Dimerization of NP-1 is important for inducing endothelial cell growth.

ECs developed in P-Sp cultures were stained with anti–PECAM-1 mAb. PECAM-1⁺ cells are visualized as dark blue products. Upon the addition of L cells that possessed only the “a” domain (data not shown) or “a” and “b” domains of the NP-1 protein (A-C) to an NP-1^−/− P-Sp culture (2 × 10³ cells per well) on the fourth P-Sp culture day, the defective vascularity was not rescued at either the areas that were adherent to the L cells (red dashed line [B]) or the areas that were not adherent to the L cells (C). Small numbers of ECs make cordlike structures, indicated by arrow in panels B and C. Panels B and C are higher magnifications of the areas indicated by the boxes in panel A. On the other hand, upon the addition of L cells that possessed the “a,” “b,” and “c” domains (D-F) to an NP-1^−/− P-Sp culture (2 × 10³ cells per well) on the fourth P-Sp culture day, the defective vascularity was rescued at the areas that were adherent to the L cells (red dashed line [E]); however, it was not rescued at the areas that were not adherent to the L cells (F). Sheetlike structures of ECs (white dashed line) and network formation of ECs (yellow dashed line) are observed (E). Panels E and F are higher magnifications of the areas indicated by the boxes in panel D. Scale bar indicates 400 μm (A,D) and 200 μm (B-C,E-F).