Search

Purpose: Corneal injury (CI) resulting in corneal opacity remains a clinical challenge. Exosomes (Exos) derived from bone marrow mesenchymal stem cells (BMSCs) have been proven effective in repairing various tissue injuries and are also considered excellent drug carriers due to their biological properties. Recently, microRNA-29b (miR-29b) was found to play an important role in the autophagy regulation which correlates with cell inflammation and fibrosis. However, the effects of miR-29b and autophagy on CI remain unclear. To find better treatments for CI, we used Exos to carry miR-29b and investigated its effects in the treatment of CI., Methods: BMSCs were transfected with miR-29b-3p agomir/antagomir and negative controls (NCs) to obtain Exos-29b-ago, Exos-29b-anta, and Exos-NC. C57BL/6J mice that underwent CI surgeries were treated with Exos-29b-ago, Exos-29b-anta, Exos-NC, or PBS. The autophagy, inflammation, and fibrosis of the cornea were estimated by slit-lamp, hematoxylin and eosin (H&E) staining, immunofluorescence, RT‒qPCR, and Western blot. The effects of miR-29b-3p on autophagy and inflammation in immortalized human corneal epithelial cells (iHCECs) were also investigated., Results: Compared to PBS, Exos-29b-ago, Exos-29b-anta, and Exos-NC all could ameliorate corneal inflammation and fibrosis. However, Exos-29b-ago, which accumulated a large amount of miR-29b-3p, exerted excellent potency via autophagy activation by inhibiting the PI3K/AKT/mTOR pathway and further inhibited corneal inflammation via the mTOR/NF-κB/IL-1β pathway. After Exos-29b-ago treatment, the expressions of collagen type III, α-smooth muscle actin, fibronectin, and vimentin were significantly decreased than in other groups. In addition, overexpression of miR-29b-3p prevented iHCECs from autophagy impairment and inflammatory injury., Conclusions: Exos from BMSCs carrying miR-29b-3p can significantly improve the therapeutic effect on CI via activating autophagy and further inhibiting corneal inflammation and fibrosis.

Purpose: This study aimed to investigate the therapeutic effects and underlying mechanisms of locally delivered regulatory T cells (Tregs) on acute corneal wound healing after alkali burn., Methods: After corneal alkali burn, the mice were injected subconjunctivally with regulatory T cells (Tregs) isolated from syngeneic mice. The wound healing process was monitored by clinical manifestation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). As amphiregulin (Areg) was significantly upregulated, its reparative function in injured corneas was suggested. The hypothesis was further verified via loss- and gain-of-function experiments by administrating the antibody of Areg (anti-Areg) and recombinant Areg (rmAreg)., Results: Subconjunctivally injected Tregs rapidly migrated to injured corneas. The mice treated with Tregs showed prominently reduced corneal opacity, alleviated edema, and faster re-epithelialization compared with the control group. Mechanistically, Treg treatment led to suppressed infiltration of inflammatory cells, along with improved proliferation and inhibited apoptosis of corneal epithelial cells. Tregs expressed upregulated functional markers, including Areg. Expectantly, the levels of Areg in corneas were dramatically higher in the Treg injection group, in line with better corneal restoration. Additional experiments showed that the administration of anti-Areg blunted the reparative effect of Tregs, while exogenous Areg enhanced it. Treg-treated corneas also exhibited less neovascularization and fibrosis at a later reconstruction stage of corneal repair., Conclusions: The findings showed that the subconjunctival injection of Tregs effectively promoted corneal wound healing by inhibiting excessive inflammation and enhancing epithelial regeneration, with an indispensable reparative role of Areg. Subsequent complications of corneal vascularization and fibrosis were therefore reduced.

