Exosomes From Human Umbilical Cord Mesenchymal Stem Cells Treat Corneal Injury via Autophagy Activation

Abstract

Corneal injury (CI) affects corneal integrity and transparency, deteriorating the patient’s quality of life. This study aimed to explore the molecular mechanisms by which exosomes secreted from human umbilical cord mesenchymal stem cells (hucMSC-Exos) affect autophagy in human corneal epithelial cells (HCECs) and CI models. We isolated and identified hucMSC-Exos using nanoparticle tracking analysis, transmission electron microscopy, and western blotting. The effects of hucMSC-Exos combined with autophagy regulators on HCECs and CI mice were assessed using cell viability assays, scratch assay, cell cycle assay, apoptosis assay, corneal fluorescein staining, haze grades, pathological examinations, western blotting, and quantitative polymerase chain reaction (qPCR). In vitro results indicated that hucMSC-Exos combined with the autophagy activator had positive effects in promoting the cell proliferation, migration capacity, and the cell cycle by upregulating the proportions of cells in the S phase and the expression of PCNA, Cyclin A, Cyclin E, and CDK2. Meanwhile, the combination treatment reduced the apoptotic rate of HCECs. In vivo results indicated that hucMSC-Exos especially combined them with the autophagy activator significantly alleviated corneal epithelial defects and stromal opacity, reduced the levels of the apoptotic markers Bax and cleaved Caspase-3, reduced the inflammatory response products TNF-α, IL-1β, IL-6, and CXCL-2, and increased the Bcl-2. This was achieved by upregulating pAMPK/AMPK and pULK1/ULK1 ratios, and Beclin-1 and LC3B II/I, and by downregulating the pmTOR/mTOR ratio and p62. In contrast, clinical indications, apoptosis, and inflammation were aggravated after the application of the autophagy inhibitor. HucMSC-Exos combined with an autophagy activator significantly enhanced HCECs functions and alleviated corneal defects, apoptosis, and inflammation by activating the autophagy signaling pathway, AMPK-mTOR-ULK1, providing a new biological therapy for corneal wound healing and ocular surface regeneration.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells

“…Moreover, EVs have been found to be secreted in the cornea following wounding [ 41 ] and it is known that exosomes secreted by corneal epithelial cells can pass through the BM if impaired by an injury [ 41 ]. These results all pointed to a potential role of exosomes in corneal wound healing, which has recently been the focus of a few studies [ 41 , 42 , 45 , 46 , 47 , 48 ]. However, characteristics, functional roles and mechanisms of action of corneal exosomes remain to be fully investigated.…”

Research progress of exosomes in pathogenesis, diagnosis, and treatment of ocular diseases

“…Several studies have demonstrated the therapeutic effect of stem cell-derived exosomes on the corneal injury ( Hertsenberg et al, 2017 ; Shojaati et al, 2019 ; Ma et al, 2022 ). In the corneal injury mouse model, combined with an autophagy activator, human umbilical cord MSC-derived exosomes (MSC-exos) activated the autophagy AMPK-mTOR-ULK1 signaling pathway, reduced the levels of the apoptotic markers, Bax and cleaved Caspase-3, and decreased the expression of the inflammatory response products, TNF-α, IL-1β, IL-6, and CXCL-2, thereby reducing corneal epithelial defects and stromal opacity ( Figure 4 ).…”

“…In the corneal injury mouse model, combined with an autophagy activator, human umbilical cord MSC-derived exosomes (MSC-exos) activated the autophagy AMPK-mTOR-ULK1 signaling pathway, reduced the levels of the apoptotic markers, Bax and cleaved Caspase-3, and decreased the expression of the inflammatory response products, TNF-α, IL-1β, IL-6, and CXCL-2, thereby reducing corneal epithelial defects and stromal opacity ( Figure 4 ). In vitro experiments showed that the combination treatment significantly increased the viability of human corneal epithelial cells and promoted cell proliferation ( Ma et al, 2022 ). Hertsenberg et al (2017) demonstrated that exosomes from human corneal stromal stem cells through a TSG-6-dependent pathway, down-regulate the expression of the fibrosis markers myostatin C, Acta2, and Col3a1, inhibit neutrophil infiltration, inhibit corneal inflammation, stimulate regeneration of transparent stroma, and reduce corneal scar formation and fibrosis.…”

Advances in Biomaterials for Corneal Regeneration

“…Zhong and coworkers showed that when exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) were combined with an autophagy activator, they positively affected the repair of a corneal injury (CI). The repair process was regulated by accelerated cell proliferation and migration while inhibiting apoptosis and inflammation by activating the AMPKmTOR-ULK1 autophagy pathway [73]. Han et al [74] examined the possible role of exosomes in corneal wound healing and neovascularization.…”