Investigating The Downstream Effectors Of The Ccm Pathway: A Role For Adamts Proteases And Versican Cleavage

Courtney Hong, University of Pennsylvania


Cerebral cavernous malformations (CCMs) are vascular malformations that develop in the central nervous system and affect 0.1-0.5% of the general population. CCMs are caused by loss of function mutations in three genes: KRIT1 (Krev interaction trapped 1), CCM2, and PDCD10 (programmed cell death 10), that encode components of a heterotrimeric adaptor complex. Disruption of this CCM complex in endothelial cells results in activation of mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling and overexpression of Kruppel-like factor 2 (KLF2) and Kruppel-like factor 4 (KLF4) transcription factors. In the developing heart, increased A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) protease activity and the degradation of its primary substrate, the extracellular matrix proteoglycan, versican, are causal for developmental cardiac defects that arise due to endocardial loss of the CCM complex. However, whether these molecules are the critical downstream MEKK3-KLF2/4 targets that drive CCM lesion formation in the brain remains unknown. Here, using a series of combinatorial genetic approaches, we demonstrated that endothelial loss of ADAMTS5 leads to increased versican in the cerebellar white matter, where CCMs form, and reduces lesion formation in a neonatal mouse model of CCM disease. Versican haploinsufficiency similarly reduces lesion burden, indicating that versican is the relevant ADAMTS5 substrate and that cleavage and not clearance of this matrix protein is important for CCM formation. Additionally, we demonstrated that overexpression of ADAMTS5 alone can confer early lesion genesis in the absence of MEKK3-KLF2/4 signaling, but together with loss of the CCM complex leads to significant amplification of lesion formation, suggesting that ADAMTS5 may have interactions with other downstream effectors. Using RNA-sequencing, we identified PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin) signaling as a significantly altered pathway in CCM-deficient animals. With further in vivo genetic experiments, we showed that PI3K functions downstream of KLF2/4 and synergizes with ADAMTS5 to generate large malformations. Together, these studies identify endothelial secretion of ADAMTS5 and cleavage of versican as key downstream events in CCM formation and reveal synergistic interactions between the KLFs, ADAMTS5, and PI3K, providing new mechanistic insights into CCM disease pathogenesis.