Atg7 Regulates The Expression Of Proinflammatory Cytokines Of Endothelial Cells And Is Involved In The Mechanism Of Ischemic Stroke

Background and ObjectiveIschemic stroke is caused by cerebral artery occlusion and insufficient blood flow and accounts for 87%in all stroke.Ischemia/reperfusion injury is caused by the recovery or reacquisition of blood supply in the tissue cells.A variety of causes contribute to ischemic injury such as inflammatory response,excitotoxicity,Ca²⁺and Zn²⁺overload,spreading depolarization and released free radicals.These causes can lead to negative consequences,such as endothelial cells of the blood-brain barrier damage,immune cell infiltration and neuronal apoptosis,etc.Inflammatory response is the main event that occurs after brain injury.It can not only increase the ischemia/reperfusion injury,but also help to promote the recovery after injury.In the early stages of inflammatory response,released proinflammatory cytokines and harmful free radicals can accelerate cell damage.When BBB integrity is compromised in diseases or injury states,proinflammatory factors produced within the injured brain may cross the BBB to attract circulating immune cells,pathogens,and toxins in the blood,they may penetrate the brain parenchyma and elicit a secondary injury cascade.The blood-brain barrier（BBB）is a highly specialized neurovascular unit,which is comprised by brain microvascular endothelial cells,pericytes,astrocytes and microglial cells.The barrier constitutes the interactional structure in the regulation of blood materials in and out of the brain,and can keep the brain microenvironment homeostasis.A literature reported that BBB rupture may be a cause rather than a consequence of parenchymal cell injury.With the discovery and more and more research of autophagy process,the important role of autophagy related protein 7 has been widely concerned.Atg7 plays important roles in autophagic and non autophagic processes.Atg7,E1-like activating enzyme,is the core protein of the autophagic ubiquitin system,so Atg7 has always been a hub of research.In order to reveal the function of Atg7,the conditioned knockout mice with different tissues Atg7 have been widely studied.Pharmacological inhibitors of autophagy or knockdown of the essential autophagy genes Atg7 inhibits the in vitro secretion of VWF.Mice with endothelial-specific deletion of Atg7 exhibit impaired epinephrine-stimulated VWF release,reduced levels of high–molecular weight VWF multimers and a corresponding prolongation of bleeding times.This is a promising strategy for transient prevention of thrombosis.Moreover,Atg7-null endothelial cells also confer susceptibility to bleomycin-induced pulmonary fibrosis in vivo by endothelial-to-mesenchymal transition（EMT）.In our lab,we found that the microvessel density in mice brains with endothelial-specific knockout of Atg7（Atg7 eKO）was significantly decreased compared to wild-type littermate control.Consistently,in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells.Further results indicated that knockdown of Atg7 reduced interleukin-6 expression in brain microvascular endothelial cells,which is mediated by NF-κB dependent transcriptional control.Interestingly,exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown.These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.As brain tissue is very sensitive to ischemia and hypoxia,how to protect the function of cerebral tissues against ischemic insult has been a hot topic in the study of cerebrovascular diseases for a long time.In this work,we mainly focused on the functions and mechanisms of endothelial-specific knockout of Atg7 in ischemic stroke.Methods1.The knockout of Atg7 in the endothelial cells resulted in decreased cerebral infarction area and improved neurological behavioral changes in mice with ischemia/reperfusion.The expressions of Atg7 in endothelial cells of Atg7 eKO and WT mice were detected by Immunohistochemistry-frozen section（IHC-Fr）.The resistance of Atg7 knockout in the endothelial cells to cerebral ischemia/reperfusion injury