PI3-kinase/Akt signaling pathway, but not cAMP, is involved in ROCK suppression-induced expansion of ECs. Flk1⁺ MPCs were incubated with the indicated agents for 2 days, and analyses were performed on day 6.5. (A) Representative FACS analysis showing CD144⁺ ECs or α-SMA⁺ MCs incubated with PBS (Control) or Y27632 (10μM) in the absence or presence of VEGF-A (50 ng/mL) and VEGF-Trap (5 μg/mL). (B-C) Percentage of CD31⁺/CD144⁺ EC population and α-SMA⁺ MC population. (B) Each group, n = 3. *P < .01 versus Control. (C) Each group, n = 4. *P < .01 versus each Control. (D) Immunoblotting for pPTEN, PTEN, pAkt, Akt, pERK, ERK, pMyPT, MyPT, pMLC, MLC, α-SMA, and GAPDH in Flk1⁺ MPCs treated with PBS (Control), Y27632 (10μM), VEGF-A (50 ng/mL; VA), or VA plus Y27632. The Flk1⁺ MPCs were cultured for 12 hours, starved for 6 hours with or without Y27632, and then treated with VEGF-A (50 ng/mL) for 10 minutes. Results from 4 independent trials were similar to each other. (E) Representative FACS analysis of CD31⁺/CD144⁺ ECs incubated with 0.1% DMSO or LY294002 (20μM; LY) in the presence of PBS (Control) or Y27632 (10μM; Y). (F) Percentage of CD31⁺/CD144⁺ EC population. Each group, n = 3. *P < .01 versus DMSO; #P < .01 versus Y27632. (G) Representative FACS analysis of α-SMA⁺ MCs or CD144⁺ ECs incubated with 0. 1% DMSO or ML7 (5μM) in the presence of PBS (Control) or Y27632 (10μM). (H) Percentage of α-SMA⁺ MC population. Each group, n = 3. *P < .01 versus DMSO. n.s represents statistically nonsignificant. (I) Schematic diagram showing the negative involvement of ROCK in VEGF-A/VEGFR2/PI3-kinase/AKT signaling pathway via PTEN in differentiation of ECs and the positive involvement of ROCK in differentiation of MCs via MLC in the Flk1⁺ MPCs. Specific inhibitors for each signaling molecules are indicated. (J) Representative FACS analysis of CD31⁺/CD144⁺ ECs incubated with PBS (Control) or 8br-cAMP (0.5mM) in the presence of PBS (Control) or Y27632 (10μM; Y). (K) Percentage of CD31⁺/CD144⁺ EC population. Each group, n = 4. *P < .01 versus Control. #P < .01 versus 8br-cAMP or Y27632.