Figure 4: rLECT2 protein suppresses VEGF-induced angiogenic responses.

(a) Proliferation ratios for HUVECs seeded in a 96-well plate and treated with VEGF₁₆₅ (50 ng/mL) alone or combined with various concentrations of rLECT2 protein (1.25, 2.50, and 5.00 nM) as indicated for 24 and 48 h. Cell growth was measured using an MTT assay. (b) A confluent HUVEC monolayer was wounded with a blue pipette tip and then exposed to fresh M199 medium (control) or a medium containing VEGF₁₆₅ (50 ng/mL) with various concentrations of rLECT2 protein (0–5 nM) for 14 h. The width of the wound on the monolayer was measured to determine migration ability of HUVECs. Images of migration HUVECs were obtained and analyzed using the Image-Pro Plus software program (version 4.5). (c) HUVECs were seeded onto a Matrigel layer in a 24-well plate and treated with VEGF₁₆₅ (50 ng/mL) combined with various concentrations of rLECT2 protein as indicated for 6 h. Tube formation was determined by manual counting of the tubular structures in low-power fields (40×). (d) CAM blood vessel formation. CAMs of 9-day-old chicken embryos were incubated with VEGF₁₆₅ alone (50 ng/mL) or combined with various concentrations of rLECT2 protein as indicated for 1–4 days and then photographed. (e) A Matrigel mixture containing VEGF alone or combined with various concentrations of rLECT2 protein as indicated was injected subcutaneously into nude mice at sites lateral to the abdominal midline. Matrigel plugs were recovered from the mice and photographed immediately 10 days later. The hemoglobin absorbance was measured to determine hemoglobin levels in the plugs. The data are presented as the mean ± SD. Each treatment was performed in triplicate, and the assays were repeated at least three times. *P < 0.05; **P < 0.01.