Synergistic TOR and ERK inhibition mitigates the hereditary haemorrhagic telangiectasia-like phenotype and excesskugelformation in endoglin mutant zebrafish

Rationale Hereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin ( ENG ) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling. Objective We therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling. Methods and Results We used light sheet fluorescence microscopy to visualise the vasculature in eng mu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, eng mu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial “kugeln” on cerebral vessels. Adult eng mu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in eng mu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in eng mu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways. Conclusions These results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT. Graphical Abstract