Transcriptome analysis reveals coagulation and hypoxia in CCM pathogenesis. (A) Diagram illustrating the study design of the transcriptome analysis of cerebellar brain endothelial cells (EC) isolated from wild-type and Ccm3-iECKO mice (n = 3 mice per group). (B) Bar plot showing the number of significantly DEGs between wild-type and Ccm3-iECKO mice; 600 genes were upregulated and 441 were downregulated in Ccm3-iECKO mice. Importantly, 87 upregulated genes and 34 downregulated genes were related to coagulation and hypoxia. (C) Bar plot of a Gene Ontology (GO) analysis of the upregulated genes in Ccm3-iECKO mice showing the top 20 overrepresented GO terms ranked by the number of DEGs in each group. GO terms related to coagulation are highlighted in red and marked with a star. (D) Heat map showing the expression levels (z score of regularized log [rlog]-transformed counts) of significantly upregulated DEGs (adjusted P value < .05 & |log2foldchange|>.5; blue: low; red: high) in Ccm3-iECKO mice. The first annotation after the biological replicates (wild-type and Ccm3-iECKO mice 1-3) indicates the differential expression in log₂-fold change (Log2FC, red: high; white: low). The second to fifth annotations indicate the DEGs associated with enriched GO terms from the overrepresentation analysis in panel C. The genes labeled in red are described in this study. (E) Gene set enrichment analysis with hallmark enrichment plots demonstrating that genes related to coagulation and hypoxia were more expressed in endothelial cells isolated from Ccm3-deficient mice than endothelial cells of wild-type mice (NES > 2 and FDR < 5%). (F) Heat map showing the log-fold change of selected genes (related to coagulation and hypoxia, selected from panel D) in different endothelial subtypes of Ccm3-iECKO mice. On the right side the number of DEGs related to coagulation and hypoxia are listed for each endothelial subtype (upregulated in red, downregulated in blue; adjusted P value < .05). Only genes that were significantly differentially expressed between Ccm3-iECKO and wild-type mice in at least 1 endothelial cell subtype are shown. The identify of each endothelial cell cluster (C) is as follows: Cap, capillary (C0); Tip, tip cells (C1, C6); Mit Ven, mitotic/venous capillary (C2, C7); Art Cap, arterial capillary (C3, C5); Ven Cap, venous capillary (C4); Art, arterial (C8); Ven, venous/venous capillary (C9); Cap Tip, capillary/tip cells (C12, C14). Extr., extrinsic; neg., negative; path., pathway; pos., positive; reg., regulation; sig., signaling; rcp., receptors.