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Effect of D-dimer and Nogo-B on Macrophages during Atherosclerosis

Author: XiongQingHui
Tutor: ZuoYaoCheng；YangPengYuan
School: Second Military Medical University
Course: Pharmacology
Keywords: Atherosclerosis Oxidized low-density lipoprotein Macrophages D-dimer Nogo-B
CLC: R543
Type: Master's thesis
Year: 2010
Downloads: 64
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Abstract

Background: atherosclerosis (Atherosclerosis, AS) is a chronic inflammatory response, monocyte-macrophage cells play an important role in the development of the disease occurred: AS early monocyte adhesion to damaged endothelial cell surface and migrated to subendocardial differentiate into macrophages, which engulf lipid to form foam cells; foam cells through the secretion of pro-inflammatory cytokines induced inflammatory reaction further occurrence of plaque area; AS late, foam cell necrosis extracellular lipid accumulation formation of necrotic core; macrophages secrete matrix metalloproteinase related with plaque instability. Lectin-like oxidized low-density lipoprotein receptor -1 (Lectin-like oxLDL receptor-1, LOX-1) were expressed at the cell surface involved in the AS: LOX-1-mediated oxidation of low-density lipoprotein (Oxidized Low Density Lipoprotein , oxLDL)-induced endothelial injury and adhesion molecule expression, to promote adhesion of mononuclear macrophages and endothelial lipid uptake and foam cell formation. Many pathological factors may be involved in the development of AS by upregulating the expression of LOX-1. D-dimer is a fibrin degradation products, as an independent clinical indicators used in the diagnosis of the disease. Plasma D-dimer levels can indicate the severity of AS. Gene chip, D-dimer Macrophages LOX-1 expression, suggesting that D-dimer may be involved in AS. Nogo-B protein play a dramatic role in the vascular remodeling process, and studies have shown that the protein associated with AS. Objective: To study the D-dimer macrophage oxLDL receptor LOX-1 expression, macrophage-derived foam cell formation and its function. Nogo-B expression in macrophage-derived foam cells change its affect foam cell functions. Methods and Results: 1. Treated with D-dimer RAW264.7 cells, cells collected at different time points total RNA was extracted and total protein sample LOX-1 mRNA and protein expression was measured Realtime PCR and Western blot, respectively. The results showed that D-dimer was time-dependent induction of LOX-1 mRNA expression after stimulation of the 12h that raised the maximum effect occurs at 24-36h 48 hours after induction of weakening. LOX-1 protein expression is most evident in the 24-48h. With different concentrations of D-dimer RAW264.7 cells were incubated LOX-1 mRNA and protein expression was measured again. The results showed that the D-dimer of LOX-1 induced a dose-dependent manner, 1μg/mL D-dimer can be induced LOX-1 expression, respectively, with a D-dimer (2μg/mL), oxLDL (10μg/mL) macrophages were incubated for 48 hours, then the expression of CD36 cells was detected by Western blot, the results showed that the concentration of D-dimer not upregulate CD36 protein expression, oxLDL also can not promote CD36 expression was significantly elevated. But the cells were treated with the same concentration of D-dimer 24h and then with oxLDL (10μg/mL) and incubated 24h Western blot results showed that CD36 protein expression compared to the negative control group and oxLDL treatment group were significantly elevated. 3. D-dimer were incubated for macrophages after 24h with the LOX-1 antibody blocking cell surface receptor, then DiI-oxLDL stimulated cells, to observe the fluorescence intensity difference of the cells in each group. The results show that the fluorescence intensity of the D-dimer treated cells compared to the untreated group was significantly enhanced, while the cells treated with the LOX-1 antibody, the fluorescence intensity decreased. Tip D-dimer may phagocytic macrophages by inducing the expression of LOX-1 promotion oxLDL, oxLDL-stimulated cells pre-treated with D-dimer, the Realtime PCR detection of inflammatory cytokines TNF-α and IL-6 expression. Results: D-dimer can enhance oxLDL induced by inflammatory cytokines. 4. D-dimer were incubated macrophages 24h, Western blot, the expression of the cells constitutively PKC protein, results showed that D-dimer may increase the expression of the protein. PKC inhibitor pretreatment macrophages, and then the incubation of D-dimer, Western blot detection cell LOX-1 protein expression, and results showed that treatment with the PKC inhibitor induced expression of LOX-1, D-dimer weakened. With different concentrations of DiI-oxLDL induced macrophage lipid formation of foam cells, and fluorescence intensity indicates the amount of lipid phagocytosis. 48h after cell lysis, Western blot was used to detect the expression of Nogo-B in the foam cells. The fluorescence intensity that macrophages is proportional to the amount of lipid phagocytosis and extracellular concentration of DiI-oxLDL Western blot results show an increase in foam cells in Nogo-B protein expression, and low concentrations of oxLDL (≤ 10μg/mL) can not be induced Nogo-B protein expression. Nogo-B siRNA transfection Raw264.7 cells 48 hours after detection of Nogo-B protein expression in the cells, the results showed that siRNA can effectively reduce the expression of Nogo-B protein in macrophages, silent efficiency of up to 70% . Transfection with siRNA macrophages with oxLDL stimulation, Realtime PCR detection of cytokines TNF-α, IL-1β, IL-6 expression: reduce Nogo-B protein expression can foam cells express more proinflammatory cytokines IL-1β, TNF-α and 1L-6 had no significant effect. Conclusion: D-dimer can be induced by increasing macrophages activated PKC content of LOX-1 expression in macrophages promote oxLDL phagocytosis and the expression of inflammatory cytokines, thus speeding up the AS course development. Nogo-B protein in oxLDL-induced macrophage-derived foam cells express higher and may slow down the development of AS 1L-1β expression by inhibiting inflammatory cytokines.

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