Tissue preparation.
At the end of the procedure, mice were euthanized under anesthesia (Isoflurane, Abbott laboratories, Madrid, Spain). Aortic aneurysm presence was evaluated in all mice at the end point before further process of the aortic tissue. Aortas were collected, placed in cold (4°C) Krebs-Henseleit solution (KHS) (115 mmol/L NaCl, 25 mmol/L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4.7H2O, 2.5 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 11.1 mmol/L glucose, and 0.01 mmol/L Na2EDTA, pH7.4). Aortas were processed in different conditions depending on the specific experiment. Due to the low amount of tissue and the different experimental approaches, different experimental groups were used for histological studies, protein evaluation techniques (MRM-MS, Western Blot (WB) and zymography) and RNA-seq studies. For histological studies, suprarenal aortic segments were buffered in 4% paraformaldehyde (PFA, pH= 7.4) and embedded in paraffin. For protein or gene expression studies, tissue was immediately frozen in liquid nitrogen and kept at -80ºC until analysis. All the evaluations were done in aortic segments of at least 5 mice per group.