Fig. 6

Depletion of macrophages using liposomes with clodronate abolishes the therapeutic effect induced by hADSCs. The immunofluorescence analyzes of blood vessels and macrophages 7 days after the artery ligation and hADSCs and clodronate liposomes administration (hADSCs+clodronate liposomes) and control group (hADSCs+PBS liposomes). The representative images of M2 macrophages (F4/80⁺ (red)/CD206⁺ (green)) (a, b, c) and blood vessels CD31⁺ (green) (d, e, f). The graph of the total area of F4/80 macrophages (g) and capillaries (CD31) (h) in collected muscles. The images and the graph show that liposomes containing clodronate effectively deplete F4/80⁺ and F4/80⁺CD206⁺ cells in mouse gastrocnemius muscles in vivo (c), (g). After the depletion of macrophages an increase in the number of blood vessels was not observed in muscles where hADSC were administrated (f), (h) (n = 10; 10 muscles per group were analyzed; 5 (for the total area of F4/80) or 10 (for the number of capillaries) pictures of each muscle were taken). *p < 0.05, ***p < 0.001 evaluated with Kruskal-Wallis one-way analysis of variance and multiple comparison of mean ranks for all groups. Nuclei stained with DAPI (blue), scale bars = 50 μm