Corneal Epithelial Cells Alter Phenotype By Metabolic Reprogramming To Promote Corneal Neovascularization

Background:Under normal circumstances,the cornea is an avascular,transparent connective tissue,as well as the mechanical barrier and anterior refractive surface of the eye.Corneal neovascularization is a disease that threatens visionand can be caused by infection,chemical damage,autoimmune diseases,corneal transplantation,and inflammation associated with traumatic diseases,etc.However,the pathogenesis of corneal neovascularization is unclear.Our previous studies have shown that corieal stromal cells degrade the extracellular matrix by secreting metalloproteinase 13,providing space for vascular endothelial cells to enter the corieal stroma and promote corneal neovascularization.Although the normal corneal epithelium plays an important role in maintaining the avascular state of the cornea,the specific pathogenesis of how corneal epithelium promote corneal neovascularization remains unkown.Purpose:To investigate whether metabolic reprogramming promote corneal neovascularization(CNV)through altering phenotype of corneal epithelial cells.Methods:Keratin-12-Cre and Keratin-14-Cre mice were bred to mT/mG dual reporter mice to target green fluorescent protein(GFP)within Keratin-12 cells or Keratin-14 cells,whereas other cells expressed tomato red.Four different injury models were used,including alkali burn,big range of scratch corneal epithelium,small range of scratch corneal epithelium and corneal suture.Whole-mounted staining and digital camera under fluorescence excitation were used to observe the change of keratin.Slit lamp microscope and digital camera were used to observe corneal neovascularization and the repair of corneal epithelium.The expression of Keratin-5(K5),keratin-12(K 12),keratin-13(K 13),keratin-14(K 14),Carnitine palmitoyltransferase IA(CPTIA)and Hexokinase 2(HK2)was determined via quantitative(q)RT-PCR and immunohistochemistry.Results:Slit lamp results showed that alkali burns and big range of scratch corneal epithelium would generate corneal neovascularization which do not regress.Small range of scratch corneal epithelium did not generate corneal neovascularization.Corneal suture induced corneal neovascularization,while removing the suture the blood vessels would regress.None of the above four injure models can induce corneal neovascularization in chicken cornea.The results of the digital camera under fluorescence excitation and the whole-mounted staining showed that the expression of K12 decreased during corneal neovascularization.There is no K12 change,there is no CNV.Quantitative(q)RT-PCR and immunohistochemistry show that with the formation of corneal neovascularization,the expression of CPT1A decreased and HK2 increased,thereby changing the phenotype of corneal epithelial cell and the expression of VEGF.The expression of keratin-12 and keratin-5 were decreased,while the expression of keratin-13,keratin-14,VEGFa and VEGFc were increased.Changes of metabolic related genes and epithelial phenotype occurred before angiogenesis.Conclusions:Durung corneal injury,metabolic reprogramming occurred in epithelial cells.Metabolic reprogramming alters the phenotype of corneal epithelial cells and the secretion of VEGF,thereby promoting corneal neovascularization.We suggest that targeting corneal epithelial cells metabolism might be a promising strategy for CNV treatment.