include Cyp4a1, Cpt1B, and Lpl. More generally, the pathways activated include: <ul> <li>Genes involved in Metabolism of lipids and lipoproteins</li> <li>Fatty acid metabolism</li> <li>Genes involved in Fatty acid, triacylglycerol, and ketone body metabolism</li> <li>PPAR signaling pathway</li> <li>Peroxisome</li> <li>Genes involved in Cell Cycle</li> </ul> Biological state The Peroxisome Proliferator Activated receptor α (PPARα) belongs to the <a href="/wiki/index.php/Peroxisome_Proliferator_Activated_receptors_(PPARs;_NR1C)" title="Peroxisome Proliferator Activated receptors (PPARs; NR1C)">Peroxisome Proliferator Activated receptors (PPARs; NR1C)</a> steroid/thyroid/retinoid receptor superfamily of transcription factors. Biological compartments PPARα is expressed in high levels in tissues that perform significant catabolism of fatty acids (FAs), such as brown adipose tissue, liver, heart, kidney, and intestine (Michalik et al. 2006). The receptor is present also in skeletal muscle, intestine, pancreas, lung, placenta and testes (Mukherjee et al. 1997), (Schultz et al. 1999). General role in biology PPARs are activated by fatty acids and their derivatives; they are sensors of dietary lipids and are involved in lipid and carbohydrate metabolism, immune response and peroxisome proliferation (Wahli and Desvergne 1999), (Evans, Barish, & Wang, 2004). PAPRα is a also a target of hypothalamic hormone signalling and was found to play a role in embryonic development (Yessoufou and Wahli 2010). Fibrates, activators of PPARα, are commonly used to treat hypertriglyceridemia and other dyslipidemic states as they have been shown to decrease circulating lipid levels (Lefebvre et al. 2006).

Binding of ligands to PPARα is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from transactivation assays (e.g. transactivation assay with reporter gene) that demonstrate functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change of nuclear receptor that promotes binding to transcriptional co-activators. Conversely, binding of antagonists results in a conformation that favours the binding of co-repressors (Yu and Reddy 2007), (Viswakarma et al. 2010). Transactivation assays are performed using transient or stably transfected cells with the PPARα expression plasmid and a reporter plasmid, respectively. There are also other methods that have been used to measure PPARα activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits, see Table 1. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to an adverse outcome (LeBlanc, Norris, and Kloas 2011). A recent study characterized the PPARα ligand binding domain for the purpose of next-generation metabolic disease drugs (Kamata et al. 2020). The most direct measure of this MIE is microarray profiling from large gene expression databases TG-GATEs and DrugMatrix coupled with t statistical analysis of whole genome expression profiles (Svoboda et al., 2019; Igarashi et al., 2015) From these data, A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature is a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test (Corton et al., 2020; Hill et al., 2020; Kupershmidt et al., 2010; Lewis et al., 2020; Rooney et al., 2018). A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature would be a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test (Kupershmidt et al., 2010; Rooney et al., 2018; Corton et al., 2020). For all substances, MIE activation does not rise monotonically over dose or time. These fluctuations are likely due to variations in cofactor availability or access to the site of transcription (Gaillard et al., 2006; Koppen et al., 2009; Kupershmidt et al., 2010; Ong et al., 2010; Chow et al., 2011; De Vos et al., 2011; Simon et al., 2015). . <table align="left" border="1" cellpadding="1" cellspacing="1" style="height:3px; width:100px"> <caption>Measurements of PPARα Activation</caption> <thead> <tr> <th scope="row">Method/Test</th> <th scope="col">Test Principle</th> <th scope="col">Test Environment</th> <th scope="col">Test Outcome</th> <th scope="col">Assay Type/Domain</th> </tr> </thead> <tbody> <tr> <th scope="row"> molecular modelling; docking simulation </th> <td>Computational simulation of  ligand binding </td> <td>In silico</td> <td>Prediction off binding interaction </td> <td>Quantitative virtual screeings</td> </tr> <tr> <th scope="row">Scintillation proximity binding assay</th> <td>Direct binding of ligand</td> <td>In vitro</td> <td>Identifies compouds that bind to PPARα</td> <td>Qualitative in vitro screening</td> </tr> <tr> <th scope="row">PPARα reporter gene assay</th> <td>Quantify changes in in PPARα activation via a sensitive surrogate </td> <td>In vitro, Ex vivo</td> <td>Measures changes in activity of genes linked to a PPARα receptor element</td> <td>Quantitative in vitro screening</td> </tr> <tr> <th scope="row">Electrophoretic Band Shift</th> <td>determines if a protein or protein mixture will bind to a specific DNA or RNA sequence</td> <td>In vitro</td> <td>Measures cofactor binding by changes in gel mobility</td> <td>Quantitative in vitro screening</td> </tr> <tr> <th scope="row">Microarray profiling</th> <td>Develop MIE-specific sets of gene expression biomarkers</td> <td>In vivo</td> <td>Classification of PPARα biomarker genes with statistical methods</td> <td>Quantitative in vivo screening</td> </tr> </tbody> </table>

PPARα has been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).

