Figure 4.

Up-regulation of VEGF-B mediated by RTEF-1. (A) Western blots showed an increase in the protein level of RTEF-1 in HMEC-1 over-expressing RTEF-1 (upper panel). Cells were starved and then treated with normoxia or hypoxia for 12 h. VEGF-B was increased in both cell lysates and conditioned medium from HMEC-1/RTEF-1 (lower panel). VEGF-A and FGF2 were also detected in the conditioned medium. Vinculin was used as loading control. HMEC-1 transfected with pBMN vector was used as negative control. (B) Isolated endothelial cells from hearts of WT and VE-Cad/RTEF-1 mice were starved and then treated with normoxia or hypoxia for 12 h. VEGF-B was increased and further elevated by hypoxia in the lysates of VE-Cad/RTEF-1 compared with WT endothelial cells. (C) Endothelial cells were transfected with RTEF-1 siRNA. Western blot (left) and qPCR (right) indicated a significant knock down of RTEF-1 expression, accompanied by a significant reduction of VEGF-B expression in both normoxic and hypoxic conditions. qPCR data were from three independent experiments performed in triplicate and normalized to 36b4 content (n = 3). (D) VEGF-B promoter construct was co-transfected with various combinations of control vector (PXJ40) and RTEF-1 into HEK293 cells. Note that VEGF-B promoter activity was increased as the amount of RTEF-1 increased. The data are expressed as the means ± SEM of three independent experiments. (E) HEK293 cells were transfected for 24 h with PXJ40 or an equal amount of RTEF-1 and VEGF promoter constructs. Luciferase activity was determined after 6 h incubation of transfected cells in normoxic or hypoxic conditions. The data are expressed as the means ± SEM of three independent experiments. (F) Chromosomal immunoprecipitation assay showing co-immunoprecipitation of VEGF-B promoter with RTEF-1 antibody. The arrows in the schematic representation indicate the primers on the VEGF-B promoter sequence. Primers for actin promoter were used as controls. *P < 0.05 and **P < 0.01.