Exosomal miR-125b-5p derived from adipose-derived mesenchymal stem cells enhance diabetic hindlimb ischemia repair via targeting alkaline ceramidase 2

Abstract

Introduction: Ischemic diseases caused by diabetes continue to pose a major health challenge and effective treatments are in high demand. Mesenchymal stem cells (MSCs) derived exosomes have aroused broad attention as a cell-free treatment for ischemic diseases. However, the efficacy of exosomes from adipose-derived mesenchymal stem cells (ADSC-Exos) in treating diabetic lower limb ischemic injury remains unclear.

Methods: Exosomes were isolated from ADSCs culture supernatants by differential ultracentrifugation and their effect on C2C12 cells and HUVECs was assessed by EdU, Transwell, and in vitro tube formation assays separately. The recovery of limb function after ADSC-Exos treatment was evaluated by Laser-Doppler perfusion imaging, limb function score, and histological analysis. Subsequently, miRNA sequencing and rescue experiments were performed to figure out the responsible miRNA for the protective role of ADSC-Exos on diabetic hindlimb ischemic injury. Finally, the direct target of miRNA in C2C12 cells was confirmed by bioinformatic analysis and dual-luciferase report gene assay.

Results: ADSC-Exos have the potential to promote proliferation and migration of C2C12 cells and to promote HUVECs angiogenesis. In vivo experiments have shown that ADSC-Exos can protect ischemic skeletal muscle, promote the repair of muscle injury, and accelerate vascular regeneration. Combined with bioinformatics analysis, miR-125b-5p may be a key molecule in this process. Transfer of miR-125b-5p into C2C12 cells was able to promote cell proliferation and migration by suppressing ACER2 overexpression.

Conclusion: The findings revealed that miR-125b-5p derived from ADSC-Exos may play a critical role in ischemic muscle reparation by targeting ACER2. In conclusion, our study may provide new insights into the potential of ADSC-Exos as a treatment option for diabetic lower limb ischemia.

Keywords: Adipose-derived mesenchymal stem cells; Alkaline ceramidase 2; Bioinformatics analysis; Exosomes; Hindlimb ischemia; miR-125b-5p.

Fig. 1

Identification of ADSCs and ADSC-Exos. a Flow cytometry analysis showed that ADSC were highly positive for CD73, CD90 and CD44, but negative for CD34 and HLA-DR. b The morphology of ADSC (Scar bar, 100 μm). c, d The typical phenotypes of osteocytes (stained with Alizarin Red) and adipocytes (stained with Oil Red O) (Scar bar, 200 μm). e Exosomes isolated from ADSC culture media were observed by electron microscope (Scale bar: 100 nm). f Measurement of ADSC-Exos population using nanoparticle tracking analysis (NTA) demonstrated a single-peaked pattern. g Surface markers of ADSC-Exos were confirmed by western blotting (CD9, CD63, and CD81)

Fig. 2

The promotion effect of ADSC-Exos. a Confocal images of C2C12 cells incubated with 20 μg/mL PKH26-labeled ADSC-Exos (red) for 24 h (Scale bar: 50 μm). b Representative micrographs show that C2C12 cells were stained with Hoechst (blue) and EdU (green) (Scale bar: 50 μm). c Qualification analysis of the proliferation rate. The data were collected as the six randomly chosen fields from three independent experiments (n = 5). d Images of migrated C2C12 cells (Scale bar: 50 μm). e Quantitative analysis of the migration rate of C2C12 cells. The data are expressed as six randomly chosen fields from three independent experiments (n = 5). f HUVECs tube formation in a Matrigel assay (Scale bar, 100 μm). g Qualification of the closed tubular structure shown in f (n = 3). h Protein levels in C2C12 cells treated with 20 μg/mL ADSC-Exos. *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 3

ADSC-Exos accelerated diabetic hindlimb ischemia (HLI) repair. a Flow chart for diabetic HLI model construction. b Representative image of blood perfusion in ischemic limbs. c Quantification of blood reperfusion ratio in four groups (n = 5, **p < 0.01, ***p < 0.001, ###p < 0.001 versus PBS). d Quantification of limb motor score (n = 5, *p < 0.05, #p < 0.05 versus PBS). e Quantification of limb salvage score (n = 5). f Schematic of ADSC-Exos therapy on diabetic HLI

Fig. 4

ADSC-Exos prevented the inflammatory response and enhanced neovascularization in vivo. a Representative HE of gastrocnemius muscles derived from the ischemic hindlimb (Scale bar: 20 µm, red arrows point to inflammatory infiltration). b Quantification of the number of centronuclear myofibers within the ischemic muscle at days 14 and 21 (n = 5). c Representative Masson staining of gastrocnemius muscles derived from ischemic hindlimb (Scale bar: 100 µm). d Quantification of collagen deposition at days 14 and 21 after ischemic injury (n = 5). e Immunofluorescent staining of gastrocnemius muscles given the different treatments at day 14 post-ischemia. Smooth muscle cells (a-SMA), endothelial cells (CD34), and cell nuclei (DAPI) were stained with red, green, and blue colors (Scale bar, 100 μm). f Quantification of newly formed vessels stained with green color (n = 5). g Quantification of mature vessels stained with red colors (n = 5, scale bar, 100 μm). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

miR-125b derived from ADSC-Exos could be delivered to C2C12 cells. a The miR-125b-5p expression in C2C12 cells treated with ADSC-Exos. b The miRNA expression in C2C12 cells treated with ADSC-Exos under hypoxic conditions. c The miR-125b-5p expression level in C2C12 cells under hypoxic conditions. d, e EdU assay of C2C12 cells and qualification analysis of the proliferation rate (n = 5, scale bar: 50 μm). *p < 0.05, ***p < 0.001

Fig. 6

miR-125b regulated the proliferation and migration of C2C12 cells. a The miR-125b-5p expression in C2C12 cells treated with miR-125b-5p mimic/ inhibitor. b Western blotting analysis of AMPK and bcl-2 protein expression in C2C12 cells in four groups. c EdU assay of the proliferation rate of C2C12 cells in each group (Scale bar: 50 μm). d Qualification analysis of the proliferation rate (n = 5). e Images of migrated cells in each group (n = 5, scale bar: 50 μm). f Quantitative analysis of the migration rate of C2C12 cells. g Images depicted at 24 h after scratch (Scale bar: 50 μm). h Qualification analysis of the wound healing rate (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

miR-125b-5p directly regulated the expression of ACER2 in C2C12 cells. a Venn diagram representing the number of predicted targets of miR-125b-5p. b Schematic of the putative binding sites or mutations of miR-125b-5p in ACER2 mRNA 3’UTR. c Luciferase reporter assay determined ACER2 as the target of miR-125b-5p (n = 3). d The mRNA expression of ACER2 in each group (n = 4). e, f Western blotting analysis of ACER2 protein expression in C2C12 cells. **p < 0.01, ***p < 0.001

Fig. 8

miR-125b-5p/ ACER2 axis regulated C2C12 cell function. a EdU assay of the proliferation rate of C2C12 cells in each group (Scale bar: 50 μm). b Images depicted at 24 h after scratch (Scale bar: 50 μm). c Qualification analysis of the proliferation rate (n = 5). d Qualification analysis of the wound healing rate (n = 5). **p < 0.01, ***p < 0.001

Fig. 9

The mechanism of ADSC-Exos modulating diabetic hindlimb ischemia process (figure was created with BioRender.com)