VEGFR2 activation mediates the proangiogenic activity of gremlin in vitro and in vivo. Serum-starved HUVECs were stimulated for 0-30 minutes with 50 ng/mL gremlin (A) or 30 ng/mL VEGF-A (B) or with 50 ng/mL gremlin or 30 ng/mL VEGF-A for 15 minutes in the absence or in the presence of 5.0μM SU5416 (C). At the end of incubation, 50 μg of cell extracts were probed by Western blotting with a monoclonal anti–phospho-VEGFR2 antibody (pY1175; Cell Signaling Technology). Uniform loading of gels was confirmed by incubation of the membranes with anti–focal adhesion kinase (FAK) antibodies (Santa Cruz Biotechnology). Capture ELISA assay ruled out a significant VEGF-A contamination of the recombinant gremlin preparations (≤ 0.1 pg of VEGF-A per nanogram of protein). Accordingly, gremlin-induced VEGFR2 autophosphorylation in ECs was not affected by coincubation with a neutralizing anti–VEGF-A antibody (data not shown). (D) Serum-starved HUVECs were stimulated for 0 or 5 minutes with 50 ng/mL gremlin or 30 ng/mL VEGF-A and immunostained with rabbit monoclonal anti–phospho-VEGFR2 antibody (pY1175) followed by Alexa Fluor 488 anti–rabbit IgG (Invitrogen). Samples were analyzed using a Zeiss LSM510Meta confocal microscope equipped with Plan-Apochromat 63×/1.4 numerical aperture oil objective and LSM 510 Meta Software Version 3.5 (Carl Zeiss). Note the presence of the phosphorylated receptor both on HUVEC membrane and in submembrane vesicles in stimulated cells. (E) HUVEC spheroids prepared in medium supplemented with carboxymethylcellulose were embedded in type I collagen gel and treated with vehicle or 30 ng/mL gremlin or VEGF-A in the absence or in the presence of 3.0μM cyclo-VEGI. Formation of radially growing cell sprouts was observed during the next 48 hours.3 Sprouts were photographed at 40× magnification with an IX51 inverted microscope equipped with a 4×/0.10 numerical aperture objective and a Camedia C-4040 digital camera (Olympus). Data are expressed as total number of sprouts measured in 50 spheroids. *Statistically different from the stimulus in the absence of any inhibitor (P < .01, Student t test). (F) One-millimeter human umbilical artery rings were embedded in fibrin gel and incubated with 50 ng/mL gremlin, 30 ng/mL VEGF-A, or vehicle alone in absence or in the presence of 5.0μM SU5416 or 20nM VEGFR2 kinase inhibitor I (VEGFR2I). After 3 days, EC sprouts, morphologically distinguishable from scattering fibroblasts/smooth muscle cells, were counted at 100× magnification with an IX51 inverted microscope equipped with a Plan A chromatic phase contrast 10×/0.25PhP numerical aperture objective (Olympus). *Statistically different from gremlin or VEGF-A–treated rings (P < .01, Student t test). (G) Alginate beads containing vehicle, 100 ng of gremlin, or VEGF-A were implanted on the top of chick embryo CAMs at day 11 of development. When indicated, pellets also contained 150 ng of sVEGFR2 or 5.0μM SU5416. 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 (Carl Zeiss). Data are expressed as mean ± SEM (n = 6-8). *Statistically different from the stimulus in the absence of any inhibitor (P < .01, Student t test).
