Figure 6.

TUFM derived from hDPSC-apoVs activate EC autophagy and promote EC angiogenesis via the TFEB-induced autophagy-lysosome pathway

(A) Western blotting analysis shows the presence of TUFM in apoVs derived from hDPSCs, si-NC-treated hDPSCs, and si-TUFM-treated hDPSCs. (B) Western blot analysis shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after incubation with apoVs or si-TUFM-apoVs. The angiogenic capacity of ECs, including tube formation (C), migration (D), and proliferation (E) were detected after treatment with apoVs or si-TUFM-apoVs. Scale bar, 100 μm. (F) Representative confocal microscopy images show the number of lysosomes in ECs after treatment with apoVs or Torin 1. Scale bar, 50 μm. (G) Western blot analysis shows lysosome-associated gene (LAMP1 and CLCN7) and TFEB expressions in ECs after incubation with apoVs at concentrations of 10, 20, or 30 μg/mL. (H) Expression of TFEB in cytosolic (Cyt.) and nuclear (Nuc.) fractions was detected by western blots after treatment with apoVs or Torin 1. (I) Representative confocal microscopy images show the locations of TFEB in ECs after treatment with apoVs or Torin1. Scale bar, 25 μm. (J) Western blot analysis shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after decreased expression of TFEB. (K) Western blot analysis shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after increased expression of TFEB. After transfection with si-TFEB, the angiogenic capacity of ECs, including tube formation (L), migration (M), and proliferation (N) were detected following coculture with apoVs (20 μg/mL). Scale bar, 100 μm; n = 3-5 per group. Data are presented as mean ± SD. Statistical analyses were performed by Student’s t test (two-tailed) for two-group comparisons and one-way ANOVA with Tukey’s post hoc test or Welch’s ANOVA with Games-Howell post hoc test for multiple-group comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, p > 0.05.