| Summary: | 碩士 === 中國醫藥大學 === 生物科技學系碩士班 === 99 === Background: Atherosclerosis is a chronic inflammatory disease. An important early pro-inflammatory event is the initiation of atherosclerosis in the adhesion of circulating monocytes to vascular endothelial cells mediated by cell adhesion molecules. Bacterial lipopolysaccharide (LPS), a potent endotoxin, aggravates atherosclerosis by inducing a strong inflammatory response in the arterial vascular wall. Protease-activated receptor-2 (PAR-2) is a member of G protein-couple receptor family, activated by serine proteasess. PAR-2 is an important mediator in innate immune and plays a role in propagation of pro-inflammation response. PAR-2 is abundantly expressed on endothelial cells. The activation of endothelial PAR-2 is the consequence of the local-released serine proteases accompanied with tissue inflammation or injury. However, the relationship between PAR-2 signaling pathway and LPS-activated pro-inflammation response remains unclear. The aim of the present study is to investigate whether LPS activates PAR-2 expression and in turn, enhances the protease (trypsin)-induced PAR-2 signaling and its association with adhesion molecule expression in human vascular
endothelial cells.
Methods and Results: In cultured umbilical vein endothelial cell lines (EA.hy926 cells), treated with various dosage of LPS (0, 0.125, 0.25, 0.5, 1, 2, 5 and 10 μg/ml respectively) for 24 hours, the morphology of endothelial cells was observed by microphotography and the cells were counted by MTT assay. Intracellular calcium imaging was used to determine the activity of PAR-2 induced by it’s protease agonists. Stimulation with trypsin (2μg/ml) increased intracellular calcium level in endothelial cells. This calcium influx was augmented in cells pre-treated with high dose LPS (10μg/ml).Western blotting was employed to determine the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule, MCP-1 in endothelial cells. Twenty-four hours after endothelial cells treated with LPS, there was no obvious change of the cell counts and morphology. Western blotting analysis reveals that LPS up-regulated the expression of PAR-2 and enhanced phosphorylation of ERK and MCP-1 protein level in response
to PAR-2 agonist, trypsin.
Conclusion: Our findings provide the first evidence that endotoxin, LPS potentiates calcium mobilization and activation of ERK pathway, and subsequently leads to expression of pro-inflammatory chemokine, MCP-1 via induction of PAR-2 gene expression and its activity in vascular endothelial cells. The present results implicated the cooperative functions of proteases and the inflammatory stimuli in the amplification of vascular inflammation. Therefore, PAR-2 antagonists may have the therapeutic
potential in the treatment of atherosclerosis.
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