M1 macrophage-derived extracellular vesicle containing tsRNA-5006c promotes osteogenic differentiation of aortic valve interstitial cells through regulating mitophagy

Figures

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  Figure 1. Identification of EV-derived from M1 macrophages. (A) Representative image of the immunofluorescence of CD86 in RAW 264.7 macrophage induced by LPS/IFN-γ (RAW-M1 group) or PBS (RAW-NC group). Scale bar: 50 µm. (B) The expression of TNF-α, IL-1β, and iNOS of M1 polarization markers was detected by RT-qPCR. (C) The concentration of TNF- α, IL-1β, and IL-6 of M1 polarization markers was detected by ELISA assays. (D) The concentration of IL-10, IL-12, and IL-13 of M1 polarization markers was detected by ELISA assays. (E) Representative image of EVs of TEM. EVs isolated from RAW 264.7 macrophage induced by LPS/IFN-γ (M1-EVs group) or PBS (NC-EVs group). (F) Surface marker of CD9 of EVs was detected by western blot. * P < 0.05, ** P < 0.01.

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  Figure 2. M1-EVs promote osteogenic differentiation of AVICs. (A) Representative image of DiI-labeled M1-EVs was internalized by AVICs. Scale bar: 50 µm. (B) Representative image of Alizarin red staining of AVICs pretreated with M1-EVs or NC- EVs. Scale bar: 50 µm. (C) The mRNA expression of RUNX2, BMP2, and SPP1 of osteogenesis-related genes in AVICs after incubated with M1-EVs was detected by RT-qPCR. (D) The protein expression of RUNX2, BMP2, and osteopontin of osteogenesis-related genes in AVICs after incubated with M1-EVs was detected by western blot. (E) The protein expression of α-SMA and Collagen I of fibrotic markers in AVICs after incubated with M1-EVs was detected by western blot. ns P > 0.05, * P < 0.05, ** P < 0.01.

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  Figure 3. tsRNA profile alters in M1-EVs. Volcano plots (A) and heat map (B) are presenting the differentially expressed tsRNAs (DEtsRNAs). Red are up-regulated and green are down-regulated DEtsRNAs in the M1-EVs group compared with the NC- EVs group, respectively. (C) Top 20 of GO enrichment DEtsRNAs. (D) Top 20 of KEGG enrichment DEtsRNAs.

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  Figure 4. tsRNA-5006c may be responsible for the M1-EVs effects. (A) RT-qPCR was used to validate RNA sequencing results on the five DEtsRNAs. (B) The regulatory network of tsRNA-5006c _target genes_pathway. (C) The interference efficiency of tsRNA-5006c in M1 macrophages was detected by RT-qPCR. (D) The interference efficiency of tsRNA-5006c in M1-EVs was detected by RT-qPCR. ns P > 0.05, ** P < 0.01, *** P < 0.001.

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  Figure 5. M1-EVs tsRNA-5006c inhibitor suppress osteogenic differentiation of AVICs. (A) Representative image of Alizarin red staining of AVICs pretreated with M1-EVs after tsRNA-5006c silencing. Scale bar: 50 µm. (B) The mRNA expression of RUNX2, BMP2, and SPP1 of osteogenesis-related genes in AVICs pretreated with M1-EVs after tsRNA-5006c silencing was detected by RT-qPCR. (C) The protein expression of RUNX2, BMP2, and osteopontin of osteogenesis-related genes in AVICs pretreated with M1-EVs after tsRNA-5006c silencing was detected by and western blot. (D) The protein expression of α-SMA and Collagen I of fibrotic markers in AVICs-pretreated with M1-EVs after tsRNA-5006c silencing was detected by western blot. NC is the negative control of tsRNA-5006c inhibitor. * P < 0.05, ** P < 0.01.

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  Figure 6. M1-EVs tsRNA-5006c enhance osteogenic differentiation of AVICs through mitophagy. (A) Co-location of Mito-Tracker Green and Lyso-Tracker Red imaging in AVICs to indicate mitophagy puncta. Scale bar: 50 µm. (B) MMP of AVICs was measured by flow cytometry using TMRM staining. (C) The expression of LC3-II, BINP3, and PGC1 α of hallmarks of mitophagy activation was detected by western blot. NC is the negative control of tsRNA-5006c inhibitor. ns P > 0.05, * P < 0.05, *** P < 0.001.

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  Figure 7. Simulation diagram. EVs is the medium of communication between M1-polarized macrophage and AVICs, and M1-EVs triggered osteogenic differentiation through delivering tsRNA-5006c to enhance mitophagy of AVICs.