The Function And Mechanism Research Of Notch Signaling Pathway Induce Atherosclerosis In Macrophages

Objective:To explore the expression of Notch signal and cytokines by oxidized low-density lipoprotein (ox-LDL) in macrophages of human acute monocytic leukemia cell line (THP1) and to search for possible mechanism of atherosclerosis (AS).Methods:Human macrophage from THPI transform by phorbol12-myristate13-acetate (PMA) were cultured with final concentration of50mg/L ox-LDL for6h. Four receptors and five ligands of Notch signaling pathway were inspected.Dynamic changes in terms of cell shape were observed by phase contrast microscopy. Notchl, DIL4and Jaggedl were given to25mg/L,50mg/L,100mg/L of three different concentrations of ox-LDL stimulation for48hours. The best concentration was50mg/L. Real time-PCR (RT-PCR) detection of Notchl, DIL4and Jaggedl mRNA expression levels of different time points after macrophages co-cultured with50mg/L ox-LDL for Oh,3h,6h,12h,24h,48h.Notchl, DIL4and Jaggedl protein expressions were determined by Western-blot of different concentrations and different time points. Vascular cell adhesive molecule-1(VCAM-1) and monocyte chemoattractant protein-1(MCP-1) expression were determined by enzyme-linked immunosorbent assay (ELISA).Lipofectamine2000transfected toll-like receptor2small interfering RNA (TLR2siRNA) which silence effect was most obvious among TLR2siRNAs on THP-I derived macrophages for24h. Macrophages with TLR2siRNA transfected previously co-cultured ox-LDL50mg/L for48h. RT-PCR was used to detect the blank control group and transfection of TLR2siRNA group Notchl,DIL4and Jaggedl and the expression of inflammatory factor VCAM-1, MCP-1. Western-blot and ELIS A detected Notchl, DIL4, Jaggedl, VCAM-1and MCP-1protein expressions respectively.Results:Macrophages which inducted by different concentrations of ox-LDL for48h occured dendritic-like cell (DC) shape change. Compared with the control group DC shape change ratio was significantly increased (P<0.05),50mg/L concentration effect was the highest. Macrophages joining ox-LDL stimulated of Notchl expression was significantly elevated (P<0.05) compared to control group. Notch2and Notch3expressions decreased slightly, the expression of Notch4increased slightly, but not statistically significant. Ligands in DIL4and Jaggedl expression were significantly elevated (P<0.05), the expression of DILI mildly elevated, D1L3and Jaggde2expressions decreased slightly, not reached statistical significance. Detected Notch receptors expression by Western-blot, we found that Notchl expression was significantly elevated, Notch4not change, Notch2and Notch3decreased. With different concentrations of ox-LDL stimulation of macrophages Notchl, D1L4and Jagged1expressions were elevated (P<0.05),the effect on the concentration of50mg/L increased most obviously. At different time points after ox-LDL stimulation Notchl, DIL4and Jaggedl expressions in macrophages were elevated (P<0.05), the most obvious effect was got at6h. Compared with the control group ox-LDL enhanced VCAM-1and MCP-1expressions (P<0.05), the most obvious effect on the ox-LDL was got at concentration of50mg/L. Screening out TLR2siRNA-1could appear obvious silencing effect on the Notchl, D1L4and Jaggedl expressions with RT-PCR, Western-blot and ELISA increased significantly suppressed in the transfection of siRNA group which THP-1derived macrophages transfected by application of Iipofectamine2000,the expressions of inflammatory factor VCAM-1and MCP-1were elevated significantly suppressed.Conclusion:Our data showed that with ox-LDL challenge the expressions of Notch1,DIL4Jagged1and the levels of VCAM-1, MCP-1significantly increased in macrophages in a dose-time-dependent manner within some extent compared with that in the control group. Notch signal and TLR pathway had synergistic expression effect. Notch signaling was activated by ox-LDL stimulation and may partially mediate atherogenic effect with macrophage function. Part II Expression of Notch signaling and inflammatory cytokines in peripheral blood mononuclear-macrophages of patients with the acute coronary syndromeObjective:To explore the differential expression of Notch signaling and inflammatory cytokines in human peripheral blood mononuclear-macrophages of patients with the acute coronary syndrome (ACS).Methods:Peripheral blood mononuclears of patients with coronary arteriosclerosis disease (CAD) were separated by density gradient centrifugation in which included15patients with unstable angina pectoris(UA),21patients with acute myocardial infarction(AMI) and14with stable angina (SA) were transformed by PMA to macrophages,10without CAD as healthy control.Forty eight hours later, cells and supernate were collected separately.The expression of Notch I mRNA was measured by RT-PCR and protein measured by Western-blot. The levels of VCAM-1and MCP-1in supernate were determined by ELISA. According to the first part of the experimental results TLR2siRNA-1was used as transfection primer. The macrophages derived from peripheral blood monocyte with CAD were gived TLR2siRNA and transfected for24h. Notch1and inflammatory cytokine VCAM-1, MCP-1mRNA were detected in healthy controls, patients with CAD before and after TLR2siRNA transfection respectively.Results:RT-PCR showed compared with healthy controls, Notch1gene expression level was increased among SA, UA and AMI. The UA group and AMI group increased significantly (P<0.05), SA group showed no significance (P>O.05). Notch2, Notch3expressions decreased slightly and Notch4mildly elevated, but had no statistical significance (P>0.05). VCAM-1and MCP-1gene expression level of SA, UA and AMI group significantly increased (P<0.05). With RT-PCR results, Western-blot test shows:compared with healthy controls, SA, UA and AMI group Notchl protein expression were increased, UA and AMI significantly higher than those in control group (P<0.05). SA, UA and AMI in three groups of Notch2and Notch3protein expression decreased slightly, the expression of Notch4protein mild hypertension, but not reach statistical standard (P>0.05). ELISA results showed that compared with the control group, SA group, UA group and AMI group of three groups of VCAM-land MCP-1protein expression were significantly increased (P<0.05). Compared with healthy controls, TLR2siRNA transfection after pretreatment inhibited the Notch1increased gene expression of ACS with peripheral blood monocyte derived macrophages,Notchl mRNA reduced significantly (P<0.05).TLR2siRNA transfection pretreatment inhibited VCAM-1and MCP-1enhanced role of expression in monocyte derived macrophages with ACS. The expression of VCAM-land MCP-1mRNA were significantly decreased (P<0.05).Conclusion:Patients with CAD increased Notch1expression induced by peripheral blood monocyte derived macrophages, the role in ACS patients was significant. At the same time atherosclerosis cytokine secretion of VCAM-1and MCP-1increased. Notch1expression and TLR pathway had a synergistic effect. ACS induced by macrophages proatherogenic effect may be partially mediated by Notch signaling. Notch signal pathway and its mediated macrophage innate immune response in atherosclerosis vulnerable plaque in the occurrence and development played an important regulatory role.