Aortic ring assay. After mice were killed with an overdose of anesthetic, the thoracic aorta was dissected from the posterior mediastinum, placed in serum-free EBM-2 endothelial basal medium (Cambrex), and cleaned of blood and fibroadipose tissue under a stereoscopic microscope using fine forceps and scissors. The vessel was then cut into 1-mm-long rings, which were subjected to 8 consecutive washes with serum-free EBM-2. The aortic rings were then embedded in thick collagen gel (Cellmatrix Type I-A; Nitta Gelatin Inc.) and cultured for 7 days, with or without recombinant hVEGF (50 ng/ml; R&D Systems) supplement. The capillaries that sprouted from the edges of the rings were analyzed (44).