Hypoxia increases neuroinflammation and angiogenesis gene programs in CCM disease

The CCM endothelium is hypersensitive to angiogenesis and can induce a hypoxic program associated with changes in angiogenesis, inflammation, and endothelial-cell metabolism under normoxic conditions. However, the role of active drivers of angiogenesis as CCM disease modifiers in human disease remains unclear. To examine if hypoxia, a driver of angiogenesis, may contribute to CCM exacerbation, we performed bulk RNA-seq of brain tissue from P50 Pdcd10BECKO mice under normoxia and hypoxia. P50 Pdcd10fl/fl littermate controls under normoxia and hypoxia were used as controls. Brain endothelial cell-specific conditional Pdcd10-knockout mice were generated by crossing a brain endothelial tamoxifen-regulated Cre recombinase (Slco1c1-iCreERT2) strain with loxP-flanked Pdcd10 (Slco1c1-iCreERT2; Pdcd10fl/fl). On a postnatal day 1 (P1), mice were administered 50 μg of 4-hydroxy-tamoxifen (H7904, Sigma-Aldrich) by intragastric injection to induce genetic inactivation of endothelial CCM3 gene in littermates with Slco1c1-iCreERT2;Pdcd10fl/fl (Pdcd10BECKO) and Pdcd10fl/fl mice were used as littermate controls. Non-injected animals with Cre recombinase were also used as littermate controls, whose gene expression and histology were the same as in Pdcd10fl/fl mice. Hypoxic conditions consisted in 12% O2, for seven days and normoxic conditions consisted in 21% O2. Total RNA from cerebral tissue was isolated by TRIzol method according to the manufacturer’s instructions (Thermo Fisher Scientific). Total RNA was assessed for quality using an Agilent Tapestation 4200, and 50 nanograms of RNA from samples with an RNA Integrity Number (RIN) greater than 8.0 were used to generate RNA-seq libraries using the Illumina® Stranded mRNA Prep (Illumina, San Diego, CA). Samples were processed following manufacturer’s instructions.