| Purpose: Retinal neovascularization(RNV)is a common pathological feature in many kinds of fundus oculi diseases.Sometimes RNV can even lead to severe vision loss.Oxidative injury is one of the main predisposing factors for RNV occurrence and development.The specific mechanism may be closely related to the special structural tissues of the retina.Retinal astrocytes(RACs)are glial cells located in the retinal neuroepithelial layer.RACs have an intimate anatomical relationship with microvascular endothelial cells.They have a variety of functions,but little is known about the mechanisms by which RACs regulate the function of endothelial cells.The molecules secreted by RACs,such as exosomes,have recently received a lot of attention and may provide potential clues to address the RAC‐mediated modulation of endothelial cells.In this study,we aimed to preliminarily explore the mechanisms of how RAC exosomes generated under oxidative stress are involved in the regulation of endothelial function.Methods: 1.Effects of oxidative stress injury on RACs(1)CCK8 and LDH were used to verify the cell survival rate after RACs were injured by t BHP,and the model concentration was determined.(2)Flow cytometry and annexin V-FITC staining were used to detect the apoptosis of RACs after t BHP treatment.(3)In order to further confirm that this damage is caused by oxidative stress,we tested the GSH concentration and the cell MDA level.These two substances can evaluate the level of antioxidant products and membrane lipid peroxidation,respectively.DCHA-DA staining was detected by flow cytometry to determine intracellular ROS levels.(4)Western blot,immunofluorescence,electron microscopy and confocal detection of RACs apoptosis and autophagy levels after oxidative stress injury.2.The effects of autophagy levels of RACs on the proliferation,migration and tube formation of HUVECs in co-culture systems(1)RACs autophagy levels were verified by Western blot.Grouped into: control group,t BHP treatment group,t BHP + 3MA treatment group,t BHP + DMSO treatment group.(2)The effects of different treatments on the proliferation,migration,and tube formation of HUCECs were tested by scratch and tube formation experiments.The groups were: ECGS group,ECGS + control RACs co-culture group,no ECGS group,no ECGS + t BHP-treated RACs co-culture group,and no ECGS + t BHP + 3MA-treated RACs co-culture group.(3)Western blot and immunofluorescence detection of HUVECs proliferation and migration-related pathway expression levels under different treatments.3.Extract exosomes secreted by RACs and test the effects of RACs-derived exosomes on the proliferation,migration and tube formation of HUVECs under different conditions(1)The exosomes secreted by RACs were extracted by ultra-high speed centrifugation,and the exosomes were extracted from the aspects of concentration,particle diameter,morphology,and expression of labeled proteins by Nanosight,electron microscope scanning,confocal,western blot Identification.(2)Western blot was used to detect the effects of different treatments on RACs autophagy level.Scratch experiments and tube formation experiments were used to detect the effects of RACs-derived exosomes on HUVECs proliferation,migration and tube formation under different conditions.(3)Western blot and immunofluorescence were used to detect the expression levels of proliferation and migration-related pathways of HUVECs treated with RACs-derived exosomes under different conditions.Results: 1.Oxidative stress induced RACs apoptosis and autophagy(1)CCK8 and LDH results showed that 15 and 30 u M t BHP treated RACs resulted in a cell survival rate of 75% and 55%,respectively.The trypan blue staining and bright field images of RACs showed that after t BHP treatment,the number of positive staining cells increased significantly,and the cell morphology also changed significantly(p <0.01).(2)Using annexin-V FITC staining and flow cytometry to detect cell apoptosis after low and high doses of t BHP treatment,showing a significant increase in apoptosis level after t BHP treatment(p <0.01).(3)The results of MDA and GSH concentration in RAC cells after t BHP treatment showed that MDA increased with the increase of t BHP concentration,and GSH decreased with the increase of drug concentration.The results of DCHA-DA staining and flow cytometry showed that the intracellular ROS level increased significantly with the increase of t BHP concentration(p <0.01).