Cerebral Cavernous Malformations Develop through Clonal Expansion of Mutant Endothelial Cells

AbstractRationaleVascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations.ObjectiveWe utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multi-color fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation.Methods and ResultsWe combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with one of four possible colors. We acquired Z-series confocal images from serial brain sections and created 3D reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3D distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (p<0.001). Many of the small (<100μm diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100μm diameter) CCMs contained both the clonally dominant mutant cells and wildtype ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wildtype ECs to increase the size of the malformation.ConclusionsThis is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wildtype ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.

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Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells

Circulation Research ◽

10.1161/circresaha.118.313970 ◽

2018 ◽

Vol 123 (10) ◽

pp. 1143-1151 ◽

Cited By ~ 28

Author(s):

Matthew R. Detter ◽

Daniel A. Snellings ◽

Douglas A. Marchuk

Keyword(s):

Endothelial Cells ◽

Clonal Expansion ◽

Cerebral Cavernous Malformations ◽

Cavernous Malformations

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Transcriptome-wide Profiling of Cerebral Cavernous Malformations Patients Reveal Important Long noncoding RNA molecular signatures

Scientific Reports ◽

10.1038/s41598-019-54845-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Santhilal Subhash ◽

Norman Kalmbach ◽

Florian Wegner ◽

Susanne Petri ◽

Torsten Glomb ◽

...

Keyword(s):

Noncoding Rna ◽

Vascular Malformations ◽

Cerebrovascular Disorders ◽

Gene Set Enrichment Analysis ◽

Differentially Expressed ◽

Low Flow ◽

Pcr Analysis ◽

Cerebral Cavernous Malformations ◽

Cavernous Malformations ◽

Non Coding Rnas

AbstractCerebral cavernous malformations (CCMs) are low-flow vascular malformations in the brain associated with recurrent hemorrhage and seizures. The current treatment of CCMs relies solely on surgical intervention. Henceforth, alternative non-invasive therapies are urgently needed to help prevent subsequent hemorrhagic episodes. Long non-coding RNAs (lncRNAs) belong to the class of non-coding RNAs and are known to regulate gene transcription and involved in chromatin remodeling via various mechanism. Despite accumulating evidence demonstrating the role of lncRNAs in cerebrovascular disorders, their identification in CCMs pathology remains unknown. The objective of the current study was to identify lncRNAs associated with CCMs pathogenesis using patient cohorts having 10 CCM patients and 4 controls from brain. Executing next generation sequencing, we performed whole transcriptome sequencing (RNA-seq) analysis and identified 1,967 lncRNAs and 4,928 protein coding genes (PCGs) to be differentially expressed in CCMs patients. Among these, we selected top 6 differentially expressed lncRNAs each having significant correlative expression with more than 100 differentially expressed PCGs. The differential expression status of the top lncRNAs, SMIM25 and LBX2-AS1 in CCMs was further confirmed by qRT-PCR analysis. Additionally, gene set enrichment analysis of correlated PCGs revealed critical pathways related to vascular signaling and important biological processes relevant to CCMs pathophysiology. Here, by transcriptome-wide approach we demonstrate that lncRNAs are prevalent in CCMs disease and are likely to play critical roles in regulating important signaling pathways involved in the disease progression. We believe, that detailed future investigations on this set of identified lncRNAs can provide useful insights into the biology and, ultimately, contribute in preventing this debilitating disease.

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Novel Chronic Mouse Model of Cerebral Cavernous Malformations

Stroke ◽

10.1161/strokeaha.119.027207 ◽

2020 ◽

Vol 51 (4) ◽

pp. 1272-1278 ◽

Cited By ~ 3

Author(s):

Cécile Cardoso ◽

Minh Arnould ◽

Coralie De Luca ◽

Cécile Otten ◽

Salim Abdelilah-Seyfried ◽

...

Keyword(s):

Mouse Model ◽

Cerebral Cavernous Malformations ◽

Cavernous Malformations

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Endothelial cell clonal expansion in the development of cerebral cavernous malformations

Nature Communications ◽

10.1038/s41467-019-10707-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 25

Author(s):

Matteo Malinverno ◽

Claudio Maderna ◽

Abdallah Abu Taha ◽

Monica Corada ◽

Fabrizio Orsenigo ◽

...

