Aorta Ring Assay
1. Aortas were removed from 6-week-old mice killed by CO2 asphyxiation and immediately transferred to a culture dish containing ice-cold endothelial cell basal medium (EGM-2; Cambrex Bio Science, Walkersville, MD).
2. The periaortic fibroadipose tissue was carefully removed, paying special attention not to damage the aortic wall.
3. 1 mm long aortic rings were sectioned and rinsed extensively in 8 consecutive washes of EGM-2.
4. The rings were then individually embedded in 48-well plates previously coated with 50 µL synthetic basement membrane (Matrigel; BD Biosciences, Bedford, MA) per well.
5. Next, an additional 50 µL of Matrigel was placed over each ring. After 1 hour, 500 µL EGM-2 was added to each well, and the cultures were incubated at 37°C for 5 days.
6. The culture medium was changed on day 3 and the test compounds and vehicle were added.
7. The aortic rings were photographed on day 5 at 4x magnification with an inverted microscope. For neovessel-regression experiments, the rings were cultured without drugs until day 6, after which the rings were treated with the test compound and allowed to grow until day 7.