EphB4 forward signalling mediates angiogenesis caused by CCM3/PDCD10-ablation.

Chao You,Philipp Dammann,Ilonka Kreitschmann‐Andermahr,Yuan Zhu,Ulrich Sure,Kai Zhao,Kathy Keyvani

doi:10.1111/jcmm.13105

Abstract

CCM3, also named as PDCD10, is a ubiquitous protein expressed in nearly all tissues and in various types of cells. It is essential for vascular development and post‐natal vessel maturation. Loss‐of‐function mutation of CCM3 predisposes for the familial form of cerebral cavernous malformation (CCM). We have previously shown that knock‐down of CCM3 stimulated endothelial angiogenesis via impairing DLL4‐Notch signalling; moreover, loss of endothelial CCM3 stimulated tumour angiogenesis and promoted tumour growth. The present study was designed to further elucidate the inside signalling pathway involved in CCM3‐ablation‐mediated angiogenesis. Here we report for the first time that silencing endothelial CCM3 led to a significant up‐regulation of EphB4 mRNA and protein expression and to an increased kinase activity of EphB4, concomitantly accompanied by an activation of Erk1/2, which was reversed by treatment with the specific EphB4 kinase inhibitor NVP‐BHG712 (NVP), indicating that silencing CCM3 activates EphB4 kinase forward signalling. Furthermore, treatment with NVP rescued the hyper‐angiogenic phenotype induced by knock‐down of endothelial CCM3 in vitro and in vivo. Additional study demonstrated that the activation of EphB4 forward signalling in endothelial cells under basal condition and after CCM3‐silence was modulated by DLL4/Notch signalling, relying EphB4 at downstream of DLL4/Notch signalling. We conclude that angiogenesis induced by CCM3‐silence is mediated by the activation of EphB4 forward signalling. The identified endothelial signalling pathway of CCM3‐DLL4/Notch‐EphB4‐Erk1/2 may provide an insight into mechanism of CCM3‐ablation‐mediated angiogenesis and could potentially contribute to novel therapeutic concepts for disrupting aberrant angiogenesis in CCM and in hyper‐vascularized tumours.

Highlights

Cerebral cavernous malformation 3 (CCM3) is named programmed cell death 10 (PDCD10)
We have previously demonstrated that CCM3 is deficient in the endothelial cells of the cerebral cavernous malformation (CCM) lesion derived from CCM3-mutation carriers and that loss of endothelial CCM3 stimulates angiogenesis via impairing DLL4-Notch signalling [21, 22]
The present study was designed to explore whether EphB4 forward signalling is involved in angiogenesis mediated by ablation of endothelial CCM3, and if so, how EphB4 and DLL4-Notch signalling, which we have identified in our previous study to be targeted by CCM3-silence [22], coordinate each other in angiogenesis resulting from CCM3-ablation

Summary

Introduction

Cerebral cavernous malformation 3 (CCM3) is named programmed cell death 10 (PDCD10). Loss-of-function mutation of CCM3 gene causes cerebral cavernous malformation (CCM), one of the most common vascular disorders involving aberrant angiogenesis in the central nervous system [1]. It is known that CCM3 can act in a protein complex of CCM1–CCM2–CCM3, thereby sharing common signalling pathways [2, 3]. Apart from that, CCM3 displays distinct functions. CCM3 is involved in angiogenesis, vessel permeability, apoptosis and senescence, oxidative metabolism and Golgi complex polarization [4] and is essential for the neuron-glial unit [5]. We and others have identified a variety of

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