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.

Therapeutic Applications of Stem Cell-Derived Exosomes

“…The exosome-rich medium can then be harvested and centrifuged to remove residual cells and debris. An ultracentrifugation process helps pellet the exosomes, which are resuspended and further refined using techniques like size-exclusion chromatography, ensuring a high-quality exosome yield [ 76 , 78 , 79 ], as illustrated in Figure 2 .…”

“…They exhibit a distinctive cup-like shape when viewed under transmission electron microscopy (TEM). Research focusing on these exosomes in the context of lower limb ischemia identified the presence of exosomal markers CD9, CD63, and CD81, while markers GM130 and β-tubulin were found to be absent [ 78 , 79 ]. Interestingly, a recent study has highlighted the role of the microRNA miR-125b-5p, found in ADSC-exos, in repairing muscle tissue damaged by ischemia.…”

“…Laboratory tests on living organisms have shown that ADSC-exos can safeguard ischemic skeletal muscle, expedite muscle injury healing, and foster vascular regeneration. The pivotal molecule in these processes appears to be miR-125b-5p [ 78 ]. Another investigative study exploring the influence of ADSC-exos on nonunion bone fracture healing in diabetic rats found that ADSC-exos notably advanced the bone healing process.…”

“… Boost angiogenesis in HUVECs. CD9, CD63, and CD81 [ 76 , 77 , 78 , 79 ]. Exosomes from Bone Marrow-Derived Mesenchymal Stem Cells Cardioprotection against doxorubicin-induced toxicity.…”

Multifunctional ADM hydrogel containing endothelial cell-exosomes for diabetic wound healing

“…Exosomes (Exos), small vesicles with 50–150 nm diameters, play a crucial role in regulating cell-to-cell communication by transferring proteins, lipids, DNA, RNA, and other bioactive substances [ 17 , 18 ]. Various studies have demonstrated the potential of stem cell-derived exosomes in promoting wound repair through different mechanisms, such as down-regulating transforming growth factor (TGF-β) expression, activating the AKT/eNOS pathway for angiogenesis, and enhancing cell function while inhibiting apoptosis [ [19] , [20] , [21] ].…”