Autophagy Resists EMT Process To Maintain The Homeostasis Of Retinal Pigment Epithelium

Objective:Proliferative vitreoretinopathy?PVR?is the most serious complication of rhegmatogenous retinal detachment?RD?and the main cause of surgical failure of RD.Since its initial detailed description,there is no effective relevant clinical treatment progress.Although PVR can occur preoperatively,it still has a higher incidence after any kind of intraocular RD surgery,and the incidence of PVR after all RD case is 5-10%.The proliferative membranes on both surfaces of the posterior membrane of vitreous and on the detached retina are the pathological features of PVR.The retinal distortion and continuous detachment caused by posterior contraction of these membranes transforms RD into tractional retinal detachment.In this pathological process,retinal pigment epithelium?RPE?loses epithelial phenotype through epithelial-mesenchymal transition?EMT?to obtain mesenchymal cell phenotype,which increases cells the ability of migration,invasion,resistance to apoptosis,and extracellular matrix production,transforming RPE into fibroblast-like cells.Base on the most important cytological feature of PVR,many researchers have spent more than 40 years trying to solve this problem,and yet have not found effective treatment.This makes us have to pay attention to some other mechanisms involved in RD and in PVR.Autophagy is an intracellular catabolic process that regulates the degradation of non-essential or dysfunctional organelles or proteins.By autophagy,cells can digest damaged protein molecules or organelles,thus ensuring the maintenance of intracellular environmental homeostasis.However,autophagy is also considered a strategy for cells to fight intracellular or environmental stress.This includes nutrient deprivation,hypoxia and drug effects.Degradation of organelles is an alternative mechanism for cells to obtain energy supplies and basic metabolites.In the eye,from the anterior cornea to the posterior RPE that provides a protective barrier to the retina,almost all cell types rely on one or more types of autophagy to maintain normal structural and physiological function.Moreover,the expression of autophagy-related proteins in different cells in the eye also sheds light on the importance of autophagy progression in maintaining healthy visual function.In contrast,mutations in related autophagy genes can also directly contribute to the development of ocular diseases.In the meantime,intraocular cell homeostasis also depends on the regulation of the autophagy pathway induced by the interaction of basal and pressure.In retinal RPE cells and photoreceptor cells,autophagy is highly activated,and impairment of autophagy can lead to early degeneration of RPE cells.These characteristics of RPE strongly associate autophagy with retinal degenerative diseases caused by retinal senescent diseases and photodamage.This makes the research of autophagy and retinal diseases focused on degenerative diseases such as age-related macular degeneration?AMD?.Autophagy and EMT could not be linked for a long time in the past because of their independent mechanisms of action leading to alienated associations.However,recent research reveals that these two important processes can be regulated with each other through complex relationships.We found that in the development of retinal diseases,autophagy can protect RPE cells from stress stimuli such as oxidative stress and prevent retinal degeneration.Autophagy is also involved in the EMT process of RPE cells.We speculate that autophagy pathway may play an important role in the pathogenesis of PVR,and the elucidation of this regulatory mechanism will provide new ideas for the prevention and treatment of PVR.In the TGF-?induced EMT process of RPE,the autophagy behavior of cells is promoted.However,the mechanism of autophagy behavior caused by non-survival pressure is still unclear and needs further demonstration.This is also the key issue to solved in this study.Methods:1.Construction of autophagy-associated gene 7?Atg7?-deficient retinal pigment epithelial cell line;Detection of occludin and ZO-1 expression in cells by Western blot;Observe the growth state in the cell plane and three-dimensional space;Detection of changes in?-SMA expression and localization;Preliminary study on the relationship between autophagy and EMT in RPE cells.2.To establish TGF-?-induced RPE cell EMT model:The human retinal pigment epithelial cell line ARPE-19 was cultured in vitro and stimulated with different concentrations or time courses of TGF-?.The epithelial phenotype marker:Claudin-1,mesenchymal phenotype marker:N-cadherin,and cytoskeletal protein,stress fibrin:?