Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis

Abstract

Background:Corneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.Methods:ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40–200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = −82.742, P < 0.01; collagen II: t = −72.818, P < 0.01; collagen III: t = −104.452, P < 0.01; collagen IV: t = −133.426, P < 0.01, and collagen V: t = −294.019, P < 0.01; and fibronectin: t = −92.491, P < 0.01, respectively).Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing

“… 20 Exosomes contain complex molecular components, which include general and cell type specific lipids, proteins, mRNA, and miRNA, enabling them to function as vectorized, multisignaling devices. 27 – 29 …”

“…The bi-lipid membrane of exosomes can maintain the encapsulated proteins, messenger RNA (mRNA), and microRNA (miRNA) under stable conditions to exert lasting effect. 27 – 29 Therefore, they can be formulated as a topical gel or drop and can be locally administered. They can also be reprogrammed to be carriers for therapeutic agents.…”

Extracellular Vesicles and Matrix Remodeling Enzymes: The Emerging Roles in Extracellular Matrix Remodeling, Progression of Diseases and Tissue Repair

“…Likewise, the treatment of MSC-derived EVs to corneal stromal cells (CSCs) induces the upregulation of MMPs and downregulation of ECM-related proteins such as collagens and fibronectin in CSCs. The findings indicate that adipose-MSCs might play an important role in the regulation of CSC viability through ECM remodeling, via EVs [ 195 ].…”

Therapeutic Advances of Stem Cell-Derived Extracellular Vesicles in Regenerative Medicine

“…In a mouse model of hyperoxic-induced retinopathy, intravitreal injection of MSC-EXs partially preserved retinal vascular blood flow and improved the symptoms of retinal ischemia without immunosuppression [40]. Corneal stromal cells (CSCs) are involved in the formation of a functional corneal endothelium and they play a remarkable role in moderating the corneal endothelial microenvironment [41]. Shen et al indicated that AdMSC-EX treatment obviously promoted CSC proliferation in vitro, inhibited its apoptosis, triggered higher expression of collagen and fibronectin, and caused lower expression of matrix metalloproteinases [41].…”

“…Corneal stromal cells (CSCs) are involved in the formation of a functional corneal endothelium and they play a remarkable role in moderating the corneal endothelial microenvironment [41]. Shen et al indicated that AdMSC-EX treatment obviously promoted CSC proliferation in vitro, inhibited its apoptosis, triggered higher expression of collagen and fibronectin, and caused lower expression of matrix metalloproteinases [41]. Samaeekia et al demonstrated that human corneal MSC-derived EXs significantly increased the corneal wound healing rate (Ex vs. Control; 77.5% vs. 41.6%) in a murine model [42], providing a promising non-cellular therapeutic approach for ocular surface injury.…”