Cerebral cavernous malformation (CCM) is a rare neurovascular disease that is characterized by enlarged and irregular blood vessels that often lead to cerebral hemorrhage. Loss-of-function mutations to any of three genes results in CCM lesion formation; namely, KRIT1, CCM2, and PDCD10 (CCM3). Here, we report for the first time in-depth single-cell RNA sequencing, combined with spatial transcriptomics and immunohistochemistry, to comprehensively characterize subclasses of brain endothelial cells (ECs) under both normal conditions and after deletion of Pdcd10 (Ccm3) in a mouse model of CCM. Integrated single-cell analysis identifies arterial ECs as refractory to CCM transformation. Conversely, a subset of angiogenic venous capillary ECs and respective resident endothelial progenitors appear to be at the origin of CCM lesions. These data are relevant for the understanding of the plasticity of the brain vascular system and provide novel insights into the molecular basis of CCM disease at the single cell level.

Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies; but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA-sequencing to investigate mouse brain endothelial cells with specific Ccm3 gene deletion (Ccm3-iECKO). We found that Ccm3 deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared to wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. Lastly, we created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3, expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCM. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.

Background and Purpose: Cerebral cavernous malformations (CCM) present as mulberry-like malformations of the microvasculature of the central nervous system. Current medical treatment of CCM lesions is limited to surgical removal of the vascular malformations. It is, therefore, important to identify therapeutic drug treatments for patients with CCM. Propranolol has shown great benefit in the treatment of infantile hemangioma. In addition, patients with CCM who receive propranolol have demonstrated a reduction of their lesions. Our investigation set out to provide preclinical data to support propranolol as a therapeutic treatment. Methods: An inducible endothelial-specific Ccm3 knockout murine model (CCM3 iECKO ) was used, with assessment of lesion quantity and size following oral treatment with propranolol. Scanning and transmission electron microscopy were used to characterize the CCM3 iECKO lesions and the effects of propranolol on the disease. Immunofluorescent imaging was used to investigate pericyte coverage in the propranolol-treated CCM3 iECKO mice. Results: With propranolol treatment, the lesion quantity, size, and volume decreased in both the brain and retina in the CCM3 iECKO model. Novel characteristics of the CCM3 iECKO lesions were discovered using electron microscopy, including plasmalemmal pits and thickening of the endothelial-pericyte basal membrane. These characteristics were absent with propranolol treatment. Pericyte coverage of the CCM3 iECKO lesions increased after propranolol treatment, and vascular leakage was reduced. Conclusions: This study supports the concept that propranolol can be used to reduce and stabilize vascular lesions and can, therefore, be suggested as a pharmaceutical treatment for CCM.

Trophoblast stem (TS) cells in the mouse derive from the polar trophectoderm of the blastocyst and persist through early gestation (to E8.5) to support placental development. Further development and growth is proposed to rely on layer-restricted progenitor cells. Stem cell antigen (Sca) -1 is a member of the Ly6 gene family and a known marker of stem cells in both hematopoietic and non-hematopoietic mouse tissues. Having identified that Sca-1 mRNA was highly expressed in mouse TS cells in culture, we found that it was also expressed in a subset of trophoblast within the chorion and labyrinth layer of the mouse placenta. Isolation and in vitro culture of Sca-1+ trophoblast cells from both differentiated TS cell cultures and dissected mouse placentae resulted in proliferating colonies that expressed known markers of TS cells. Furthermore, these cells could be stimulated to differentiate and expressed markers of both junctional zone and labyrinth trophoblast subtypes in a manner comparable to established mouse TS cell lines. Our results suggest that we have identified a subpopulation of TS cell-like cells that persist in the mid- to late- gestation mouse placenta as well as a cell surface protein that can be used to identify and isolate these cells.

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas  of patients with CCM confirms the clinical relevance of NETs in CCM.