The Role Of Autophagy In The Stroke-induced Impairment Of Blood-brain Barrier

Blood-brain barrier(BBB)dysfunction is a common event in the clinical manifestations of stroke,which is a key step in the pathological process of stroke.BBB dysfunction promotes peripheral blood to infiltrate the brain parenchyma,which in turn causes secondary damage to the central nervous system(CNS).However,the role of BBB in the pathological process of stroke has rarely been reported.BBB is a physiological barrier between peripheral blood and brain tissue,and it regulates regulates influx and efflux of molecules through tight junction for maintaining the homeostasis of the CNS.Claudin-5(Cldn5),as an important member of the tight junction proteins,is a key regulatory protein of BBB permeability.Therefore,exploring the potential mechanism on the expression and localization of Cldn5 is identified as a new and promising avenue for the prevention and potentially treatment of strokeAutophagy is a lysosomal degradation pathway that is essential for cellular survival and is activated in endothelial cells after stroke.However,the role of autophagy on BBB is not fully understood.Hence,the role of autophagy on the hypoxia(or serum starvation)-induced BBB impairment and its potential mechanism is explored in this study.Part 1:A model of BBB in hypoxia-induced impairmentObjective:To investigate the role and mechanism of hypoxia on BBB.Methods:In this study,endothelial cell lines and zebrafish were treated with CoCl2 or oxygen-deficient air to generate a model of BBB injury under hypoxia.Subsequently,the influence of hypoxia on BBB permeability was measured by means of trans-endothelial electrical resistance(TEER),endothelial cell barrier permeability and dye diffusion of cerebral vascular in zebrafish.Furthermore,The effect of hypoxia on the TJs expression and localization was analyzed by immunological technique(immunofluorescence,western blot,immune electron microscopy),super-resolution optical imaging(stimulated emission depletion)and flow cytometry.Results:BBB dysfunction induced by hypoxia was manifested by extensive permeability of BBB,which is resulted from and the internalization and decreased level of Cldn5.Conclusion:Internalization and the loss of membrane Cldn5 are the main causes of hypoxia-induced BBB impairment.Part 2:Preliminary study on the correlation between hypoxia-induced autophagy and BBBObjective:To explore the correlation between hypoxia-induced autophagy and BBB Methods:Autophagy flux in cerebrovascular endothelial cells was observed by transmission electron microscopy(TEM),and was further confirmed by transfection with LC3-GFP vector in endothelial cells lines.The localization of Cldn5 and LC3 was imaged by immunoassay.Results:Autophagy was activated by hypoxia.The aggregated Cldn5 co-localized with LC3 in cerebrovascular endothelial cells.Conclusion:Hypoxia-induced autophagy might be involved in regulation of BBB integrity.Part 3:The role of autophagy induced by hypoxia on BBB integrityObjective:To investigate the role of endothelial autophagy on BBB integrity.Methods:Autophagy inhibitors or activators were combined with hypoxia to treat endothelial cells or zebrafish to observe the effect of autophagy on its permeability.The effects of autophagy on scavenging reactive oxygen species(ROS),Cldn5 localization and apoptosis were performed by immunological technique and flow analysis.Results:Autophagy induced by short-term hypoxia maintained BBB function by inhibiting the redistribution of Cldn5 and scavenging the aggregated Cldn5.However,the prolonged hypoxia-induced autophagy was identified to enhance the permeability of BBBConclusion:Autophagy induced by short-term(not long-term)hypoxia protected the integrity of BBB.Part 4:The role of autophagy induced by starvation in the integrity of endothelial barrierObjective:To investigate the effect and mechanism of autophagy on endothelial barrier integrity under starvation.Methods:This study analyzed the relationship between Akt-mTOR-p70S6K signaling pathway and the serum starvation-induced autophagy by western blot.Furthermore,the role of autophagy on the endothelial barrier function and the localization of Cldn5 were performed by immunological technique and super-resolution optical imaging.Results:Under serum starvation,autophagy was activated by inhibition of phosphorylation of Akt,mTOR and p70S6K,and showed a role in maintaining the structure and function of endothelial barrier.The degradation of Cldn5 was mediated by autophagy.Conclusion:Autophagy induced by serum starvation protected the endothelial barrier by the degradation of the aggregated Cldn5 in cytosol.