Macrophage-Derived miRNA-Containing Exosomes Induce Peritendinous Fibrosis after Tendon Injury through the miR-21-5p/Smad7 Pathway

Abstract

Following tendon injury, the development of fibrotic healing response impairs tendon function and restricts tendon motion. Peritendinous tissue fibrosis poses a major clinical problem in hand surgery. Communication between macrophages and tendon cells has a critical role in regulating the tendon-healing process. Yet, the mechanisms employed by macrophages to control peritendinous fibrosis are not fully understood. Here we analyze the role of macrophages in tendon adhesion in mice by pharmacologically depleting them. Such macrophage-depleted mice have less peritendinous fibrosis formation around the injured tendon compared with wild-type littermates. Macrophage-depleted mice restart fibrotic tendon healing by treatment with bone marrow macrophage-derived exosomes. We show that bone marrow macrophages secrete exosomal miR-21-5p that directly targets Smad7, leading to the activation of fibrogenesis in tendon cells. These results demonstrate that intercellular crosstalk between bone marrow macrophages and tendon cells is mediated by macrophage-derived miR-21-5p-containing exosomes that control the fibrotic healing response, providing potential targets for the prevention and treatment of tendon adhesion.

Keywords: Smad7; bone marrow macrophage; exosome; miR-21-5p; peritendinous adhesion.

Graphical abstract

Figure 1

Depletion of Macrophages Reduces Tendon Adhesion In Vivo (A) Experimental scheme showing the application of Clo- or PBS-containing liposomes in mice with TI or SO. (B) Macroscopic images of FDL tendons and adhesion grading score (n = 5 per group). (C) Maximal tensile strength and MTP joint flexion ROM (n = 5 per group). (D) Immunostaining of macrophages (F4/80⁺ cells, green) in each group; cell nuclei were stained with DAPI (blue). T, tendon; M, muscle. White arrows indicate F4/80-positive cells. Dotted lines indicate the border of FDL tendon or surrounding tissue. Scale bars, 50 μm. (E and F) Histology of adhesion. T, tendon; B, bone; M, muscle. Arrows indicate tendon adhesion in (E) H&E and (F) Masson staining. Dotted lines indicate the border of FDL tendon or surrounding tissue. Scale bars, 50 μm. (G) Histological adhesion score and histological healing score (n = 5 per group). (H) Western blot analysis and quantification of the levels of COL I, COL III, α-SMA, and TGF-β1 expression (n = 3 per group). *p < 0.05 and **p < 0.01.

Figure 2

Characteristics of BMDM-Derived Exosomes and Internalization (A) Representative electron microscopy pictures for exosomes secreted by BMDM. Scale bar, 100 nm. (B) Size of exosomes was measured by NTA. (C) Western blotting analysis of exosome markers CD9, TSG101, CD63, ALIX and macrophage marker CD68 (n = 3 per group). (D) Exosome production by macrophages. The data are shown as the mean ± SD (n = 5 per group). (E) BMDMs transfected with a Cy3-labeled miR-223 mimic were co-cultured with tenocytes or fibroblasts in a transwell, and the expression levels of miR-223 in tenocytes or fibroblasts are shown (n = 5 per group). Scale bars, 20 μm. (F) Representative immunofluorescence images showing the internalization of PKH67-labeled BMDM-derived exosomes (green) by fibroblasts or tenocytes stained with phalloidine (red) at 6, 12, and 24 hr. Cell nuclei were stained with DAPI (blue). White arrows indicate exosomes (green). Scale bars, 100 μm. *p < 0.05 and **p < 0.01.

Figure 3

BMDM-Derived Exosomes Promote Proliferation, Migration, and Fibrotic Activity of Fibroblasts and Tenocytes (A) Proliferation of fibroblasts treated with or without BMDM exosomes (n = 5 per group). (B) Migration and quantification of migrating fibroblasts (n = 5 per group). Scale bars, 100 μm. (C) mRNA expression of COL I, COL III, α-SMA, and TGF-β1 in fibroblasts (n = 3 per group). (D) Representative immunofluorescence images and quantification of COL I, COL III, α-SMA, and TGF-β1 protein expression in fibroblasts (n = 8 per group). Scale bars, 50 μm. (E) Proliferation of tenocytes treated with or without BMDM exosomes (n = 5 per group). (F) Migration and quantification of migrating tenocytes (n = 5 per group). Scale bars, 100 μm. (G) mRNA expression of COL I, COL III, α-SMA, and TGF-β1 in tenocytes (n = 5 per group). (H) Representative immunofluorescence images and quantification of COL I, COL III, α-SMA, and TGF-β1 protein expression in tenocytes (n = 8 per group). Scale bars, 50 μm. *p < 0.05 and **p < 0.01.

