The Effects Of OxLDL And OxHDL In Regulating Expression Of TLR2 And TLR4 On ECV304 Endothelial Cell And Changes On Human Monocyte Chemotaxis

BACGROUNDIt has become evident that atherosclerosis is an inflammatory disease with immune responses during initiation and progression of this disease. The innate immune response is the first line of defense and is limited to recognize highly conserved pathogen motifs, entitled pathogen-associated molecular patterns(PAMPs). In the first line of defense, the receptors capable of recognizing these PAMPs are toll-like receptors (TLRs) and scavenger receptors. The expression of TLR2 and TLR4 was markedly enhanced in human atherosclerotic plaques. TLR2 and TLR4 associated with inflammatory activation in human atherosclerotic lesions, and they encourage further exploration of innate immunity in the pathogenesis of atherosclerosis. Atherosclerosis is characterized by the accumulation of lipid and inflammatory cells in the vascular wall Atherosclerosis develops when circulating lipoproteins are retained, aggregated and modified in the intimal space of the vessel wall. This triggers infiltration of inflammatory cells and unregulated lipid uptake by macrophages. Activation of TLR2 and TLR4 was shown to elicit the production and releasing of cytokines and chemokines,such as MCP-1, IL-8 and some adhesion molecules. Many kinds of these molecules are associated with the onset of atherosclerosis. In addition, many roles of the lipoprotein in atherosclerosis desease are certified. But the roles of the immune response in this process are not clearly. Thus, the study is aimed to investigate the role of modified lipoprotein on expression of TLR2 and TLR4 on endothelial cell, and the chemotatic effect of monocyte to endothelial cell treated by modified lipoprotein. OBJECTIVETo investigate the effects of TLR2 and TLR4 expression on endothelial cell by oxLDL and oxHDL, L, and then the effects of THP-1 chemotaxis.METHODSChange the medium for 24 hours before experiment. ECV304 endothelial cell were treated with 0 mg/L,25 mg/L,50 mg/L,100 mg/L oxLDL and oxHDL for 24 hours respectively. And then choose a proper concentration of oxLDL and oxHDL for treating ECV304 endothelial cell through the determining TLR2 and TLR4 mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). Afer treating ECV304 endothelial cell with the proper concentration of oxLDL and oxHDL for 24 hours, TLR2 and TLR4 mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. Treat ECV304 endothelial cell in lower compartment of transwell by oxLDL and oxHDL in proper concentration for 24 hours. And then, put the THP-1 monocyte in upper compartment for 4 hours until counting the THP-1 cells in the lower compartment. Moreover, 5μmol/LEGCG was added in the lower compartment to suppress the signals of TLRs, and count the THP-1 monocyte in lower compartment to observe the chemotaxis[0].RESULTSThe expression of TLR2 and TLR4 mRNA on ECV304 endothelial cell was enhanced with higher concentrations of oxLDL and oxHDL after the endothelial cells treated with concentrations of oxLDL and oxHDL for 24 hours respectively. By 100 mg/L oxLDL and 100 mg/L oxHDL the levels of TLR2 and TLR4 mRNA and protein were obviously elevated. The effect of THP-1 monocyte chemotaxis[0] to ECV304 endothelial cell was also enhanced significantly by culturing the ECV304 endothelial cell in lower compartment and treating it by oxLDL and oxHDL. When we suppressed the signals pathway of TLRs by 5μmol/L EGCG, the effect of THP-1 monocyte chemotaxis was obviously attenuated.CONCLUSIONS(1) The TLR2 and TLR4 can express on the ECV304 endothelial cell.(2) OxLDL and oxHDL can upregulate the tow receptors on the level of mRNA and protein.(3) It can encourage THP-1 monocyte to migrate to ECV304 endothelial cell when oxLDL and oxHDL upregulates the expression of the TLR2 and TLR4 on endothelial cell. Moreover, this effect can be suppressed by EGCG.