CCM2L (Cerebral Cavernous Malformation 2 Like) Deletion Aggravates Cerebral Cavernous Malformation Through Map3k3-KLF Signaling Pathway

Abstract

Background and Purpose: Cerebral cavernous malformation (CCM) is a common cerebrovascular disease. CCMs are major causes of stroke, cerebral hemorrhage, and neurological deficits in young individuals. Loss-of-function mutations in CCM1 , CCM2 , and CCM3 have been identified to cause CCM in humans. Ccm2-like ( Ccm2l ) is a paralog of Ccm2 and is predominantly expressed in endothelial cells (ECs). CCM2L (CCM2-like) competes with CCM2 for binding to CCM1 and has been shown to have an antagonistic function to that of CCM2 during vascular development. The role of CCM2L in CCM pathogenesis is unknown. Methods: We isolated brain ECs from the inducible-CCM mouse models for gene expression analysis. Micro-computed tomography imaging was used to analyze CCM lesion burden from the genetic cross of Ccm2l knockout mice ( Ccm2l −/− ) with Ccm1 or Ccm2 -deficient mice to determine the role of Ccm2l in CCM pathogenesis. Genetic crosses with Map3k3 fl/fl mice were used to determine the role of Map3k3 in Ccm2l -facilitated CCM formation. Results: We demonstrated increased Ccm2l expression in brain ECs of Ccm2 -deficient mice. Analysis of RNA-seq data from CCM patient samples revealed a trend of increased CCM2L expression and its positive correlation with Kruppel-like factor 2/4 (KLF2/4 ) expression. Micro-computed tomography revealed that the deletion of Ccm2l in Ccm2 -deficient mice increased CCM lesion volume compared with that of controls but had no effect on lesion numbers. Correlating to the increased lesion burden, Klf2/4 mRNA expressions in brain ECs were significantly increased in double knockouts ( Ccm2 - and Ccm2l -deficient mice) compared with that of controls ( Ccm2 deficient). Hemizygous deletion of Map3k3 in ECs relieved CCM lesion burden in the double knockouts. These results suggest that CCM2L regulates the Map3k3-KLF signaling pathway in CCM pathogenesis. Conclusions: Loss of CCM2L aggravates CCM lesion formation in the Ccm2 -deficient mouse model through increased Map3k3-KLF signaling. Our data suggest that increased Ccm2l expression is a compensatory mechanism in CCM pathogenesis.

Cerebral Cavernous Malformation: From Mechanism to Therapy

“…Genetic interaction studies in zebrafish embryos support a redundant role with CCM2 in heart development. 80–82 In contrast, genetic studies in developing mice revealed no phenotype with loss of CCM2L and instead found that loss of CCM2L rescued lethal cardiovascular defects observed in Heg −/− ;Ccm2 +/− embryos. These findings suggested opposing roles for CCM2 and CCM2L, a conclusion also supported by biochemical studies demonstrating that CCM2L does not bind PDCD10, an essential component of the CCM complex.…”

The Involvement of Krüppel-like Factors in Cardiovascular Diseases

“…All of which participate in the development of nonhomologous cytoplasmic proteins of the signaling complex [ 86 , 87 ]. The CCM2 paralog, known as Ccml2-like , is a gen largely expressed in endothelial cells that has been shown to aggravate the CCM lesion in mice due to an increased expression of Map3k3-KLF signaling, particularly KLF-2 and KLF-4 [ 88 ]. The mutation leads to the activation of MEKK3 kinase cascade, which is an underlying cause of CCM lesion development and the expression of KLF-2 , KLF-4 , and Adam4/5 genes [ 89 ].…”

Genotype‐phenotype correlations in multiple lesions of familial cerebral cavernous malformations concerning phosphatidylinositol 3‐kinase catalytic subunit alpha mutations

“…C57BL/6 mice with endothelial cell‐specific deletion of Krit1 ( Krit1 iECKO ) were induced by 4‐hydroxytamoxifen injection at postnatal day 1 (P1) as described previously (Figure S8 ). 9 Then, adeno‐associated virus (AAV)‐control or AAV‐ PIK3CA H1047R was administered to Krit1 iECKO or Krit1 fl/fl mice at P60 via retro‐orbital sinus injection. Brain MRI and histologic examination were performed at P90 (Figure 3B ).…”