UBERON:0002107

UBERON liver

CL:0000255

CL eukaryotic cell

High High High

Bhattacharya, Nandini, Jannette M Dufour, My-Nuong Vo, Janice Okita, Richard Okita, and Kwan Hee Kim. 2005. “Differential Effects of Phthalates on the Testis and the Liver.” Biology of Reproduction 72 (3) (March): 745–54. doi:10.1095/biolreprod.104.031583. Bility, Moses T, Jerry T Thompson, Richard H McKee, Raymond M David, John H Butala, John P Vanden Heuvel, and Jeffrey M Peters. 2004. “Activation of Mouse and Human Peroxisome Proliferator-Activated Receptors (PPARs) by Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 82 (1) (November): 170–82. doi:10.1093/toxsci/kfh253. Chow, C. C., Ong, K. M., Dougherty, E. J., & Simons, S. S. (2011). Inferring mechanisms from dose-response curves. Methods Enzymol, 487, 465-483. https://doi.org/10.1016/B978-0-12-381270-4.00016-0 Corton, J. C., Hill, T., Sutherland, J. J., Stevens, J. L., & Rooney, J. (2020). A Set of Six Gene Expression Biomarkers Identify Rat Liver Tumorigens in Short-Term Assays. Toxicol Sci. https://doi.org/10.1093/toxsci/kfaa101 De Vos, D., Bruggeman, F. J., Westerhoff, H. V., & Bakker, B. M. (2011). How molecular competition influences fluxes in gene expression networks. PLoS One, 6(12), e28494. https://doi.org/10.1371/journal.pone.0028494 Dufour, Jannette M, My-Nuong Vo, Nandini Bhattacharya, Janice Okita, Richard Okita, and Kwan Hee Kim. 2003. “Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis.” Biology of Reproduction 68 (4) (April): 1215–24. doi:10.1095/biolreprod.102.010488. Feige, Jérôme N, Laurent Gelman, Daniel Rossi, Vincent Zoete, Raphaël Métivier, Cicerone Tudor, Silvia I Anghel, et al. 2007. “The Endocrine Disruptor Monoethyl-Hexyl-Phthalate Is a Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator That Promotes Adipogenesis.” The Journal of Biological Chemistry 282 (26) (June 29): 19152–66. doi:10.1074/jbc.M702724200. Gaillard, S., Grasfeder, L. L., Haeffele, C. L., Lobenhofer, E. K., Chu, T.-M., Wolfinger, R., Kazmin, D., Koves, T. R., Muoio, D. M., Chang, C.-y., & McDonnell, D. P. (2006). Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs. Mol Cell, 24(5), 797-803. https://doi.org/10.1016/j.molcel.2006.10.012 Hill, T., Rooney, J., Abedini, J., El-Masri, H., Wood, C. E., & Corton, J. C. (2020). Gene Expression Thresholds Derived From Short-Term Exposures Identify Rat Liver Tumorigens. Toxicol Sci. https://doi.org/10.1093/toxsci/kfaa102 Hurst, Christopher H, and David J Waxman. 2003. “Activation of PPARalpha and PPARgamma by Environmental Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 74 (2) (August): 297–308. doi:10.1093/toxsci/kfg145. Igarashi, Y., Nakatsu, N., Yamashita, T., Ono, A., Ohno, Y., Urushidani, T., & Yamada, H. (2015). Open TG-GATEs: a large-scale toxicogenomics database. Nucleic Acids Res, 43(Database issue), D921-7. https://doi.org/10.1093/nar/gku955 Kamata S, Oyama T, Saito K, Honda A, Yamamoto Y, Suda K, Ishikawa R, Itoh T, Watanabe Y, Shibata T, Uchida K, Suematsu M, Ishii I. PPARα Ligand-Binding Domain Structures with Endogenous Fatty Acids and Fibrates. iScience. 2020;23(11):101727. 10.1016/j.isci.2020.101727 Kaya, Taner, Scott C Mohr, David J Waxman, and Sandor Vajda. 2006. “Computational Screening of Phthalate Monoesters for Binding to PPARgamma.” Chemical Research in Toxicology 19 (8) (August): 999–1009. doi:10.1021/tx050301s. Koppen, A., Houtman, R., Pijnenburg, D., Jeninga, E. H., Ruijtenbeek, R., & Kalkhoven, E. (2009). Nuclear receptor-coregulator interaction profiling identifies TRIP3 as a novel peroxisome proliferator-activated receptor gamma cofactor. Mol Cell Proteomics, 8(10), 2212-2226. https://doi.org/10.1074/mcp.M900209-MCP200 Kupershmidt, I., Su, Q. J., Grewal, A., Sundaresh, S., Halperin, I., Flynn, J., Shekar, M., Wang, H., Park, J., Cui, W., Wall, G. D., Wisotzkey, R., Alag, S., Akhtari, S., & Ronaghi, M. (2010). Ontology-based meta-analysis of global collections of high-throughput public data. PLoS One, 5(9). https://doi.org/10.1371/journal.pone.0013066 Lampen, Alfonso, Susan Zimnik, and Heinz Nau. 2003. “Teratogenic Phthalate Esters and Metabolites Activate the Nuclear Receptors PPARs and Induce Differentiation of F9 Cells.” Toxicology and Applied Pharmacology 188 (1) (April): 14–23. doi:10.1016/S0041-008X(03)00014-0. Lapinskas, Paula J., Sherri Brown, Lisa M. Leesnitzer, Steven Blanchard, Cyndi Swanson, Russell C. Cattley, and J. Christopher Corton. 2005. “Role of PPARα in Mediating the Effects of Phthalates and Metabolites in the Liver.” Toxicology 207 (1): 149–163. Le Maire, Albane, Marina Grimaldi, Dominique Roecklin, Sonia Dagnino, Valérie Vivat-Hannah, Patrick Balaguer, and William Bourguet. 2009. “Activation of RXR-PPAR Heterodimers by Organotin Environmental Endocrine Disruptors.” EMBO Reports 10 (4) (April): 367–73. doi:10.1038/embor.2009.8. LeBlanc, GA, DO Norris, and W Kloas. 2011. “Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors” (178). Lefebvre, Philippe, Giulia Chinetti, Jean-Charles Fruchart, and Bart Staels. 