Fig. 4: Hypoxic TEC-derived sEV facilitate endothelial cell proliferation via VEGF-A signaling in vitro.

a Size distribution of sEV derived from HK-2 under normoxia (Ctrl-sEV) or hypoxia (Hypo-sEV). Representative TEM image of Hypo-sEV. b Quantification of the particle concentrations of Ctrl-sEV and Hypo-sEV (n = 3). c Western blotting analysis of sEV markers (Alix, CD63, and CD9) and VEGF-A in Ctrl-sEV and Hypo-sEV (n = 3). d Effects of purified Ctrl-sEV or Hypo-sEV (15 μg/ml) on the proliferation of HUVECs (n = 3). e Effects of the culture medium from hypoxic HK-2 cells with or without Hypo-sEV on the proliferation of HUVECs (n = 8). f HK-2 cells were transfected with siVEGF-A and were then exposed to hypoxia for 24 h. The conditioned Hypo-sEV with VEGF-A knockdown were purified for the analysis of their proliferative effects on HUVECs (n = 5). g A transwell culture system was employed to observe the sEV-mediated communication between HK-2 cells (upper chamber, DIO-stained) and HUVECs (lower chamber). Representative confocal image showing the uptake of DIO-labeled vesicles by HUVECs. Scale bar, 20 μm. h Flow cytometry analysis of the internalization of DIO-labeled Hypo-sEV by HUVECs with or without VEGFR1/VEGFR2 downregulation (n = 3). i Effects of VEGFR1/VEGFR2 knockdown of the recipient cells on the Hypo-sEV-mediated proliferation of HUVECs (n = 4 or 5). Data are presented as means ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. Ctrl-sEV (b–d), Hypo-CM (e), NC-Hypo-sEV (f) or NC (h, i); Two-tailed Student’s t test (b–f), One-way ANOVA (h, i).