Fig. 7.

Exosomes derived from melatonin-treated ECs alleviated vascular calcification and vascular aging in 5/6 NTP mice model. (A) Spectrum in vivo imaging results of mice injected with PBS, fluorescent dye DIR, and DIR-labeled exosome via tail vein, respectively (n = 6). (B) Spectrum imaging results of organs harvested from mice injected intravenously with PBS, and DIR-labeled exosome for 4h, 8h, 16h, 24h, respectively (n = 6). (C) Quantitation of relative florescent ROI between organs from Veh-ECs-Exos and MT-Exo infused mice at 0h, 8h, 16h, 24h (n = 6). (D) The thoracic aorta was obtained to analyze the uptake of endothelial exosomes in the slices after injection 24h. Blue fluorescence (DAPI)-labeled cell nuclei, green fluorescence (Alexa 488)-labeled α-SMA, and red fluorescence (Alexa 555)-labeled CD81 indicating exosomes (n = 6, scale bar = 50 μm). (E–H) Veh-ECs-Exos, MT-ECs-Exos, and MT-ECs-Exos transfected with miR-302d-5p inhibitor (MT-ECs-Exos + miR-302d-5p-I) were injected to 5/6 NTP mice. (E) Calcium content of thoracic aorta of mice was measured by o-cresolphthalein method. (F) The ALP activity level among four groups measured by ALP kit (n = 6). (G) thoracic aorta calcification was analyzed by Alizarin Red S staining (n = 6). (H) SA-β-gal-stained micrographs were presented to reveal aorta senescence (n = 6). (I) Immunohistochemistry analysis of Runx2, P21and Wnt3 in thoracic aorta(n = 5). Scare bar 50 μm (Red) and 500 μm (Blue). (J) Quantitation of middle OD of IHC. Results were represented by mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.