EphB4 forward signalling mediates angiogenesis caused by CCM 3/ PDCD 10 ‐ablation

CCM 3, also named as PDCD 10, 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 CCM 3 predisposes for the familial form of cerebral cavernous malformation ( CCM ). We have previously shown that knock‐down of CCM 3 stimulated endothelial angiogenesis via impairing DLL 4‐Notch signalling; moreover, loss of endothelial CCM 3 stimulated tumour angiogenesis and promoted tumour growth. The present study was designed to further elucidate the inside signalling pathway involved in CCM 3‐ ablation‐mediated angiogenesis. Here we report for the first time that silencing endothelial CCM 3 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 ‐ BHG 712 ( NVP ), indicating that silencing CCM 3 activates EphB4 kinase forward signalling. Furthermore, treatment with NVP rescued the hyper‐angiogenic phenotype induced by knock‐down of endothelial CCM 3 in vitro and in vivo . Additional study demonstrated that the activation of EphB4 forward signalling in endothelial cells under basal condition and after CCM 3 ‐silence was modulated by DLL 4/Notch signalling, relying EphB4 at downstream of DLL 4/Notch signalling. We conclude that angiogenesis induced by CCM 3 ‐silence is mediated by the activation of EphB4 forward signalling. The identified endothelial signalling pathway of CCM 3‐ DLL 4/Notch‐EphB4‐Erk1/2 may provide an insight into mechanism of CCM 3‐ ablation‐mediated angiogenesis and could potentially contribute to novel therapeutic concepts for disrupting aberrant angiogenesis in CCM and in hyper‐vascularized tumours.