miRNA-15a and -16 target endothelial cells in vitro and in vivo. (A) VEGF concentrations were measured in triplicate, by ELISA, in conditioned media obtained from MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S). (B) HUVECs were cultured in presence or absence of MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S). Nontransfected MM.1S cells were used as control (ctrl). Whole cell lysates were subjected to Western blotting using anti–phospho(p)-AKT, -AKT, p-ERK, -ERK, and -actin antibodies. (C) MMECs were seeded on top of ECM-matrix, in presence of CM obtained from either control probe–, pre-miRNA-15a, or -16-1–transfected MM.1S cells. Serum-free medium (SFM) was used as control. Tube formation was assessed using an inverted light microscope. Photographs are representative of 3 independent experiments. (D) Angiogenic responses induced by gelatin sponges loaded with CM obtained from either control probe–, pre-miRNA-15a, or -16-1–transfected MM.1S cells, in the in vivo CAM model. SFM and VEGF (10 ng/mL) were used as negative and positive control, respectively. Original magnification: ×50. Vessel counts at the sponge-CAM boundary are indicated below each image.