was detected by transient focal cerebral ischemia（tFCI）model and TTC staining.The different effects of cerebral ischemia/reperfusion injury on Atg7 eKO and WT mice were investigated by using the neurological deficit scoring method and motor deficiencies tests.2.Decreased apoptotic neurons and fewer infiltration of neutrophils into brain parenchyma were detected in the Atg7 eKO mice after cerebral I/R injury.The apoptotic cells of Atg7 eKO and WT mice were examined by TUNEL.The infiltration of neutrophils in Atg7 eKO and WT mice were detected by IHC with a FITC labeled Ly6B.2⁺antibody.3.The knockout of Atg7 in endothelial cells in vivo resulted in the lower pretein level of proinflammatory cytokines（IL-6,IL-1β,IL-8 and TNF-α）in the cerebral striatal or cortical homogenates.The bilateral cerebral striatal or cortical protein homogenates of Atg7 eKO and WT mice were prepared after tFCI injury.Furthermore,the protein levels of proinflammatory cytokines（IL-6,IL-1β,IL-8 and TNF-α）were detected by ELISA.The mRNA levels of proinflammatory cytokines（IL-6,IL-1β,IL-8 and TNF-α）of contralateral or ipsilateral cerebral tissue of striatun or cortex after tFCI injury were detected by real-time PCR.4.The knockdown of Atg7 reduced the secretion and intracellular protein level of proinflammatory cytokines in HBMECs.The establishment of stable cell lines with knockdown of Atg7 in HBMECs by the specific small hairpin RNA sequences targeting human Atg7 was constructed.A non-silencing shRNA sequence was used as a contol.The oxygen-glucose deprivation/reoxygenation model in vitro mimics cerebral ischemia/reperfusion injury model.The secretion and intracellular protein level of proinflammatory cytokine（IL-6,IL-1β,IL-8 and TNF-α）of Atg7 knockdown and control cell lines under normoxia and hypoxia were determined by ELISA.Under normoxia and OGD4hr/R24hr,the relative mRNA expression of proinflammatory cytokine（IL-6,IL-1β,IL-8 and TNF-α）of Atg7 shRNA and non-silencing shRNA cell lines were detected by real-time PCR.5.In normoxia and hypoxia,Atg7 regulates the production of proinflammatory cytokines（IL-6,IL-1β,IL-8 and TNF-α）through NF-κB signaling pathway rather than HIF-1α.The phosphorylations of IKKβand IκBαof Atg7 knockdown cell lines and controls were detected by WB under OGD4hr/Re24hr,normoxia used as a control.The translocation of p65 in Atg7 knockdown was detected by WB and IF in normoxia and hypoxia.The cells were incubated with NF-κB agonist betulinic acid（10μg/mL）for 24hr.The mRNA levels of IL-6,1β,8 and TNF-αin the HBMECs transfected with Atg7shRNA were determined by real-time PCR,with HBMECs transfected with non-silencing shRNA as a control.The total protein level and nuclear HIF-1αof Atg7 shRNA were determined by WB and IF in normoxia and hypoxia,with non-silencing shRNA as a control.6.The specific expressions of proinflammatory factors by Atg7 via NF-κB pathway.The protein and mRNA expressions of IL-6,1β,8 and TNF-αof Atg7 siRNA transfected HBMECs were determined by ELISA and real-time PCR.The abilities of p65 binding the promoter regions of IL-6,1β,8 and TNF-αwere detected by ChIP-qPCR respectively in Atg7 siRNA transfected HBMECs.The influences of pharmacological inhibitors（3-MA and chloroquine）of autophagy to the expression of proinflammatory factors were measured.7.Summary model of the down-regulation of proinflammatory factors in the HBMECs with Atg7 shRNA via IKKβ-IκBα-NF-κB-p65 pathway.Draw the summary model.Results1.Endothelial-specific knockout of Atg7（Atg7 eKO）mice showed decreased infarct volume and superior functional recovery than WT littermate controls.Atg7 eKO mice do not show the protein expression of Atg7 in the endothelial cells,which is obvious in ECs of WT littermate mice.After cerebral ischemia/reperfusion injury,the volume of cerebral infarction in Atg7eKO mice was smaller than that of WT mice.After cerebral ischemia/reperfusion injury,Atg7 eKO mice showed superior motor function outcomes.2.Atg7 eKO mice showed reduction in neuronal apoptosis and reduction in neutrophils invasion.Atg7 eKO mice demonstrated