The Function Of Exosomes From Endothelial Cells In Neurovascular Unit Towards Neuronal Injury After Ischemic Stroke

BackgroundAfter the onset of ischemic stroke(IS)the pathological processes of ischemia and reperfusion injury(IRI)including excitotoxicity,oxidative stress,nitrification stress,inflammatory and apoptosis are activated which could bring irreversible damage to brain tissue.The neurovascular unit(NVU)constitutes the basic functional structure in brain in which neurons and endothelial cells are the chief components.In spatial distribution,endothelial cells which form the blood-brain barrier(BBB)are adjacent to neurons.In functional association,the communications between endothelial cells and neurons could respond to ischemic stroke and participate in the progression and recovery of neuronal injury.As a small extracellular vesicle(sEV)derived from the multivesicular body(MVB),exosomes could function as a vital mediator of the cellular communication.In the assembling process of the exosome,a variety of bioactive materials such as proteins,peptides,lipids,mRNAs,ncRNAs could be sorted into intraluminal vesicles(ILVs)in a specific manner and are eventually secreted to the extracellular space as exosomes.The exosomes outside the cells can either interact with adjacent cells through paracrine or be transferred to a distant target through blood or transcytosis.At last,exosomes could be internalized by recipient cells and function by releasing their unique cargoes into the cytosol.Compared with peptides,proteins,nucleotides directly secreted by cells,exosomes can not only protect their cargoes from degradation via their lipidic membrane but also cross some impenetrable barriers such as BBB and placental barrier easily via transcytosis.As most of the secretory compounds are blocked by pericytes,basal membrane and the end-feet of astrocytes,endothelial cells and neurons can hardly interact with each other.As a result,exosomes might be a crucial method of cellular communication between them.At present,endothelial cell-derived exosomes(EC-EXOs)have been identified to ameliorate neuronal damage from IS.However,the exact exosomal contents and mechanisms mediating the neuroprotective effects are still unknown.Elucidating these will help to improve our understanding of the pathological processes of ischemic stroke and facilitate the progress in discovering a novel therapeutic target.Objectives1.Confirm the protective function of the EC-EXOs towards IRI neurons2.Depict the compositions of EC-EXOs and determine the neuroprotective mediator among them3.Determine EC-EXOs’ neuroprotective mechanism while treating IRI neuronsMethods1.Isolation and identification of exosomesWe utilized PEG chemical precipitation protocol to isolate exosomes from the supernatant of endothelial cells and identified the characteristics of the isolated sample including marker protein expression,diameter distribution and morphological features.2.Confirm the neuroprotective function of EC-EXOs in vivoAfter the establishment of the transient middle cerebral artery occlusion/reperfusion(tMCAO/R)model on mice,we first utilized mNSS and TTC staining to confirm the successful establishment.Next,we stereotactically injected EC-EXOs to the injured site in brain and evaluated the neurological defect recovery by mNSS.Finally,we stained apoptotic cells in the injured brain to which the EC-EXOs were injected to detect the apoptosis level.3.Confirm the uptake of EC-EXOs by neurons in vitroAfter the isolation and culture of mouse primary hippocampal neurons,we first identified the neuronal characteristics of the isolated cells.Subsequently,we labeled ECEXO with fluorescent dye-DiI and incubated labeled exosomes with neurons in vitro.Finally,we determined the uptake process via live-cell imaging.4.Confirm the neuroprotective function of EC-EXOs in vitroWe first established the oxygen and glucose deprivation/reperfusion(OGD/R)model on primary hippocampal neurons.Next,we detected the apoptosis level of the injured neurons cocultured with endothelial cells or coincubated with EC-EXOs.5.Analyze the microRNA contents of EC-EXOsWe first analyzed microRNA sequence data of EC-EXOs and screened the candidate microRNAs related to the apoptosis pathway.Next,we constructed and transfected mimics of candidate microRNAs to neurons.After establishing the OGD/R model on neurons,the apoptosis level of the transfected neurons injured by OGD/R was examined.6.Verify the change of EC-EXO internalization ability in neurons after IRI in vitroAfter establishing the OGD/R model on primary hippocampal neurons,we detected mean fluorescent intensity(MFI)and the levels of marker microRNAs of EC-EXOs in neurons after incubated with EC-EXOs to evaluate internalization ability.7.Determine the association between Cav-1 and exosomes’ internalization ability in vitro and in vivoAfter establishing the IRI model on primary hippocampal neurons and on mice,we detected changes in the expression level of endocytic molecules in the brain and primary neurons.Next,we constructed and transfected siRNAs of Cav-1 to neurons.After incubated with fluorescently labeled EC-EXO,MFI and marker microRNAs in the transfected neurons were detected to evaluate internalization ability.8.Determine the regulatory role of Cav-1 in EC-EXOs’ neuroprotective functionWe first transfected Cav-1 siRNAs to primary hippocampal neurons and established the OGD/R model on neurons.After incubated with EC-EXO,the apoptosis level of transfected neurons injured by OGD/R was examined.Results1.Exosomes isolated from HUVECs’ supernatant following PEG chemical precipitation protocol matched the characteristics of typical exosomes including protein markers,diameter distribution and morphological features.The microRNA expression pattern of PEG-isolated HUVECs-derived exosomes was similar to those isolated by ultracentrifugation.2.Stereotactic injection of HUVECs-derived exosomes to the site adjacent to hippocampus injured by tMCAO/R reduced neuronal apoptotic rate in the hippocampus but failed to ameliorate defects of the neurological function.3.HUVECs-derived exosomes could be internalized by neurons in vitro.In this process,exosomes could be internalized by dendrite and transferred in neurons through anterograde transport,or by axon terminal and transferred in neurons through retrograde transport.4.HUVECs-derived exosomes reduced the apoptosis level of IRI primary hippocampal neurons in vitro significantly.miR-1290,a microRNA highly enriched in HUVECsderived exosomes,could mediate the protective effect and significantly reduce the apoptosis level of IRI primary hippocampal neurons.5.With the upregulation of Cav-1 in IRI primary hippocampal neurons,the internalization of HUVECs-derived exosomes was promoted in such neurons.Additionally,knocking down Cav-1 in neurons could attenuate the internalization.6.Knockdown of Cav-1 in primary hippocampal neurons could cancel the protective function of HUVECs-derived exosomes.ConclusionsIn conclusion,after isolating exosomes from HUVECs’ supernatant by PEG chemical precipitation protocol,we confirmed that HUVECs-derived exosomes could reduce the apoptosis level of IRI neurons via miR-1290.Furthermore,the IRI neurons could upregulate Cav-1 to enhance the internalization of exosomes.With more internalized HUVECs-derived exosomes,neurons eventually acquired an enhanced resistance to the IRI insult.Our study not only confirmed the neuroprotective role of EC-EXO and its contents,but also put forward a novel mechanism by which neurons could resist insult from IRI.These findings provide further support for the therapeutic use of exosomes and a promising intervention target in the treatment of IS.