EMMPRIN up-regulates VEGF and VEGFR-2 through HIF stimulation. (A) EMMPRIN up-regulates HIF-1α and HIF-2α under normoxic conditions. (i) HMEC-1 cells were transfected with HIF-1α, HIF-2α siRNA, or scrambled siRNA before treatment with 20 μg/mL Emp for 1 hour. HIF-1α and HIF-2α transcripts were quantified by qRT-PCR. (ii) The cells were incubated with Emp or desferrioxamine (DFO). The HIF-1α and HIF-2α levels were detected by Western blotting. (B) EMMPRIN up-regulates VEGFR-2 and soluble VEGF isoforms via HIF-2α. HMEC-1 cells were transfected with HIF-1α, HIF-2α siRNA, or scrambled siRNA before treatment with Emp. VEGFR-2 and VEGF₁₂₁ transcripts were quantified by qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD. *P < .05. (C) HMEC-1 and HUVECs cocultured with EMMPRIN- or mock-transfected CHO cells. Total RNA was extracted from cocultured cells. mRNA levels for HIF-1α and HIF-2α were quantified using qRT-PCR. Columns indicate means of relative expression to TBP housekeeping gene of at least 3 independent experiments; and bars, SD. *P < .05. Cocultured cell lysates were immunoblotted for HIF-2α (β-actin was used as loading control). Representative blots of 3 independent experiments.