Inflammation and immunothrombosis in cerebral cavernous malformation : Novel molecular targets for the treatment of an incurable disease
Abstract: Cerebral cavernous malformation (CCM) is a vascular disease that causes mulberry-like lesions (cavernomas) in the central nervous system (CNS). Cavernomas are fragile, leaky and prone to rupture which may cause symptoms such as epileptic seizures, focal neurological deficits and hemorrhagic strokes. CCM lesions can appear sporadically in 0.5% of the general population. Alternatively, CCM lesions appear as a consequence of a loss-of-function germline mutation in either CCM1, CCM2, or CCM3 in endothelial cells of the CNS. Inherited CCM is termed familial, and it affects approximately one in ten thousand individuals in an autosomal dominant manner. The aim of this thesis project is to identify novel cell and molecular mechanisms that contribute to the development and progression of cavernous malformations. Additionally, this thesis project aims to identify and validate inhibitors that may reduce lesions and alleviate the side effects in CCM. In this thesis project, two inducible endothelial specific Ccm3 deficient mouse models (acute and chronic) were evaluated with methods such as RNA-sequencing, immunofluorescence, ELISA, scanning and transmission electron microscopy, flow cytometry, and in situ hybridization. Moreover, in vitro cell cultures were used with methods such as immunofluorescence, qPCR, and western blot. Importantly, sporadic and familial human CCM samples were used to show the clinical relevance of our studies. In paper I we focused on the role and kinetics of inflammation in CCM. We analyzed the transcriptome of healthy and Ccm3 deficient mouse brain endothelial cells and found that genes related to inflammation were upregulated in CCM pathology. We identified various inflammatory cytokines in vivo and also identified neutrophils as the most prominent immune cell subtype in CCM. Moreover, we found that neutrophils in CCM produce neutrophil extracellular traps (NETs), that can be inhibited with DNase I. The inhibition of NETs stabilized cavernoma vasculature by reducing fibrinogen and IgG leakage. In paper II we re-used the acute RNA-seq database from paper 1 and focused on endothelial hemostasis and hypoxia. We found that genes related to procoagulation, anticoagulation, and hypoxia were highly upregulated in Ccm3 deficient mice. We validated the findings in vivo and found that the hemostatic system in CCM is dysregulated and that it causes, bleeding, thrombosis, and cerebral hypoxia. In paper III we evaluated the effect of propranolol in CCM. We treated chronic CCM mice with the beta-blocker propranolol and found that propranolol was able to reduce lesions in the brains and retinas of CCM mice as well as reduce cadaverine leakage. Importantly, we identified endothelial plasmalemmal pits and a thick basal membrane between endothelial cells and pericytes, pathological features which reduced upon propranolol treatment. Altogether this thesis significantly contributes to the CCM field as it identified pathological features of cavernomas such as neutrophils with NETs, endothelial plasmalemmal pits, and polyhedrocytes. This thesis work also evaluated pharmacological inhibitors (DNase I and propranolol) in mouse models of CCM and supports the use of anticoagulant therapies in patients with CCM.
CLICK HERE TO DOWNLOAD THE WHOLE DISSERTATION. (in PDF format)