Changes in angiopoietins and Tie2 expressions following treatment with retinoid. (A) Effect of atRA and VEGF on differentiation of Flk1⁺ cells into ECs/MCs and formation of vascular tree–like structures. Flk1⁺ cells were cultured on collagen type 1–coated dishes for 5 days or spheroids formed by Flk1⁺ cells were cultured for 5 days in collagen gels in the absence and presence of atRA and VEGF. In 2D cultures, ECs and MCs were immunostained with anti–PECAM-1 antibody (red) and anti-αSMA antibody (green), respectively (i-iv). White arrowheads indicate margins of MC-EC interaction. In 3D cultures, ECs and MCs were immunostained with anti–PECAM-1 antibody (purple) and anti-αSMA antibody (brown), respectively (v-viii). Black arrowheads indicate the points showing dissociation of ECs from MCs. i, vehicle (0.5% ethanol); ii, 1 μM atRA; iii, 50 ng/mL VEGF; iv, 1 μM atRA plus 50 ng/mL VEGF. Original magnification, × 400. Scale bar, 10 μm. Representative micrographs from 3 different fields (n = 3) are presented. (B) Changes in angiopoietins and Tie2 expressions. Spheroids formed by Flk1⁺ cells were cultured for 5 days in collagen gels. Flk1⁺ cells were treated with vehicle (0.5% ethanol; lane 1), 1 μM atRA (lane 2), 50 ng/mL VEGF (lane 3), or 1 μM atRA plus 50 ng/mL VEGF (lane 4). Total RNA was isolated and mRNA levels of each of the indicated factors were assessed by RT-PCR and quantified by quantitative PCR as described in “Study design.” Representative data from 3 different experiments with similar results are presented. Relative changes were calculated after normalization to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels and presented in parentheses under each corresponding band. (C) Effect of atRA treatment on Ang-2 mRNA levels in microvasculature layer within CAM. The 4.5-day-old CAMs were treated with vehicle (1% ethanol; lane 1), 100 ng atRA (lane 2), or 1 μg Ro41-5253 (lane 3) for 48 hours. CAM tissue was harvested from embryos, and vertical sections (15 μm) were prepared. Regions underneath the chorionic epithelium (50 pieces for one sample; each piece corresponded to about 25 μm × 500 μm) were collected using the microdissection system and total RNA was isolated. Changes in mRNA levels of the indicated factors were assessed by RT-PCR and quantitated as described in “Study design.” Relative changes were calculated after normalization to GAPDH mRNA levels and presented in parentheses under each corresponding band.