2006. “Sorting out the Roles of PPAR Alpha in Energy Metabolism and Vascular Homeostasis.” The Journal of Clinical Investigation 116 (3) (March): 571–80. doi:10.1172/JCI27989. Lewis, R. W., Hill, T., & Corton, J. C. (2020). A set of six Gene expression biomarkers and their thresholds identify rat liver tumorigens in short-term assays. Toxicology, 443, 152547. https://doi.org/10.1016/j.tox.2020.152547 Maloney, Erin K., and David J. Waxman. 1999. “Trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals.” Toxicology and Applied Pharmacology 161 (2): 209–218. Michalik, Liliane, Johan Auwerx, Joel P Berger, V Krishna Chatterjee, Christopher K Glass, Frank J Gonzalez, Paul A Grimaldi, et al. 2006. “International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors.” Pharmacological Reviews 58 (4) (December): 726–41. doi:10.1124/pr.58.4.5. Mukherjee, R, L Jow, G E Croston, and J R Paterniti. 1997. “Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-Activated Receptor (PPAR) Isoforms PPARgamma2 versus PPARgamma1 and Activation with Retinoid X Receptor Agonists and Antagonists.” The Journal of Biological Chemistry 272 (12) (March 21): 8071–6. Ong, K. M., Blackford, J. A., Kagan, B. L., Simons, S. S., & Chow, C. C. (2010). A theoretical framework for gene induction and experimental comparisons. Proc Natl Acad Sci U S A, 107(15), 7107-7112. https://doi.org/10.1073/pnas.0911095107 Rooney, J., Hill, T., Qin, C., Sistare, F. D., & Corton, J. C. (2018). Adverse outcome pathway-driven identification of rat liver tumorigens in short-term assays. Toxicol Appl Pharmacol, 356, 99-113. https://doi.org/10.1016/j.taap.2018.07.023 Schultz, R, W Yan, J Toppari, A Völkl, J A Gustafsson, and M Pelto-Huikko. 1999. “Expression of Peroxisome Proliferator-Activated Receptor Alpha Messenger Ribonucleic Acid and Protein in Human and Rat Testis.” Endocrinology 140 (7) (July): 2968–75. doi:10.1210/endo.140.7.6858. Simon, T. W., Budinsky, R. A., & Rowlands, J. C. (2015). A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators. PLoS One, 10(6), e0127952. https://doi.org/10.1371/journal.pone.0127952. Staels, B., J. Dallongeville, J. Auwerx, K. Schoonjans, E. Leitersdorf, and J.-C. Fruchart. 1998. “Mechanism of Action of Fibrates on Lipid and Lipoprotein Metabolism.” Circulation 98 (19) (November 10): 2088–2093. doi:10.1161/01.CIR.98.19.2088. Svoboda, D. L., Saddler, T., & Auerbach, S. S. (2019). An Overview of National Toxicology Program’s Toxicogenomic Applications: DrugMatrix and ToxFX.  In Advances in Computational Toxicology (pp. 141-157). Springer. https://link.springer.com/chapter/10.1007/978-3-030-16443-0_8 ToxCastTM Data. “ToxCastTM Data.” US Environmental Protection Agency. <a class="external free" href="http://www.epa.gov/ncct/toxcast/data.html" rel="nofollow" target="_blank">http://www.epa.gov/ncct/toxcast/data.html</a> Vanden Heuvel, John P, Jerry T Thompson, Steven R Frame, and Peter J Gillies. 2006. “Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor-Alpha, -Beta, and -Gamma, Liver X Receptor-Beta, and Retinoid X Rec.” Toxicological Sciences : An Official Journal of the Society of Toxicology 92 (2) (August): 476–89. doi:10.1093/toxsci/kfl014. Venkata, Nagaraj Gopisetty, Jodie a Robinson, Peter J Cabot, Barbara Davis, Greg R Monteith, and Sarah J Roberts-Thomson. 2006. “Mono(2-Ethylhexyl)phthalate and Mono-N-Butyl Phthalate Activation of Peroxisome Proliferator Activated-Receptors Alpha and Gamma in Breast.” Toxicology Letters 163 (3) (June 1): 224–34. doi:10.1016/j.toxlet.2005.11.001. Viswakarma, Navin, Yuzhi Jia, Liang Bai, Aurore Vluggens, Jayme Borensztajn, Jianming Xu, and Janardan K Reddy. 2010. “Coactivators in PPAR-Regulated Gene Expression.” PPAR Research 2010 (January). doi:10.1155/2010/250126. Wahli, Walter, and B Desvergne. 1999. “Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism.” Endocrine Reviews 20 (5) (October): 649–88. Wu, Bin, Jie Gao, and Ming-wei Wang. 2005. “Development of a Complex Scintillation Proximity Assay for High-Throughput Screening of PPARgamma Modulators.” Acta Pharmacologica Sinica 26 (3) (March): 339–44. doi:10.1111/j.1745-7254.2005.00040.x. Xu, Chuan, Ji-An Chen, Zhiqun Qiu, Qing Zhao, Jiaohua Luo, Lan Yang, Hui Zeng, et al. 2010. “Ovotoxicity and PPAR-Mediated Aromatase Downregulation in Female Sprague-Dawley Rats Following Combined Oral Exposure to Benzo[a]pyrene and Di-(2-Ethylhexyl) Phthalate.” Toxicology Letters 199 (3) (December 15): 323–32. doi:10.1016/j.toxlet.2010.09.015. Yessoufou, a, and W Wahli. 2010. “Multifaceted Roles of Peroxisome Proliferator-Activated Receptors (PPARs) at the Cellular and Whole Organism Levels.” Swiss Medical Weekly 140 (September) (January): w13071. doi:10.4414/smw.2010.13071. Yu, Songtao, and Janardan K Reddy. 2007. “Transcription Coactivators for Peroxisome Proliferator-Activated Receptors.” Biochimica et Biophysica Acta 1771 (8) (August): 936–51. doi:10.1016/j.bbalip.2007.01.008. AOPWiki 2016-11-29T18:41:23