Keyword(s):

Endothelial Cell ◽

Clonal Expansion ◽

Cerebral Cavernous Malformations ◽

Cavernous Malformations ◽

Cell Clonal Expansion

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The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510495112 ◽

2015 ◽

Vol 112 (46) ◽

pp. 14284-14289 ◽

Cited By ~ 35

Author(s):

Xavier Cullere ◽

Eva Plovie ◽

Paul M. Bennett ◽

Calum A. MacRae ◽

Tanya N. Mayadas

Keyword(s):

Endothelial Cells ◽

Cavernous Malformation ◽

Body Axis ◽

Cerebral Cavernous Malformations ◽

Loss Of Function ◽

Cavernous Malformations ◽

Downstream Target ◽

Density Flow

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe “big heart” and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

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Are cavernous sinus hemangiomas and cavernous malformations different entities?

Neurosurgical FOCUS ◽

10.3171/foc.2006.21.1.7 ◽

2006 ◽

Vol 21 (1) ◽

pp. 1-5 ◽

Cited By ~ 42

Author(s):

L. Fernando Gonzalez ◽

Gregory P. Lekovic ◽

Jennifer Eschbacher ◽

Stephen Coons ◽

Randall W. Porter ◽

...

Keyword(s):

Cavernous Sinus ◽

Cavernous Malformation ◽

Vascular Malformations ◽

Response To Treatment ◽

Vascular Tumors ◽

Cerebral Cavernous Malformations ◽

Imaging Studies ◽

Cavernous Malformations ◽

Cavernous Hemangiomas

✓Cavernous hemangiomas that occur within the cavernous sinus (CS) are different from cerebral cavernous malformations (CMs) clinically, on imaging studies, and in their response to treatment. Moreover, CMs are true vascular malformations, whereas hemangiomas are benign vascular tumors. Because of these differences, the authors suggest that these two entities be analyzed and grouped separately. Unfortunately, despite these differences, much confusion exists in the literature as to the nature, behavior, and classification of these two distinct lesions. This confusion is exacerbated by subtle histological differences and the inconsistent use of nomenclature. The authors use the term “cavernous malformation” to refer to intraaxial lesions only; they prefer to use the term “cavernous sinus hemangioma” to refer to extraaxial, intradural hemangiomas of the CS.

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Developmental timing of CCM2 loss influences cerebral cavernous malformations in mice

Journal of Experimental Medicine ◽

10.1084/jem.20110571 ◽

2011 ◽

Vol 208 (9) ◽

pp. 1835-1847 ◽

Cited By ~ 78

Author(s):

Gwénola Boulday ◽

Noemi Rudini ◽

Luigi Maddaluno ◽

Anne Blécon ◽

Minh Arnould ◽

...

Keyword(s):

Autosomal Dominant ◽

Vascular Malformations ◽

Vascular Lesions ◽

Developmental Timing ◽

Cerebral Cavernous Malformations ◽

Loss Of Function ◽

Cavernous Malformations ◽

The Central Nervous System ◽

Dominant Condition

Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model.

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A Novel CCM2 Missense Variant Caused Cerebral Cavernous Malformations in a Chinese Family

Frontiers in Neuroscience ◽

10.3389/fnins.2020.604350 ◽

2021 ◽

Vol 14 ◽

Author(s):

Guoqing Han ◽

Li Ma ◽

Huanhuan Qiao ◽

Lin Han ◽

Qiaoli Wu ◽

...

Keyword(s):

Vascular Malformations ◽

Case Reports ◽

Chinese Family ◽

Incomplete Penetrance ◽

Cerebral Cavernous Malformations ◽

Missense Mutations ◽

Inherited Disease ◽

Cavernous Malformations ◽

Direct Dna Sequencing ◽

Number Of Patients

Cerebral cavernous malformations (CCMs) are common vascular malformations in the central nervous system. Familial CCMs (FCCMs) are autosomal dominant inherited disease with incomplete penetrance and variable symptoms. Mutations in the KRIT1, CCM2, and PDCD10 genes cause the development of FCCM. Approximately 476 mutations of three CCM-related genes have been reported, most of which were case reports, and lack of data in stable inheritance. In addition, only a small number of causative missense mutations had been identified in patients. Here, we reported that 8/20 members of a Chinese family were diagnosed with CCMs. By direct DNA sequencing, we found a novel variant c.331G > C (p.A111P) in exon 4 of the CCM2 gene, which was a heterozygous exonic variant, in 7/20 family members. We consider this variant to be causative of disease due to a weaken the protein–protein interaction between KRIT1 and CCM2. In addition, we also found the exon 13 deletion in KRIT1 coexisting with the CCM2 mutation in patient IV-2, and this was inherited from her father (patient III-1H). This study of a Chinese family with a large number of patients with CCMs and stable inheritance of a CCM2 mutation contributes to better understanding the spectrum of gene mutations in CCMs.

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