| Objectives:The incidence of atherosclerosis(AS) has increased in developed countries, even in China. Proliferation and migration of vascular smooth muscle cells play an important role in the development of AS. A variety of extracellular stimulating factors plays a significant role on the migration of VSMC, such as vasoactive substances, growth factors and extracellular matrix. Platelet-derived growth factor (PDGF) is a major principle in promoting cell division, the main factors that caused atherosclerosis, its main role is to promote smooth muscle cell migration from the middle to the inner. The migration of vascular smooth muscle cells involves transmembrane of signals promoting migration, cascade activation of signaling molecules in cell, cytoskeletal proteins shrinkage and cell deformation. PDGF is a family of disulfide-bonded homo-and heterodimers, with PDGF-AA, PDGF-BB and PDGF-AB in three forms. This factor was not expressed in normal arteries, but expression of PDGF has increased after angioplasty or atherosclerosis induced endothelial cell injury. Accordingly, PDGF may act as the media play an important role in migration. PDGF-BB come from platelets, endothelial cells, macrophages, acting on smooth muscle cells could stimulate chemotaxis and proliferation of smooth muscle cells. PDGF-BB, as one type of PDGF, is known as the strongest chemotatic agent. PDGF-BB could control VSMC migration by cell migration pathway of direct control, but the related mechanism of intracellular signal transduction is not yet clear. This study is aimed to examine the molecular mechanism in the migration of vascular smooth muscle cell induced by PDGF-BB.Methods:(1)To determine the best concentration of PDGF-BB induced migration of vascular smooth muscle cells, using wound healing assay, with different concentrations (1,5,10,20 and 50ng/ml) PDGF-BB as inducer, observe cell migration distance after 24h.(2)Using the ROCK inhibitor Y27632 and MLCK specific inhibitor ML7 to observe the effects on cell migration. (3)Using immunofluorescence, we observed morphological changes of F-actin and vinculin through the different concentrations of PDGF-BB induced, and the morphological changes of F-actin and vinculin after adding inhibitor ML7 and Y27632. (4)To detect expression levels of ROCKâ… , ROCKâ…¡and MLC phosphorylation levels induced by PDGF-BB, Western bloting was used.(5)To test the level of MLC phosphorylation, RNA interference technology was used to make ROCKâ… and ROCKâ…¡protein decreased; Using the ROCK inhibitor Y27632 and MLCK specific inhibitor ML7 to observe the effect on MLC phosphorylation.Results:(1)Wound healing assay to detect cell migration:The optimum concentration of PDGF-BB induced cell migration is 10ng/ml; Y27632 and ML7 inhibited the cell migration. (2)Immunofluorescence assay to observe cell morphology:Cell morphology was observed after induced by different concentrations of PDGF-BB, pseudopod of F-actin increased, form a stress filaments; Expression of vinculin was decreased, more obvious in the lOng/ml; Pseudopod of F-actin decreased, expression of vinculin increased after adding the inhibitor ML7; The cells change greatly, but no F-actin stress fiber form after adding the inhibitor Y27632. (3)Western bloting:The expression of ROCKâ… ,ROCKâ…¡was increased after induced by different concentrations of PDGF-BB, showing a concentration dependent manner. (4)Glycerol gel to detect MLC phosphorylation levels:MLC phosphoryla-tion level did not change significantly after induced by lOng/ml of PDGF-BB at different times; MLC phosphorylation levels were reduced after down regulation of ROCK gene and inhibitor Y27632, ML7 added.Conclusions:(1)PDGF-BB induce cell migration through both Rho kinase and MLCK signaling pathway. (2)Cell morphology was changed when cell migration was induced by PDGF-BB, F-actin pseudopod increased, expression of vinculin decreased. (3)The MLC phosphorylation pathway, downstream of MLCK and ROCK, was not go through when cell migration was induced by PDGF-BB.
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