EMMPRIN up-regulates VEGF₁₂₁, VEGF₁₆₅, and VEGFR-2, but not VEGF₁₈₉ and VEGFR-1 expression in endothelial cells. HMEC-1 cells were incubated with 20 μg/mL Emp in serum-free medium. (A) After 24 and 48 hours, CM were collected and soluble VEGF production was measured by ELISA. Results were normalized to the cell number and expressed as pg/10⁶ cells. Columns indicate means of 3 independent experiments; and bars, SD. *P < .05. (B) Total RNA was extracted from HMEC-1 cells treated with Emp or with 10 ng/mL bFGF for 4 hours. mRNA levels for VEGF₁₂₁, VEGF_165, VEGF₁₈₉, VEGFR-2, and VEGFR-1 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. (C) Cell lysates were immunoblotted for VEGF isoforms and VEGFR-2 (β-actin was used as loading control). Concentrated CM were immunoblotted with anti-VEGF antibody. Band densities were expressed relative to total protein concentration of the corresponding cell lysates. Representative blots of 3 independent experiments. (D) HMEC-1 and HUVECs cocultured with EMMPRIN- or mock-transfected CHO cells. Total RNA was extracted from cocultured cells. mRNA levels for VEGF₁₂₁, VEGF₁₆₅, VEGFR-2, and VEGFR-1 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 VEGF isoforms and VEGFR-2 (β-actin was used as loading control). Representative blots of 3 independent experiments. (E) EMMPRIN regulates mouse VEGFR-2 in xenograph tumors in vivo. Tumors grown in nude mice obtained from EMMPRIN- or mock-transfected mammary tumor sections (NS2T2A) were analyzed for mouse VEGFR-2 expression by both qRT-PCR and immunohistochemistry. Mouse VEGFR-2 transcripts were quantified using qRT-PCR. Columns indicate means of relative expression to mouse TBP housekeeping gene of at least 3 independent experiments. Bars indicate SD. *P < .05. Mouse flk-1/VEGFR-2 protein was analyzed by immunohistochemistry.