Fig 2: Ultrastructural analysis of cell–cell interaction between macrophages (Mfs) [interleukin (IL)-10 + IL-18] and endothelia. SEM images were obtained at 4 h after coculture of b.End5 with Mfs (IL-10 + IL-18) on Matrigel. Lower images are magnified regions from red rectangles in the corresponding upper panels. Magnification and scale bars: (A) upper, ×1.0 K, 50 µm; Lower, ×5.0 K, 10 µm; (B) upper, ×1.0 K, 50 µm; lower, ×2.0 K, 20 µm; (C) upper, ×1.0 K, 50 µm; lower, ×8.0 K, 5 µm; (D) upper, ×1.0 K, 50 µm; lower, ×3.0 K, 10 µm; (E) upper, ×1.0 K, 50 µm; lower, ×5.0 K, 10 µm; (F) upper, ×1.0 K, 50 µm; lower, ×4.0 K, 10 µm; (G) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (H) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (I) upper, ×1.0 K, 50 µm; lower, ×3.5 K, 10 µm; respectively. Black or white arrows indicate Mfs or endothelial cells, respectively. Red arrowheads indicate pseudopodia of Mfs interacting with endothelial cells.

Fig 3: Characteristic behavior of macrophages (Mfs) during angiogenesis. (A) Representative series of time-lapse images at 4 h intervals from 0 to 16 h extracted from Video S1 which shows live-cell imaging of Matrigel tube formation assay where endothelial cells (green) and Mfs [interleukin (IL)-10 + IL-18] (red) were cocultured. Scale bar represents 50 µm. (B) Higher magnification images of white rectangle region in panel (A) were reconstructed from 4 h 00 min to 7 h 00 min in Video S5 in Supplementary Material. The time elapsed after starting the movie is indicated in hours:minutes in the bottom left of each panel. White arrowheads highlight the characteristic behavior of Mf (IL-10 + IL-18) as well as the cell–cell interaction with endothelium in respective image. Scale bar represents 10 µm.

Fig 4: CD163 is a critical factor for determining the angiogenic capacity of macrophage (Mf). (A) Upper; representative confocal laser scanning immunofluorescence overlay images of CD163 (green) and DAPI (blue) in Mf (–) and Mf [interleukin (IL)-10 + IL-18]. Scale bar represents 20 µm. Lower; Three-dimensional images of each upper panel. Higher magnification image in the panel of Mf (IL-10 + IL-18) is from white rectangle region. Scale bar represents 10 µm. White arrowheads indicate CD163 highly expressed and localized at pseudopodia. (B) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 were cocultured with each Mf subset. An anti-CD163 antibody (Ab) and its isotype-matched control Ab were used at 4 µg/mL, n = 3 (***p < 0.001, *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). (C) Representative images (upper) and corresponding three-dimensional images (lower) of tube-like structures as well as (D) total areas and lengths of tubular structure where b.End5 (green) and Mfs (IL-10 + IL-18) (red) were cocultured on Matrigel for 16 h with an anti-CD163 Ab or its isotype-matched control Ab. Scale bar represents 100 µm, n = 8 (***p < 0.001 vs. untreated, ###p < 0.001 vs. IL-10 + IL-18). All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.

Fig 5: Thrombin contributes to macrophage (Mf) M2 polarization and angiogenic capacity through proteolytic modification for osteopontin (OPN). (A) The mRNA expression level of Prothrombin relative to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was analyzed by reverse transcription polymerase chain reaction in each Mf subset and were normalized to Mf (–), n = 7 [***p < 0.001, **p < 0.01 vs. untreated, ##p < 0.01 vs. interleukin (IL)-10 alone]. (B,C) The protein expression levels of (B) thrombin or (C) OPN N-Half relative to GAPDH were measured by western blotting in each Mf subset and were normalized to Mf (–). Lower panels are typical images of each protein. (B) n = 8 (***p < 0.001, *p < 0.05 vs. untreated, #p < 0.05 vs. IL-10 alone). (C) n = 16 (***p < 0.001, *p < 0.05 vs. untreated, ##p < 0.01, #p < 0.05 vs. IL-10 alone, †††p < 0.001 vs. IL-10 + IL-18). (D) Representative confocal laser scanning immunofluorescence images of OPN (red), thrombin (green), and their merge with DAPI (blue) in each Mf subset. Scale bar represents 20 µm. Higher magnification images are from the white rectangle region in merged panel of Mf (IL-10 + IL-18). Scale bar represents 10 µm. (E) Relative mean fluorescence intensity (MFI) of CD163 was measured by FACS analysis in each Mf subset. Hirudin, a specific thrombin inhibitor, was used at 1 µg/mL, n = 3 (***p < 0.001 vs. untreated, ###p < 0.001 vs. IL-10 alone, †††p < 0.001 vs. IL-10 + IL-18). (F) The total areas and lengths of tube-like structures were determined by the Matrigel tube formation assay where b.End5 were cocultured with each Mf subset. Hirudin was used at 1 µg/mL, n = 6 (***p < 0.001, **p < 0.01 vs. untreated, ##p < 0.01, #p < 0.05 vs. IL-10 alone, †††p < 0.001 vs. IL-10 + IL-18). All data are expressed as means ± SEM and were analyzed by a one-way ANOVA followed by Tukey’s test.