Therapeutic Effect And Mechanism Of Human Amniotic Epithelial Cell-derived Extracellular Vesicles On Corneal Alkali Burn

Purpose: Corneal alkali burns are prone to cause serious consequences suchas corneal epithelial defects,severe corneal inflammation,and scarring damage to the cornea,whichcan lead to chronic complications and lifelong disability,making the injury one of the most common and devastating ophthalmic emergencies.Amniotic membrane transplantation is an ideal surgical treatment for ocular surface reconstruction after alkali burns.However,amniotic membrane grafts have disadvantages suchas complex surgical manipulation,the need for prolonged suturing operations,rapid early postoperative degradation,increased risk of ocular surface infection,and adverse calcification of the transplanted amniotic membrane,whichare clinically insurmountable problems.Human amniotic epithelial cells(hAECs)are one of the perinatal stem cells whichpossess embryonic stem cell-like differentiation capability and mesenchymal stem cells-like immunomodulatory properties.Extracellular vesicles(EVs)isolated from stem cells are considered to stem cell-based,cell-free drugs that contain a variety of bioactive cargo,including cells that secrete nucleic acids,proteins,and lipids.Therefore,we focused on the effect and mechanism of human amniotic epithelial cell-derived extracellular vesicles(hAEC-EVs),the effective component of the amniotic membrane,on a rabbit corneal alkali burn model.We are dedicated to investigating formulations of hAEC-EVs to provide new options for clinical intervention in corneal alkali burns.Methods:Part 1: The amnion was incubated withtrypsin-EDTA,and then the hAECs were mechanically detached from the amnion.The extracted hAECs were identified by the microscope,immunofluorescence assay and fluorescence-activated cell sorting analysis(FACS).HAEC-EVs were isolated from the hAECs culture medium using ultracentrifugation.The hAEC-EV particle sizes were determined using nanoparticle tracking analysis(NTA).Transmission electron microscopy(TEM)and Western blot analysis were performed to characterize the morphological and surface markers of hAEC-EVs.Part 2: For the model of corneal alkali burn,ring-shaped sterile Whatman filter paper containing Na OH solution was placed on the center of the cornea of rabbits.The hAEC-EVs treatment was prescribed eye drops in association withsubconjunctival injection of hAEC-EVs.For the ophthalmology examination,the cornea was viewed and imaged witha slit lamp.After 14 days of consecutive treatment,animals were euthanized withan overdose of intravenous pentobarbital,and the corneal tissue was immediately obtained.Hematoxylin and eosin(H&E)staining and immunofluorescence staining were used to analyze the pathologic morphology of the cornea.Part 3: To explore the mechanism of hAEC-EVs in the treatment of corneal alkali burns,we performed micro RNA sequencing and liquid chromatograph-tandem mass spectrometry(LC-MS/MS)proteomics on the contents of hAEC-EVs,and the results were bioinformatically analyzed to explore the key mi RNAs and proteins in hAECs that function to treat corneal alkali burns.In vivo experiments,we carried out Masson staining of rabbit corneal tissue to observe the arrangement of collagen fibers in the corneal stroma,immunofluorescence staining,and Western blot analysis to detect the expression of α-smoothmuscle actin(αSMA)in corneal tissue.Western blot analysis was performed to detect the expression of key proteins in corneal tissue associated withinjury healing.In vitro experiments,the 5-Ethynyl-2’-deoxyuridine(Ed U)cell proliferation assay,wound healing migration assay and Transwell chamber assay were applied to evaluate the effects of hAEC-EVs on the proliferation and migration properties of human corneal epithelial cells(HCECs)and human corneal stromal cells(HCSCs).Western blot analysis was performed to detect the expression of extracellular matrix(ECM)and focal adhesion-related proteins in HCECs and HCSCs.Results:Part 1: The hAECs were successfully isolated and cultured in vitro.HAECs isolated from the human amniotic membrane showed a confluent and tightly connected monolayer morphology in the hAEC medium.The hAECs expressed hAEC-specific markers,including CK19,E-cadherin and SSEA-4.Additionally,the hAECs were positive for CD29,CD73,CD326 and SSEA-4 and negative for HLA-ABC.The diameters of the hAEC-EVs ranged from 105 nm to 195 nm and were approximately154.5 nm on average.The morphological assessment of hAEC-EVs by TEM revealed a typical cup-shaped morphology.And compared to the normal hAECs,the EV-specific markers CD9,CD81,and ALIX were highly enriched in hAEC-EVs,while the expression of calnexin and GAPDH were negatively detected.The above data indicated that hAEC-EVs were successfully isolated from cultured hAECs and showed a typical EV characteristic and status.Part 2: A slit-lamp analysis of the ocular surfaces revealed alkali burn-induced central epithelial loss and stromal haze of the cornea,obscuring the iris and pupil.After two weeks of continuous treatment withhAEC-EVs,slit-lamp examination and histological findings indicated that hAEC-EVs facilitated re-epithelialization of the cornea after alkali burns,reduced scar formation and promoted the restoration of corneal tissue transparency.Part 3: Multi-omics analysis showed that extracellular matrix reorganization,inhibiting excessive myofibroblasts,and promoting target cell adhesion to the ECM are hAEC-EVs primary functions.The hAEC-EVs provide the ECM-related components to trigger the early focal adhesion signaling of corneal cells and attenuate the excessive activation of TGFβ-SMAD2/3 signaling pathways to promote the corneal wound healing from corneal injury rabbits.HAEC-EVs effectively promoted the proliferation and migration of HCECs and HCSCs in vitro and activated the focal adhesion signaling pathway.Conclusion: HAEC-EVs provided functional mi RNAs and proteins to the wound area of alkali-burned rabbit corneas.The hAEC-EVs promote re-epithelialization and stromal layer reconstruction of the cornea after alkali burns by providing the ECMrelated components to trigger the focal adhesion signaling of corneal cells.The hAECEVs also reduce scar formation and promote the restoration of corneal tissue transparency via attenuating the excessive activation of TGFβ-SMAD2/3 signaling pathways.We demonstrated that hAEC-EVs are an excellent cell-free candidate drug for the treatment of corneal injuries,including corneal alkali burns.