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).
The periaortic fibroadipose tissue was carefully removed, paying special attention not to damage the aortic wall.
1 mm long aortic rings were sectioned and rinsed extensively in 8 consecutive washes of EGM-2.
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.
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.
The culture medium was changed on day 3 and the test compounds and vehicle were added.
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.
The angiogenic response was determined by measuring the area of neovessel formation on computer (Image Pro Plus software; Media Cybernetics, Inc.).