The mobility of LECs was analysed in 24-well plates (Corning) using Cell Culture Inserts (Corning) with a 8-µm pore size. The interior of the Cell Culture Inserts was coated with collagen I ([Cellmatrix Type I-C]; Nitta Gelatin, Osaka, Japan) following the manufacturer’s protocol. LECs were cultured in ECGM-MV2 with 5% FBS, and after 6 h of serum-free starvation, 1 × 105 cells suspended in 200 µl of ECBM/0.5% FBS were seeded onto upper chambers. Lower chambers were filled with 700 µl of ECBM/0.5% FBS with or without growth factors. In the control condition, only ECBM/0.5% FBS was placed in lower chambers. In other groups, VEGF-A (10 ng/mL), -C (50 ng/mL) or -D (100 ng/mL) were added to ECBM/0.5% FBS as a chemoattractant. Cells were also dissociated by 0.05% trypsin/EDTA (Thermo Fisher Scientific), incubated with MAZ51 (10 μM) or a monoclonal anti-human integrin α9-neutralizing antibody (50 μg/mL), and seeded in the upper chamber.

After 4 h, the non-migrating cells on the surface of the upper chamber were removed with cotton swabs. The migrating cells at the bottom of the membrane were fixed using a Diff-Quik Stain Kit (Sysmex Corporation, Kobe, Japan) according to the manufacturer’s protocol. The number of migrated cells were counted on 4 randomly selected images under a bright-field light microscope. Data were expressed as means ± SD.

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