Figure 1.
Figure 1. Impaired vascular remodeling by retinoid induced in CAM. The 4.5-day-old CAMs were implanted with a silicon ring and charged with different amounts of atRA or Ro41-5253. (A) Effect of retinoids on blood vessel formation within CAM. After 48 hours, developing new blood vessels were observed under a microscope and photographed. i, vehicle (1% ethanol); ii, 100 ng atRA; iii, 1 μg Ro41-5253. Original magnification, ×35; scale bar, 1 mm. The total area and the number of paths of blood vessels were analyzed using angiogenesis measuring software. Their relative changes are shown in parentheses. Each set of data represents the average ± SD (n = 3). Asterisks represent significant difference (P < .05) from control. (B) Structural changes induced in CAM tissues following treatment with retinoids. Vertical sections (5 μm) were stained with hematoxylin and eosin (i-iii), vertical sections (10 μm) were prepared and double-stained with a combination of either anti–Queck-1 antibody (for ECs, red) and anti-αSMA antibody (for MCs, green) (iv-vi), or anti-PCNA antibody (red) and anti-αSMA antibody (green) (vii-ix). The 6.5-day-old CAM was composed of 4 different layers, including a thin chorionic epithelium (ce), microvasculatures (mv), a thick mesenchymal layer (fb) consisting of sparsely distributed fibroblasts and a few small blood vessels (bv), and a thin allantoic epithelium (ae). Original magnification, × 200. Scale bar, 50 μm. (C) Effect of simultaneous treatment with atRA, Ang-1, and Ang-2 on blood vessel formation. The 4.5-day-old CAMs were treated with vehicle (1% ethanol; i), 100 ng atRA (ii), 300 ng human recombinant Ang-2 (iii), 300 ng human recombinant Ang-1 (iv), and combinations of atRA and Ang-1 (v), or Ang-2 and Ang-1 (vi). After 48 hours, developing new blood vessels were observed under a microscope and photographed. Original magnification, ×35; scale bar, 1 mm. The number of paths of blood vessels was analyzed as described above and presented underneath each picture with relative changes in parenthesis. Symbols *, †, and ‡ represent significant difference (P < .05) obtained by comparing to control samples (i), samples treated with atRA (ii), and samples treated with Ang-2 (iii), respectively. For panels A to C, a total of 15 eggs (n = 5 × 3 times) for each experimental group were evaluated and representative results are shown.

Impaired vascular remodeling by retinoid induced in CAM. The 4.5-day-old CAMs were implanted with a silicon ring and charged with different amounts of atRA or Ro41-5253. (A) Effect of retinoids on blood vessel formation within CAM. After 48 hours, developing new blood vessels were observed under a microscope and photographed. i, vehicle (1% ethanol); ii, 100 ng atRA; iii, 1 μg Ro41-5253. Original magnification, ×35; scale bar, 1 mm. The total area and the number of paths of blood vessels were analyzed using angiogenesis measuring software. Their relative changes are shown in parentheses. Each set of data represents the average ± SD (n = 3). Asterisks represent significant difference (P < .05) from control. (B) Structural changes induced in CAM tissues following treatment with retinoids. Vertical sections (5 μm) were stained with hematoxylin and eosin (i-iii), vertical sections (10 μm) were prepared and double-stained with a combination of either anti–Queck-1 antibody (for ECs, red) and anti-αSMA antibody (for MCs, green) (iv-vi), or anti-PCNA antibody (red) and anti-αSMA antibody (green) (vii-ix). The 6.5-day-old CAM was composed of 4 different layers, including a thin chorionic epithelium (ce), microvasculatures (mv), a thick mesenchymal layer (fb) consisting of sparsely distributed fibroblasts and a few small blood vessels (bv), and a thin allantoic epithelium (ae). Original magnification, × 200. Scale bar, 50 μm. (C) Effect of simultaneous treatment with atRA, Ang-1, and Ang-2 on blood vessel formation. The 4.5-day-old CAMs were treated with vehicle (1% ethanol; i), 100 ng atRA (ii), 300 ng human recombinant Ang-2 (iii), 300 ng human recombinant Ang-1 (iv), and combinations of atRA and Ang-1 (v), or Ang-2 and Ang-1 (vi). After 48 hours, developing new blood vessels were observed under a microscope and photographed. Original magnification, ×35; scale bar, 1 mm. The number of paths of blood vessels was analyzed as described above and presented underneath each picture with relative changes in parenthesis. Symbols *, †, and ‡ represent significant difference (P < .05) obtained by comparing to control samples (i), samples treated with atRA (ii), and samples treated with Ang-2 (iii), respectively. For panels A to C, a total of 15 eggs (n = 5 × 3 times) for each experimental group were evaluated and representative results are shown.

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