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Cerebral cavernous malformation (CCM) is a neurovascular disease distinguished by clusters of leaky, mulberry-like blood vessels. KRIT1 bi-allelic loss-of-function mutations in endothelial cells are known to trigger brain cavernomas; however, human preclinical models are needed to unveil the importance of germline KRIT1 heterozygous mutations in CCM pathogenesis. We generated three induced pluripotent stem cells (iPSCs) from patients with CCM with hereditary KRIT1 heterozygous mutations. Patient-derived vascularized organoids exhibited intricate and abnormal vascular structures with cavernoma-like morphology, and iPSC-derived endothelial cells displayed phenotypic abnormalities at the junctional and transcriptional levels. Upon injection into brain explants, CCM endothelial cells integrated into the normal vasculature and created vascular anomalies. Lastly, transcriptional analysis showed that the endothelial progenitor marker paternally expressed gene 3 (PEG3) was highly expressed in iPSC-derived CCM endothelial cells, and this was further confirmed in familial and sporadic cavernoma biopsies. Overall, our study sheds light on the molecular consequence of KRIT1 heterozygous mutations in endothelial cells and the potential implications in cavernoma pathogenesis.

Cerebral cavernous malformation (CCM) is a neurovascular disease with symptoms such as strokes, hemorrhages and neurological deficits. With surgery being the only treatment strategy, understanding the molecular mechanisms of CCM is crucial in finding alternative therapeutic options for CCM. Neutrophil extracellular traps (NETs) were recently reported in CCM, and NETs were shown to have positive or negative effects in different disease contexts. In this study, we investigated the roles of NETs in CCM by pharmacologically inhibiting NET formation using Cl-amidine (a peptidyl arginine deiminase inhibitor). We show here that Cl-amidine treatment reduced lesion burden, coagulation and endothelial-to-mesenchymal transition. Furthermore, NETs promoted the activation of microglia and fibroblasts, leading to increased neuroinflammation and a chronic wound microenvironment in CCM. The inhibition of NET formation caused endothelial quiescence and promoted a healthier microenvironment. Our study suggests the inhibition of NETs as a potential therapeutic strategy in CCM. Onyeogaziri et al. show that the formation of neutrophil extracellular traps contributes to a chronic wound state in cerebral cavernous malformation, while inhibition of these traps with CI-amidine establishes a healthier microenvironment and promotes endothelial cell quiescence, suggesting use of CI-amidine as a potential therapeutic strategy.