2020-12-28T12:48:16

reduced production, VEGF

Cellular

Vascular endothelial growth factors (VEGFs) are a family of homodimeric glycoproteins that stimulate vasculogenesis and angiogenesis in various tissues[1].  They play vital roles in fetal development and increased oxygen supply in response to tissue injury and hypoxic stress[1,2].  VEGFs signal through cell surface receptor tyrosine kinases: VEGFR-1, VEGFR-2 and VEGFR-3 (Figure 1), which play critical roles in haematopoietic cell development, vascular endothelial cell development and lymphatic endothelial cell development, respectively[3].  The mammalian VEGF-A family has been extensively studied, and includes multiple splice variants, with VEGF165 being the most abundantly expressed[1].    <img alt="" class="mw-mmv-dialog-is-open mw-mmv-final-image png" src="https://upload.wikimedia.org/wikipedia/commons/c/c8/VEGF_receptors.png" /> Figure 1: VEGF family members and their respective receptors (Häggström, Mikael (2014). "<a class="external text" href="https://en.wikiversity.org/wiki/WikiJournal_of_Medicine/Medical_gallery_of_Mikael_H%C3%A4ggstr%C3%B6m_2014">Medical gallery of Mikael Häggström 2014</a>". WikiJournal of Medicine 1 (2). <a class="extiw" href="https://en.wikipedia.org/wiki/Digital_object_identifier" title="w:Digital object identifier">DOI</a>:<a class="external text" href="https://doi.org/10.15347/wjm/2014.008" rel="nofollow">10.15347/wjm/2014.008</a>. <a class="extiw" href="https://en.wikipedia.org/wiki/International_Standard_Serial_Number" title="en:International Standard Serial Number">ISSN</a> <a class="external text" href="http://www.worldcat.org/issn/2002-4436" rel="nofollow">2002-4436</a>. <a class="external text" href="https://creativecommons.org/publicdomain/zero/1.0/deed.en" rel="nofollow">Public Domain</a>. Retrieved 24/05/2017)

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? VEGF protein can be measured by enzyme-linked immunosorbent assay (Ivnitski-Steele et al. (2005), immunihistochemistry or western blot (Li et al. 2016). VEGF gene expression, which is directly correlated with protein levels, can be measured by quantitative real-time polymerase chain reaction (QPCR) (Medford et al. 2009).

VEGF proteins have been isolated and characterized in multiple species including mammals[1,2,4], chicken[4], Japanese quail[6], Xenopus laevis[7] and zebrafish[4,5,7]; VEGF165 in particular is highly conserved among species with >95% homology between the human transcript and bovine, ovine and murine variants[1].  The avian and amphibian VEGF proteins are highly homologous to the mammalian VEGFs, wheres the fish homologue is less similar[7]. Invertebrates, such as C. elegans and Drosophila also contain a VEGFR-like receptor[7].

