The Impact Of Chemokine Fractalkine And Its Receptor On Atherogenesis And The Therapeutic Intervention By Fluvastatin/Probucol

BackgroundA preponderance of evidence from clinical and experimental studies supports the notion that inflammation plays an important role in the pathogenesis of atherosclerosis(AS).It is a critical step in inflammation that mononuclear cells activate and migrate from the blood stream to the vessel wall.This step happens during all the phases from plaque formation,plaque rupture to thrombokinesis.Circulating mononuclear cells are the precursors of foam cells during atherosclerotic injuries. Chemokines are vital in regulating the recruitment and infiltration of mononuclear cells(MC).The chemokines are a family of small secreted proteins(molecular mass is about 8_~12kD)that function as potent mediators of inflammation by their ability to recruit and activate specific leukocyte subpopulations. So far more than 50 chemokines and 20 of their ligands have been identified,and they are subdivided into 4 subfamilies,C-,CC-,CXC-, and CX3C-chemokines,according to the number and pacing of the first 2 cysteines in a conserved cysteine structural motif.Fractalkine(FKN, CX3CL1)is the only CX3C-chemokine to have been described.It exists in two forms:the membrane-anchored form and the soluble form,each mediating distinct biological actions in regulating the trafficking and adhesion of leucocytes,functioning as both a chemokine and a adhesion molecule.In contrast to other chemokines,FKN is a transmembrane glycoprotein;it may have its unique biological features and play a distinctive role in the pathogenesis.CX3CR1 is the specific and high affinity receptor of human FKN,expressed on neutrophils,MC,T lymphocytes,natural killer(NK)cells.Crossing CX3CR1-/- into the apoE-/- background results in decreased atherosclerotic lesion formation with reduced macrophage accumulation.Gene polymorphisms of human CX3CR1 have been reported to be a genetic risk factor for coronary artery disease.FKN mRNA expression is reportedly upregulated in human atherosclerotic injuries,and it is also observed that FKN and CX3CR1 are overexpressed on damaged endothelial cells(EC),macrophage-derived foam cells and other cells as well as atherosclerotic plaques.Nuclear factor-Kappa B(NF-Kappa B)is a transcription factor functioning in multiple regulations of gene transcription.It is reported that NF-KappaB exists on the main component cells of AS such as MC,EC and vascular smooth muscular cells(SMC),and previous studies have shown that NF-Kappa B can damage the endothelial structure and functions,so as to participate in initiating the vascular atherosclerotic plaque formation.Statins and probucol are two commonly used lipid-regulating agents. But people now focuse more attention on their effects independent of lipid-lowering.Clinical studies have shown that statins inhibit the expression of FKN and CX3CR1,probucol can clean oxygen free radicals,reduce the contents of oxidized lipid and cholesterol,and decrease the area of necrotic lipid core.Both agents have been reported to be able to inhibit the activation of NF-KappaB.But so far it remains unclear of the roles of NF-KappaB,FKN and CX3CR1 on the atherosclerosis induced by high-fat dietary and oxidized low density lipoprotein(Ox-LDL),and few studies were done on the therapeutic effects of stains and probucol intervention on the above-mentioned inflammation cytokines.Therefore it is of meaningful importance to elaborate the possible new mechanism by which statins and probucol have their anti-AS potency through investigating their effects on expression of NF-KappaB,FKN and CX3CR1 during atherogenesis.ObjectiveThis study is aimed to elaborate the possible new mechanisms by which Fluvastatin/Probucol have their anti-AS potency,through observing their interventional impact on FKN/NF-KappaB expression in mice AS plaque,and investigating their therapeutic effects on Ox-LDL induced FKN expression on Human Umbilical Vein Endothelial Cells(HUVEC)and CX3CR1 expression on THP-1 