Figure 10.

Effect of apelin on the P-Sp culture system. (A) Effect of apelin on the network-like structure of ECs in the P-Sp culture system. P-Sp explants from E9.5 mouse embryos were cultured for 7 days on OP9/vector (a, b) or OP9/apelin, in the presence of B220 control mAbs (c, d) or anti-apelin mAbs (e, f), and then stained with anti-CD31 mAb. (b), (d) and (f) are higher magnifications of areas indicated by the box in (a), (c) and (e), respectively. Arrows indicate network-forming ECs. Scale bar indicates 1 mm (a, c, e) or 200 μm (b, d, f). (g) Quantitative evaluation of the vascular network area cultured as above. Endothelial space per 500 μm length of network-forming ECs was measured in 10 random fields. ^*P<0.001. (B) Expression of APJ in ECs of P-Sp culture. Cells on culture plates were stained with anti-CD31 (red) and anti-APJ (green) antibodies. Tip, tip EC. Dotted line indicates the border of the vascular bed (vb). Note APJ expressed on ECs forming a network-like structure. Scale bar indicates 100 μm. (C) Network-forming ECs derived from P-Sp explants cultured on OP9/vector (a, b), OP9/apelin in the presence of control B220 mAbs (c, d) or OP9/apelin in the presence of anti-apelin mAbs (e, f) for 7 days were stained with anti-VE-cadherin (red) mAbs. Scale bar indicates 20 μm. Because nuclear staining cannot distinguish the nuclei of ECs from those of OP9 cells, only VE-cadherin expression was revealed. Therefore, the EC-to-EC boundary expressed by VE-cadherin is presented (b, d, f). Tip, tip EC. Migration direction of tip EC is indicated by the arrow.