CL:0000566

CL angioblastic mesenchymal cell

High Unspecific

High

Embryo

High

Development

High

Adult

High High High High High

1. Cecilia Y. Cheung (1997) Vascular Endothelial Growth Factor: Possible Role in Fetal Development and Placental Function. J Soc Gynecol Invest. 4: 169-77 2. Ahluwalia, A., and Tarnawski, A. S. (2012). Critical role of hypoxia sensor--HIF-1alpha in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr. Med. Chem. 19(1), 90-97. 3. Holmes, K., Roberts, O. L., Thomas, A. M., and Cross, M. J. (2007). Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal. 19(10), 2003-2012. 4. Ivnitski-Steele, I. D., Friggens, M., Chavez, M., and Walker, M. K. (2005). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A). Birth Defects Res. A Clin Mol. Teratol. 73(6), 440-446. 5. Zhu, D., Fang Y., Gao,  K., Shen, J., Zhong, T.P., and Li,  F. (2017) Vegfa Impacts Early Myocardium Development in Zebrafish. Int J Mol Sci. 18(2): 444. 6. Eichmann, A., Marcelle, C., Breant, C., and Le Douarin, N.M. (1996). Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules. Gene 174, 3–8. 7. Masabumi Shibuya (2002) Vascular Endothelial Growth Factor Receptor Family Genes: When Did the Three Genes Phylogenetically Segregate? Biol. Chem., 383: 1573 – 1579. 8. Li, X.; Liu, X.; Guo, H.; Zhao, Z.; Li, Y.S. and Chen, G. (2016) The significance of the increased expression of phosphorylated MeCP2 in the membranes from patients with proliferative diabetic retinopathy. Scientific Reports, volume 6, Article number: 32850. 10.1038/srep32850 9. Medford, A. R., Douglas, S. K., Godinho, S. I., Uppington, K. M., Armstrong, L., Gillespie, K. M., van Zyl, B., Tetley, T.D., Ibrahim, N.B.N. and Millar, A. B. (2009). Vascular Endothelial Growth Factor (VEGF) isoform expression and activity in human and murine lung injury. Respiratory Research, 10(1), 27. http://doi.org/10.1186/1465-9921-10-27   AOPWiki 2016-11-29T18:41:28

2018-03-28T11:48:00

Reduction, Angiogenesis

Molecular

<div> 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 (<a href="http://www.informatics.jax.org/vocab/gene_ontology/" style="color:#2b3ecd; text-decoration:underline">http://www.informatics.jax.org/vocab/gene_ontology/</a>, 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. </div>

<div> 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.   <div> 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]. </div> </div>

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. [2016] 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.

CL:0000499

CL stromal cell

Abbott, B. D. and Buckalew, A. R. (2000). Placental defects in arnt-knockout conceptus correlate with localized decreases in vegf-r2, ang-1, and tie-2. Developmental dynamics : an official publication of the American Association of Anatomists 219, 526-538. doi:10.1002/1097-0177(2000)9999:9999<::AID-DVDY1080>3.0.CO;2-N. PMID:11084652 Argraves, W. S., Larue, A. C., Fleming, P. A. et al. (2002). Vegf signaling is required for the assembly but not the maintenance of embryonic blood vessels. Developmental dynamics : an official publication of the American Association of Anatomists 225, 298-304. doi:10.1002/dvdy.10162. PMID:12412012 Belair, D. G., Whisler, J. A., Valdez, J. et al. (2015). Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells. Stem cell reviews and reports 11, 511-525. doi:10.1007/s12015-014-9549-5. PMID:25190668 Belair, D. G., Miller, M. J., Wang, S. et al. (2016). Differential regulation of angiogenesis using degradable vegf-binding microspheres. Biomaterials 93, 27-37. doi:10.1016/j.biomaterials.2016.03.021. PMID:27061268 Belair, D. G., Schwartz, M. P., Knudsen, T. et al. (2016). Human ipsc-derived endothelial cell sprouting assay in synthetic hydrogel arrays. Acta biomaterialia 39, 44554-44554. doi:10.1016/j.actbio.2016.05.020. PMID:27181878 Blanco, R. and Gerhardt, H. (2013). Vegf and notch in tip and stalk cell selection. Cold Spring Harbor Perpect Med 3, a006569-a006569. doi:10.1101/cshperspect.a006569. PMID:23085847 Bowers, S. L. K., Kemp, S. S., Aguera, K. N. et al. (2020). Defining an upstream vegf (vascular endothelial growth factor) priming signature for downstream factor-induced endothelial cell-pericyte tube network coassembly. Arteriosclerosis, thrombosis, and vascular biology 40, 2891-2909. doi:10.1161/ATVBAHA.120.314517. PMID:33086871 Drake, C. J., Fleming, P. A. and Argraves, W. S. (2007). The genetics of vasculogenesis. Novartis Foundation symposium 283, 61-71; discussion 71. doi:10.1002/9780470319413.ch6. PMID:18300414 Eilken, H. M. and Adams, R. H. (2010). Dynamics of endothelial cell behavior in sprouting angiogenesis. Current opinion in cell biology 22, 617-625. doi:10.1016/j.ceb.2010.08.010. PMID:20817428 Fong, G. H., Rossant, J., Gertsenstein, M. et al. (1995). Role of the flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66-70. doi:10.1038/376066a0. PMID:7596436 Herbert, S. P. and Stainier, D. Y. (2011). Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nature reviews. Molecular cell biology 12, 551-564. doi:10.1038/nrm3176. PMID:21860391 Jin, D., Zhu, D., Fang, Y. et al. (2017). Vegfa signaling regulates diverse artery/vein formation in vertebrate vasculatures. Journal of genetics and genomics = Yi chuan xue bao 44, 483-492. doi:10.1016/j.jgg.2017.07.005. PMID:29037991 Kleinstreuer, N., Dix, D., Rountree, M. et al. (2013). A computational model predicting disruption of blood vessel development. PLoS computational biology 9, e1002996-e1002996. doi:10.1371/journal.pcbi.1002996. PMID:23592958 Knudsen, T. B. and Kleinstreuer, N. C. (2011). Disruption of embryonic vascular development in predictive toxicology. Birth defects research. Part C, Embryo today : reviews 93, 312-323. doi:10.1002/bdrc.20223. PMID:22271680 Nguyen, E. H., Daly, W. T., Le, N. N. T. et al. (2017). Versatile synthetic alternatives to matrigel for vascular toxicity screening and stem cell expansion. Nature biomedical engineering 1, doi:10.1038/s41551-017-0096. PMID:29104816 Oladipupo, S., Hu, S., Kovalski, J. et al. (2011). Vegf is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting. Proceedings of the National Academy of Sciences of the United States of America 108, 13264-13269. doi:10.1073/pnas.1101321108. PMID:21784979 Palm, M. M., Dallinga, M. G., van Dijk, E. et al. (2016). Computational screening of tip and stalk cell behavior proposes a role for apelin signaling in sprout progression. PloS one 11, e0159478-e0159478. doi:10.1371/journal.pone.0159478. PMID:27828952 Roberts, D. M., Kearney, J. B., Johnson, J. H. et al. (2004). The vascular endothelial growth factor (vegf) receptor flt-1 (vegfr-1) modulates flk-1 (vegfr-2) signaling during blood vessel formation. The American journal of pathology 164, 1531-1535. doi:10.1016/S0002-9440(10)63711-X. PMID:15111299 Shirinifard, A., McCollum, C. W., Bolin, M. B. et al. (2013). 3d quantitative analyses of angiogenic sprout growth dynamics. Developmental dynamics : an official publication of the American Association of Anatomists 242, 518-526. doi:10.1002/dvdy.23946. PMID:23417958 Tal, T., Kilty, C., Smith, A. et al. (2017). Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity. Reproductive toxicology (Elmsford, N.Y.) 70, 70-81. doi:10.1016/j.reprotox.2016.12.004. PMID:28007540 van Duinen, V., Stam, W., Borgdorff, V. et al. (2019). Standardized and scalable assay to study perfused 3d angiogenic sprouting of ipsc-derived endothelial cells in vitro. Journal of visualized experiments : JoVE doi:10.3791/59678. PMID:31762444 van Duinen, V., Stam, W., Mulder, E. et al. (2020). Robust and scalable angiogenesis assay of perfused 3d human ipsc-derived endothelium for anti-angiogenic drug screening. International journal of molecular sciences 21, doi:10.3390/ijms21134804. PMID:32645937 Venkatraman, L., Regan, E. R. and Bentley, K. (2016). Time to decide? Dynamical analysis predicts partial tip/stalk patterning states arise during angiogenesis. PloS one 11, e0166489-e0166489. doi:10.1371/journal.pone.0166489. PMID:27846305 Williams, C. K., Li, J. L., Murga, M. et al. (2006). Up-regulation of the notch ligand delta-like 4 inhibits vegf-induced endothelial cell function. Blood 107, 931-939. doi:10.1182/blood-2005-03-1000. PMID:16219802   AOPWiki 2016-11-29T18:41:22