(4)Western blot,tuningl staining and immunofluorescence results showed that with the increase of t BHP treatment concentration,the apoptosis pathway-related proteins Bcl-2,Bax,and caspase 3 of RACs increased significantly,while m TOR,P62,LC3 related to autophagy pathway Both also increased significantly(p <0.01).Confocal images showed that autophagolysosomes were significantly increased in RACs cells treated with t BHP,and LC3β and LAMP1 were positive.Electron microscopy images showed that with the increase of t BHP treatment concentration,the number of normal mitochondria in the cells decreased and the number of autophagosomes increased(p <0.01).2.In the co-culture system,enhanced autophagy level of RACs promoted the proliferation,migration and tube-formation of HUVECs(1)Western blot results showed that t BHP-treated RACs significantly increased the expression of LC3(p <0.01)and decreased the expression of P62(p <0.05).When 3MA was added to t BHP,the expression of autophagy-related proteins was reversed(p <0.01).(2)In the co-culture system,the results of the scratch and tube formation experiments showed that HUVECs migration and tube formation were inhibited after co-culture with normal RACs(p <0.05),and HUVECs Migration and tube formation were promoted(p <0.01).After adding 3MA to RACs on the basis of t BHP,its promotion effect on HUVECs migration and tube formation was reversed(p <0.05).(3)In co-culture systems,normal RACs highly inhibited the pathways related to proliferation and migration in HUVEC,while RACs with higher levels of autophagy enhanced these pathways.Western blot and immunofluorescence results showed that compared with the ECGS group,co-culture with normal RACs reduced the activity of Src,FAK,Akt,m TOR and the expression of cyclin D1,VE cadherin in HUVECs(p <0.05).Co-culture with RACs treated with t BHP significantly increased the activity of Src,FAK,Akt,m TOR and the expression of cyclin D1 and VE cadherin compared with the ECGS-free group(p <0.01).In addition,3MA could block the effects of t BHP-treated RAC co-culture on HUVECs-related pathways(p <0.01).3.Under oxidative stress condition,RACs-derived exosomes could promote HUVECs proliferation,migration,and tube-formation(1)In RAC confocal images after t BHP treatment,autophagic endosomes co-labeled with exosomal marker protein CD63 and autophagy marker protein LC3β can be observed.The results of Nanosight and transmission electron microscopy showed that the exosomes produced by normal RACs,RACs treated with t BHP,and RACs treated with t BHP + 3MA were different.Normal RACs-derived exosomes are smaller and higher in concentration than RACs-derived exosomes after t BHP treatment.The addition of 3MA to t BHP can further increase the size and concentration of exosomes.Western blot analysis of markers in cells and exosomes showed that HSP70,TGS101,CD81 and CD63 had significantly different expressions in controls,t BHP and t BHP + 3MA exosomes.(2)Exosomes isolated from normal and t BHP-treated RACs have different effects on HUVEC migration and tube formation,and GW4869 can significantly reduce this effect.Western blot results showed that GW4869 did not affect the LC3β / LC3α ratio or p62 expression in t BHP-induced RACs.Migration and tube formation experiments showed that exosomes produced by normal RACs could inhibit the improvement of HUVECs function induced by ECGS(p <0.05).However,exosomes obtained from RACs under oxidative stress can significantly promote HUVEC migration and tube formation(p <0.01).After GW4869 inhibited the release of exosomes,the promotion effect of RACs-derived exosomes on HUVECs after t BHP treatment was significantly reduced(p <0.01).(3)Western blot and immunofluorescence results show that normal RACs exosomes can inhibit the pathways related to proliferation and migration of HUVECs,while exosomes from RACs with higher autophagy levels enhance these pathways.Compared with the ECGS treatment group,exosomes derived from normal RACs reduced Src,FAK,Akt,m TOR activity and expression of cyclin D1 and VE cadherin in HUVECs.However,compared with the ECGS-free group,RACs exosomes after t BHP treatment can significantly increase the expression of cyclin D1 and VE cadherin and Src,FAK,Akt,m TOR activity in HUVECs.In addition,GW4869 can block the effects of RACs-derived exosomes on the proliferation and migration-related pathways of HUVECs after t BHP treatment.Conclusions:1.Oxidative stress can induce RACs apoptosis and enhanced autophagy.2.In the co-culture system of RACs and HUVECs,the autophagy status of RACs can promote the migration and tube formation of HUVECs and the related pathways,this promotion can be weakened by the autophagy inhibitor 3MA.3.Autophagy endosomes can be labeled by both LC3 and exosomal marker CD63.RACs-derived exosomes differ in size,concentration and contents under different conditions.4.RACs-derived exosomes with increased level of autophagy can promote HUVECs migration,tube formation and related pathways,this promotion can be attenuated by the exosomal inhibitor GW4869. |
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