Fig. 1: Cl-amidine treatment attenuated NETs and lesion formation in CCM.

a, Representative images of the cerebellum of Ccm3-iECKO vehicle-treated mice (upper panel) and Cl-amidine-treated (10 mg kg⁻¹ d⁻¹) mice (lower panel) stained with DAPI (blue), MPO (green) and citH3 (magenta). Insets (right) are zoomed-in regions. b,c, Quantification of citH3⁺MPO⁺DAPI⁺ NETs (b) and Ly6G (c) in the cerebellum of Ccm3-iECKO vehicle-treated (n = 13) and Cl-amidine-treated (10 mg kg⁻¹ d⁻¹; n = 8) mice. d, Representative images of mouse brains used in the study and the output from the semi-automated quantification of lesion burden in the brain of Ccm3-iECKO vehicle-treated and Cl-amidine-treated (10 mg kg⁻¹ d⁻¹) mice. e,f, Quantification of lesion area (e) and lesion burden (f) within the cerebellum of Ccm3-iECKO vehicle-treated (n = 13) and Cl-amidine-treated (10 mg kg⁻¹ d⁻¹; n = 8) mice. In the graphs, each data point represents one biological replicate; the bar indicates the median of each group; and the error bars represent the IQR. Statistical significance was determined using a Mann–Whitney U-test (two-tailed). g, Schematic depicting the process of NET formation (left) and the pathways affected by NETs and CCM. NETs were reported to induce endothelial activation⁴³. Endothelial activation facilitates the binding of platelets^42,47, leading to increased thrombi formation. NETs can also directly induce platelet activation²⁷. Continuous thrombi formation would lead to ischemia/hypoxia within the brain. EndMT is a known driver of CCM lesion progression⁴⁶ and was reported to be promoted by NETosis⁴⁵. Endothelial cells that undergo EndMT become mesenchymal and, consequently, more proliferative and highly mobile⁴⁶, leading to lesion growth. Reactive astrogliosis was shown to promote CCM pathogenesis directly through HIF1/VEGF hypoxia pathway¹⁰ and indirectly by attracting microglial cells via CX3CR1 (ref. ¹⁴). This figure was created with BioRender. cb, cerebellum.

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