2022-07-20T13:04:45

Increase, Hemopericardium

Organ

AOPWiki 2025-10-29T17:05:33

2025-10-29T17:05:33

Increase, Early Life Stage Mortality

Individual

Increased early life stage mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time. In Birds: Early life stage mortality occurs at any stage in development prior to birth/hatch and is considered embryolethal. In Fishes: Early Life Stage Mortality refers to death prior to yolk sac adsorption and swim-up.

In birds it may be identified as failure to hatch or lack of movement within the egg when candled; heartbeat monitors are available for identifying viable avian and reptillian eggs (ex. Avitronic's Buddy monitor). In mammals, stillborn or mummified offspring, or an increased rate of resorptions early in pregnancy are all considered embryolethal, and can be detected using ultra-high frequency ultrasound (30-70 MHz; a.k.a. ultrasound biomicroscopy) (Flores et al. 2014). In fishes, mortality is typically measured by observation. Lack of any heart beat, gill movement, and body movement are typical signs of death used in the evaluation of mortality.

All members of the subphylum vertebrata are susceptible to early life stage death (Weinstein 1999).

High Unspecific

High

Embryo

High

Foetal

High

Development

High

1. Flores, L.E., Hildebrandt, T.B., Kuhl, A.A., and Drews, B. (2014) Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy. Reproductive Biology and Endocrinology 12(38). DOI: 10.1186/1477-7827-12-38 2. Weinstein, B. M. (1999). What guides early embryonic blood vessel formation? Dev. Dyn. 215(1), 2-11. Doering, J.A.; Giesy, J.P.; Wiseman S.; Hecker, M. (2013). Predicting the sensivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environmental Science and Pollution Research. 20 (3), 1219-1224. AOPWiki 2016-11-29T18:41:28

2018-03-22T10:23:28

<upstream-id>33523cf3-6e07-486d-b423-81f46622f8be</upstream-id> <downstream-id>4fac3fa2-0340-4a83-94c8-e2dcaf858550</downstream-id>

AOPWiki 2025-10-30T04:33:52

2025-10-30T04:33:52

<upstream-id>4fac3fa2-0340-4a83-94c8-e2dcaf858550</upstream-id> <downstream-id>ac45bf76-bad2-4e30-9b94-66b59a265195</downstream-id>

AOPWiki 2025-10-30T04:34:00

2025-10-30T04:34:00

<upstream-id>ac45bf76-bad2-4e30-9b94-66b59a265195</upstream-id> <downstream-id>31662b90-af54-40d0-b9b1-08cafd5d3a45</downstream-id>

