| BackgroundAcumulating data indicated that lectin-like oxidized low density lipoprotein receptor-1(LOX-1)contributes to atherosclerotic plaque formation and progression.All the evidences based on both clinical studies and animal diseases models.LOX-1 is increasingly viewed as a cardiovascular disease biomarker and a potential therapeutic target.It is beneficial for anti-atherogenesis by suppressing expression of LOX-1. RNA interference(RNAi)is a novel technique of gene silence by transfection of exogenous or endogenous double strand RNA(dsRNA) into cells,which is characterized by specificity and stability.RNAi is becoming an important tool of gene study.Circulating monocytes adhere to endothelium play an important role in atherogenesis,which occurs at the earliest stage of atherosclerosis. This process is mediated by adhesion molecules and chemokines Oxidized low-density lipoprotein(Ox-LDL)is a well-known coronary risk factor,which can induce expressions of adhesion molecules and chemokines in endothelial cells.However,it remains unclear whether Ox-LDL can induce expression of Fractalkine or not in endothelial cells, as well as its signal pathway.Monocytes adhere to the vessel wall and are recruited into the subendothelial space where they differentiate into macrophages and subsequently develop into foam cells Macrophages uptake Ox-LDL mediated by scavenger receptors play a critical role in the formation of foam cells,which is the hallmark of atherosclerosis.LOX-1 and CD36 are both receptors of Ox-LDL in macrophages.However,some evidences implied that LOX-1 and CD36 may play different role in the process of foam cell formation.Ox-LDL up-regulate CD36 expression in macrophages,which facilitate uptake of Ox-LDL.It remains unclear if LOX-1 involved in the up-regulation process of CD36 induced by Ox-LDL in macrophages.Objective(1)To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to human LOX-1 gene and to observe its silencing effect on LOX-1 in human umbilical vascular endothelial cells(HUVECs)(2)To observe expressions of Fractalkine and monocyte chemoattractant protein-1(MCP-1)induced by Ox-LDL in HUVECs,and to investigate its signal pathway.(3)To investigate the role of LOX-1 for expression of CD36 induced by Ox-LDL in THP-1-derived macrophages.Methods(1)The pGenesil-1LOX-1shRNA expression vector was constructed by gene recombination,then transfected into the cultured HUVECs.At five days after transfection,the stable transfection and expression of LOX-1 mRNA in HUVECs were determined by semi-quantitative RT-PCR and the protein level were determined by western-blot.(2)HUVECs were cultured and treated with Ox-LDL of 3 different concentrations(25 microg/L,50 microg/L,and 100 microg/L)for 24 hours.We determined mRNA expressions of lectin-like oxidized-LDL receptor-1(LOX-1),Fractalkine and MCP-1 by semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR).Protein expressions of LOX-1 and Fractalkine were assayed by western blot analysis.Concentration of MCP-1 protein in medium was measured using the ELISA method.(3)pGenesil-1LOX-1shRNA were transfected into HUVECs and then cells were treated with Ox-LDL(50 microg/L)for 24 hours.We determined mRNA expressions of LOX-1,Fractalkine and MCP-1 by semi-quantitative RT-PCR.Protein expressions of LOX-1 and Fractalkine were assayed by western blot analysis.Concentration of MCP-1 protein in medium was measured using the ELISA method.(4)Pretreated HUVECs with PD98059(A specific inhibitor of ERK1/2)1 hour before adding Ox-LDL,then cells were treated with Ox-LDL(50 microg/L)for 2 hours,mRNA expressions of LOX-1,Fractalkine and MCP-1 were determined by semi-quantitative RT-PCR.Protein expressions of ERK1/2 and p-ERK1/2 were assayed by western blot analysis.(5)THP-1 cell line were incubated with phorbol 12-myristate 13-acetate(PMA)(100ng/ml)for 72 hours.