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Cerebral cavernous malformation (CCM) is a neurovascular disease that affects 0.5% of the general population. For a long time, CCM research focused on genetic mutations, endothelial junctions and proliferation, but recently, transcriptome and proteome studies have revealed that the hemostatic system and neuroinflammation play a crucial role in the development and severity of cavernomas, with some of these publications coming from our group. The aim of this review is to give an overview of the latest molecular insights into the interaction between CCM-deficient endothelial cells with blood components and the neurovascular unit. Specifically, we underscore how endothelial dysfunction can result in dysregulated hemostasis, bleeding, hypoxia and neurological symptoms. We conducted a thorough review of the literature and found a field that is increasingly poised to regard CCM as a hemostatic disease, which may have implications for therapy.

Maintaining blood vessel integrity is an active process, necessary for their proper functioning and adaptation to changing conditions. Aberrations in any of these vascular processes results in consequences exemplified in cerebral cavernous malformation (CCM). Patients with this neurovascular disease can experience neurological symptoms and hemorrhages; with surgical resection as the main treatment. The exact mechanisms through which lesions form and progress are not fully understood. This thesis aimed to elucidate novel mechanisms in the initiation and progression of CCM. In paper I, the molecular mechanisms underlying hemostasis in CCM were studied. We showed that hemostasis is dysregulated in murine and human CCM, leading to clot formation and hypoxia. Additionally, we observed that CCM lesions simultaneously express pro and anticoagulant proteins. However, we observed heterogeneity in the vascular expression of these pro and anticoagulant proteins which may potentially affect response to therapies. Paper II focused on inflammation in CCM, where we demonstrated that a chronic inflammatory microenvironment exists in CCM. We also showed that neutrophils produced neutrophil extracellular traps (NETs) in human and murine CCM, and contribute negatively to CCM pathogenesis. Paper III focused on the therapeutic inhibition of NETs in murine CCM using Cl-amidine. We found that NETs contribute to coagulation, endothelial dysfunction and neuroinflammation in CCM. Additionally, we observed activation of fibroblasts and microglia cells which promoted CCM pathogenesis. In Paper IV, we presented a novel proteomic approach for human CCMs, which allowed high-throughput protein screening. Our studies showed a dysregulation of the neurovascular unit’s components in CCM. Additionally, we report CCM-relevant brain proteins, which can serve as disease biomarkers. In Paper V, we present a proof-of-concept approach using CCM paraffin-embedded biopsy samples for transcriptomic analysis. Using spatial transcriptomics, we elucidated thrombomodulin heterogeneity in CCM lesions. Collectively, our studies demonstrate that immunothrombosis and neuroinflammation occur in CCM and that CCM pathogenesis goes beyond endothelial dysfunction, affecting other cells of the neurovascular unit. Furthermore, we highlight vascular heterogeneity in CCM and its implications for CCM therapy development. Our studies have increased the understanding of CCM pathogenesis and offer potential pathways that can be targeted for treatment in CCM patients.

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 ( Ccm3 iECKO ), we show that endothelial cells from Ccm3 iECKO 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 Ccm3 iECKO 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 Ccm3 iECKO 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.