Fig 1: Intergins a4/a9 are responsible for the action of osteopontin (OPN) in the augmentation of macrophage (Mf) M2 polarization and angiogenic capacity. (A) Relative mean fluorescence intensities (MFIs) of integrins a4/a9 were measured by FACS analysis in each Mf subset. Anti-integrin a4 or a9 antibodies (Abs) and each isotype-matched control Ab were used at 10 µg/mL, n = 4 [***p < 0.001 vs. untreated, ###p < 0.001 vs. interleukin (IL)-10 alone]. (B) Representative confocal laser scanning immunofluorescence overlay images of integrin a4 (green) and DAPI (blue), as well as those of integrin a9 (red) and DAPI (blue) in Mf (–) and Mf (IL-10 + IL-18). Scale bar represents 20 µm. (C) Relative MFI of CD163 was measured by FACS analysis in each Mf subset. Anti-integrin a4 or a9 Abs and each isotype-matched control Ab were used at 10 µg/mL, n = 4 (***p < 0.001 vs. untreated, ###p < 0.001 vs. IL-10 alone, †††p < 0.001 vs. IL-10 + IL-18). (D) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 was cocultured with each Mf subset. Anti-integrin a4 or a9 Abs and each isotype-matched control Ab were used at 10 µg/mL, n = 6 (***p < 0.001, **p < 0.01, *p < 0.05 vs. untreated, ###p < 0.001, ##p < 0.01, #p < 0.05 vs. IL-10 alone, †††p < 0.001 vs. IL-10 + IL-18). Integrin a4 = ITGA4; Integrin a9 = ITGA9. All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.