Activation Of OX40Ligand Reverse Singal Potentiates The Expression Of Atherosclerosis Plaque Related Factors Of Cultured Aortic Endothelial Cells From C57BL/J6Mice

BackgroundInflammation plays an important role in all stages of atherosclerotic process. The development of a vulnerable atherosclerotic lesion composed of the progression of endothelial dysfunction, the migration of the mononuclear macrophages into the arterial wall and the production of plaque rupture factors. Of these processes, endothelial dysfunction is the initial key point.OX40ligangd (OX40L), a kind of costimulatory moleculars, participates the formation and development of atherosclerostic plaque. OX40L is mainly expressed on the antigen cells, including endothelial cells. OX40L plays an important role of atherosclerotic process. Anti-OX40L antibodies can prevent the formation and enhance the stability of atherosclerostic plaque. Endothelial cells express aboundent OX40L in the position of aortic plaque of atherosclerostic mice. The high expression of endothelial OX40L whether or not plays an role in the formation and eruption of atherosclerostic plaque is unkown.The reverse signal is a common figure of co-stimulators. In deed, OX40L can activates the signal patheway of OX40and on the other hand OX40L can also stimulate the activation of its receptor, OX40. Activation of OX40L reverse signal mediates adhesion of activated T cells to the endothelial layer directly. The accumulation of large amount of T-cells inside the plaque and high expression of OX40L of endothelial cells near the plaque would be able to promotes the interaction between endothelial cells and T-cells, which maybe resulting in the mis-equilibrium of the expression or release of pro-atherosclerotic factors.AimThe present study was designed to investigate whether or not the activation of OX40L reverse singal of cultured aortic endothelial cells can promote the expression of atherosclerotic plaque related factors and the possible mechanism underlying.Methods1. The isolated aortic endothelial cells from C57BL/6J mice were cultured and passsaged to second generation. With immunocytochemistry, the cultured endothelial cells were identified and the epression of OX40L was confirmed on the passaged endothelial cells. The cultured endotehlial cells were incubated with0.lmg/L soluble OX40for48hours. The cells were collected. The changes of mRNA level of atherosclerotic related factors of the collected cells were exmined by real-time PCR, including adhesion molecules (Pecam-1, E-selectin, ICAM-1and VCAM-1), inflammatory factors (COX-1, COX-2, IL-6and IL-8) and matrix metalloproteinases (MMP-1, MMP-2and MMP-9). The effect of0.1mg/L monoclonal antibody against OX40L on the mRNA expression of the above factors were checked.2. The effect of soluble OX40on intracellular calcium concentration of the cultured endothelial cells was accessed by fura-2fluorescent calcium imaging. The adminstration of monoclonal antibody against OX40L was used to identify the sepcific effect of soluble OX40on the intracellular calcium concentration. The effects of calcium chelator, Bapta-AM, on changes of gene and protein expression level of OX40L on cultured endothelial cells induced by soluble OX40were examined by Real-time PCR and Western Blot methods. The changes of the activety of MMP-2and MMP-9were checked by gelatin-zymography as well as the effect of Bapta-AM and COX inhibitors. ResultsThe primary cultured were passaged to second generation. The cells were identified to be endothelial cells by immunocytochemistry with PEC AM-1monoclonal antibody. The passaged cells expression of OX40L was confirmed by immunocytochemistry with monoclonal antibody against OX40L. After the stimulation with soluble OX40for48hours, the mRNA levels of COX-1, COX-2, MMP-2, MMP-9and Pecam-1of the cultured cells were significantly higher than those of un-treated cells. This effect of soluble OX40was inhibited by the aplication of monoclonal antibody against OX40L.The intracellular calcium concentration of the cultured endothelial celll was significantly increased by the stimulatiuon of soluble OX40, which could be blocked by the aplication of either monoclonal antibody against OX40L or calcium chelator, Bapta-AM. Meanwhile, the elevation of protein expression level of COX-1, MMP-2and MMP-9induced by soluble OX40was decreased with Bapta-AM. Soluble OX40induced augmentation of MMP-9activity was significantly inhibited by selective COX-1inhibitor, SC-560, and non-selective COX inhibitor,indomethacin.ConclusionThe present results indicates that the activation of OX40L reverse signal of cultured aortic endothelial cells from C57BL/J6mice can promote the expression of atherosclerotic plaque related factors through the increasing of intracellular calcium concentration of the cultured endothelial cells.