AOPWiki 2025-10-30T04:34:08

2025-10-30T04:34:08

<upstream-id>31662b90-af54-40d0-b9b1-08cafd5d3a45</upstream-id> <downstream-id>7755426f-2dcc-4f39-8a82-7f4c9299d315</downstream-id>

AOPWiki 2025-10-30T04:19:36

2025-10-30T04:19:36

Peroxisome proliferator-activated receptor alpha activation leading to early life stage mortality via reduced vascular endothelial growth factor

PPARα activation leading to ELS mortality via reduced VEGF

Evgeniia Kazymova

You Song Norwegian Institute for Water Research (NIVA), Oslo, Norway

BY-SA

2.7

This Adverse Outcome Pathway (AOP) describes the mechanistic sequence through which activation of peroxisome proliferator-activated receptor alpha (PPARα) disrupts vascular endothelial growth factor (VEGF) signaling and angiogenesis, ultimately resulting in hemopericardium and increased early life stage mortality in fish. The molecular initiating event (MIE) is PPARα activation, which downregulates VEGF expression, reduces angiogenic sprouting, compromises vascular development, and leads to cardiac failure and death. The AOP integrates molecular regulatory pathways controlling lipid metabolism and vascular growth with developmental toxicity endpoints, providing a mechanistically coherent foundation for assessing cardiovascular and embryotoxic effects of peroxisome proliferators, particularly per- and polyfluoroalkyl substances (PFAS). The AOP supports applications in chemical screening, hazard prioritization, and development of non-animal testing strategies under next-generation risk assessment (NGRA) frameworks. This AOP was developed to elucidate a vascular-specific mechanistic route connecting PPARα activation to developmental toxicity. Activation of PPARα, a key transcription factor in lipid metabolism, modulates numerous downstream targets, including suppression of hypoxia-inducible factor 1-alpha (HIF1α) and VEGF expression, both critical regulators of angiogenesis. Impairment of VEGF signaling and reduced vascular branching are frequently observed in zebrafish embryos exposed to PPARα agonists and PFAS. The resulting loss of vascular integrity contributes to hemopericardium and early developmental mortality. This AOP complements existing PPARα- and mitochondrial-centered AOPs by defining a distinct, angiogenesis-focused mechanistic branch.

<ul> <li> Objective: Identify and evaluate mechanistic evidence linking PPARα activation to impaired angiogenesis and embryonic lethality. </li> <li> Data sources: Literature screening in PubMed, AOP-Wiki, OECD AOP-KB, and Web of Science (2010–2025). </li> <li> Key search terms: PPARα activation, VEGF suppression, angiogenesis, vascular development, hemopericardium, zebrafish embryo, PFAS, fibrates. </li> <li> Selection criteria: Experimental studies demonstrating sequential causal relationships, dose–response concordance, or temporal consistency between events. </li> <li> Evaluation: Weight-of-evidence assessment based on OECD (2018) AOP guidance principles—biological plausibility, essentiality, and empirical support. </li> </ul>

Fibrates are ligands of PPARα (Staels et al. 1998). Phthalates MHEP (CAS 4376-20-9) directly binds in vitro to PPARα (Lapinskas et al. 2005) and activates this receptor in transactivation assays PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999), (Hurst and Waxman 2003), (Bility et al. 2004), (Lampen, Zimnik, and Nau 2003), (Venkata et al. 2006) ]. DEHP (CAS 117-81-7) has not been found to bind and activate PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999). However, the recent studies shown activation of PPARα (ToxCastTM Data). Notably, PPARα are responsive to DEHP in vitro as they are translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005). Expression of PPARα [mRNA and protein] has been reported to be also modulated by phthtalates: (to be up-regulated in vivo upon DEHP treatment (Xu et al. 2010) and down-regulated by Diisobutyl phthalate (DiBP) (Boberg et al. 2008)). Perfluorooctanoic Acid (PFOA) is known to activate PPARα (Vanden Heuvel et al. 2006). Organotin Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)

Poor early life stage survival is an endpoint of major relevance to environmental regulators, as it is likely to lead to population decline.  Early-life stage, acute and chronic test guidelines have been established by the Organisation for Economic Co-operation and Development (OECD), U.S. Environmental Protection Agency (EPA) and Environment and Climate Change Canada (ECCC), and are currently used in risk assessments to set limits for safe exposures.  Aquatic test guidlines are most prevalent and include OECD210, OECD229, EPA850.1400 and ECCC  EPS 1/RM/28 for fish and OECD241 for frogs.

adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified Unspecific

Not Specified

Embryo

Not Specified

Juvenile

Not Specified

The AOP is supported by strong biological plausibility and moderate empirical evidence. Upstream events (PPARα activation and VEGF suppression) are mechanistically well established. Evidence for downstream outcomes (impaired angiogenesis, hemopericardium, mortality) is consistent across fish species and multiple PPARα agonists. Quantitative understanding of the intermediate key event relationships (KERs) is moderate but growing, with transcriptomic and imaging data supporting causal linkages. The overall confidence level is moderate-to-high, making this AOP suitable for chemical screening, read-across, and mechanistic risk assessment applications. <table> <thead> <tr> <th>Aspect</th> <th>Applicability</th> </tr> </thead> <tbody> <tr> <td>Taxa</td> <td>Teleost fish (e.g., Danio rerio, Oryzias latipes); mechanism likely conserved among vertebrates.</td> </tr> <tr> <td>Life stage</td> <td>Embryonic and early larval stages.</td> </tr> <tr> <td>Sex</td> <td>Non-sex-specific (pre-differentiation).</td> </tr> <tr> <td>Biological systems</td> <td>Hepatic (PPARα activation), vascular endothelium (VEGF signaling), and cardiovascular system (angiogenesis and cardiac integrity).</td> </tr> </tbody> </table>

