Corneal Neovascularization Is Caused By Reduced Epithelial Cells Expressing Muscarinic Acetylcholine Receptor M5

Purpose:To investigate the role of the epithelial cells expressing CHRM5 in corneal neovascularization.Methods:The models of traumatic corneal neovascularization in mice were established by corneal alkali burn,extensive corneal epithelial debridement and suture,respectively,and the Corn1 mice with spontaneous corneal neovascularization were used in the study.The mouse corneas of each group were taken for the extraction of RNA and protein,and fixed for frozen and paraffin sections and transmission electron microscopy after the ocular was examined by slit lamp microscope and stereofluorescence microscope.The expression of CHRM5,ADRA1A,KRT12,KRT14,GSTA4,HMGN1,HK2,IL-1β,FGF2,VEGFA and CPT1 A in the cornea was determined by qRT-PCR,Western blotting,immunofluorescence or immunohistochemistry.Transmission electron microscopy was used to observe the changes in the ultrastructure.Phenotypic changes of corneal epithelium and their relationship with neovascularization were verified by a variety of transgenic mouse models,the changes of corneal glucose uptake were detected by 18F-FDG-PET.Results:The expression of mRNA and protein of CHRM5 decreased significantly in injured and Corn1 mice cornea.Immunofluorescence showed that CHRM5 was mainly located in corneal epithelium and endothelial cells.The CHRM5 positive cells decreased significantly,while ADRA1A positive cells increased in corneal epithelium of injured and Corn1 mice.The decrease of CHRM5 positive cells leads to the phenotypic change of corneal epithelial cells,which is characterized by a decrease in the number of cells expressing KRT12 and an increase in the number of cells expressing KRT14.The extensive debridement of corneal epithelium in Krt12Cre-Tomatof/fGFP and Krt14Cre-Tomatof/fGFP mice directly showed that the formation of corneal neovascularization was accompanied by the disappearance of KRT12 positive epithelial cells and the repair of KRT14 positive epithelial cells,and Krt12Cre-Tomatof/fGFP-Corn1 mice also proved that corneal neovascularization was due to the significant decrease of KRT12 positive epithelial cells.In the injured cornea with phenotypic changes of epithelial cells and Corn1 mouse cornea,neovascularization was not accompanied by a corresponding increase in the expression of IL-1β,FGF2 and VEGFA,but showed the immaturity of epithelial cells and increased utilization of glucose,which was characterized by decreased expression of GSTA4 and HMGN1,increased expression of HK2 and increased uptake of 18F-FDG.The expression of CPT1 A was decreased and there were a large number of high electronic densities which may be unsaturated fatty acids in corneal epithelium under transmission electron microscope.Conclusions:The decrease of CHRM5 positive cells in corneal epithelium leads to immature phenotypic changes and metabolic changes in corneal epithelial cells,which causing the formation of corneal neovascularization.The changes of corneal epithelial cell metabolism may become a new target for corneal neovascularization therapy.