| Purpose:To investigate cell phenotype and mechanism of rabbit corneal endothelial wound healing process as well as validate and regulate the key factors.Methods:A rabbit corneal endothelial injury model was established by mechanical scraping.At four time points of D0,D3,D7 and D14 after injury,we performed slitlamp observation to evaluate corneal edema,optical coherence tomography(OCT)to measure corneal thickness,and in vivo confocal microscopy(IVCM)to observe corneal endothelium.Samples were taken at the above four time points for alizarin red S staining to observe cell morphology and immunofluorescence staining(IF)to detect the expression of cellular markers like ZO-1,N-cadherin,α-SMA,and Vimentin.Morever,samples were taken for transcriptome sequencing(RNA-seq).The quantitative analysis of gene expression,sample correlation analysis,differentially expressed gene analysis,GO functional annotation and classification,and KEGG functional signaling pathway were performed according to RNA-seq.The analyzed results of RNA-seq were validated by quantitative RT-PCR(qRT-PCR)in an ex vivo injured model and the related signaling pathways were regulated to observe their effects.Results:Normal rabbit corneas remained transparent.On D3 after injury,the cornea showed significant edema and thickening,which was relieved on D7 and then returned to transparecny and normal thickness on D14.IVCM showed that normal endothelium exhibited regularly hexagonal arrangement.On D3 and D7 after injury,the cells around the injured area continuously migrated to the center and changed from hexagonal shape to spindle shape.Morever,the alizarin red S staining showed the cells were unevenly stained with unclear or even disappeared cell boundaries.While on D14,the cells returned to regularly hexagonal shape with uniform alizarin red S staining and clear cell borders.IF results showed that norrmal endothelium expressed intact junction markers of ZO-1 and N-cadherin,without expression of mesenchymal markers of αSMA and Vimentin.On D3 and D7 after injury,the integrity of ZO-1 and N-cadherin was disrupted to varying degrees,and the expression of α-SMA and Vimentin was detected.On D14,ZO-1 and N-cadherin returned to normal pattern,while α-SMA and Vimentin were undetected.In RNA-seq results,analysis of differentially expressed genes revealed that mesenchymal markers of α-SMA,Vimentin,Vitronectin1,Snail1,ZEB1,MMP2,Laminin α5 and Integrin α5 showed a trend of first upregulation and then downregulation;while ZO-1 and N-cadherin showed a trend of first downregulation and then upregulation during the process.GO functional annotation and classification showed that the enriched factors involved included:intercellular adhesion and connection;component changes of cytoskeleton or extracellular matrix(ECM);interaction between ECM and its receptors;regulation of cell cycle,proliferation,migration;activation and regulation of signal transduction pathways.KEGG functional analysis showed that Wnt,TGF-β and Notch signaling pathways were important.In ex vivo injury model,qRT-PCR results showed that the expression trend of key genes was consistent with RNA-seq results.Morever,the activation of Wnt or TGF-β signaling pathway could promote EndMT,while inhibition of Wnt,TGF-β or Notch signaling pathways could inhibit EndMT.Conclusions:Rabbit corneal endothelium could undergo self-repair after injury.The cells involved in wound healing firstly transformed from endothelial to mesenchymal phenotype,and then transformed back into endothelial phenotype,so as to maintain its normal structures and functions.In this process,intercellular adhesion and connection;components of cytoskeleton and ECM;interactions between ECM and its receptors;regulation of cell cycle,proliferation,migration as well as Wnt,TGF-βand Notch signaling pathways played important roles.By activating or inhibiting Wnt,TGF-β and Notch signaling pathways,the transition between endothelial phenotype and mesenchymal phenotype could be modulated. |
