| IntroductionThe occurrence, development and the process from stability to rapture of atherosclerotic plaques are associated with number of factors such as the disorder of lipid metabolism, involvement of immune system, endothelial dysfunction and genetic background. The exact mechanism is still under investigation. OX40 Ligand (OX40L/CD134L/gp34), a kind of co-stimulator molecules, belongs to the TNF super family. It acts as the auxiliary factor involving the process of inflammation. OX40L is mainly expressed on surface of the antigen presented cells, vascular endothelial cells and smooth muscle cells. The reverse signal pathway is an important factor of co-stimulatory molecules. It means that OX40L can also transducer the signal from its receptor, OX40L, to intracellular pathway. OX40L is highly expressed in the atherosclerotic plaque. The ligation of OX40 and OX40L can induce increased expression of c-jun gene and RANTES/CCL5 in human umbilicus veins endothelial cells , directly induces the adhesion of T-cells or OX40L+T cells to endothelial cells. Previous study find that the C57BL/6 mice are atherosclerosis susceptible, while the C3H/ He mice are atherosclerosis resistant. They foud the first atherosclerosis susceptible gene including OX40L gene by the mothed of quantitative trait locus. The dysfunction of endothelial cells not only is the initial step of development of atherosclerosis but also accelerate the formation of the atherosclerosis lesions. While the atherosclerosis lesions are relieve from the protection of endothelial cells The administration of OX40L monoclonal antibody reverses the plaque in atherosclerotic mice. These data suggests a pivotal role of OX40L in atherosclerosis. C-reactive protein (CRP) is an acute-phase reactant and serves as a pattern-recognition molecule in the innate immune system. It is regarded as an inflammatory marker atherosclerosis. Our previous data showed that CRP can induce the injury of cardiac myocytes. In fact, the high expression of CRP level is seen in the blood of patients with coronary artery disease. Whether the increase of CRP in the peripheral circulation influences the expression of OX40L in endothelial cells is unknown.ObjectiveThe present study was designed to compare the expression of OX40L in tissues from normal atherosclerosis susceptible C57BL/6 mice with normal atherosclerosis resistant BALB/c mice. Investigate the effect of CRP on OX40L expression in cultured C57BL/6 mice aorta endothelial cells.Methods1.Eight C57BL/6 mice and eight BALB/c mice at the age of eight weeks were sacrificed, and the total mRNA as well as tissue homogenates were isolated from the heart, brain, kidney, skeletal muscle and spleen. The OX40L mRNA expression level detected by RT-PCR and the protein expression level was detected by Western Blot.2.CRP was purified by Immobilized p-Aminophenyl Phosphoryl Choline Gel. The purified CRP was analysis by SDS-PAGE and identified by Western Blot.3. BALB/c mice were sacrificed and the aorta was dissected. The generation of cultured endothelial cells was utilized by collagenaseⅡdigestion of the aorta. The identification of the endothelial cells carried out by immunostaining with special antibody factorⅧantibody. When the endothelial cells grow to about 60~70% of the dish, 100ug/ml CRP was applied to medium. 48 hours later, the endothelial cells were collected and the total RNA and protein homogenates were isolated. The gene expression and protein expression of OX40L were measured by RT-PCR and Western Blot respectively.Results1. The OX40L mRNA expression level in the heart of C57BL/6 mice was significantly higher than that of BALB/c mice (0.79±0.10 vs 0.60±0.05, P <0.05, n=8). There was no significant difference between BALB/c mice and C57BL/6 mice in brain, kidney and skeletal muscle. The OX40L mRNA level was augmented significantly in the spleen from BALB/c mice than that from C57BL/6 mice (0.39±0.17 vs 0.25±0.04, P <0.05, n=8). The protein levels of OX40L expressed significantly higher in the heart, brain and kidney from and was C57BL/6 mice than those from BALB/c mice, respectively (0.56±0.01 vs 0.19±0.07, 0.18±0.07 vs 0.01±0.01, 0.31±0.13 vs 0.03±0.01, P<0.05, n=8). No significant difference was obtained in skeletal muscle and spleen between these two kinds of mouse.2. The purified CRP showed more than 95% purity by SDS-PAGE. The purified CRP binded specifically to CRP monoclonal antibody.3. After 48 hours treatment of 100 ug/ml CRP, the expressions of OX40L mRNA or protein level, in the treated cultured endothelial cells were significantly augmented than those in the untreated cultured endothelial cells (0.09±0.04 vs 0.76±0.12 and 0.07±0.02 vs 0.21±0.06,P<0.05, n=3).ConclusionsThe present study showed the different expression level of OX40L tissues from BALB/c mice and C57BL/6 mice. CRP can induce the expression of OX40L in mouse aortic endothelial cells. These data suggested a role underlying the mechanism of OX40L in atherosclerosis. |
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