Dabigatran Ameliorates Human Airway Smooth Muscle Cells Remodeling By Modulating YAP

Background and objective:Thrombin,the core protease of the coagulation cascade,in addition to exerting coagulation function,it may also be related to the pathogenesis of asthma.Our previous research found that dabigatran,a direct thrombin inhibitor for clinical anti coagulation,effectively attenuated the pathological changes of airway remodeling in asthma mouse models induced by ovalbumin(OVA).However,the effect of dabigatran on thrombin-induced remodeling of human airway smooth muscle cells is unclear.Yes-associated protein(YAP)is a key effector of the Hippo pathway that regulates organ size and tissue homeostasis by inducing cell proliferation and differentiation.Recent studies showed that YAP was upregulated in airway smooth muscle in a mouse model of asthma,but whether YAP is involved in thrombin-induced remodeling of human airway smooth muscle cells has not been reported.Therefore,the purpose of this study was to investigate the effects of dabigatran on thrombin-induced functional changes in HASMCs,the role of YAP in thrombin-induced functional changes in HASMCs,and the underlying molecular mechanism that regulates YAP by thrombin.Methods:The subjects were primary human airway smooth muscle cells.CCK-8 and EdU experiments were used to detect cell proliferation;scratch assay was used to test cell migration;qRT-PCR and Western Blot were conducted to measure mRNA and protein expression of Collagen I,?-SMA,CTGF and Cyclin D1;Western Blot was conducted to measure the protein expression of relative signal pathway;Immunofluorescence was applied to observe the location of YAP in cells and changes in F-actin cytoskeleton;immunofluorescence double staining and CoIP were conducted to detect the binding of YAP to SMAD2 and their interaction.Results:1.Effects of dabigatran on thrombin-induced HASMCs remodeling(1)Dabigatran inhibited thrombin-induced cell proliferation in a concentration-and time-dependent manner,with the greatest inhibitory effect at a concentration of 3000ng/ml and a 48h time point;(2)Dabigatran inhibited thrombin-induced cell migration;(3)Dabigatran inhibited thrombin-induced up-regulation of Collagen I,?-SMA,CTGF,and Cyclin D1.2.Effect of dabigatran on thrombin-induced YAP activation(1)Thrombin promoted YAP dephosphorylation and nuclear translocation in a time-dependent manner,with the greatest activation effect at 1 h;(2)Dabigatran inhibited thrombin-induced YAP dephosphorylation and nuclear translocation3.Effects of YAP on thrombin-induced HASMCs remodeling(1)YAP knockdown or verteporfin(YAP inhibitor)inhibited thrombin-induced cell proliferation;(2)YAP knockdown or verteporfin(YAP inhibitor)inhibited thrombin-induced cell migration;(3)YAP knockdown or verteporfin(YAP inhibitor)inhibited thrombin-induced up-regulation of Collagen I,?-SMA,CTGF,and Cyclin D14.Investigation on the mechanism of thrombin to activate YAP(1)PAR1 inhibitor SCH79797 inhibited thrombin-induced up-regulation of RhoA,ROCK,p-MLC2,F-actin polymerization and YAP dephosphorylation and nuclear translocation;(2)The RhoA inhibitor Y-16 inhibited thrombin-induced up-regulation of ROCK,p-MLC2,F-actin polymerization and YAP dephosphorylation and nuclear translocation;(3)ROCK inhibitor Y-27632 inhibited thrombin-induced up-regulation of p-MLC2,F-actin polymerization and YAP dephosphorylation and nuclear translocation;(4)Cytochalasin D,an F-actin depolymerizer,inhibited thrombin-induced F-actin polymerization and YAP dephosphorylation and nuclear translocation;(5)Thrombin induced the binding of YAP to SMAD2 and their nuclear translocation.Conclusions:1.Dabigatran ameliorated thrombin-induced HASMCs remodeling by inhibiting YAP;2.Thrombin promoted actin stress fiber polymerization through the PAR1/RhoA/ROCK/MLC2 axis to activate YAP,then interacted with the transcription factor SMAD2 to induce downstream target genes.