Loss of CCM3 impairs DLL4-Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations

J Cell Mol Med. 2013 Mar;17(3):407-18. doi: 10.1111/jcmm.12022. Epub 2013 Feb 7.

Abstract

CCM3, a product of the cerebral cavernous malformation 3 or programmed cell death 10 gene (CCM3/PDCD10), is broadly expressed throughout development in both vertebrates and invertebrates. Increasing evidence indicates a crucial role of CCM3 in vascular development and in regulation of angiogenesis and apoptosis. Furthermore, loss of CCM3 causes inherited (familial) cerebral cavernous malformation (CCM), a common brain vascular anomaly involving aberrant angiogenesis. This study focused on signalling pathways underlying the angiogenic functions of CCM3. Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways. Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression. Importantly, restoration of DLL4-Notch signalling entirely rescued the hyper-angiogenic phenotype induced by CCM3 silence. A concomitant loss of CCM3 and the core components of DLL4-Notch signalling were also demonstrated in CCM3-deficient endothelial cells derived from human CCM lesions (CCMEC) and in a CCM3 germline mutation carrier. This study defined DLL4 as a key downstream target of CCM3 in endothelial cells. CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Adult
  • Apoptosis
  • Apoptosis Regulatory Proteins / antagonists & inhibitors*
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Blotting, Western
  • Calcium-Binding Proteins
  • Cell Adhesion
  • Cell Movement
  • Cell Proliferation
  • Cells, Cultured
  • Child
  • Endothelium, Vascular / metabolism
  • Endothelium, Vascular / pathology*
  • Female
  • Fluorescent Antibody Technique
  • Genetic Predisposition to Disease
  • Hemangioma, Cavernous, Central Nervous System / blood supply
  • Hemangioma, Cavernous, Central Nervous System / metabolism
  • Hemangioma, Cavernous, Central Nervous System / pathology*
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • MAP Kinase Signaling System
  • Male
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Neovascularization, Pathologic*
  • Proto-Oncogene Proteins / antagonists & inhibitors*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Receptor, Notch4
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Transcription Factor HES-1
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • Basic Helix-Loop-Helix Transcription Factors
  • Calcium-Binding Proteins
  • DLL4 protein, human
  • HEY2 protein, human
  • Homeodomain Proteins
  • Intercellular Signaling Peptides and Proteins
  • Membrane Proteins
  • NOTCH4 protein, human
  • PDCD10 protein, human
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • Receptor, Notch4
  • Receptors, Notch
  • Repressor Proteins
  • Transcription Factor HES-1
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • HES1 protein, human