Mechanism And Significance Of β-amyloid Beta Up-regulating CCR5 Expression In Human Brain Microvascular Endothelial Cells

PurposeAlzheimer's disease(AD) is the most common cause of dementia in the elderly, and it is the fourth leading cause of death in the United States.Its clinical features include progressive dementia with gradual loss of cognitive function.Its main neuropathological features include neurofibrillary tangles,senile plaques,and loss of neurons and synapses.The main component that deposits in senile plaques and brain vascular is amyloid beta(Aβ),composed of 39-43 amino acids peptide,and it is generated through amyloid precursor protein shearing.The accumulation of Aβis thought to be an early feature of AD.Vascular disfunction in brain can promote gradual loss of cognitive function and happen of dementia.The blood-brain barrier(BBB) is composed of brain microvascular endothelial cell,inner basement membrane and neuroglia membrane.The BBB is a physical and metabolic barrier between the brain and the systemic circulation,which functions to protect the brain from circulating drugs,toxins,and xenobiotics.The human brain microvascular endothelial cell that is separated and cultured in vitro becomes the ideal object for the study of BBB.Recently,more and more evidences show that the mechanism of inflammation plays the crucial role in the pathogenesis of AD.Chemokine belongs to a family of cytokines,the primary function of which is recruitment of leukocytes to the site of inflammation.Chemokine receptor5(CCR5) is one of chemokine receptors,and immunohistochemical studies show increased expression of CCR5 on reactive microglia associated with amyloid deposits in AD,suggesting that CCR5 may play the role in the regulation of the immune response.In previous studies,we found that Aβcan upregulate the expression of CCR5 protein inHBMECsS,but its mechanism and significance are not clear,and this research is ready to answer the above problem.BBB transports Aβthrough receptor for advanced glycation end products(RAGE) and low-density lipoprotein receptor-related protein 1(LRP-1).RAGE is a multiligand receptor in the immunoglobulin(Ig) superfamily of cell surface molecules,which interacts with Aβ,a key protein in AD.RAGE mediates transcytosis of plasma-derived Aβacross brain endothelium in vivo.In AD,RAGE protein expression in brain is increased,and then Aβinto brain is increased and deposited,which is related with pathogenesis.Recently,additional RAGE isoforms that encode several truncated forms of RAGE lacking the transmembrane region and the cytosolic tail were identified,and these isoforms can combine with ligands of RAGE,and then inhibit the combination between RAGE and its ligands,and finally prevent the signal transduction of ligand-receptor axis.How does Aβupregulate CCR5 expression inHBMECs? Does RAGE take part in the upregulation of CCR5? What role does the upregulation of CCR5 inHBMECs play in AD? These questions are related with the pathogenesis of AD.We hope that answers for these questions can provide some basal knowledge for investigating the pathogenesis of AD and then looking for methods to cure and prevent AD.So I did some relevant experiments to resolve above questions.Methods1.In study of signaling pathways by which Aβup-regulating CCR5 inHBMECs(1) Cell culture:HBMECs is cultured in RPMI1640,supplemented with 10% Nu-serum etc.(2) CCR5 mRNA expression is detected by Real-time PCR,whenHBMECs is administered with Aβin different doses and different times.(3) CCR5 protein expression is detected when Aβis administered in different doses and different timse by western-blot.(4) MAPK/PI3K signal molecules are detected when Aβis administered by western-blot.(5) Egr-1 protein expression is detected when Aβis administered inHBMECs by western-blot.(6) The combination of Egr-1 and CCR5 promoter is detected by chromatin immunoprecipitation method.(7) Egr-1 siRNAs are stably transfected intoHBMECs and then the stable cell line is established.(8) Then CCR5 protein expression an MAPK/AKT expression are detected when Aβis administered in transfected cells by western-blot inHBMECs that is transfected by Egr-1 siRNAs by western-blot.2.The role of RAGE in the up-regulation of CCR5 expression by Aβ(1)RT-PCR:RAGE mRNA expression is detected when Aβis administered inHBMECs.(2) Western-blot:a RAGE protein expression is detected when Aβis administered inHBMECs.b CCR5 protein expression is detected when RAGE neutralizing antibody is administered ahead of AβinHBMECs.(3) The construction of eukaryotic expression plasmid and the establishment of HBMEC ^RAGE+cell line by stable transfection.a.The construction of pUCmT -RAGE plasmid.b.The construction of eukaryotic expression plasmid.c.Stable transfection.