HDAC inhibitors tune miRNAs in Extracellular Vesicles of Dystrophic Muscle-Resident Mesenchymal Cells (2020)
Figure 4

Fig. 4: FAPs derived EVs promote regeneration and inhibit fibrosis and inflammation in dystrophic muscles of mdx mice.

(A) Flow cytometry analysis of mdx muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA. The left panel shows the percentage of PKH-67 positive cells in whole muscle. The right panel shows the percentage of the PKH-67 positive MuSCs.

(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm.

(C) Graph showing the relative expression of miR-206-3p in MuSCs isolated as described in (A and B) and in MuSCs from muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA, and then transfected or not with Drosha siRNA (siRNA Drosha) (n=4). Star (*) means significance relative to MuSCs that not uptake PKH67-labled EVs, **p < 0.01. Hash (#) means significance compared to MuSCs that uptake EVs-FAPs TSA, ##p < 0.01. § indicates Anova analysis, §§p<0,01.

(D) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05, **p<0.01

(E) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs-derived EVs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05.

(F-M) Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (F) Representative images of immunofluorescence for embryonic myosin heavy chain (eMyHC-red) and laminin (Lam-cyan) stainings. Scale bar = 50 μm. (G) Graph showing the quantification of cross-sectional area (CSA). (H) Graph showing the quantification of muscle regeneration (eMyHC). (I) Representative images of Masson's Trichrome staining. Scale bar = 50 μm. (J) Graph showing the quantifications of muscle fiber area fraction (MFAF). (K) Graph showing the quantifications of fibrotic area. (L) Representative images of myeloperoxidase staining (MPO-red). Scale bar = 50 μm. (M) Graph showing the quantifications of inflammation (MPO).

Data information: Nuclei were counterstained with DAPI (blue). Statistical analysis by Anova and Tukey test. All data correspond to the average ± SEM. In (A-H) star (*) means significance compared to CTR, *p<0,05, **p< 0.01; ***p<0,001; ***p<0,0001; while hash (#) means significance compared to EVs-FAPs CTR -, #p<0,05, ##p<0,01, and (†) means significance to EV-FAPs TSA -, † p<0,05, ††p< 0.01, †††† p<0,0001 . § indicates significance by Anova test; §§§ p<0,001; §§§§ p<0,0001