Ang-1 modulates the proangiogenic activity of rDrm. (A) Cell aggregates from naive VEGFR2-MAE cells (−) or from VEGFR2-MAE cells transfected with fluorescein-conjugated control siRNA (c) or 4 different Ang-1 siRNA (1-3, 3 distinct single oligonucleotides, [Qiagen, Valencia, CA]; 4, one pool of 3 different oligonucleotides, Santa Cruz Biotechnology) were embedded in fibrin gel. Then, 50 ng/mL of rDrm was added on the top of the gel in medium containing 10 μg/mL of aprotinin. In addition, some rDrm-treated aggregates were incubated in the presence of 1.0 μg/mL of neutralizing anti–Ang-1 antibodies or irrelevant IgGs. Formation of radially growing cell sprouts was observed during the next 48 hours.⁸  Sprouts were photographed at ×40 magnification with an IX51 inverted microscope equipped with a 4×/0.10 NA objective and a Camedia C-4040 digital camera (Olympus, Melville, NY). Sprouting was quantified by computerized analysis of the digitalized images.⁸  Data are expressed as means plus or minus SEM (n = 10). * Statistically different from rDrm-treated naive cells (P < .01, Student t test). Conditioned media from the corresponding transfectants were analyzed for Ang-1 protein content by Western blotting (inset). (B) HUVEC spheroids prepared in medium supplemented with carboxymethylcellulose were embedded in type I collagen gel and treated with vehicle or 30 ng/mL of rDrm in the absence or in the presence of 100 ng/mL sTie-2 (Relia-Tech, Woburn, MA). After 24 hours, cell sprouts were photographed at ×200 magnification using an Axiovert 200M microscope equipped with a 20× objective (LD A PLAN 20×/0.30PH1, Zeiss, Jena, Germany). Aggregates were fixed in 4% paraformaldehyde, permeabilized in TBS-0.2% Triton X100, and costained with anti–Ang-1 antibodies/antirabbit Alexa594 (Invitrogen) (in red) and counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) (in blue). Images were acquired using a LSM510Meta confocal microscope equipped with objective LD A PLAN 32×/0.40PH1 and processed using the LSM software (Zeiss). (C) One-millimeter human umbilical artery rings were embedded in fibrin gel and incubated with the indicated concentrations of rDrm in the absence or in the presence of 1.0 μg/mL of neutralizing anti-Ang-1 antibodies or 100 ng/mL of sTie-2. After 3 days, EC sprouts were counted under an IX51 inverted microscope at ×10 magnification. * Statistically different from rDrm-treated rings (P < .01, Student t test). (D) Alginate beads containing vehicle, 100 ng of rDrm, parental ([GRAPHIC]) or Drm-overexpressing-COS cells (10 000 cells/embryo) were implanted on the top of chick embryo CAMs at day 11 of development. When indicated, pellets contained also 1.0 μg of neutralizing anti–Ang-1 antibodies or 100 ng of sTie-2. After 72 hours, newly formed blood vessels converging toward the implant were counted in ovo at ×5 magnification using a STEMI SR stereomicroscope equipped with an objective f equal to 100 mm with adapter ring 475070 (Zeiss). Data are expressed as mean plus or minus SEM (n = 6-8); * Statistically different from the corresponding stimulus in the absence of any inhibitor (P < .01, Student t test).