Cyclic adenosine monophosphate/protein kinase A pathway plays a critical role in vascular development. (A-D) Enhancement of EC induction by cyclic adenosine monophosphate (cAMP) through protein kinase A (PKA) at Flk-d3. (A) Fluorescent staining for CD31 (green). (Left panel) VEGF treatment alone (50 ng/mL). (Middle panel) VEGF with 8bromo-cAMP (0.5 mM). (Right panel) VEGF with 8bromo-cAMP and PKA inhibitor, PKI (10 μM). Nuclei are stained with DAPI (blue). Scale bars represent 250 μm. (B) Flow cytometry. x-axis: CD31; y-axis: side scatter (SSC). Percentages of CD31⁺ ECs in total Flk1⁺ cell–derived cells are indicated. (C-D) Quantitative evaluation of the effect of PKA inhibitors on CD31⁺ EC induction from Flk1⁺ cells by FACS. (C) Percentages of CD31⁺ cell population in total Flk1⁺ cell–derived cells. VEGF (50 ng/mL; n = 16); VEGF and 8bromo-cAMP (0.5 mM; n = 16); VEGF, cAMP, and PKI (10 μM; n = 7); and VEGF, cAMP, and H89 (10 μM; n = 6) treatment are shown (**P < .01 vs VEGF and 8bromo-cAMP). (D) CD31⁺ cell number that appeared from 1.5 × 10⁵ Flk1⁺ cells. VEGF (50 ng/mL; n = 4); VEGF and 8bromo-cAMP (0.5 mM; n = 4); VEGF, cAMP, and PKI (10 μM; n = 4); and VEGF, cAMP, and H89 (10 μM; n = 4) treatment are shown (**P < .01 vs VEGF and 8bromo-cAMP). (E-F) Role of PKA in vascular formation in the embryo. (E) Representative results of ex vivo culture of mouse embryo. Isolated E6.75 concepti were cultured in the absence (control, left panels) or presence (right panels) of H89 (30 μM) for 3 days. Vasculature in yolk sacs of concepti was immunostained for CD31 (purple). Bottom panels correspond to boxed regions in control and H89, respectively. Apparent reduction of CD31⁺ vascular formation was induced by H89 treatment. Scale bar represents 1 mm. (F) Quantitative evaluation of CD31⁺ vasculature formation in yolk sacs of concepti. The ratio of CD31⁺/whole yolk sac area was evaluated (n = 3; *P < .05 vs control).