Figure 5.

Exosomes from hypoxia-treated mouse lung ECs stimulate recruitment of αSMA-positive cells in the fibrin gel implanted on the mouse lungs. (A) IB analysis of CD63, Flotillin-1, and GM130 in exosomes collected from conditioned media of ECs isolated from tamoxifen-induced p16^fl/fl or p16^iΔEC mouse lungs treated with normoxia or hypoxia for 3 weeks. (B) Size distribution and particle concentration of isolated exosomes analyzed using NTA. (C) TEM image of exosome morphology. Scale bar: 150 nm. Arrows indicate exosomes. (D) List of top 20 BP GO terms of proteins differentially enriched in exosomes isolated from conditioned media of mouse lung ECs isolated from C57BL6 mouse lungs treated with normoxia or hypoxia for 3 weeks. (E) Graph showing EdU-positive PASMCs treated with exosomes (10 μg/ml) collected from conditioned media of ECs isolated from tamoxifen-induced p16^fl/fl or p16^iΔEC mouse lungs treated with normoxia or hypoxia for 3 weeks (n = 3, mean ± SEM, *p < 0.05). (F) Graph showing PASMCs migrating toward medium containing exosomes (10 μg/ml) collected from conditioned media of ECs isolated from tamoxifen-induced p16^fl/fl or p16^iΔEC mouse lungs treated with normoxia or hypoxia for 3 weeks (left, n = 6, mean ± SEM, *p < 0.05). Representative micrographs showing PASMCs migrating toward medium containing exosomes (10 μg/ml) collected from conditioned media of ECs isolated from tamoxifen-induced p16^fl/fl or p16^iΔEC mouse lungs treated with normoxia or hypoxia for 3 weeks (right, Wright Giemsa staining). Scale bar, 50 μm. (G) IF micrographs of αSMA expression and DAPI in the fibrin gel supplemented with exosomes collected from conditioned media of ECs isolated from tamoxifen-induced p16^fl/fl or p16^iΔEC mouse lungs treated with normoxia or hypoxia for 3 weeks and implanted on the p16^fl/fl mouse lung for 7 days. Scale bar, 50 μm. Graph showing integrated fluorescent density of αSMA (n = 6, mean ± SEM, *p < 0.05).