Activation of cAMP/PKA signaling rescues the inhibitory effects of KOR on EC induction from Flk1⁺ vascular progenitors. (A) Double fluorescent staining for CD31 and αSMA at Flk-d3. Left, CD31 (pan-ECs, green) and DAPI (blue); middle, αSMA (MCs, red) and DAPI (blue); and right, merged. Flk1⁺ cells stimulated with VEGF (50 ng/mL), U50,488H (10μM), TRK820 (10μM), 8br-cAMP (0.5mM), and 6-Bnz-cAMP (0.1mM) as indicated. Scale bars, 200 μm. (B) Flow cytometry. X-axis: CD31; y-axis: SSC. Percentages of CD31⁺ ECs among total Flk1⁺ cell–derived cells are indicated. (C) Three-dimensional culture of Flk1⁺ cell aggregates in type I collagen gel. In-gel immunostaining for CD31 (green). Scale bars, 200 μm.