Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation

Abstract

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.

Dysregulated Hemostasis and Immunothrombosis in Cerebral Cavernous Malformations

“…This creates a positive feedback loop that promotes the release of vWF from activated platelets. Interestingly, vWF accumulates in the lumen of human cavernomas, and in vitro models of CCM show that vWF increases in endothelial cells and forms vWF-strings in the absence of CCM1 or CCM3 [ 17 , 31 ]. These data suggest that vWF might be released directly after CCM genes are inactivated; therefore, we hypothesize that CCM-deficient endothelial cells recruit and activate platelets in the early phases of lesion development.…”

“…Tissue factor, a key molecule involved in the activation of the extrinsic pathway of the coagulation cascade, is expressed by parenchymal cells surrounding the lesions of CCM3-null mice [ 17 ]. When TF comes in contact with blood, it initiates the coagulation cascade by binding and activating coagulation factor VIIa (FVII/FVIIa).…”

“…Furthermore, acute hemorrhage, which is a serious symptom in patients, is rarely seen with the commonly used mouse strains. With regards to thrombi observed in CCM mouse models, a recent article identified fibrin clots with fibronectin, vWF ( Table 1 ) and activated platelets [ 17 ]. These data suggest that blood clots in CCM are stable, as blood glycoproteins, such as fibronectin and vWF, can stabilize blood clots in vivo [ 41 , 42 , 43 ].…”

“…For many years the leaky and fragile nature of endothelial junctions was thought to be the main factor underlying intracranial bleeding. Nevertheless, transcriptome and proteome studies have revealed that the hemostatic system and inflammation play crucial roles in the development and severity of cavernomas [ 12 , 13 , 14 , 15 , 16 , 17 ]. Notably, in 2019, Lopez-Ramirez et al reported that the anticoagulant properties of endothelial cells in CCM promoted intracranial hemorrhage [ 12 ].…”

“…Notably, in 2019, Lopez-Ramirez et al reported that the anticoagulant properties of endothelial cells in CCM promoted intracranial hemorrhage [ 12 ]. Our group has also worked on this topic extensively and has recently reported that individual CCM lesions can contain both anticoagulant and procoagulant regions that contribute to hemorrhage, thrombi and cerebral hypoxia [ 17 ]. These studies raise the possibility that CCM, at least to some extent, can be termed a hemostatic disease.…”