Aquaporin 5 Regulates Wnt/β-Catenin Pathway To Maintain Corneal Epithelial Homeostasis

Purpose:Disruption of the corneal epithelium can lead to vision loss and pathogen invasion.Aquaporin 5（AQP5）is involved in maintaining corneal transparency and barrier function.The purpose of this paper is to investigate the maintenance of corneal epithelial phenotype by AQP5and its mechanism.Methods:Aqp5 knockout(Aqp5^-/-)mice based on C57BL/6N mice were constructed by CRISPR/Cas9 technology.Slit lamp microscopy,fluorescein sodium staining,hematoxylin-eosin（H&E）staining,immunofluorescence staining,fluorescein sodium staining and quantitative real time polymerase chain reaction（q RT-PCR）were used to evaluate the pathological changes of the cornea of Aqp5^-/-mice,and to observe the abnormal changes of the corneal phenotype.Structural changes in corneal epithelial cells were analyzed by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）.A model of corneal epithelial injury was used to investigate the role of Aqp5 deficiency in accelerating injury-induced corneal haze,neovascularization and inflammation.Differentially expressed genes（DEGs）in the corneal epithelium of Aqp5^+/+and Aqp5^-/-mice were identified and screened by m RNA sequencing.The expression of Wnt/β-catenin signaling pathway-related proteins in corneal epithelial cells was detected by immunofluorescence staining assays,q RT-PCR and western blot.Wnt antagonist（C59）and DKK1 inhibitor（IIIC3）were applied to interfere with the Wnt/β-catenin signaling pathway to explore its role in Aqp5^-/-corneas and primary corneal epithelial cells.Results:（1）Aqp5 deficiency significantly increased corneal neovascularization,inflammatory cell infiltration and haze in the cornea of Aqp5^-/-mice aged 6 months and older.（2）Compared with Aqp5^+/+mice,the expression of K1 and K14 was up-regulated,the expression of K12 was down-regulated in the cornea of Aqp5^-/-mice,and the ultrastructure of corneal epithelial cells was abnormal.（3）The results of m RNA sequencing,q RT-PCR and western blotting showed that Wnt2 and Wnt6 expression was significantly reduced and BMP4 expression was significantly increased in corneal epithelium of Aqp5^-/-mice.（4）Compared with Aqp5^+/+mice,the expression of K1,K14 and BMP4 was up-regulated,the expression of K12 was down-regulated,the expression of Wnt2,Wnt6,Pax6 and ABCG2 was significantly decreased in Aqp5^-/-mouse corneal epithelial cells,and the accumulation ofβ-catenin in the cells was decreased.（5）Mice were subjected to corneal epithelial scrape,and Aqp5 deletion aggravated corneal neovascularization,inflammation,and opacification induced by corneal epithelial injury in Aqp5^-/-mice compared with Aqp5^+/+mice.In Aqp5^-/-mouse corneal epithelium,the expression of K1,K10 and K14 was up-regulated,the expression of K12 was down-regulated,the expression of BMP4 was up-regulated,the expression of Wnt2,Wnt6,Pax6 and ABCG2 was down-regulated,and the accumulation ofβ-catenin in cells was decreased.（6）Pretreatment of Aqp5^+/+primary corneal epithelial cells with C59 decreased the expression of Wnt2,Wnt6,K12,Pax6 and ABCG2,increased the expression of K1,K10,K14 and BMP4,and decreased the accumulation ofβ-catenin in the cells.（7）Pretreatment of Aqp5^-/-primary corneal epithelial cells with IIIC3 decreased the expression of K1,K10 and K14,increased the expression of K12,Pax6 and ABCG2,and increased the accumulation ofβ-catenin in the cells.（8）Intervention of exogenously administered IIIC3 to the corneas of Aqp5^-/-mice,IIIC3significantly inhibited corneal neovascularization,inflammatory response,and maintained corneal epithelial transparency in Aqp5^-/-mice.The decreased expression of K1,K10,K14 and BMP4,the increased expression of K12,Pax6 and ABCG2 in the corneal epithelium,and the increased accumulation ofβ-catenin in the cells,reversed the abnormal corneal phenotype of Aqp5^-/-mice.Conclusion:（1）With the increase of age（about 6 months）,the cornea of Aqp5^-/-mice showed obvious pathological changes such as neovascularization and haze.（2）AQP5 may be involved in the maintenance of corneal epithelial cell phenotype through Wnt/β-catenin signaling pathway.（3）Wnt agonist（IIIC3）can significantly alleviate the neovascularization and inflammatory response in the cornea of Aqp5^-/-mice.These findings demonstrate that AQP5 participates in corneal epithelial homeostasis maintenance and AQP5 may be a potential target for the therapy of corneal epithelial injury healing.