UNC5b Fucosylation Suppressed Macrophage Derived Foam Cells To Emigrate Endangium And Promote Atherosclerosis

Foamed macrophage retention plaque had become a hallmark event in atherosclerotic damage.In our study,ox-LDL induced macrophages foaming.Afterwards,the netrin-1,a member of neuronal guidance cue and its receptor UNC5 b were up-regulated in foamed macrophages,and it caused the reduction of the the migration ability of foam cells.These foam cells in the plaque contributed to the development of atherosclerosis.However,it is unclear how netrin-1and UNC5 b impaired the migrating ability of macrophage derived foam cells.UNC5 b as glycosylated membrane protein,foam of macrophages regulated its fucosylation level.Previously,foam of macrophages regulated the fucosylation level of UNC5 b.In this study,the techniques of glycobiology and molecular biology was employed to analysis effect of UNC5 b fucosylation on the migration of foam cells.Furthermore,clarify the relationship between UNC5 b glycosylation and the motor capacity of macrophage derived foam cells,and the pathway of macrophage derived foam cells emigrate endangium to promote plaque regression.To provide a new therapeutic target and idea for further elucidation of the inflammatory inducing mechanism of atherosclerosis and treatment of vascular inflammatory injury disease.Part1 Alternative expression of UNC5 b and migration after treatment of macrophages with ox-LDL to form foam cellsAims:Raw264.7 macrophages and peritoneal macrophages were used as the research objects.Ox-LDL induced foam cell model was used to study whether the neuronal guidance cue receptor UNC5 b could inhibit the migration of macrophages and establish a follow-up experimental cell model.Methods:1.Cholesterol ester quantitation assays/oil red O staining and cholesterol were used to detect the lipid droplets in the cytoplasm and contents of intracellular cholesterol ester after treated with ox-LDL respectively.2.Transwell assay and wound healing assay were utilized to detect the foam cell migration.3.The UNC5 b and netrin-1 expression in Raw264.7 cells was detected by western blot,RT-PCR and immunofluorescence staining after treatment with ox-LDL.4.Pretreated with si UNC5 b or UNC5b-p CMV6-Entry plasmid in Raw264.7 macrophages for silencing and overexpressing UNC5 b.The effect of UNC5 b expression on the migration of foam cells was detected.Results:1.50 ?g/ml ox-LDL for 24 h significantly increased the number of intracellular lipid droplets and the content of cholesterol ester in a dose and time dependent manner.These results demonstrated that ox-LDL significantly promoted foam cell formation at 50 ?g/ml for 24 h.2.The result of transwell assay revealed that the numbers of migrated cells per field in ox-LDL-treated group were less than that in the control group(p<0.05);the result of wound healing assay revealed that the speed of cell migration was much slower in the ox-LDL-treated group(p<0.05).3.To determine whether ox-LDL affects the level of UNC5 b expression,the UNC5 b protein and m RNA levels in Raw264.7 cells were analyzed by western blot and real-time RT-PCR.The UNC5 b protein and m RNA level increased in response to raised concentrations and prolonged treatment with ox-LDL(p<0.05).The same results were observed after primary peritoneal macrophages were treated with ox-LDL(p<0.05).4.Pretreated Raw264.7 macrophages with CHX to inhibit protein translation,combination of CHX and ox-LDL did not change the UNC5 b protein levels compared to CHX treatment only,suggesting that the ox-LDL mediated increase in UNC5 b protein expression was not due to decreased stability of the protein but the process of synthesis.Next,AMD was used to test UNC5 b m RNA stability.Combination of AMD and UNC5 b did not increase the UNC5 b m RNA level compared to AMD treatment only.These results demonstrated that ox-LDL could affect the transcriptional level of UNC5 b.5.Dual-luciferase Assay was performed to verify the effect of ox-LDL on UNC5 b promoter.We investigated whether NF-k B contributes to the upregulation of UNC5 b.Using UNC5 b promoterluciferase reporter gene,we demonstrate that UNC5 b promoter activity was induced by ox LDL and this was reduced by the NF-k B inhibitor BAY 11-7082 Collectively,these data demonstrate that ox-LDL of Raw264.7 with increases expression of UNC5 b.6.Pretreated with si UNC5 b or UNC5b-p CMV6-Entry plasmid in Raw264.7 macrophages,the migration of macrophages by transwell assay and wound healing assay.Pretreated with the UNC5b-p CMV6-Entry plasmid followed by ox-LDL,there was less migration cells on the underside and speed of cell migration were much slower compared with the ox-LDL group(p<0.05).Pretreated with the si UNC5 b,we found that the number of migrated cells on the undersidewas much more than that in ox-LDL group(p<0.05).Cell migration was faster after knockdown UNC5 b expression.Conclusion:Ox-LDL induced macrophags to form foam cells activation,activated NF-? B pathway,enhanced UNC5 b promoter activity and enhanced UNC5 b expression,leading a decrease in migration migration after macrophags to form foam cells.Part2 Fucosylation of UNC5 b are correlated with the migration of foam cellsAims:The changes of glycosyltransferases and glycosylation were detected and the relationships between the decreased mobility of foam cells.UNC5 b fucosylation on the migration of foam cells.Methods:1.After treated with ox-LDL,change of glycosylation and glycosyltransferases were most significantly.2.Test the fucosyltransferase(FUT)expression in Raw264.7 cells by western blot and RT-PCR after treatment with ox-LDL.3.The UNC5 b fucosylation in Raw264.7 cells was detected by western blot,RT-PCR,immunofluorescence staining,IP and point mutation assay after treatment with ox-LDL.7.Transwell test and scratch test were used to detect the effect of FUT8 expression induced by ox-LDL on macrophage foam.Results:1.The results of lectin blot showed that the AAL(total fucosylation)and LCA(?