Study On The Mechanism Of Accelerating Atherosclerotic Progression Via Inhibiting Foam Cell Migration By CML/CD36

Objective To investigate the mechanism of accelerating atherosclerotic progression via inhibiting foam cell migration by CML/CD36.Methods(1)In in vivo study,male apo E-/-mice were rendered diabetic at six weeks by intraperitoneal injection of streptozotocin(STZ,40 mg/kg/day)for five days in succession,the mice with blood glucose levels of ?300 mg/d L were considered diabetic after the STZ administration for two weeks.Then the mice were divided into four groups: control group(normal diet,n=8),model group(high fat diet,n=8),CML group(high fat diet + the tail vein injection of CML 10 mg/kg/day,n=8),and anti-CD36 group(high fat diet + the tail vein injection of CML 10 mg/kg/day + the tail vein injection of anti-CD36 antibody 100 ?g/week,n=8).The mice were euthanized after four months and performed for serology analysis(serum lipid,serum glucose,serum CML level),morphological analysis(HE staining,Oil red O staining,immunohistochemical staining or immunofluorescence staining),and molecular biology detection.(2)In in vitro study,foam cell model was formed by RAW264.7 macrophages loaded with 40?g/m L ox-LDL,the following study were investigated based on the foam cell: At first,the effect of CML on the foam cell lipid accumulation and CD36 expression and the interaction of CML with CD36,then the effect of CML on the foam cell migration and actin polymerization,and then the role of CD36 in the foam cell migration induced by CML,finally,the related pathway of CML/CD36 in foam cell migration were investigated.Then the related analyses such as Oil red O staining,the enzyme method of cellular cholesterol contents,transwell migration assay and wound-healing assay,immunoprecipitation,immunofluorescence staining,DCF metheod of ROS generation,western blot assay and quantitative real-time PCR were performed.Results(1)HE staining suggested that CML can significantly increase the atherosclerotic plaque areas,the needle cholesterol crystals in fibrous cap of apo E-/-mice,while the areas of vascular plaque in anti-CD36 group were significantly smaller than CML group,and the cholesterol crystals were also reduced.(2)Oil red O staining and total cholesterol content detection showed that lipid accumulation and total cholesterol content in atherosclerotic plaques decreased in anti-CD36 group when compared with CML group.While in para-aorta lymph node,lipid accumulation and total cholesterol content increased in anti-CD36 group compared with CML group.(3)Immunofluorescence staining and western blot assay illustrated that CD68 protein expression of aortic atherosclerotic plaques in anti-CD36 group was obviously lower than that of CML group,but CD68 protein expression of para-aorta lymph nodes in anti-CD36 group was significantly higher than that of CML group.(4)CML promoted the lipid accumulaton,upregulated the CD36 expression,inhibited cells migration,and facilitated actin polymerization of foam cells.(5)CML inhibited foam cells migration via interacting with CD36.(6)CML/CD36 inhibiting foam cells migration was related with free cholesterol generation,ROS production,the activation of phosphorylated focal adhesion kinase(FAK),Arp 2/3 complex and actin polymerization.Conclusions(1)CML/CD36 inhibited foam cells of plague migrating to para-aorta lymph nodes,accelerating atherosclerotic progression in apo E-/-mice.(2)The corresponding mechanism of CML/CD36 inhibiting the migration of foam cells may be via free cholesterol,ROS generation,p-FAK,Arp 2/3,F-actin.