Effect Of ANO1 Inhibitor On Migration Of Vascular Smooth Muscle Cells Induced By Endothelin-1 And Possible Mechanism

ObjectiveVascular remodeling(VR)is a change in the structure and function of blood vessels resulting from various causes.Vascular smooth muscle cells(VSMCs)located in the middle layer of blood vessels have many functions,including vascular contraction,having migration and proliferation capacity,releasing a variety of active substances and inflammatory factors,and secreting extracellular matrix(ECM).The abnormal function of VSMCs is recognized as a key pathogenic mechanism of vascular remodeling.ANO1(anoctamin 1)was found to be the molecular basis of calcium actived chloride channel in 2008,and widely distributed in the cardiovascular system.ANO1 has been shown to be involved in a series of normal physiological functions,such as vasodilation,blood pressure regulation and cardiomyocyte action potential formation.It is also associated with various diseases,including hypertension,myocardial ischemia-reperfusion injury,myocardial fibrosis.In spontaneously hypertensive rats(SHR)and pulmonary hypertensive(PH)rats,over-expression of ANO1 of VSMCs promotes abnormal proliferation and migration,thus promoting vascular remodeling and hypertension formation.However,the regulatory mechanism of ANO1 expression in VSMCs was not clearly understood.ET-1,a 21 amino acid peptide,is synthesized by endothelial cells and is a powerful vasoconstrictor.Under pathological conditions,excessive ET-1 also participates in VR by promoting proliferation and hypertrophy of VSMCs,inducing VSMCs to synthesize ECM and various inflammation-related substances,damaging vascular endothelium,and promoting platelet aggregation.Our previous study found that ET-1 enchanced ANO1 expression in normal rat VSMCs,while application of ANO1specific inhibitor T16A(inh)A01(A01)inhibited ET-1-induced cell proliferation.In this study,by using the primarily cultured VSMCs,we further observed the effect of A01 on ET-1-induced cell migration and explored the possible mechanism of A01,by observing cell phenotypic transformation,matrix metalloproteinase-2(MMP-2)and MMP-9 expression,and changes of RhoAassociated kinase(ROCK)signaling pathway.In addition,this study also observed the effect of A01 on ET-1-induced inflammatory response of VSMCs.Content1.The effect of ET-1 on expression of ANO1 of VSMCs was observed.2.Using the Transwell assay,the effect of ANO1 inhibitor A01 on ET-1 induced cell migration was examined.3.To explore the possible mechanism of A01,the expression of α-smooth muscle actin(αSMA)and osteopontin(OPN),MMP-2 and MMP-9 and ROCK signaling pathway proteins,was observed.4.The effects of A01 on ET-1 induced inflammation-related proteins,such as inhibitor of NF-κB(IKB),inductible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2),were also determined.MethodsThe primary culture of VSMCs from normal Wistar rat thoracic aorta was performed,the changes in cell migration ability were observed by the Transwell method,and the protein expression was examined by Western blot(WB).Results1.The changes in ANO1 protein expression were observed after different concentration of ET-1(0.1,1,10 nmol/L)were added to VSMCs for different time(1,6,12,24,48 h).The results showed that 1 nmol/L ET-1 significantly promoted the protein expression of ANO1(P<0.01),the change of ANO1 protein level was the most significant after 24 h of ANO1 treatment(P<0.01).These results suggested that ET-1 had a significant promoting effect on ANO1 expression.In subsequent experiments,the cells were treated with 1 nmol/L ET-1 for 24 h.2.The effect of A01 on ET-1 induced cell migration was examined by the Transwell assay.The results showed that ET-1(1 nmol/L,24 h)increased the numbers of migrating cells(P<0.01).However,after co-treatment of 20 μmol/L A01 with ET-1,the migrating cells were significantly reduced(P<0.05).Thus,ET-1 clearly promoted the cell migration,while A01 blocked this effect.3.Phenotype transformation of VSMCs plays a vital role in cell migration.This experiment observed the changes of α-SMA(contractile type)and OPN(synthetic type)to explore the inhibitory mechanism of A01 on cell migration induced by ET-1.The results showed that ET-1 treatement significantly reduced the expression of α-SMA(P<0.01)but elevated the expression of OPN(P<0.05).After adding A01,the above changes were reversed(P<0.05),suggesting that A01 might prevent ET-1-induced cell migration by phenotypic transformation from the synthetic phenotype to the contactile phenotype.4.We also observed the effect of A01 on ET-1-induced protein expression of MMP-2 and MMP-9,two important enzymes for ECM metabolism.The results showed that ET-1 treatment elevated the protein levels of both MMP-2 and MMP-9(P<0.05),these alterations were counteracted by A01 treatment.Thus,the inhibitory effect of A01 might be associated with the reduced protein levels of MMP-2 and MMP-9.5.The effects of A01 on the expression of ROCK pathway proteins(RhoA,ROCK1 and pMYPT1)were also studied.The results showed that ET-1 markedly increased the protein expression of RhoA,ROCK1 and p-MYPT1(P<0.05-0.01),indicating the activation of ROCK signalling pathway.As expected,A01 blocked the activation of ROCK signaling pathway,as evidenced by signifcanlty reduced expression of three proteins(P<0.05-0.01).We hypothesized that inhibiton of ROCK pathway might be involved in the inhibotory effect of A01.6.Nuclear factor kappa-B(NF-κB)is the most important signaling pathway related with inflammation of VSMCs.Phosphalation of IKB(a key inhibitory protein)results in translocation of NF-κB to the nucleus,promoting the expression of target genes.In this study,we examined the effect of A01 on p-IKB expression.The results showed that ET-1 elevated the p-IKB level(P<0.01),and this change was blocked by A01(P<0.01).7.This study also observed the effect of A01 on ET-1-induced protein epression of iNOS and COX-2(P<0.05-0.01),which respectivley synthesize NO and PGE2,two important inflammatory factors.The results showed that ET-1 up-regulated of both iNOS and COX2,these changes were also counteracted by A01 treatment(P<0.05-0.01).ConclusionIn conclusion,ET-1 promotes the protein expression of ANO1 of VSMCs.The ANO1 specific inhibitor A01 significantly inhibited the migration of ET-1-induced cell migration.This inhibitory effect of A01 might be due to the phenotypic transformation,reduced MMP2/9 expression and inhibition of ROCK signaling pathway.Moreover,A01 also inhibited the ET-1-induced inflammatory response of VSMCs,the latter might be associated with dysfunction of VSMCs.