Article
Loss of CCM3 activated angiogenesis through impairing endothelial Dll4-Notch signalling: implication in familial cerebral cavernous malformation
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Published: | May 21, 2013 |
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Objective: Germline mutations in CCM1, CCM2 or CCM3 genes have been frequently detected in familial cerebral cavernous malformations (CCMs). Numerous studies have demonstrated that loss of any of three CCM genes leads to aberrant angiogenesis and to CCMs. However, the underlying signalling pathways are not yet fully understood. Dll4-Notch is a critical signalling negatively regulating endothelial angiogenesis under various pathological conditions. The present study attempted to identify whether Dll4-Notch pathway is affected in CCM3-mutated familial CCM and in CCM3 silenced endothelial cells.
Method: The expression of the Notch ligand Dll4, Notch receptors (Notch1 and Notch4) and Notch target (Hey1) was examined in CCM lesions derived from a familial CCM3-mutation carrier and in CCM3-silenced endothelial cells by real-time RT-PCR, Western blot and immunostaining. The angiogenic phenotype including proliferation, migration and sprouting as well as the possible affected downstream signalling VEGF and p-Erk1/2 were studied in CCM3-silenced endothelial cells in the absence and the presence of the recombinant DLL4 (rhDll4).
Results: Western blot revealed a 5-fold and a 7-fold lower protein level of CCM3 and Dll4, respectively, in the familial CCM tissue in comparison to the control (sporadic CCM tissue). In cultured endothelial cells, CCM3-silencing resulted in a significant decrease in the expression of Dll4 (5-fold), Notch4 (3.5-fold), Hey1 (2-fold) accompanied with a hyper-angiogenic phenotype. Interestingly, CCM3-silence mediated activation of proliferation, migration and sprouting was markedly inhibited by rhDLL4. Indeed, rhDLL4 also reversed the upregulation of VEGF and p-Erk1,2 caused by CCM3 silencing.
Conclusions: CCM3 gene deficiency led to hyper-angiogenesis and to inactivation of Dll4-Notch signalling. Restoration of Dll4 rescued the hyper-angiogenic phenotype most likely through inhibition of VEGF and Erk1,2 pathways. These findings highlight Dll4-Notch signalling as a potential therapy target for familial CCM harbouring CCM3 mutations.