Effect Of ANO1 Inhibitor On AngⅡ-induced Migration Of Vascular Smooth Muscle Cells And Its Mechanism

Objective Essential hypertension is the most common cardiovascular disease.Long term of hypertension might cause abnormal structure and function of the blood vessels(vascular remodeling),which plays a vital role in occurrence and development of hypertension and target organ injury.Angiotensin Ⅱ(AngⅡ)is the main active component of renin-angiotensin system(RAS).The activation of RAS increased the local levels of AngⅡ in blood vessles,inducing abnormal proliferation,migration,phenotype transformation and extracellular matrix(ECM)metabolism of VSMCs.TMEM16A/ANO1(ANO1)has been considered as a calcium-activated chloride channel of VSMCs.ANO1 was found to promote vasoconstriction and be involved int the regulation of blood pressure,but few studies were carried out to observe the relationship between ANO1 and vascular remodeling.In our previous studies,AngⅡ was found to induce expression of ANO1 of VSMCs,but the receptor of AngⅡ is not clear;ANO1 was also found to participate in AngⅡ-induced proliferation of VSMCs by regulating expression of cyclin(p21,p27)as well as AKT and ERK signaling pathways.In the present study,VSMCs from rat’s thorac aorta were used to clarify the subtype of AngⅡ receptor responsible for ANO1 over-expression,then a selective ANO1 inhibitor T16Ainh-A01(A01)was used to observe its influence on AngⅡ-induced migration of VSMCs,and its related mechanism was also studied.Contents 1.To observe the effect of AngⅡ on ANO1 expression and its related receptor.2.LY294002 and PD98059 were used to explore whether AKT and ERK signaling pathways participate in AngⅡ-induced expression of ANO1.3.To observe the effect of ANO1 inhibitor(A01)on AngⅡ-induced migration of VSMCs.4.To explore the related mechanism by detecting the protein levels of phenotypic marker(α-SMA,OPN)、MMP-2 and MMP-9、three proteins of ROCK signaling pathway as well as cyclin CDK4 and Cyclin D1.Methods After sacrificed,the thoracic aorta of rats were quickly isolated and the endothelial cells were removed.The primary culture of VSMCs was carried out by tissue patch method,the cell migration was estimated by the Transwell method,and the protein levels of various target proteins were determined by Western blot method.Results 1.To clarify the up-regulation of ANO1 by AngⅡ and its combined receptor,AT1 R blocker(losartan potassium,LP)or AT2 R blocker(PD123319,PD)was added before 100 nmol/L AngⅡ treatment for 24 h(This will be used in the following experiments).The results showed that compared with the control group,the ANO1 level in AngⅡ group was increased(1.37±0.09 v.s 0.96±0.08,P<0.05).Compared with AngⅡ group,the ANO1 level in the AngⅡ+LP group was significantly reduced(0.89±0.08 v.s 1.37±0.09,P<0.05),while there was no significant change in AngⅡ+PD group(1.32±0.09 v.s 1.37±0.09,P>0.05).These results suggest that AngⅡ might up-regulate the expression of ANO1 by its binding with AT1 R instead of AT2 R.2.To confirm that AKT signaling pathway might be involved in AngⅡ-induced expression of ANO1,LY294002(LY,a PI3 K inhibitor)was added before AngⅡ treatment.The results of AKT were as follows: compared with the control group,AngⅡ signficantly increased the AKT level(0.59±0.04 v.s 0.39±0.02,P<0.01).However,after pretreatment with LY,the AKT level was suppressed(0.39±0.03 v.s 0.59±0.04,P<0.01).The results of ANO1 was similar to that of AKT: AngⅡ markedly increased the expression of ANO1(1.42±0.10 v.s 1.00±0.07,P<0.05),and this effect was attenuated by pretreatment with LY(1.04±0.06 v.s 1.42±0.10,P<0.05).These results suggest that AngⅡ might activate PI3K/AKT signaling pathway to up-regulate the expression of ANO1.3.To clarify that ERK signaling pathway might be involved in AngⅡ-induced expression of ANO1,PD98059(PD,a MEK inhibitor)was added before AngⅡ treatment.The results of ERK were as follows: compared with the control group,AngⅡ signficantly increased the ERK level(1.40±0.04 v.s 1.00±0.06,P<0.05).However,after pretreatment with PD,the ERK level was suppressed(0.85±0.10 v.s 1.40±0.04,P<0.01).The results of ANO1 was similar to that of ERK: AngⅡ markedly increased the expression of ANO1(1.26±0.11 v.s 0.87±0.04,P<0.05),and this effect was attenuated by pretreatment with PD(0.89±0.11 v.s 1.26±0.11,P<0.05).The above results suggest that AngⅡ might activate MEK/ERK signaling pathway to up-regulate the