Myh11 creert2 mice

Prdm16^fl/fl mice (stock number 024992) and Myh11-CreER^T2 mice (stock number 019079) were from the Jackson Laboratory and were backcrossed to the C57BL/6J background. Prdm16^fl/fl mice were crossbred with Myh11-CreER^T2 mice (carrying the CreER cassette on the Y chromosome) to generate Prdm16^SMKO mice. Eight- to 10-week-old male mice were treated with tamoxifen (75 mg/kg/day) in corn oil for 5 consecutive days by gavage. Two weeks later, the mice were used for AAA induction by application of 100% of porcine pancreatic elastase (PPE; Sigma-Aldrich) to the adventitial surface of the suprarenal abdominal aorta. The AAA incidence rate of this model is dependent on the incubation time with PPE. We optimized the PPE incubation time and found that incubation of aortas with PPE for 10 minutes was the optimal time window in our study. Briefly, the upper abdomen of mice was opened, and the suprarenal abdominal aorta was isolated and exposed to PPE for 10 minutes, followed by washing with 0.9% sodium chloride solution 3 times, wound closure, and recovery. After 2 weeks, mice were euthanized, and AAA formation was evaluated. Verhoeff–Van Gieson staining of the abdominal aorta was performed using the Elastic Stain Kit (ab150667, Abcam). All mice were maintained under normal housing conditions (12-hour light/dark cycle, 23°C) with free access to regular chow diet and water.

MyH11-CreERT2 mice were purchased from Jackson Lab (Bar Harbor, ME, USA). IRflox/flox mice were cross-bred with MyH11-CreERT2 mice to generate MyH11-CreERT2/IRfox/flox (MyH11IRKO) mice. Insulin receptor in smooth muscle cells in MyH11IRKO mice was deleted by tamoxifen intraperitoneal injection (Sigma, St. Louis, MO, USA, 50 mg/Kg body weight) once every 24 h for a total of 10 consecutive days. The control mice were IRflox/flox mice which were also received tamoxifen intraperitoneal injection (50 mg/Kg body weight) once every 24 h for a total of 10 consecutive days.