Effect Of D-dimer And Nogo-b On Macrophages During Atherosclerosis

Background:Atherosclerosis is a chronic inflammatory disease, and monocytes or macrophages play important roles in the formation of atherosclerotic lesions. One of the earliest events in atherogenesis is monocytes attach to the surface of injured endothelial cells. Adherent monocytes migrate into the subendothelial space and differentiate into macrophages. Uptake of oxLDL via scavenger receptors leads to foam cell formation. Cytokines secreted by foam cells exert both pro-and antiatherogenic effects on each of the cellular elements of the vessel wall. Necrosis of macrophage and smooth muscle cell-derived foam cells leads to the formation of a necrotic core and accumulation of extracellular cholesterol. Macrophage secretion of matrix metalloproteinases contribute to weakening of the fibrous plaque. Plaque rupture exposes blood components to tissue factor, initiating coagulation, the recruitment of platelets, and the formation of a thrombus. lectin-like oxidized LDL receptor-1 (LOX-1) expressed on cells participated in atherogenesis. Oxidized Low Density Lipoprotein (oxLDL) play a role in endothelial cells injury, adhersion molecule expression, foam cells formation via LOX-1. Many stimulis aggrevate atherosclerosis by induction of LOX-1 expression. D-dimer is a fibrin degradation product and substantially increased in plasma after thrombotic/fibrinolytic. Nowadays D-dimer testing is frequently applied in the detection of some diseases, and its levels may reflect atherosclerosis severity. The result from gene microarray show that D-dimer induce LOX-1 expression in macrophages, suggesting D-dimer may accelerate atherosclerosis development. Nogo-B is noteable because it plays a role in vasculer remodeling. Some research show it may also associated with atherogenesis.Aim:To study on effect of D-dimer on LOX-1 expression, oxLDL uptake, foam cells formation and inflammatory cytokines expression in macrophages. The change of Nogo-B expression in macrophage-drived foam cells and the role of Nogo-B in foam cells function.Methods and Results:1. RAW264.7 cells treated with D-dimer in dose and time gradient manner. The stimulatory effect of D-dimer on cells LOX-1 mRNA expression was time- and dose-dependent, with maximal effect occuring on 2μg/mL D-dimer. Incubation of RAW264.7 cells for 0 to 48 hours with 1μg/mL D-dimer, macrophage LOX-1 gene expression increased in a time-dependent manner,.Maximal effect was observed from 24 to 36hours. Incubation of RAW264.7 cells for 48 hours with increasing D-dimer concentrations (0 to 2μg/mL), LOX-1 protein expression in these cells is enhanced in a dose-dependent manner. Treatment of RAW264.7 cells with 1μg/mL, D-dimer enhance LOX-1 protein expression in these cells.This effect was observed from 24 to 48 hours2. After treatment of Raw264.7 for 24 hours with 1μg/mL D-dimer we found D-dimer used alone cannot upregulated CD36. But effect of oxLDL increased CD36 is intensified when cell was pretreated with D-dimer for 24 hours3. To evaluate whether D-dimer enhanced uptake of oxLDL by Raw264.7 cells, these cells were treated for 24 hours with 2μg/mL D-dimer, and then were exposed for 24 hours to Dil-oxLDL (50μg/mL). To ascertain effect of D-dimer on oxLDL uptake is mediated by LOX-1 receptor, we use saturating amounts (20μg/mL) of antibodies to LOX-1 to antagonise the receptor, The photographs show D-dimer can increase uptake of oxLDL in macrophages. and effect of D-dimer is abolished by antibody of LOX-1, as assessed by fluorescence microscopy. Experiments were performed in the murine macrophage cell line RAW 264.7. These cells were incubated with or without D-dimer (1μg/mL) for 24 h and then activated with oxLDL (50μg/mL). We analyzed TNF and IL-6 mRNA levels in the RAW cells. TNF-a induction was increased approximately 5-fold in cells treated without D-dimer and 15-fold in cells had been loaded with D-dimer for 24 h. and IL-6 mRNA levels was increased approximately 1.5-fold in cells treated without D-dimer and 3-fold in cells had been loaded with D-dimer for 24 h.4. To determine the expression of the constitutive PKC in D-dimer-treated cells, Raw264.7 cells were treated for 24 h with 2μg/mL D-dimer, and expression of PKC protein was determined by Western blot analysis. D-dimer increased the expression of PKC protein To examine the signaling pathways mediating effect of D-dimer on LOX-1, Raw264.7 cells were treated for 1 h with the PKC inhibitor staurosporine (0 to 100 nmol/L) and then exposed for 48 hours to D-dimer (0 to 2μg/mL). pretreatment of the cells with c staurosporine abolished the stimulatory effect of D-dimer on LOX-1 protein expression5. To promote macrophage-drived foam cells formation, we treated Raw 264.7 cells with Dil-oxLDL(0 to 50μg/mL) for 48 h, observe cells with fluorescence microscope. The data show,oxLDL uptake is related to the concentration of extracellular oxLDL. We determined Nogo-B expression with western blot, the result show Nogo-B protein expression is upregulated in macrophage-drived foam cells. 6. Transfect siRNA of Nogo-B to RAW 264.7 cells for 48 h to ascertain the efficiency of siRNA, lysis cells and measure Nogo-B protein expression, data show that siRNA of Nogo-B is efficient. macrophages transfected with siRNA were stimulated with oxLDL for 8 h,measure TNF-a, IL-1β, IL-6 mRNA level with realtime PCR, TNF-α, IL-1βexpression does not change, but IL-1βmRNA is enhanced in Nogo-B-knockdown foam cells compare with normal foam cells.Conclusion:D-dimer induces LOX-1 express by increass actived PKC protein in macrophages, promote oxLDL uptake and foam cell formation, thus lead to AS aggravation. However, Nogo-B which is enhanced in macrophage-drived foam cells play a protective role in AS, when Nogo-B is knockdown in foam cells, oxLDL-induced IL-1βexpression will elevate.