-1,6 fucosylation)had a enhanced trend after ox-LDL treatmentfoam of macrophages regulated the ?-1,6fucosylation level of UNC5 b.Follow up experiments will focus on the relationship between UNC5 b ?-1,6 fucoylation and FUT8 and the migration ability of foam macrophages.2.To determine whether ox-LDL affects the level of UNC5 b expression,the UNC5 b protein and m RNA levels in Raw264.7 cells were analyzed by western blot and real-time RT-PCR.The FUT8 protein and m RNA level increased in response to raised concentrations and prolonged treatment with ox-LDL(p<0.05).3.IP assay confirms that after Raw264.7 macrophages induced by ox-LDL UNC5 b is a target protein for fucosylation modification.4.UNC5 b and FUT8 co-transfected 293 T cells,and the protein signals were located on endoplasmic reticulum,suggested that FUT8 catalyzed fucosylation of UNC5 b.Meanwhile,UNC5 b fluorescent signals were observed on cell membrane.However,although UNC5b-K0222,347 and FUT8 were located on endoplasmic reticulum,there were no UNC5b-K0222 and 347 on cell membrane.These result showed that fucosylation of UNC5 b took place in endoplasmic reticulum,and FUT8 catalyzed this modification.5.FUT8 regulates the migration of foam cells and FUT8 is related to the fucose level of UNC5 b.Conclusion:After ox-LDL treated macrophages to form foam cells,the level of UNC5 b fucosylation modification was increased by enhanced FUT8 expression,leading a decrease in migration after macrophags to form foam cells.Part3 Molecular mechanism of UNC5 b induced downregulation migration of macrophage derived foam cellsAims:Clarify the relationship between UNC5 b glycosylation and the motor capacity of macrophage derived foam cellsas well as molecular mechanism of UNC5 b induced downregulation of macrophage derived foam cells.Methods:1.The CCR7 expression in Raw264.7 cells was detected by western blot and RT-PCR after treatment with ox-LDL.2.To clarify the direct relationship between UNC5 b and CCR7,expression of CCR7,downregulation migration of macrophage derived foam cells was observed after knockdown or overexpression of UNC5 b.3.Immunofluorescence staining was used to detect the polymerization of F-actin in Raw264.7 macrophages induced by ox-LDL.4.Analyzing the activation status of downstream Rac1,CDC42 and PAK signaling pathways and the molecular mechanisms involved in cell movement.Results:1.Expression of CCR7 m RNA and protein were significant decreased in ox-LDL-treatment group(p<0.05),and downregulated migration of macrophage derived foam cells.2.Ox-LDL induced F-actin aggregation was significantly decreased during macrophage foaming.3.UNC5 b blocked the F-actin aggregation of foam macrophages by downregulating CDC42 and upregulating PAK,which reduced the migration and mobility of foam cells.Conclusion:UNC5b inhibited the migration of macrophage derived foam cells by downregulating the expression of CCR7.UNC5 b blocked the F-actin aggregation of foam macrophages by downregulating CDC42 and upregulating PAK,which reduced the migration and mobility of foam cells.Part4 The relationship between the expression of UNC5 b and the formation of atheromatous plaque in miceAims:To verify the effect of UNC5 b expression on the formation of atherosclerosis plaque in vivo.Methods:Male Apo E-/-mice(6-weeks-old;n=36)were randomly divided into three groups: Vehicle group(n=6),the Apo E-/-mice were kept on a normal diet for 12 weeks.Then,the mice in Vehicle group were sacrificed.Baseline group(n=6),the Apo E-/-mice were kept on a Western diet containing 0.15% cholesterol and 21% fat for 12 weeks.Then,the mice in Baseline group were sacrificed.Development group(n=6),the Apo E-/-mice were kept on a Western diet containing 0.15%cholesterol and 21% fat for 16 weeks.Then,the mice in Development group were sacrificed.Ad-NC group(n=6),Ad-sh group(n=6)and Ad-EX group(n=6),The other three groups were kept on a Western diet containing 0.15% cholesterol and 21% fat for 12 weeks.In the meantime,the mice in Ad-NC group,Ad-sh group(n=6)and Ad-EX group were given adenovirus injection into caudal vein for four weeks.Serum lipid levels(TC,TG,LDL-C and HDL-C),liver function levels and serum glucose levels were analyzed by automatic analyzer.Aortic root cryosection from each mouse was stained with Oil-red O,H&E,Immunofluorescence staining and immunohistochemical staining.Results:1.Compared to control forage,level of TC,TG and LDL-C was significantly higher in other group mice.The histology staining showed that intima in Aortic root and arch was significantly thinner in sh UNC5 b group.Oil O staining showed that plaque in aortic root increased when UNC5 b was overexpressed.On the contrary,UNC5 b knockdown decreased plaque.These results revealed that development of atherosclerosis was closely correlative with level of UNC5 b.2.Immunohistochemical staining showed a positive correlation between CD68 content and UNC5 b expression.3.Immunofluorescence staining showed that UNC5 b and FUT8 were positively correlated with atherosclerotic plaque formation.Conclusion:The expression of UNC5 b had a positive relationship with the progress of atherosclerosis plaque.