(6)THP-1-derived macrophages were pretreated with a CD36-specific blocking antibody(0.5μg/ml),then incubated with Ox-LDL of 3 different concentrations(25 microg/L,50 microg/L,and 100 microg/L) for 24 hours.We assessed mRNA expressions of LOX-1 and CD36 by semi-quantitative RT-PCR.Protein expressions of LOX-1 and CD36 were assayed by western blot analysis.(7)pGenesil-1LOX-1shRNA were transfected into cells before treating with CD36-specific blocking antibody(0.5μg/ml),and then cells were treated with Ox-LDL(50 microg/L)for 24 hours,mRNA and protein expressions of LOX-1 and CD36 were assessed by semi-quantitative RT-PCR and western blot analysis respectively.(8)Treated with CD36-specific blocking antibody(0.5μg/ml) and transfected pGenesil-1LOX-1shRNA into macrophages respectively, then cells were incubated with Ox-LDL(50 microg/L)for different time(0 hour,6 hours,12 hours,24 hours and 48 hours).mRNA expressions of LOX-1 and CD36 were assessed by semi-quantitative RT-PCR.Results(1)cDNA sequence assay confirmed that pGenesil-1LOX-1shRNA expression vector was successfully constructed.LipofectamineTM 2000-mediated gene transfection of pGenesil-1LOX-1shRNA expression vector into HUVECs down-regulated the mRNA and protein expression levels of LOX-1,as compared with the control group(p＜0.01)(2)Exposure of HUVECs to Ox-LDL(25 microg/L,50 microg/L and 100 microg /L,respectively)resulted in upregulation of LOX-1, Fractalkine and MCP-1,both mRNA and protein expressions,in a dose-dependent manner(p＜0.01).(3)Expressions of Fractalkine and MCP-1 induced by Ox-LDL (50 microg /L)were significantly suppressed by transfection with LOX- 1 specific siRNAs(p＜0.01)(4)PD98059 and LOX-1 gene silence significantly suppressed the protein expression of p-ERK1/2 induced by Ox-LDL(50 microg/L),as well as inhibited mRNA expressions of LOX-1,Fractalkine and MCP-1(p＜0.01)(5)THP-1 cell line could be differentiated successfully into macrophages treated with PMA(100ng/ml)for 72 hours.(6)Exposure of macrophages to Ox-LDL(25 microg /L,50 microg/L and 100 microg/L,respectively)resulted in upregulation of LOX-1,and CD36,both mRNA and protein expressions,in a dose-dependent manner(p＜0.01).There was no significant difference of mRNA and protein expressions,both LOX-land CD36,between the group that without adding CD36-specific blocking antibody and the control group(adding CD36-specific blocking antibody but without Ox-LDL)(p＞0.05).(7)CD36 mRNA and protein expressions induced by Ox-LDL(50 microg/L)were suppressed significantly by pGenesil-1LOX-1shRNA mediated LOX-1 gene silencing together with CD36-specific blocking antibody(p＜0.01).(8)Alter blocking CD36 by CD36-specific blocking antibody, mRNA expression of LOX-1 significantly increased 6 and 12 hours alter Ox-LDL treatment compared with 0 hour,and reached a peak level after 24 hours(p＜0.01,respectively).However,after exposure to Ox-LDL for 48 hours,mRNA expression of LOX-1 trends to decrease.As well as for CD36,expression of CD36 mRNA significantly increased 6,12 and 24 hours after Ox-LDL treatment compared with 0 hour,and reached a peak level after 48 hours(p＜0.01,respectively).After gene silencing mediated by pGenesil-1LOX-1shRNA,expression of CD36 mRNA begin to increase 12 hours after Ox-LDL treatment(compared with 0 hour, p＜0.05),and significantly increased 24 hours after Ox-LDL treatment and then reached a peak level after 48 hours(compared with 0 hour,p＜0.01).Conclusions(1)The pGenesil-1LOX-1shRNA expression vector can inhibit expressions of LOX-1 mRNA and its protein level in HUVECs, (2)Our results suggest that Ox-LDL can induce expressions of Fractalkine and MCP-1 in a dose-dependent manner in HUVECs,and this process correlated with the activation of LOX-1-ERK1/2 signal pathway.It provides a novel potential therapeutic target for atherosclerosis.(3)Ox-LDL could up-regulate expression of CD36 in a dosedependent manner in THP-1 derived macrophages,and this process might be mediated by LOX-1,especially at the earlier stage.
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