The Role Of Rho Kinase Signal Pathway In Endothelin-1 Induced Human Airway Smooth Muscle Cell Migration And Cytoskeletal Reorganization

BackgroundAsthma is characterized by chronic airway allergic inflammation as well as alteration of structural components of the airways.These changes,collectively referred to as 'airway remodeling',including thickening of subcpithelium,incrcased collagen and proteoglycan deposition,fibroblast proliferation and differentiation to myofibroblasts,and an increase in smooth muscle mass.Smooth muscle cells,which are seen in grate numbers and size,play an important role in airway remodeling,which are considered as the most important cells in the procedure of airway remodeling.The changes of ASMC like contraction,proliferation,hypertrophy and excreting ECM,which also promote the airway remodeling and AHR.In the past several years,some researches found that,like the migration of vascular smooth muscle cell(VSMC) in atherosclerosis,ASMC also possesses the capability of migrating.ASMC migrates to the tunica intima of airway and proliferate,which resulting in the airway thicken and narrow.Although there is,as yet, no direct evidence showing that ASMC migrate in vivi in the adult airway,it is possible that ASMC migration is an important aspect of the airway remodeling. Moreover,it is also possible that the migration of ASMC toword the lumen of the airway plays a role in the appearance of myofibroblasts in the lamina reticularis of the asthmatic airway.The myfibroblast has an intermediate betwen differentiated smooth muscle cells and fibroblasts.By electron microscopy,myfibroblasts of the airway are elongate cells and located in the lamina reticularis,a layer that lies between the true epithelial basement membrane and the airway smooth muscle layer.Thus,the myofibroblasts may comes from the migtated ASMC.Some research have found that cytokine,mediators of inflammation,growth factors such as PDGF,IL-1β,urokinase,LPA can induce ASMC migration,but as an important mediators of inflammation,Endothelin-1(ET-1) is one of the strongest contract substance in the airway.Airway epithelium expresses much ET-1 when it is damaged,and ET-1 can induce many types of cells such as EOS,VSMC,fibroblast,tumour cells,langerhans cells migtation and infiltration.Rho/Rho kinase signal pathway is an important path way in the ASMC,Rho kinase(ROCK) is the key element of this signal pathway,more and more evidences indicated that it participates in the pathophysiology of AHR,migration of EOS,airway remodeling and so on.But there is few research on the role of Rho/Rho kinase pathway in the migration of ASMC induced by ET-1.ObjectivesThis research will concentrate on two aspects:First,to study the effect of ET-1 on the migration of ASMC.Second,to study the role of Rho/Rho kinase in the migration of ASMC induced by ET-1.Methords1,The bronchus samples were got from the Pulmonary lobectomy,chest surgery department of nanfang hospital,after obtaining the permission of the lung cancer patients.2,ASMC was primarily cultured by explant method from the trachea of human, and identified by light inverted microscope and cell immunohistochemistry.3,Migration of ASMC was examined by using modified Boyden chambers,5ng/ml PDGF as a positive control. 4,Using different concentrations of Y-27632 to block the Rho kinase pathway,the role of the signal pathway in the migration of ASMC was studied.5,FITC-labeled phalloidine labeled ASMC cytoskeletal,Changes in actin cytoskeletal organization induced by ET-1 were observed with confocal laser scanning microscope.6,ASMC was lysed and the concentration of protein was measured,the phosphorylation of MYPT1,which is the Rho kinase downstream substrate,was examined by western blot.7,All statistical tests were operated with the program SPSS13.0,all the data was expressed by(?)±S,One-Way ANOVA was applied in the comparison of more samples.We accepted statistical significant values of P＜0.05.Results1.The cellular morphology and identification of ASMC:The cellular morphology of ASMC was observed by inverted microscope,showing fusiform shape,When they grew to near-confluent,peak-valley was seen.Immunocytochemical study revealed strong expression ofα- actin.2,ET-1 increased ASMC migration activity at 0.1nmol/L,1nmol/L,10nmol/L. 100nmol/L,the number of migrated cells was 25.2±1.56,36.03±2.94,49.80±4.47,37.96±3.59,and the maximal effect was obsvered and 10nmol/L ET-1,compared with the control group(19.08±2.06,P＜0.05),there is significant difference between the ET-1 group and the control group.PDGF(5ng/ml) can significantly induced ASMC migration(87.73±6.14),compared with control group,there is significant difference between this two groups(P＜0.01).3,Rho kinase inhibitor Y-27632 inhibited ET-1(10nmol/L) induced migration of ASMC in a dose-dependent manner,as the concentration of Y-27632 increased,the inhibition effect was more obvious.when ASMC was pretreated with Y-27632 at the concentration of 0.4μmol/L,2μmol/L,10μmol/L,the number of migrated cells was 44.26±2.50,34.56±1.67,20.93±1.84.Y-27632(10μmol/L) significantly inhibited the migration of ASMC,compared with Y-27632(0μmol/L) group(48.30±2.64),there is significant difference(P＜0.01).4,There is a little of stress fiber and no filopodia in the control group,stimulated ASMC with ET-1(10nmol/L) induced reorganization of actin cytoskeleton and formation of stress fibers in 30minues.While ASMC were pretreated with Y-27632(10μmol/L ),then stimulated with ET-1(10nmol/L),the changes of actin cytoskeleton and stress fibers were inhibited.5,Western blot:The expression of p-MYPT1 protein in ASMC after stimulation with ET-1(10nmol/L) in 15min,30min,1h,2h,6h,12h,24h was as follows: 0.68±0.04,0.66±0.10,0.44±0.06,0.48±0.14,0.43±0.13,0.39±0.10,0.28±0.05.Compared with the control group(0.37±0.03),stimulation with ET-1 in 15min and 30min can significantly enhance the p-MYPT1 expression(P＜0.01),while the time prolonged,the expression of p-MYPT1 was decreased,compared with control group,there is no significant difference(P＞0.05).ConclusionIn certain concentrations range,Endothelin-1 possesses the capability of inducing ASMC migration,while Rho kinase inhibitor Y-27632 significantly inhibited the migration and cytoskeletal reorganization of ASMC induced by ET-1.ET-1 can activate the Rho kinase signal pathway,which indicated that Rho kinase pathway play an important role in ASMC's migration.Thus,Rho kinase signal path way plays an important role in the airway smooth muscle cell migration induced by Endothelin-1.