Differential induction of MGP and CV2 by BMP-4 and BMP-9. (A) HAECs were treated with BMP-4 (40 ng/mL) or control vehicle and ALK1-Fc (300 ng/mL). RNA samples were collected every 2 hours for up to 48 hours, and expression of ALK2, ALK1, MGP, and CV2 (left panels), and ALK5 and VEGF (right panels) was determined. (B) HAECs were pretreated with BMP-4 and ALK1-Fc for 8 hours, and then treated with (top panels) control vehicle, BMP-4 (40 ng/mL), BMP-9 (10 ng/mL), or both BMP-4 and BMP-9, or (bottom panels) nonspecific IgG (300 ng/mL), anti–BMP-4 Abs (300 ng/mL), anti–BMP-9 Abs (300 ng/mL), or CV2 (100 ng/mL). RNA was collected every 2 hours for up to 48 hours. Expression of (left panels) MGP and (right panels) CV2 was determined by real-time PCR. (C) HAECs were pretreated with BMP-4 and ALK1-Fc for 8 hours, and then treated with (top panels) control vehicle, BMP-4, or BMP-9, or (bottom panels) nonspecific IgG, anti–BMP-4 Abs, anti–BMP-9 Abs, CV2, or MGP. RNA was collected every 2 hours for up to 48 hours. Expression of ALK5 (left panels) and VEGF (left panels) was determined by real-time PCR. (D) HAECs were pretreated for 8 hours with control vehicle (left) or BMP-4 (right), plated, and treated as indicated in the figure. Cell proliferation was determined after 48 hours by ³H-thymidine incorporation. (E) HAECs were pretreated for 8 hours with (left) control vehicle or (right) BMP-4, plated in Matrigel, and treated as indicated in the figure. Images were obtained after 6 hours. Asterisks indicate statistically significant differences compared with control treatment. *P < .05, **P < .01, ***P < .001, Tukey test.