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.

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.

Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.

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.

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