-SMA,Vimentin,were detected by Western blot assay and ICC.The suitable concentration and time points of EMT induced by TGF-?in RPE cells were determined,and the markers of epithelial phenotype and mesenchymal phenotype of RPE cells were determined.3.To detect changes in autophagic flux in EMT process of RPE:Using Western blot to analysis the expression of expression under different time gradients of TGF-?stimulation.To investigate the effects of autophagic flux and autophagosome formation by detecting changes of p62 and LC3.The GFP-LC3eukaryotic expression vector was constructed and transformed into ARPE-19.Using The fluorescence inversion microscope to observe and record the GFP-LC3 spot aggregation.The lysosomal inhibitor CQ was used to block the autophagy-lysosomal complex degradation,and the effect of TGF-?on autophagic flux was confirmed by Western blot.4.To detect the role of autophagy in regulating EMT progression in RPE:4.1 Serum starvation and rapamycin were used to induce autophagy and the expression of Atg7,p62 and LC3 were detected.Using Western blot and ICC to detect the expression of Claudin-1 and N-cadherin.4.2 The Atg7 RNAi and control RNAi interference vectors were constructed and purified by plasmid extraction.The lentiviral vector packaging system was co-transfected into 293T to infect RPE cells.To construct stable sliencing Atg7 gene ARPE-19 cells.The expression of Claudin-1 and N-cadherin was detected.Transwell assay was used to detect cell migration ability.Gel contraction assay was used to detect the contractility of RPE cells.4.3 Myc-Atg7 plasmid expression vector was constructed,which was transfected into Atg7 shRNA cells to detect the expression of Claudin-1 and N-cadherin.The regulation of autophagy on EMT was clarified by rescue experiments.4.4 The rescue effect of rapamycin was detected by Western blot and ICC under the treatment of TGF-?combined with rapamycin;the change of cell migration ability was detected by Transwell assay;the contraction ability of RPE cells was detected by gel contraction assay in vitro.5.To explore the molecular mechanism of autophagy in regulating EMT:Mouse WT MEF and Atg7^-/-MEF cells were extracted,and the binding of Twist to p62 was detected by immunoprecipitation under the stimulation of TGF-?and HBSS conditions.Results:1.Atg7 deletion leads to RPE cell fibrosis.2.The EMT model of RPE layer was successfully constructed.Under TGF-?stimulation,the expression of tight junction protein Claudin-1 decreased,and the expression of mesenchymal markers:N-cadherin,?-SMA and Vimentin increased.3.In the EMT process of RPE,autophagy is activated,p62 expression is decreased,LC3-II expression is increased,and intracellular GFP-LC3 is aggregated.4.The eukaryotic expression vector of Atg7 was successfully constructed.The ARPE-19cell line with Atg7 stable knockdown was constructed,and the knockdown efficiency of Atg7 was over 70%.5.In autophagy deficient RPE cells,Claudin-1 expression was decreased,and the expression of mesenchymal markers:N-cadherin,?-SMA,Vimentin was increased;cell migration ability and contractility were enhanced.6.Overexpression of Atg7 in autophagy deficient RPE cells increased the expression of Claudin-1,while the expression of N-cadherin,?-SMA and Vimentin decreased;serum-free starvation in RPE cells enhanced epithelial characteristics.7.In the RPE EMT process,using rapamycin to promote autophagy can reverse the decrease of Claudin-1 induced by TGF-?,prevent the expression of N-cadherin,?-SMA and Vimentin,and reduce the cell migration and contraction ability.8.In Atg7 knockout MEF cells,p62 was significantly accumulated and the expression of LC3-II was significantly decreased compared with wild-type MEF cells.Meanwhile,the expression of mesenchymal proteins was significantly increased.9.Wild-type MEF cells were stimulated with HBSS or serum-free starvation,showing a marked decrease of mesenchymal markers.Compared with Atg7^-/-MEF cells,under serum-free starvation,WT MEF cells decreased mesenchymal proteins,while autophagic deficient cells had no significant changes.10.The results of co-immunoprecipitation experiments revealed that Twist did not bind to p62 under the stimulation of TGF-?,but the combination of Twist and p62 increased significantly under HBSS starvation.Conclusion:1.Autophagy is the key pathway to maintain the normal epithelial morphology of RPE.2.Promoting autophagy can protect RPE against EMT stress,maintain normal cell tight junction structure,and reverse fibrosis.3.The underlying mechanism is to promote the degradation of Twsit through p62-mediated selective autophagy,thereby increasing the expression of tight junction proteins between cells and inhibiting the progression of EMT.Our study provides a more complete understanding of the relationship between autophagy and intraocular RPE fibrotic disease,and a new perspective for autophagy as a therapeutic target for PVR.