expression of ANO1.4.The Transwell method was used to detect the effect of A01 on the migration of VSMCs induced by AngⅡ.Compared with the control group,the number of migrated cells in AngⅡ group was markedly increased(64±6 v.s 35±4,P<0.05),while the number of migrated cells in A01+AngⅡ group was reduced(38±4 v.s 64±6,P<0.05).These suggest that A01 effectively inhibit AngⅡ-induced migration of VSMCs.5.This experiment was carried out to observe the effect of A01 on AngⅡ-induced phenotypic transformation by detecting α-SMA(a contractile phenotype)and OPN(a synthetic phenotype).Compared with the control group,the protein level of α-SMA was decreased after AngⅡ treatment(0.79±0.03 v.s 1.22±0.04,P<0.01),while the expression of α-SMA was elevated in A01+AngⅡ(1.10±0.03 v.s 0.79±0.03,P<0.05).The expression of OPN was opposite to that of α-SMA: AngⅡ induced higher expression of OPN(1.00±0.04 v.s 0.71±0.06,P<0.01),and this effect was attenuated by A01(0.74±0.07 v.s 1.00±0.04,P<0.05).These data showed that A01 might inhibit AngⅡ-induced cell migration by phenotypic transformation from synthetic to contractile phenotype.6.MMP-2 and MMP-9 might participate in the migration of VSMCs by promoting the degradation of ECM.This study observed the effect of A01 on AngⅡ-induced expression of MMP-2 and MMP-9.The results showed that compared with the control group,MMP-2 protein was increased(0.96±0.05 v.s 0.76±0.04,P<0.05)after AngⅡ treatment,while MMP-2 in A01+AngⅡ group was decreased(0.73±0.05 v.s 0.96±0.05,P<0.01).The changes of MMP-9 were similar to that of MMP-2: AngⅡ increased the MMP-2 level(0.84±0.04 v.s 0.58±0.04,P<0.01),and this effect was suppressed by A01(0.67±0.01 v.s 0.84±0.04,P<0.05).The above results suggest that A01 markedly inhibited AngⅡ-induced expression of MMP-2 and MMP-9,two enzymes closely related with cell migration.7.This experiment to observe the effect of A01 on ROCK signaling pathway by detecting protein levels of Rho A,ROCK1 and MYPT1.The results of Rho A showed that compared with the control group,the Rho A protein was increased(1.13±0.07 v.s 0.69±0.11,P<0.05)after AngⅡ treatment,while the Rho A expression in A01+AngⅡ group was decreased(0.75±0.03 v.s 1.13±0.07,P<0.05).The change of ROCK1 protein was similar to that of Rho A.Compared with the control group,the ROCK1 level of AngⅡ group was increased(0.90±0.04 v.s 0.66±0.07,P<0.01).After adding A01,the ROCK1 level was decreased(0.64±0.04 v.s 0.90±0.04,P<0.01).In addition,this experiment also detected the changes of the downstream substrate MYPT1.Compared with the control group,the level of MYPT1 in the AngⅡ group was increased(1.64±0.08 v.s 0.94±0.15,P<0.05).After adding A01,the protein level of MYPT1 was decreased(1.07±0.15 v.s 1.64±0.08,P<0.05).The above results suggestthat A01 markedly inhibted AngⅡ-induced activation of ROCK signaling pathway,which is closely related with both constriction and migration of VSMCs.8.This experiment explored the effect of A01 on AngⅡ-induced proliferation by detecting the levels of cyclin CDK4 and Cyclin D1.The results showed that compared with the control group,the CDK4 protein level was increased after AngⅡ treatment.(1.00±0.05 v.s 0.70±0.03,P<0.01).Compared with the AngⅡ group,the CDK4 protein in the A01+AngⅡ group was reduced(0.82±0.04 v.s 1.00±0.05,P<0.05).The changes of Cyclin D1 were similar to CDK4: AngⅡ induced the increased expression of CDK4(1.01±0.04 v.s 0.76±0.02,P<0.01),while this alteration was attenuated by A01(0.81±0.05 v.s 1.01±0.04,P<0.05).These results suggested that AngⅡ can accelerate the cell cycle process by promoting the expression of CDK4 and Cyclin D1,while A01 can inhibit this effect.Conclusions In conclusion,the combination of AngⅡ with ATIR might activate AKT and ERK signaling pathways,leading to up-regulation of ANO1 of VSMCs.The ANO1 inhibitor A01 significantly reduced the migrated cells induced by AngⅡ,suggesting that ANO1 might be involved in this process.The inhibitory effect of A01 might be due to the following mechanism:(1)Promoting the phenotypic transformation from a synthetic phenotype to a contractile phenotype;(2)Reducing the expression of MMP-2 and MMP-9,two important enzymes involved in degradation of ECM;(3)Inhibiting the activation of ROCK signaling pathway.In addition,A01 was also found to decrease the expression of CDK4 and Cyclin D1,which might be responsible for the inhibitory effect of A01 in AngⅡ-induced proliferation of VSMCs.