LSS-dependent canonical BMP signaling triggers MSX1 expression in ECs. (A) HUAECs were transfected with control siRNA or siRNA against SMAD1/5, seeded in fibronectin-coated chambers, and subsequently kept static or subjected for 1 h to an LSS of 20 dynes/cm². MSX1 expression was analyzed and represented relative to the control static condition. n = 7. (B) Corresponding representative Western blot analysis for MSX1, pSMAD1/5/8, SMAD1, and GAPDH. (C) HUAECs were pretreated for 1 h with DMSO or 2.5-µM DMH1 and subjected to static or LSS (20 dynes/cm²) conditions for 1 h in the presence of DMSO or DMH1. MSX1 expression was analyzed and represented relative to the DMSO condition. n = 3. (D–F) Cells transfected with control siRNA or siRNA against ALK6 (n = 5), BMPR2 (n = 6), or ENG (n = 4) were exposed to static or flow conditions and were analyzed for MSX1 expression. *, P < 0.05 versus the indicated condition. (G) qRT-PCR analysis of HUAECs kept in static conditions or exposed to LSS (20 dynes/cm² for the indicated times). mRNA expression is represented relative to the static condition. n = 3. (A, C, and D–F) *, P < 0.05 versus static condition. ud, undetectable. (H) qRT-PCR analysis of HUAECs and SMCs serum starved for 1 h and exposed to 10 ng/µl BMP2, BMP6, or the corresponding amount of solute for 48 h. mRNA expression is represented relative to the control condition. n = 4 and 7. *, P < 0.05; #, P = 0.13 versus corresponding solute condition. Error bars represent the SEM.