<table> <thead> <tr> <th>Key Event</th> <th>Essentiality Evidence</th> <th>Type of Evidence</th> </tr> </thead> <tbody> <tr> <td>Event 227: PPARα activation (MIE)</td> <td>Knockdown of ppara in zebrafish prevents VEGF suppression and vascular abnormalities following PPARα agonist exposure.</td> <td>Direct</td> </tr> <tr> <td>Event 948: VEGF production (Decrease)</td> <td>VEGF mRNA and protein levels are reduced by PPARα agonists; VEGF mRNA rescue restores angiogenesis.</td> <td>Direct</td> </tr> <tr> <td>Event 28: Angiogenesis (Decrease)</td> <td>Anti-angiogenic phenotypes (reduced intersegmental vessels, impaired sprouting) observed following VEGF suppression.</td> <td>Direct</td> </tr> <tr> <td>Event 2383: Hemopericardium (Increase)</td> <td>Occurs secondary to vascular malformation and cardiac congestion; attenuated by VEGF rescue.</td> <td>Indirect</td> </tr> <tr> <td>Event 947: Early life stage mortality (Increase)</td> <td>Final apical outcome following cumulative vascular collapse.</td> <td>Outcome</td> </tr> </tbody> </table>

<h4>Biological Plausibility</h4> High. PPARα activation interferes with the HIF1α–VEGF signaling axis, reducing vascular endothelial proliferation and vessel formation. Reduced VEGF signaling has well-documented consequences for angiogenesis and vascular integrity, which align mechanistically with observed cardiac and mortality endpoints in developing fish embryos. <h4>Empirical Support</h4> Moderate to high. <ul> <li> Zebrafish and medaka exposed to PPARα agonists (e.g., clofibrate, PFOA, HFPO-DA) show consistent suppression of vegfa transcripts. </li> <li> Inhibition of VEGF signaling recapitulates vascular and cardiac phenotypes identical to those caused by PPARα activation. </li> <li> Temporal concordance is observed: VEGF downregulation → reduced vessel branching → hemopericardium → mortality. </li> <li> Dose–response relationships are evident between PPARα activation markers, VEGF suppression, and observed apical outcomes. </li> </ul> <h4>Quantitative Understanding</h4> Moderate. Transcriptomic dose–response data demonstrate ≥40% reduction in vegfa expression correlates with reduced intersegmental vessel count and >50% mortality at high exposure levels. Quantitative models for VEGF-to-angiogenesis linkage are emerging but not yet standardized across taxa.

<table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) Involved</th> </tr> </thead> <tbody> <tr> <td>Oxygen availability</td> <td>Hypoxia upregulates HIF1α–VEGF signaling, counteracting PPARα-mediated VEGF suppression.</td> <td>PPARα activation → VEGF decrease</td> </tr> <tr> <td>Nutritional status</td> <td>Lipid levels influence PPARα activation strength and downstream vascular effects.</td> <td>PPARα activation → VEGF decrease</td> </tr> <tr> <td>Antioxidant capacity</td> <td>Reduces oxidative stress–mediated modulation of VEGF signaling.</td> <td>VEGF decrease → angiogenesis decrease</td> </tr> <tr> <td>Chemical lipophilicity</td> <td>Determines bioaccumulation and PPARα binding potency.</td> <td>MIE and downstream effects</td> </tr> <tr> <td>Developmental stage</td> <td>Early embryos are more sensitive due to rapid angiogenic processes.</td> <td>VEGF → angiogenesis → hemopericardium</td> </tr> </tbody> </table>

<ul> <li> Threshold effects: VEGF mRNA reduction >30–40% predicts loss of angiogenic sprouting. </li> <li> Temporal sequence: VEGF suppression within 24–36 h post-exposure precedes angiogenic defects and hemopericardium by 12–24 h. </li> <li> Model potential: Dose–response and time-series data from zebrafish enable semi-quantitative modeling for VEGF–angiogenesis relationships. </li> </ul>

<ul> <li> Regulatory screening: Supports the prioritization of chemicals with PPARα agonist activity for developmental vascular toxicity. </li> <li> Read-across: Enables grouping of PFAS, fibrates, and related lipid modulators based on shared mechanistic profiles. </li> <li> NAM integration: Aligns with transcriptomic biomarkers (PPARα target genes, vegfa repression) for use in in vitro assays. </li> <li> Test guideline refinement: May inform OECD Fish Embryo Test (TG 236) adaptations to include vascular endpoints. </li> <li> Risk assessment: Provides mechanistic rationale for linking molecular bioactivity data to early life stage lethality outcomes. </li> </ul>

AOPWiki 2025-10-29T17:14:10

2026-06-26T16:12:16