cells.Methods1.apoE-/- male rats were fed with high-fat dietay to develop AS, then Fluvastain/probucol were administered respectively,AS plaque was observed under optical microscopy,and its maximum thickness,mean thickness and percentage of plaque over lumen area were determined; FKN/NF-KappaB p65 protein expression were detected using PV-9000 two-step kit immuno-staining;FKN/NF-KappaB mRNA expression were detected by RT-PCR.2.After pretreating HUVEC with Probucol/Fuvastatin at different concentrations and pyrrolidine dithiocarbamate(PDTC),then stimulating HUVEC with Ox-LDL at different concentration,to detect FKN/NF-KappaB mRNA expression by semi-quantitative RT-PCR and FKN/NF-KappaB p65 protein expression by western blot.3.After pretreating THP-1 cells with Probucol/Fuvastatin at different concentrations and PDTC,then stimulating THP-1 cells with Ox-LDL at different concentration,to determine CX3CR1/NF-KappaB mRNA expression by semi-quantitative RT-PCR and CX3CR1/ NF-KappaB p65 protein expression by western blot.Results 1.Compared with those of ordinary dietary group,the maximum plaque thickness,mean thickness and percentage of plaque/lumen area of hgh-fat dietary increased significantly(P＜0.01);Fluvastatin group,Probucol group and Combination therapy group presented an decrement of above-mentioned parameters and FKN/NF-KappaB p65mRNA expression(P＜0.01)if compared with western-dietary group.2.①Ox-LDL at 25,50 and 100mg/L stimulated HUVEC to up-regulate FKN/NF-KappaB expression both at mRNA level(P＜0.01) and protein level(P.0.01)compared to control group;and this up-regulating expression action was concentration-dependent(100mg/L vs 25mg/L Ox-LDL goup:P＜0.05);②Probucol/Fuvastatin inhibited the Ox-LDL-induced expression(Probucol group VS control:P＜0.05; Fluvastatin group VS control group:P＜0.01);both agents presented a dose-dependent inhibitory effects,combination therapy showed a further inhibition than monotherapy(P＜0.05);③pretreated with PDTC then stimulated with OX-LDL,HUVEC showed down-regulation expression of NF-KappaB p65mRNA(P＜0.01),and significant down-regulation expression of NF-KappaB protein(P＜0.01),FKN mRNA(P＜0.01) and FKN protein(P＜0.01)were observed if compared with no PDTC pretreatment group.3.Compared with control group,Ox-LDL induced THP-1 cells to up-regulate CX3CR1/NF-KappaB expression in a concentration-dependent manner both at mRNA level(25,50,100mg/L Ox-LDL group VS Control group:P＜0.05,P＜0.01,P＜0.01;100mg/L VS 25mg/L group: P＜0.05,respectively)and protein level(25,50,100mg/L Ox-LDL group VS Control group:P＜0.01;100 VS 25mg/L Ox-LDL group: P＜0.05);②Probucol/Fluvastatin significantly inhibited Ox-LDL induced up-regulation expression in a dose-dependent tendency;Combination therapy group presented a further inhibitory effect on CX3CR1/NF-KappaB mRNA(P＜0.05)and on CX3CR1/NF-KappaB protein expression(P＜0.01)if compared with monotherapy group;③pretreated with PDTC followed by Ox-LDL stimulation,THP-1 cells showed NF-KappaB protein down-regulation expression(P＜0.01),but no CX3CR1mRNA and CX3CR1 protein down-regulation expression(P＞0.05)if compared no-PDTC pretreatment group.Conclusions1.High-fat dietary can produce AS lesions in apoE gene knockout mice,there are high expression of FKN/NF-KappaB in AS plaques, Fluvastatin,Probucol and combination therapy can inhibit the expression of FKN/NF-KappaB,and reduce the atherosclerotic lesion formation.2.Ox-LDL can induce HUVEC to express FKN via NF-KappaB signal pathway;Probucol/Fluvastatin intervention can downregulate FKN expression on Ox-LDL stimulated HUVEC by inhibiting NF-KappaB activation. 3.Ox-LDL can up-regulate CX3CR1 expression on THP-1 cells,this effect is by a way other than the NF-KappaB signal pathway;PDTC can inhibit Ox-LDL-driven NF-KappaB activation but not CX3CR1 expression on THP-1 cells;Probucol/Fluvastatin can inhibit both NF-KappaB activation and CX3CR1 expression on Ox-LD1 stimulated THP-1 cells,the inhibitory effect of Probucol/Fluvastatin on CX3CR1 is not related with inhibition of NF-KappaB activation.