Purpose: To investigate the effect and mechanism of Agrin on limbal stem cell proliferation and corneal wound healing., Methods: Limbal stem cells were isolated and treated with different concentrations of Agrin. CCK-8 and cell proliferation markers (Ki67 and pH3) were detected to evaluate cell numbers or proliferative potential of limbal stem cells. The corneal epithelium wound model was induced by debridement of central corneal epithelial, and the effects of Agrin on limbal stem cell proliferation and corneal epithelial wound healing rate were determined., Results: Agrin promoted the proliferation of cultured limbal stem cells in vitro and increased the expression level of p63α rather than keratin 12. Furthermore, Agrin accelerated the wound healing rate of corneal epithelium through activating limbal stem cell proliferation in vivo. In terms of mechanism, Agrin could facilitate the dephosphorylation of Yap1, which contributed to the nuclear translocation of Yap1 and expression of Cyclin D1, and subsequently promoted proliferation of limbal stem cells., Conclusions: Agrin promotes the proliferation of limbal stem cells and accelerates the healing rate of corneal wound through Hippo-Yap signaling pathway.

Purpose: We determine the feasibility of human conjunctival epithelial cells (hCjECs) on poly(lactic-co-glycolic) acid (PLGA) membranes for corneal epithelium regeneration. hCjECs on PLGA or polyester (PET) membranes with or without coculture of human Tenon's fibroblasts (hTFs) were compared in vitro, and to determine whether epithelial sheets grown on PLGA membranes can repair injured rabbit corneal epithelium by transplantation for 2 weeks in vivo., Methods: Primary hCjECs were cultured on PLGA or the original PET membrane-based transwell inserts with or without coculture of hTFs on the floor of the culture plate. Cell behaviors, such as proliferation and differentiation, were compared. For in vivo assessment, the corneas of rabbits were burned, and PLGA-based epithelial sheets then were transplanted for 2 weeks before histologic staining was conducted and analyzed to determine the effectiveness of the repair., Results: Primary human epithelial cells on the PLGA membrane showed an increased proliferation when cocultured with fibroblasts, which was confirmed by CCK-8 analysis, and upregulation of Ki67, with the expression of the epithelial marker CK19. The stratified squamous cell marker MUC1 and conjunctival cell marker MUC5AC also were expressed in the epithelial sheet. The epithelial defect in the burned corneas was decreased in the PLGA-based epithelial sheet treatment group (6.1% ± 1.6% of the area) compared to that in the no-treatment group (30.5% ± 6.3%) 2 weeks postoperatively., Conclusions: We developed a coculture system using a human feeder cell layer and PLGA membrane-based transwell inserts to create human conjunctival epithelial sheets. This system represents a promising strategy to regenerate corneal epithelium by transplantation.

Purpose: To evaluate morselized amniotic membrane and umbilical cord (AMUC) eye drops in promoting corneal re-epithelialization., Methods: Following a 2-mm diameter central epithelial wound in one eye of 48 normal C57BL/6 mouse corneas, 10 μL of saline with (n = 24) or without (n = 24) AMUC was applied three times a day for 6 days. The corneal epithelial defect was measured using 0.1% fluorescein, while corneal epithelial regularity was measured by assessment of a reflected light off the corneal surface. Hematoxylin and eosin and immunohistochemistry was performed for Ki-67, CD45, βIII-tubulin, and keratin12. Safety and toxicity were also assessed by monitoring physical activity and body weight., Results: Compared with the vehicle saline control, AMUC resulted in a significantly smaller corneal epithelial defect at 12 hours (P = 0.002), 1 day (P = 0.016), and 2 days (P = 0.04) post abrasion. Amniotic membrane and umbilical cord also achieved a more rapid complete epithelialization (3.15 ± 1.44 vs. 4.00 ± 1.63 days, P = 0.06) and induced a higher incidence of corneal regularity without affecting physical activity and body weight. Spearman correlation showed that epithelialization was significantly correlated with treatment groups (P < 0.001), time (P < 0.001), and corneal regularity (P = 0.04). Amniotic membrane and umbilical cord significantly decreased CD45+ cell infiltration in the peripheral cornea (P < 0.05) and promoted Ki-67+ cells in the central corneal epithelium (P < 0.05)., Conclusion: Topical AMUC significantly promotes corneal epithelialization and restores corneal regularity by reducing inflammation and promoting proliferation in a murine model of corneal abrasion without causing safety or toxicity concerns. This encouraging preclinical finding warrants a controlled human trial in the future.

Purpose: The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2., Methods: Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas., Results: Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na+/K+-ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction., Conclusions: We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.