No abstract

Rationale: Vascular 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. Objective: We 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 Results: We 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 wild type 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 wild type ECs to increase the size of the malformation. Conclusions: This is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wild type 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.

Vascular malformations may arise in any of the vascular beds present in the human body. These lesions vary in location, type, and clinical severity of the phenotype. In recent years, the genetic basis of several vascular malformations has been elucidated. This review will consider how the identification of the genetic factors contributing to different vascular malformations, with subsequent functional studies in animal models, has provided a better understanding of these factors that maintain vascular integrity in vascular beds, as well as their role in the pathogenesis of vascular malformations.

Cerebral cavernous malformations (CCMs) are ectatic capillary-venous malformations that develop in approximately 0.5% of the population. Patients with CCMs may develop headaches, focal neurologic deficits, seizures, and hemorrhages. While symptomatic CCMs, depending upon the anatomic location, can be surgically removed, there is currently no pharmaceutical therapy to treat CCMs. Several mouse models have been developed to better understand CCM pathogenesis and test therapeutics. The most common mouse models induce a large CCM burden that is anatomically restricted to the cerebellum and contributes to lethality in the early days of life. These inducible models thus have a relatively short period for drug administration. We developed an inducible CCM3 mouse model that develops CCMs after weaning and provides a longer period for potential therapeutic intervention. Using this new model, three recently proposed CCM therapies, fasudil, tempol, vitamin D 3 , and a combination of the three drugs, failed to substantially reduce CCM formation when treatment was administered for 5 weeks, from postnatal day 21 (P21) to P56. We next restricted Ccm3 deletion to the brain vasculature and provided greater time (121 days) for CCMs to develop chronic hemorrhage, recapitulating the human lesions. We also developed the first model of acute CCM hemorrhage by injecting mice harboring CCMs with lipopolysaccharide. These efficient models will enable future drug studies to more precisely target clinically relevant features of CCM disease: CCM formation, chronic hemorrhage, and acute hemorrhage.

Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10 ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10 ECKO . We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = − 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10 +/− when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets. Electronic supplementary material The online version of this article (10.1186/s40478-019-0789-0) contains supplementary material, which is available to authorized users.