Presence of vascular smooth muscle and endothelial cells in aortic ring sprouts induced by KRX-725, S1P, and VEGF. (A) Involvement of vascular smooth muscle cells in sprouts. Aortic rings of BALB/c mice were incubated with vehicle (0.1% DMSO), KRX-725 (20 μM), S1P (200 nM), and VEGF (20 ng/mL) for 8 days. The rings were then fixed with paraformaldehyde and stained with FITC-conjugated alpha smooth muscle actin antibody (left column), followed by 0.02% crystal violet (right column). (B) Involvement of endothelial cells in sprouts. Aortic rings of FVB/N-TgN(TIE2GFP)287Sato mice were incubated with vehicle (0.1% DMSO), KRX-725 (20 μM), S1P (200 nM), and VEGF (20 ng/mL) for 8 days. Fluorescent micrographs were taken prior to fixation and staining. Fluorescence emanates from expression of green fluorescent protein (GFP) under endothelial-specific promoter (tie-2). Each micrograph is a representative of 9 repeats. Micrographs were taken under × 10 objective.