Effects Of C-reactive Protein And Lipoprotein On The Expression Of Monocyte Chemoattractant Protein-1 And C-C Chemokine Receptor 2 In Human Peripheral Blood Monocytes

BackgroundAs one of the most common cardiovascular diseases,Coronary heart disease (CHD) has been seriously jeopardizing health and quality of life of human beings. The pathogenesis of CHD,However,is not thoroughly identified.It is of much importance to study the relevant risk factors of CHD.Data from several studies,such as the Framingham Heart Study,have demonstrated some independent risk factors of CHD and predictors for cardiovascular outcomes,such as elevated blood pressure, diabetes mellitus,smoking,high low-density lipoprotein,reduced high-density lipoprotein,age and gender.Recently,some researchers believe that inflammation is involved in the pathogenesis and progress of CHD.So we have paid much attention on the inflammatory mechanism of CHD and try to illustrate pathogenesis of CHD in terms of relationships among traditional risk factors such as lipid,C-reactive protein (CRP) and inflammation.Atherosclerosis(AS) is pathological basis of CHD.Since Ross raised the response-to-injury theory of AS in 1999,increasing evidence suggests AS is an inflammatory process after vascular injury.Endothelial injury and oxidative modification of low-density lipoprotein(LDL) are considered the earliest stage of AS pathogenesis.Low-density lipoprotein(LDL),which may be modified by oxidation, glycation(in diabetes),aggregation,association with proteoglycans,or incorporation into immune complexes,is a major cause of injury to the endothelium and underlying smooth muscle.When LDL particles become trapped in an artery,they can undergo progressive oxidation and be internalized by macrophages by means of the scavenger receptors on the surfaces of these cells.The internalization leads to the formation of lipid peroxides and facilitates the accumulation of cholesterol esters,resulting in the formation of foam cells.Oxidized-LDL(Ox-LDL) is chemotactic for other monocytes and can up-regulate the expression of genes for macrophage colony-stimulating factor and monocyte chemoattractant protein-1(MCP-1) derived from endothelial cells,and other inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6),vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1).These cytokines may help expand the inflammatory response by stimulating the replication and secretion of monocyte-derived macrophages and the entry of new monocytes into lesions.Those activated macrophages have enhanced secretion property leading to increased level of MCP-1,ICAM-1 and VCAM-1.Such a positive-feedback results in the aggregation and adhesion to injury endothelium of monocyte-macrophages.These macrophages transformed into foam cells and,thus,led to the AS.So,serum level of MCP-1 could partly reflect the extent and amplitude of inflammation.The C-C chemokine receptor 2,commonly known as CCR2,is the specific receptor for MCP-1 and widely expression on endothelial cells,monocytes and smooth muscle cells.MCP-1/CCR2 plays an important role in pathogenesis and progress of inflammation relevant diseases,including AS. C-reactive protein(CRP) is a sensitive marker for inflammation and tissue injury. A large number of research indicated that CRP was an independent risk factor for AS. The American heart Association(AHA) recommended the use of CRP as a prognostic marker in patients with acute coronary syndrome(ACS) in addition to other prognostic factors,including troponin levels.An evaluation of CRP levels at the time of admission should be included in the evaluation of the patient risk profile,including clinical data,associated diseases,markers of myocardial necrosis,left ventricle performance,and age.A cutoff level of 10mg/L was identified as marker of higher risk for death and possibly myocardial infarction in ACS,whereas a cutoff of 3 mg/L identifies a group of patients at intermediate risk and a high rate of recurrent events.Other researches indicated that CRP enhanced gene expression of Lectin- like oxidized low density lipoprotein receptor—1(LOX—1) leading to intracellular accumulation of cholesterol,induced apoptosis of vascular smooth muscle cell and VCAM-1 gene expression through nuclear factor-κB pathway.CRP was also found to attenuate nitric oxide synthesis in endothelial cell and repair in injured endothelium. These findings provided direct evidence for the hypothesis that CRP may not only a marker and predictor,but a direct cause for AS through upregulate inflammatory response.However,little is known about the relationship between CRP and MCP-1.The results of numerous epidemiological studies have established that high density lipoprotein(HDL) levels correlate inversely with the risk of developing cardiovascular disease.This relationship reflects several functions of HDL,the most extensively studied of which is their ability to remove excess cholesterol from cells, such