decreased number of TUNEL positive cells than WT littermate mice in brain cortex after ischemic insult.Atg7 eKO mice demonstrated decreased number of Ly6B.2 positive cells infiltrating in brain parenchyma from circling blood than WT littermate mice after ischemic insult.3.Endothelial-specific knockout of Atg7 in vivo contributed to the decrease of proinflammatory factors in cerebral striatum or cortex.After cerebral ischemia/reperfusion injury,the protein levels of proinflammatory factors such as IL-6,IL-1β,IL-8 and TNF-αin the striatum or cortex of Atg7 eKO mice were lower than that of WT mice.After cerebral ischemia/reperfusion injury,except for IL-8/CXCL-15,the mRNA relative expression levels of proinflammatory factors such as IL-6,IL-1βand TNF-αin striatum or cortex of Atg7 eKO mice were also lower than that of WT mice.4.In the human brain microvascular endothelial cells,Atg7 knockdown can reduce the expression of proinflammatory cytokines.Construct Atg7 shRNA and non-silencing shRNA cell lines and determine the interference efficiency of Atg7.In normoxia and hypoxia,Atg7 knockdown in HBMECs can reduce the secretion or intracellular expressions of proinflammatory cytokines such as IL-6,IL-1β,IL-8 and TNF-α.Atg7 knockdown in HBMECs can lead to lower mRNA relative expressions of proinflammatory factors such as IL-6,IL-1β,IL-8 and TNF-αthan non-silencing shRNA in HBMECs.5.Atg7 regulates the production of proinflammatory cytokines in HBMECs via NF-κB signal pathway,but not transcriptional factor HIF-1αpathway both normoxia and hypoxia.Atg7 knockdown in HBMECs leads to the decreases of the phosphorylations of inhibitory factor IKKβand IκBαof the NF-κB upstream under normoxia and OGD4hr/R24hr.At the same time,the nuclear translocation of p65,a functional subunit of NF-κB,is decreased by Atg7 knockdown.Betulinic acid,a kind of NF-κB signaling pathway activator,increased the relative expressions of the proinflammatory factors IL-1β,IL-6 and IL-8 mRNA,but had no effect on the expression of TNF-α.In normoxia and OGD4hr/R24hr,a decrease in the expression of proinflammatory factors with Atg7 knockown had no relevance to HIF-1α.6.Atg7 is specific to increase the expressions of proinflammatory factors via NF-κB signaling pathway.Atg7 siRNA in HBMECs can mimic the influences of stable transfected Atg7 in HBMECs to proinflammatory cytokines IL-1β,IL-6,IL-8 and TNF-α.The ChIP-qPCR of the transcriptional factor p65 to the promoter DNA of pronflammatory factors further proved the above results.The recruitments of p65 to the promoter regions of IL-1β,IL-6,IL-8 and TNF-αwere decreased in siAtg7 transfected HBMECs than in control siRNA.Autophagy inhibitor,3-MA and chloroquine cannot influence the mRNA expression of IL-1β,IL-8 and TNF-α,but they can increase the mRNA expression of IL-6.7.Summary model of that Atg7 regulates the production of proinflammatory cytokines in HBMECs by NF-κB dependent pathway.In HBMECs,when ischemia or OGD occurs,Atg7 can regulate the expressions of proinflammatory cytokines such as IL-1β,IL-6,IL-8 and TNF-αby NF-κB signaling pathway,and promote neuronal apoptosis and neutrophil recruitment and infiltration.ConclusionsAtg7 eKO mice have shown resistance to brain demage after transient ischemia caused brain ischemia/reperfusion injury.Atg7 eKO mice demonstrated superior functional recovery and showed significantly reduced infarct volumes after tMCAO compared with WT littermate mice.The number of TUNEL positive neurons in brain cortex of Atg7 eKO mice was reduced,and a smaller number of Ly6B.2⁺positive neutrophils had infiltrated into the brain parenchyma in Atg7 eKO mice.In vitro experiments,Atg7 knockdown in HBMECs decreased the secretions and protein levels of proinflammatory factors under OGD/Re,which was consistent to in vivo tFCI.Atg7 regulates the expressions of proinflammatory cytokines via IKKβ-IκBα-NF-κB-p65 signaling pathway,which has no relevance to HIF-1α,a transcriptional factor response to hypoxia.The specificity of NF-κB signaling pathway regulated by Atg7 cannot be mimiced by autophagy inhibitors.