Reactive astrocytes with inflammatory signature in CCM disease in hypoxia and normoxia conditions
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 assess whether CCM reactive astrocytes with neuroinflammatory capacity respond to hypoxia-induced CCM exacerbation, we employed the astrocyte-specific (Aldh1l1-EGFP/Rpl10a) Translational Ribosome Affinity Purification (TRAP) system in combination with a CCM mouse model (Pdcd10BECKO;Aldh1l1-EGFP/Rpl10a). TRAP protocol was performed using Slco1c1-iCreERT2;Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a mice to isolate ribosomes from astrocytes as previously described. Astrocyte-TRAP mRNAs were from cerebral tissue of mice age P50 exposed to hypoxia or normoxia conditions. Overall design: 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). To perform Translational Ribosome Affinity Purification (TRAP) in astrocytes we generated Slco1c1-iCreERT2;Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a and controls littermate Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a. 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; Aldh1l1-EGFP/Rpl10a (Pdcd10BECKO; Aldh1l1-EGFP/Rpl10a) and Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a 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; Aldh1l1-EGFP/Rpl10a mice. Hypoxic conditions consisted in 12% O2,  for seven days and normoxic conditions consisted in 21% O2.TRAP mRNAs from astrocytes was perform using the protocol and instructions as previously described. Astrocyte-TRAP mRNAs were from brains of mice age P57. 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.