Effects And Mechanisms Of Fut8 On The Macrophage-derived Foam Cell Migration

Atherosclerosis（AS）is an inflammatory disease of the medium and large-s ize arteries,characterized by lipid accumulation in the arterial wall.Capacity of macrophage foam cell to emigrate seems to be impaired in atherosclerotic plaques,contribute to build-up of necrotic pools and atherosclerosis plaques.More than half of all proteins are glycosylated,which play important roles in normal physiological processes,including protein folding,stability and function.Previous research revealed that knock out the glycosyltransferases in mice could reduce atherosclerotic plaques size and reduce inflammatory cell content when fed with high fat diet.The relationship between glycosylation and the diminished capacity of foam cell migration remains unknown.In the current study,lysophosphatidic acid would be used to induce the foam cell formation from Raw264.7 macrophages and mouse peritoneal macrophages.The foam cell migration was evaluated by Transwell and scratch assays.The changes of glycosylation and glycosyltransferases level before and after foam cell formation were assessed by flow cytometry,immunofluorescence and Western Blot.The glycosyltransferases,which had the most significant relationship with foam cell migration,was observed and the detailed regulation mechanism was further discussed using dual-luciferase assay,EMSA and Chip.In our experiments,we found that LPA promoted foam cell formation,using 200μM for 24 h.The downregulation of Fut8expression decreased the mobility of foam cell in vitro.Through the animal atherosclerosis model,we found that the level of Fut8 was significant increased in the regression of atherosclerosis plaque.And the combination of HNF1αand Fut8 promoter is the reason for the decrease of Fut8expression induced by LPA.Our results suggested that Fut8 might be an important mediator for the changes of foam cell mobility and provide a new drug therapeutic target for atherosclerosis.Part1 Lysophosphatidic acid directly induces foam cell from Raw264.7macrophagesAims:To build a new foam cell model,high concentration of LPA was used to induce the transform from Raw264.7 macrophages to foam cells and the detailed mechanism had been discussed.Methods:1.CCK8 assay was used to verify the viability of Raw264.7macrophages after treatment with LPA.2.Oil red O staining and cholesterol/cholesterol ester quantitation assays were used to detect the lipid droplets in the cytoplasm and contents of intracellular cholesterol ester after treated with LPA,respectively.3.To assess the effects of LPA on lipid transport,we utilized Western Blot and RT-PCR to observe the total proteins and mRNA expression of lipid uptake,efflux and Cholesterol synthetase in the Raw264.7macrophages after treatment with LPA.4.Activation of LPA₁ and LPA₃ receptors(LPA_1,3 receptors)were observed and receptor inhibitor was used to detect the effect on foam cell formation.5.Activation of AKT signal pathway was detected and the effects of AKT inhibitor on foam cell formation and expression of lipid transport receptors were assessed.Results:1.Cell viability was not significantly affected by LPA treatment up to a concentration of 200μM for 24 h.2.200μM LPA for 24h significantly increased the number ofintracellular lipid droplets and the content of cholesterol ester in a dose and time dependent manner.These results demonstrated that LPA significantly promoted foam cell formation at 200μM for 24 h.These results demonstrated that LPA s ignificantly promoted foam cell formation at 200μM for 24 h.3.The results of WB and RT-PCR shown that the uptake receptor SRA was upregulated and the efflux receptor ABCA1 and SRBI were downregulated while the cholesterol biosynthesis rate-limiting enzyme（HMGCR）was not influenced.These results indicated that LPA might break the imbalance between lipid uptake and efflux to promote lipid retention.4.The results of WB and RT-PCR analysis showed that the expressions of LPA₁ and LPA₃ receptors were activated.In the presence of 10μM of Ki16425(LPA_1,3 receptors inhibitor),the increased numbers of lipid droplets induced by LPA were significantly inhibited.These data indicated that the activated LPA_1,3 receptors were involved in foam cell formation.5.Treatment with LPA increased the phosphorylation of AKT in Raw264.7 macrophages,which peaked at 60 min after LPA administration.However,the LPA-induced phosphorylation of AKT was significantly decreased in the Ki16425-pretreated cells.It indicated that LPA activated AKT signal pathway by its receptors.6.Treatment with MK2206（AKT inhibitor）,phosphorylation of AKT was attenuated.Pretreated with the MK2206 for 1 h,the numbers of lipid droplets were