The spatiotemporal expression profile of VASH1. (A) Immunostaining of CD31 (red), αSMA (green), PCNA (green), and/or VASH1 (green) was performed with the indicated area of the skin flap. (B) Total RNA was isolated from each area of the skin flap. Quantitative real-time RT-PCR was performed to show mRNA levels of VASH1 in each area. Each value was standardized with β-actin. Data are expressed as mean and SDs. (C) HUVECs of sparse, subconfluent, and confluent conditions were treated with or without VEGF (1 nM) for 12 hours, and the expression of VASH1 was determined by Northern blotting.