Blockage of Notch/patched pathway attenuates while stimulation increases arterial EC differentiation of hMAPCs. (A) Q-RT-PCR analysis for arterial (EphrinB1, Hey-2, and EphrinB2) and venous markers (EphB4) on hMAPC-derived ECs (gray bars) after 14 days of differentiation using blocking (red bars) or activating (green bars) treatments (as indicated in the box). Shh blocking, Notch blocking, or a combination of both significantly reduced expression of arterial EC markers paralleled by an increase in venous marker expression compared with VEGF₁₆₅ alone (gray bars). Conversely, addition of Shh and Dll-4 to VEGF₁₆₅ further significantly increased expression of arterial marker Hey-2 while significantly decreasing venous marker EphB4. *P < .05; **P < .01 versus VEGF₁₆₅ alone; *P < .05 condition 4 versus condition 2. (B) Q-RT-PCR analysis of additional arterial (ALDH1A1, Jagged-2) and venous markers (Lefty-1, Lefty-2) in hMAPC-derived ECs cultured in VEGF₁₆₅ alone (gray bars) or combined with Shh and Dll-4 (green bars). Note the significant up-regulation of arterial markers and simultaneous down-regulation of venous markers in the combination cocktail as compared with VEGF₁₆₅ alone. The mRNA levels in all panels are expressed as mean percentage of HUAECs (arterial markers) and HUVECs (venous markers) and were normalized using GAPDH as housekeeping gene. Baseline corresponds to undifferentiated hMAPCs. The mean (± SEM) of 3 different experiments is shown. *P < .05; **P < .01 versus VEGF₁₆₅ alone.