as macrophages in the artery wall,in the first step of the reverse cholesterol transport pathway.In recent years it has become increasingly apparent that HDL also has potent anti-inflammatory properties.This has been demonstrated in vitro as well as in vivo.For example,the cytokine-induced expression of VCAM-1,ICAM-1,and E-selectin in cultured human umbilical vein endothelial cells(HUVECs) is inhibited by HDL in a concentration-dependent manner.It remains uncertain that whether HDL attenuates MCP-1 expression.In summary,we hypothesized that CRP could enhance MCP-1 and CCR2 gene expression,which result in the aggregation and activation of monocytes in early stage of AS.HDL has potent anti-inflammatory properties through inhibiting MCP-1 and CCR2 expression.Objectives:1.To determine the effects of Ox-LDL and CRP on gene and protein expression of MCP-1 and CCR2 in monocytes.2.To investigate whether HDL could regulate gene and protein expression of MCP-1 and CCR2 in monocytes which was elicited by Ox-LDL and CRP.Methods:1.Preparation of Ox-LDLWe separated and quantified the LDL from blood of healthy individuals by density gradient ultracentrifugation.Adjusted LDL to a final concentration of 500mg/L,then incubated it with 10mmol/L CuSO₄.The oxidation was ceased by adding 100μmol/L EDTA.Finally,Bradford method was utilized to detect the protein level of Ox-LDL.2.Isolation and culture of monocytesMonocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation,inoculated in RPMI-1640 medium supplemented with 1%Penicillin and 10%fetal bovine serum(FBS) and incubated under the condition of 5%CO2,37.5℃.3.Groups and cultural condition (1)Monocytes were cultured with equal RPMI-1640 medium as control.(2)Different concentration groups of Ox-LDL:Monocytes were treated with Ox-LDL (20mg/L,40mg/L and 80mg/L) for 48h,respectively.(3)Different concentration groups of CRP:Monocytes were treated with CRP (3mg/L,10mg/L,20mg/L) for 48h,respectively.(4)500mg/L HDL was added alone and cultured with monocyte to determine its role on both genes expression and MCP-1 protein level;(5)Different concentration groups of Ox-LDL and CRP as described in(2) and(3), each group added 500mg/L HDL before culture.4.Real-time quantitative PCR for MCP-1 and CCR2MCP-1 and CCR2 mRNA was examined by Fluorescent quantitative RT-PCR. Total RNA extracted from cultured cells was reverse-transcripted and amplified using specific primers for human MCP-1 and CCR2.The 2(-Delta Delta C(T)) method is utilized to analyze the relative changes in gene expression from real-time quantitative PCR experiments.5.ELISA assay for MCP-1 protein levelThis assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MCP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MCP-1 present is bound by the immobilized antibody.An enzyme-linked monoclonal antibody specific for MCP-1 is added to the wells.Following a wash to remove any unbound antibody-enzyme reagent,a substrate solution is added to the wells and color develops in proportion to the amount of MCP-1 bound in the initial step.The color development is stopped and the intensity of the color is measured.6.Data analysisSPSS 13.0 statistical software was used to analyze data.Data are presented as Mean±S.D.Statistical significance was determined in a multiple comparisons among different groups of data in which one-way ANOVA test indicated the presence of significant differences.P value＜0.05 was considered to be significant.Results:1.Effects of Ox-LDL on MCP-1 and CCR2Incubation of monocytes with Ox-LDL of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).When compared with control,MCP-1 mRNA expression increased to 1.97,3.16 and 4.07 fold and protein expression increased(P=0.000) while CCR2 mRNA expression increased to 2.12, 2.54 and 3.02 fold(P=0.009) after treated with Ox-LDL of different concentrations.2.Effects of CRP on MCP-1 and CCR2Incubation of monocytes with CRP of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).There was no significance between 3mg/L CRP group and control MCP-1 mRNA expression increased to 3.09 and 4.04 fold(P=0.000) and protein expression elevated(P=0.000) while CCR2 mRNA expression increased to 2.96 and 3.59 fold(P=0.005) after treated with CRP of different concentrations(10mg/L and 20mg/L),as compared with control.3.HDL had no direct influence on on both genes expression and MCP-1 protein level;4.Role of HDL in expression of MCP-1 and CCR2 elicited by Ox-LDL and CRPCo-incubation of monocytes with HDL significantly inhibited Ox-LDL-induced and CRP-induced expression of MCP-1(mRNA and protein) and CCR2 mRNA.Conclusions:1.Oxidized low-density lipoprotein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;2.C-reactive protein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;3.High density lipoprotein which inhibits Ox-LDL-induced and CRP-induced upregulation of MCP-1 and CCR2 may exert beneficial effect in the prevention and treatment of atherosclerosis.