Transgenic overexpression of apelin induces larger blood vessels. (A) Schematic representation of the K14-apelin expression cassette. (B) Quantitative real-time PCR analysis of apelin mRNA in the ear skin from 3-day-old WT mice and animals from 3 different founder lines of K14-apelin Tg mice. Threshold values for target genes normalized against the level of GAPDH (dCt) are shown under the graph. (C) Immunohistochemical staining of apelin expression in the dorsal skin sections from 6-week-old WT or K14-apelin Tg mice. Positive reactions are revealed by purple coloration. (D) Comparison of blood vessels in lectin-stained whole mounts of ear skin from 8-week-old WT or K14-apelin Tg mice. ECs were stained by perfusion of FITC-conjugated isolectin (green), and mural cells were stained with anti-αsmooth muscle actin (α-SMA) antibody (red). (E-F) Quantitative evaluation of the vascular diameter (E) and vascular density (F) of capillaries in the dermis from WT and K14-apelin Tg mice. Data were obtained by counting 5 random fields from 5 different mice (total 25 fields). *P < .01. (G) Immunohistochemical staining of APJ expression in the dorsal skin serial sections from 2-day-old (neonate) or 8-week-old (adult) mice. Blood vessels were detected by immunohistochemical staining with anti-CD31 Ab. Positive reactions are visualized as a purple coloration.