Inhibitory effects of KOR agonists U50,488H and TRK820 on 3D vascular formation from Flk1⁺ cells. Three-dimensional culture of Flk1⁺ cell aggregates in type I collagen gel. (A) Phase contrast images after 5 days of culture. Flk1⁺ cells were stimulated with control, VEGF (50 ng/mL), DAMGO (10μM), SNC80 (10μM), U50,488H (10μM), or TRK820 (10μM) as indicated. Scale bars, 200 μm. (B) In-gel immunostaining for CD31 (green). Scale bars, 200 μm. (C) Quantitative analysis of CD31⁺ area in 3D vascular formation (except for the aggregate area). Control (n = 3), VEGF alone (50 ng/mL; n = 3), and VEGF plus DAMGO (10μM; n = 3), SNC80 (10μM; n = 3), U50,488H (10μM; n = 3), or TRK820 (10μM; n = 3) are shown (**P < .01, *P < .05 vs control). (D) Quantitative analysis of length in CD31⁺ sprouting vessels (excluding the aggregate area; **P < .01 vs VEGF).