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

Abstract

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 engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial kugeln on cerebral vessels. Adult engmu130 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 engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 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.

An update on preclinical models of hereditary haemorrhagic telangiectasia: Insights into disease mechanisms

“…The presence of two BMP10 paralogs in zebrafish allowed for later assessment of juvenile and postnatal phenotypes; surprisingly, whereas bmp10-like mutants develop no phenotype, bmp10 mutants display vascular malformation in the skin and liver and high-output heart failure, with variable age of onset, penetrance, and expressivity ( 90 ), reminiscent of HHT. This phenotype is also similar to that seen in adult eng mutant zebrafish ( 65 , 91 ), consistent with a critical role for the BMP10-ENG-ALK1 pathway in protecting against vascular malformations. Notably, the tissue/cellular source of Bmp10 appears to be important.…”

“…In consequence, loss of BMP9/10 signalling leads to an untempered EC response to VEGF such as increased cell proliferation that would contribute to AVM formation. Targeting VEGF signalling or key players in VEGF downstream signalling pathways (pERK, PI3K/mTOR) can protect against AVM development and progression in (i) eng null zebrafish embryos ( 91 ), (ii) developing retinal vasculatures of Eng-iKOe, Alk1-iKOe , and BMP9/10 depleted neonatal mice ( 46 , 87 , 98 ), and (iii) in wounded skin of Alk1 deficient adult mice ( 99 ). Furthermore, combined low dose targeting of pERK and mTOR in eng mutant zebrafish protects against development of the HHT phenotype, suggesting synergy between these two pathways ( 91 ).…”

“…Targeting VEGF signalling or key players in VEGF downstream signalling pathways (pERK, PI3K/mTOR) can protect against AVM development and progression in (i) eng null zebrafish embryos ( 91 ), (ii) developing retinal vasculatures of Eng-iKOe, Alk1-iKOe , and BMP9/10 depleted neonatal mice ( 46 , 87 , 98 ), and (iii) in wounded skin of Alk1 deficient adult mice ( 99 ). Furthermore, combined low dose targeting of pERK and mTOR in eng mutant zebrafish protects against development of the HHT phenotype, suggesting synergy between these two pathways ( 91 ). However, a major limitation of targeting VEGF signalling during developmental angiogenesis in these neonatal mouse and zebrafish embryo HHT models is that blocking VEGF signalling inhibits progression of angiogenesis itself, making outcome analyses challenging to dissect.…”