Fig. 4.

The effects of exosomes released from RPE cells on the angiogenic behaviours of HUVECs. (A) HUVECs were plated in the upper chambers, and the lower chambers contained medium supplemented with 1% FCS with or without exosomes. Exosomes were released from unstimulated (Ex-CTL), TNF-α-stimulated (Ex-TNF), TGF-β2-stimulated (Ex-TGF), and co-stimulated (Ex-CO) ARPE-19 cells. Chemotactic cells were fixed and stained with crystal violet and DAPI. Scale bar, 500 μm. The cell number was counted by nuclei staining; the graph shows the number of translocated cells per field. The data are presented as the means ± SD in four different fields in each of three independent experiments. ***P < 0.001 (Dunnett's test). (B) HUVECs were incubated in medium supplemented with 1% FCS containing each type of exosome for 24 h and then in the additional presence of 10 μM BrdU for the last 10 h. Representative fluorescence microscopy images of cells stained with an anti-BrdU antibody (green) and of nuclei stained with DAPI. Scale bar, 100 μm. The number of BrdU-positive cells and the total number of nuclei were counted. The data are presented as the means ± SD in three different fields. ***P < 0.001 (n = 3, Dunnett's test). (C) Representative images showing tube formation in HUVECs cultured on growth factor-reduced Matrigel with or without each type of exosome released from ARPE-19 cells. We used angiogenic growth factor-containing medium (GM) as the positive control. Graphs showing the quantification of the total length of the tubes and number of branching points. Each bar indicates the mean ± SD (n = 5, *P < 0.05, **P < 0.01, Dunnett's test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)