Antiangiogenic Drug Testing Using Proangiogenic and Hypoxia Zebrafish Model

Background

Cancer angiogenesis is a process of forming abnormal ectopic neovasculatures that are essential for tumor growth and metastasis [1]. Therefore, the identification of novel antiangiogenic compounds has become an important area in cancer therapeutics. Antiangiogenic drugs can be efficiently tested in the high-throughput zebrafish angiogenesis assay [2,3]. The availability of blood vessel–specific transgenic lines such as Tg(fli1:EGFP) and Tg(kdrl:EGFP), which enable real-time visualization of vascular development and angiogenesis in live embryos, and the transparency of early larval stages make zebrafish a suitable model for antiangiogenic drug testing [4]. In zebrafish, the angiogenesis assay has been conducted in wild-type larvae by exposing the embryos to test compounds at 1 dpf and analyzing the vascular changes in the intersegmental vessels (ISVs) and subintestinal vessel (SIV) regions at 2 and 3 dpf, respectively [5,6]. The ISVs are particularly suitable for angiogenesis assessment because they normally form stereotypic secondary vessels without ectopic terminal neovasculatures. Under activated hypoxia signaling, such as in the vhl mutant, abnormal vascular sprouting and ectopic neovascularization can be readily observed in the ISVs [6,7]. Although this conventional model is useful for identifying antiangiogenic compounds that may show efficacy in normal zebrafish larvae, it may not have similar efficacy in patients experiencing hypoxia, a major driver of drug resistance.

To overcome this limitation, we generated a hypoxia zebrafish model by targeting the exon 3 (the alpha domain that helps in the formation of the Vhl complex, which is involved in the negative regulation of Hifα) of the vhl through CRISPR mutagenesis, based on our previous studies in bioinformatics analysis of identifying the most pathological and conserved position of human VHL mutation [6]. The VHL is a tumor-suppressor gene that negatively regulates hypoxia-inducible factor alpha (HIFα) by promoting its proteasomal degradation under normoxic conditions [8]. Loss of Vhl function leads to the stabilization of Hifα and binds with Hifβ, which translocates and activates the transcription of genes involved in angiogenesis, erythropoiesis, and glucose metabolism, which includes genes such as vascular endothelial growth factor (vegfa), erythropoietin (epo), glucose transporter 1 (slc2a1/glut1), and prolyl hydroxylase domain protein 3 (phd3), thereby promoting vascular growth, metabolic adaptation, and cell survival [7]. This constitutive activation of hypoxia signaling induces extensive ectopic vascular branching throughout the larval body, mimicking key features of pathological angiogenesis [6,7].

The generated vhl mutant zebrafish larvae successfully recapitulated the proangiogenic phenotype and showed drug resistance by further elevating hypoxia signaling (more than 200-fold higher expression of phd3) to overcome the antiangiogenic effect of sorafenib. Activation of hypoxia signaling in the vhl mutant can be monitored using levels of phd3 gene expression, a well-established downstream marker of HIF signaling [6,7]. This pseudo-hypoxia zebrafish model clearly showed that the efficacy of antiangiogenic drugs differed under activated hypoxia signaling conditions [6]. Validation of this model using sorafenib, a clinically approved antiangiogenic tyrosine kinase inhibitor, demonstrated significant suppression of ectopic vessel growth, targeting pathological angiogenesis [6].

The vhl mutant larvae survive up to approximately only 12 dpf (due to hyperleaky neovasculatures) [6,9], but this still provides a sufficient time to study angiogenesis development and antiangiogenic drug screening. The transparent larval stages, robust and reproducible proangiogenic phenotype, small size, rapid development, and compatibility with high-throughput screening provide important advantages for drug testing applications. Thus, this model successfully addresses the limitations of previous wild-type zebrafish angiogenesis systems and provides a more physiologically relevant platform for antiangiogenic drug testing in cancer and other angiogenesis-associated diseases.