decreased significantly and SRBI expression in Raw264.7macrophages had a reverse effect.Conclusion:Our results suggested that LPA-enhanced foam cell formation was mediated by LPA_1,3-AKT activation and subsequent SRBI expression.Part2 Effects of Fut8 on the macrophage-derived foam cell migrationAims : The changes of glycosyltransferases and glycosylation were detected and the relationships between the decreased mobility of foam cell and downregulation of Fut8 expression were illuminated.Methods:1.Wound healing assay and transwell assay were utilized to detect the foam cell migration.2.After treated with LPA,the most significant change of glycosylation and glycosyltransferases were detected by WB,RT-PCR,flow cytometer,lectin-blot and immunofluorescence staining.3.After knock-down Fut8 with s i RNA in Raw264.7 macrophages,wound healing assay and transwell assay were utilized to detect the foam cell migration.4.After overexpression Fut8 with plasmid transiently transfection in Raw264.7 macrophages,wound healing assay and transwell assay were utilized to detect the foam cell migration.5.After extraction of mouse peritoneal macrophages,the key steps were repeated.Results:1.The result of transwell assay revealed that the numbers of migrated cells per field in LPA-treated group were less than that in the control group;the result of wound healing assay revealed that the speed of cell migration was much slower in the LPA-treated group.Consistently,we observed fewer cells migrated to the lower chamber and slower speed of cell migration when mouse peritoneal macrophages were treated with LPA,indicating that the capacity of foam cell migration was significantly decreased.2.The mean fluorescence intensity（MFI）of SNA（α-2,6 sialylation）and MAL I（α-2,3 sialylation）in Raw264.7 macrophages were not significantly altered after LPA treatment.It means that LPA did not alter total protein s ialylation.The results of flow cytometer showed that the MFI of AAL（total fucosylation）had a decrease trend after LPA treatment.Also,we observed the similar result by AAL-lectin blot.These results demonstrated that total protein fucosylation levels were notably decreased after LPA treatment.3.Expression of Fut7 and Fut8 m RNA were significant decreased in LPA-treatment group.The results of flow cytometer showed that the MFI of LCA（α-1,6 fucosylation）was decreased while the MFI of LTL（α-1,3/4 fucosylation）had no obvious change in LPA-treatment group.So in the next experiment the changes of α-1,6 fucosylation would be focused.Consistently,we repeated the WB and RT-PCR experiments on the peritoneal macrophages.The protein and m RNA level of Fut8 were decreased after treated with LPA.Together,these results demonstrated that Fut8 protein and total protein α-1,6 fucosylation levels were inhibited in the process of foam cell formation.4.Pretreated with si RNA followed by LPA,there was less migration cells on the underside and speed of cell migration were much slower compared with the LPA group.We also verified the transwell assay on the peritoneal macrophages.As expected,the number of migration cells on the underside in the si RNA and LPA group was lesser than that in the LPA group.5.Pretreated with the pc DNA3.1-Fut8 plasmid,we found that the number of migrated cells on the underside was much more than that in LPA group.Speed of cell migration was faster after restoring Fut8 expression.The result of transwell assay in peritoneal macrophages was consistent with the Raw264.7 macrophages.Together,these results provided the evidence that dysfunction of Fut8 had a direct relation with migration capacity of foam cell induced by LPA.Conclusion: Diminished capacity of foam cell migration is related with the downregulation of Fut8 in the progress of foam cell formation.Part3 Relationship between Fut8 expression and the regression of atherosclerosis plaque in vivoAims: To verify the effect of Fut8 expression on the regression of atherosclerosis plaque in vivo.Methods:1.Male Apo E^-/-mice（6-weeks-old;n=18）were randomly divided into three groups: Baseline group（n=6）,Control group（n=6）and Rosuvastatin group（n=6）.All 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.The other two groups were fed on the Western diet for the next four weeks.In the meantime,the mice in Rosuvastatin group and Control group were given rosuvastatin（10 mg/kg/day,diluted in saline）or saline every day by gavage,respectively.Serum lipid levels（TC,TG,LDL-C and HDL-C）were analyzed by automatic analyzer.Aortic root cryosection from each mouse was stained with Oil-red O and H&E.2.Expression of Fut8 in the aortic root was detected by immumohistochemical staining.Results:1.TC level was extremely high in the control group（nearly 10 