(4) CCR5 protein expression is detected when Aβis administered inHBMECsS^RAGE+ by western-blot. (5) The construction of eukaryotic expression plasmid and the establishment of HBMEC^{C-truncated RAGE} and HBMEC^{N-truncated RAGE} cell line by stable transfection(6) CCR5,p-JNK,p-ERK and p-AKT protein expression are detected when Aβis administered in the above transfectedHBMEC by western-blot.3.The role of the up-regulation of CCR5 expression in T lymphocytes transmigration into brain(1) The up-regulation of CCR5 expression by Aβis related with T lymphocytes transmigration into brain by Transwell method.a.T lymphocytes of AD patients,6T-CEM and Jurkat transmigrated through normalHBMECsS monolayer that is administered to Aβin different doses.b.T lymphocytes of AD patients and 6T-CEM transmigrated through HBMEC^CCR5- andHBMEC^CCR5+ monolayer.c.T lymphocytes of AD patients and 6T-CEM transmigrated throughHBMECsS^CCR5- that is administered to Aβ.(2) RAGE is related with T lymphocytes transmigration into brain by Transwell method.a.T lymphocytes of AD patients and 6T-CEM transmigrated throughHBMECsS m-onolayer that is administered to RAGE neutralizing antibody ahead of Aβ.b.T lymphocytes of AD patients and 6T-CEM transmigrated through HBMEC^RAGE+ that is administered to Aβ.(3) MAPK signaling molecules are related with T lymphocytes transmigration into brain by Transwell method.a.JNK siRNAs and ERK siRNAs were transiently transfected into HBMECs.b.JNK,ERK and CCR5 expressions are detected by western-blot in the above transfected HBMECs.c.T lymphocytes transmigrated through the above transfectedHBMECs monolayer that is administered to Aβ.(4) PI3K/AKT signaling molecule is related with T lymphocytes transmigration into brain by Transwell method.a.CCR5 expression is detected by western-blot in PI3K/△P110HBMECs.b.T lymphocytes of AD patients and 6T-CEM transmigrated through PI3K/△P110HBMECs monolayer that is administered to Aβ.(5) Egr-1 is related with Y lymphocytes transmigration into brain by Yranswell method.T lymphocytes of AD patients and 6T-CEM transmigrated through HBMECs monolayer transfected by Egr-1 siRNA when Aβis administered.Results1.CCR5 expression is up-regulated by Aβthrough MAPK/PI3K signaling pathways(1) The up-regulation of CCR5 expression depends on time and dose of AβinHBMECs.(2) JNK,ERK and AKT take part in the up-regulation of CCR5:p-JNK,p-ERK and p-AKT expressions are increased by Aβ,and sp600125,u0126 and ly294002 can inhibit the up-regulation of CCR5 induced by Aβ.(3) Egr-1 is the critical transcription factor in the process of Aβup-regulating. CCR5 expression:a.Egr-1 expression is increased by Aβin HBMECs.b.Aβcan reinforce the combination of Egr-1 and CCR5 promoter,and ly294002 and PD98059 can inhibit the combination reinforced by Aβ.c.After Egr-1 is interfered in HBMECs,A[3 can not up-regulate CCR5 expression, but MAPK and AKT can be up-regulated by Aβ.2.CCR5 expression is up-regulated by Aβthrough RAGE in HBMECs(1) RAGE expression is increased by Aβin HBMECs.(2) RAGE neutralizing antibody can inhibit the up-regulation of CCR5 induced by Aβ.(3) RAGE neutralizing antibody can inhibit the up-regulation of some cell signaling molecules including p-JNK,p-ERK and p-AKT.(4) InHBMEC^RAGE+,Aβup-regulates CCR5 expression more than in normalHBMEC.(5) InHBMEC^{C-truncated RAGE} andHBMEC^{N-truncated RAGE},Aβcan't up-regulate CCR5 protein expression,but it can up-regulate p-JNK,p-ERK and p-AKT protein expressions.3.The up-regulation of CCR5 expression promotes T lymphocytes to transmigrate into brain(1) Aβpromotes T lymphocytes of AD patients and 6T-CEM to migrate through HBMEC monolayer.(2) The number of T lyrnphocytes of AD patients and 6T-CEM that migrate through HBMEC^CCR5+ is increased and the number of those that migrate through HBMEC^CCR5- is reduced.(3) Aβfails to promote T lymphocytes of AD patients and 6T-CEM to migrate through HBMEC^CCR5-(4)RAGE neutralizing antibody can inhibit the number of transendothelial T lymphocytes of AD patients and 6T-CEM that is incresed by Aβ.(5) AD fails to promote T lymphocytes of AD patients and 6T-CEM to migrate through HBMEC^{C-truncated RAGE} monolayer and HBMEC^{N-truncated RAGE} monolayer(6) Aβfails to promote T lymphocytes of AD patients and 6T-CEM to migrate through HBMECs that are transfected by JNK siRNAs and ERK siRNAs.(7) Aβfails to promote T lymphocytes of AD patients and 6T-CEM to migrate through PI3K△P110HBMECs monolayer.(8) Aβfails to promote T lymphocytes of AD patients and 6T-CEM to migrate through HBMECs monolayer that is transfected with Egr-1 siRNAs. CONCLUSIONSIn HBMECs,Aβcombines with RAGE,and then activates MAPK and PI3K/AKT signaling molecules,and then reinforces the combination of Egr-1 and CCR5 promoter,and then up-regulates CCR5 expression,and finally contributes to the transedothelial migration of T lymphocytes into brain.