Figure 4

Distribution of BMDM-Derived Exosomes in Tissues of Mice with Tendon Injury (A) Representative immunofluorescence images of PKH67-labeled exosomes (arrows) in the liver, muscle, spleen, and kidney of recipient mice 1 day after intravenous injection of BMDM exosome. (B) Representative immunofluorescence images of PKH67-labeled exosomes (arrows) in the TI side and SO sides of recipient mice 1 and 3 days after BMDM-exosome administration. (C) Quantitative fluorescence of exosome distribution at 1 day after administration (n = 8 per group). *p < 0.05 and **p < 0.01.

Figure 5

BMDM-Derived Exosomes Induce Peritendinous Fibrosis In Vivo (A) Experimental design scheme showing treatment of macrophage-depleted mice with BMDM exosomes or empty liposomes (control). (B) Macroscopic images of tendons after the treatment with control liposomes or BMDM exosomes and adhesion grading score (n = 5 per group). (C) Maximal tensile strength and MTP joint flexion ROM (n = 5 per group). (D and E) Histology of adhesion. T, tendon; M, muscle; B, bone. Arrows indicate tendon adhesion in (D) H&E and (E) Masson staining. Dotted lines indicate the border of FDL tendon or surrounding tissue. Scale bars, 100 μm. (F) Histological adhesion score and histological healing score (n = 5 per group). (G) Western blot analysis and quantification of the levels of COL I, COL III, α-SMA, and TGF-β1 expression (n = 3 per group). *p < 0.05 and **p < 0.01.

Figure 6

miR-21-5p Is a Predominant miRNA Component in BMDM Exosomes (A) Heatmap diagram of miRNA content in exosomal cargo, indicating high abundance of miR-21-5p in BMDM exosomes (n = 3 per group). Red, high expression; green, low expression. (B and C) Fibroblasts (B) and tenocytes (C) were treated with BMDM exosomes for 24 hr and analyzed for miR-21a-5p levels by real-time qPCR (n = 5 per group). (D) Representative fluorescence in situ hybridization (FISH) images and fluorescence quantitation of miR-21-5p expression in tendon of wild type mice (n = 5 per group). Scale bars, 100 μm. (E) Representative FISH images and fluorescence quantification of miR-21 expression in tendon of macrophage-depleted mice (n = 5 per group). Scale bars, 100 μm. *p < 0.05 and **p < 0.01.

Figure 7

miR-21-5p Promotes Proliferation, Migration, and Pro-fibrotic Activities of Fibroblasts and Tenocytes In Vitro (A and B) Representative immunofluorescence images, transfection efficacy, and qPCR of (A) fibroblasts and (B) tenocytes transfected with Cy3-labeled miR-21-5p mimic or miRNA-mimic NC for 48 hr (n = 5 per group). Scale bars, 100 μm. (C and D) Proliferation of (C) fibroblasts and (D) tenocytes (n = 5 per group). (E and F) Migration and quantification of migrating (E) fibroblasts and (F) tenocytes (n = 5 per group). Scale bars, 100 μm. (G and H) mRNA expression of COL I, COL III, α-SMA, and TGF-β1 in (G) fibroblasts and (H) tenocytes (n = 5 per group). (I and J) Representative immunofluorescence images and quantification of COL I, COL III, α-SMA, and TGF-β1 protein expression in (I) fibroblasts and (J) tenocytes (n = 8 per group). Scale bars, 50 μm. *p < 0.05 and **p < 0.01.

Figure 8

miR-21-5p Promotes Proliferation, Migration, and Pro-fibrotic Activities of Fibroblasts and Tenocytes by Targeting Smad7 (A and B) mRNA expression of Smad7 in (A) fibroblasts and (B) tenocytes transfected with miRNA-mimic NC or miR-21-5p mimic (n = 5 per group). (C and D) Western blot analysis and quantification of the levels of Smad7 expression in (C) fibroblasts and (D) tenocytes transfected with miRNA-mimic NC or miR-21-5p mimic (n = 3 per group). (E and F) Proliferation of (E) fibroblasts and (F) tenocytes transfected with miRNA-mimic NC, miR-21-5p mimic, or miR-21-5p mimic + LV-Smad7, respectively (n = 5 per group). (G and H) Migration and quantification of migrating (G) fibroblasts and (H) tenocytes (n = 5 per group). Scale bars, 100 μm. (I) Western blot analysis and quantification of the levels of COL I, COL III, α-SMA, TGF-β1, and Smad7 expression (n = 3 per group). (J) Proposed schematic diagram of BMDM-derived exosomal miR-21-5p mediating peritendinous fibrosis after tendon injury. *p < 0.05 and **p < 0.01.