mmol/L;normal,2.07 mmol/L）;whereas the rosuvastatin-treated group had lower TC level.Importantly,the rosuvastatin-treated group reduced the TG and LDL-C levels compared with the control group.Results of H&E staining of aortic valve demonstrated that the intima thickness were thicker in the control group compared with baseline group,whereas the intima thickness was decreased in the rosuvastatin group.Interestingly,the results of Oil red O staining revealed that neutral lipid contents（expected to be primarily cholesteryl esters）were also decreased in rosuvastatin group.Thus,rosuvastatin promoted the regression of atherosclerosis plaque in vivo.2.Compared with the control group,the expressions of Fut8 were up-regulated in the rosuvastatin group.Conclusion: The expression of Fut8 had a negative relationship with the progress of atherosclerosis plaque.Part4 Molecular mechanism of downregulation of Fut8 expressionAims : Through Raw264.7 macrophages and 293 T cells,the detailed mechanism of Fut8 downregulation was further illuminated.Methods:1.WB and RT-PCR were used to observe the effects of LPA1,3 receptors on Fut82.Protein translation and m RNA synthes is inhibitors（Cycloheximide,CHX;Actinomycin D,AMD）were used to definite the regulation effects of LAP on Fut8 gene transcription.3.Dual-luciferase Assay was performed to verify the effect of LPA on Fut8 promoter.A series of truncated Fut8 promoter luciferase reporter vectors were constructed and identified And they were transfected into 293 T cells to observe the possible region where have relationship with transcription factor.4.Changes of HNF1α expression was detected by WB and PCR.5.EMSA and Chip assays were used to define the directly relationship between HNF1α and Fut8 expression.6.After overexpression HNF1α,expression of Fut8 was detected.Results:1.Pretreated with 10 μM Ki16425 for 1 h,the expression of Fut8 protein and α-1,6 fucosylation levels had a significant rise compared with the LPA-treated group.The MFI and m RNA of Fut8 also had reverse effects.Together,these results concluded that the down-regulation of Fut8 expression induced by LPA was mediated by its LPA1,3 receptors.2.Pretreated Raw264.7 macrophages with CHX to inhibit protein translation,combination of CHX and LPA did not change the Fut8 protein levels compared to CHX treatment only,suggesting that the LPA mediated decrease in Fut8 protein expression was not due to decreased stability of the protein but the process of synthesis.Next,AMD was used to test Fut8 m RNA stability.Combination of AMD and LPA did not decrease the Fut8 m RNA level compared to AMD treatment only,even had an increase trend.These results demonstrated that LPA could affect the transcriptional level of Fut8 not the degradation.3.To explore the mechanism of decreased transcriptional level,we test the promoter activity of Fut8.The reporter gene p GL3-Basic harboring the promoter region（-2095/-7）of mouse Fut8 was conducted and luciferase activity was measured to estimate promoter activity.The luciferase activity was decreased significantly in the LPA-treated group in 293 T cells.To demonstrate the most important functional region in Fut8 promoter,293 T cells were co-transfected with a series of deletion constructs spanning the upstream region of the mouse Fut8 gene.A deletion from-611 to-431 bp had an apparent reverse of Fut8 promoter activity,suggesting that some element required for Fut8 promoter activity lie in the region between-611 and-431 bp.4.The whole cell lysate was obtained and the result of WB showed that the protein expression of HNF1α was decreased in the LPA-treated group.Cytoplasmic protein and nuclear protein were extracted by Nuclear and Cytoplasmic Protein Extraction Kit,respectively.Cytoplasmic protein level of HNF1α was decreased and nuclear protein level of HNF1α had more obvious decrease trends in LPA-treated group.5.To further confirm the direct binding of HNF1α and Fut8 promoter,EMSA and Chip assay were performed in Raw264.7 macrophages.The result of EMSA shown that the biotin-DNA probe had a weaker bond with HNF1α antibody after LPA treatment.And the result of Chip shown that the combination between HNF1α antibody and Fut8 promoter was significant decreased after LPA treatment.These results indicated that LPA decreased the binding of HNF1α with the Fut8 promoter.These results indicated that LPA decreased the binding of HNF1α with the Fut8 promoter.6.Overexpression of Fut8 in Raw264.7 macrophages was done by p Receier-M29 Expression Clone.The protein and m RNA expression of Fut8 had a reverse effect in overexpression group compared with LPA only.Conclusion: HNF1α had a positive effect on the Fut8 promoter region and it was weaken after treatment with LPA.