DKK1 Mediates Biomechanical Regulation Of Smooth Muscle Cell Proliferation And Migration

BackgroundCardiovascular disease is the main cause of human death,and vascular remodeling and atherosclerosis caused by various risk factors are the common pathological basis.Clinical and pathological studies have shown that atherosclerotic lesions mainly occur in vascular bifurcation,curvature and stenosis.Hypertension can cause cell proliferation and thickening of vascular wall,and abnormal blood flow after interventional therapy can cause vascular proliferation and restenosis.These factors suggest that vascular mechanics is an important inducement of vascular remodeling and atherosclerosis.The main manifestations of vascular remodeling were abnormal proliferation,apoptosis and migration of vascular smooth muscle cells(VSMCs).Vascular endothelial cells can directly sense the stimulation of blood flow shear stress and affect the function of smooth muscle cells through the intercellular interaction.The study of the molecular mechanism of vascular remodeling induced by pathologic shear stress and the interaction between ECs and SMCs is helpful to better understand the pathogenesis of cardiovascular diseases.Recent studies have shown that biomechanics plays an important role in neointimal hyperplasiaDKK1,also known as Dickkopf 1,is a secretory glycoprotein,which can act through paracrine and autocrine.On the one hand,DKK1 plays a role by binding to LRP6 and antagonizing the downstream Wnt pathway.On the other hand,DKK1 can bind type Ⅱ transmembrane cytoskeleton associated protein 4,CKAP4(P63),and then the intracellular segment recruits PI3K,activates Akt,and promotes proliferation.Previous studies have shown that DKK1 can promote the proliferation and migration of tumor cells and tumor survival by regulating the CKAP4/PI3K/Akt,non-classical Wnt/PCP/JNK and β-catenin/MMP7 pathways.In recent years,the role of DKK1 in cardiovascular disease has attracted attention,and drug development targeting DKK1 has been carried out in phase II clinical trials.It was found that DKK1 was positively correlated with carotid intima media thickness.In patients with acute coronary syndrome receiving dual antiplatelet therapy,DKK1 levels are independently associated with cardiovascular events and individual cardiovascular death.Ueland found high expression DKK1 in human carotid atherosclerotic plaques,DKK1 was involved in platelet-induced endothelial cells activation,and DKK1 could promote inflammation between endothelial cells and platelets.Our previous study found that elevated serum DKK1 level can be used as an independent predictor of recurrent adverse cardiovascular events in acute coronary syndrome,and combined with risk factors can significantly improve the predictive value.DKK1 plays an important role in regulating the function of vascular endothelial cells.Both ox-LDL and pathological shear stress can up-regulate the expression of DKK1 in vascular endothelial cells,promoting the development of atherosclerosis by antagonizing the classical Wnt pathway and activating JNK.The overexpression of lentivirus and the silencing of DKK1 in ApoE-/-mice further confirmed that DKK1 promoted endothelial cell dysfunction,promoting plaque formation and increased plaque instability.However,the effect and mechanism of DKK1 on the formation of neointima formation have not been thoroughly studied.DKK1 is regulated by shear stress,while whether the DKK1 secreted by endothelial cells under shear stress affects the function of smooth muscle cells has not been reported.The purpose of this study was to investigate the effect of shear stress induced endothelial cell derived-DKK1 on smooth muscle cell proliferation,migration and neointima formation and its potential mechanism.Objective1.To investigate whether DKK1 secreted by endothelial cells under pathological shear stress affects the function of smooth muscle cells;2.The effect of DKK1 protein deficiency in endothelial cells on the formation of neointima in the region of low shearstress was detected;3.To explore the potential mechanism of the effect of endothelial DKK1 on the neointima formation.Materials and methods1.The CRISPR/Cas9 method was used to establish the Tek-Cre-ERT/+/DKKlfl/fl mouse modelIn view of the present embryonic death of DKK1-/-mice,the TEK-CRRE-ERT/+/Tek-Cre-ERT/+/DKK1fl/fl(DKKlECKO)knockout mouse model was established by CRISPR/Cas9 method in this study.2.Mouse model of carotidartery ligation(CAL)Left carotid artery ligation was performed in DKK1ECKO mice and control littermates of 8-10 week old to prepare the low shear stress model.These mice were sacrificed after 21 days.3.Animal tissue collectionThe mice were anesthetized,weighed and fixed on a foam plate.Blood was collected from the apex of the heart with a 1ml syringe,and the levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL-C)and high density lipoprotein(HDL-C)were measured in subsequent experiments.The heart,liver,spleen,lung and kidney of mice were fully perfused with pre-cooled normal saline.The aorta and both carotid arteries were carefully dissected and stored in tissue fixation solution or liquid nitrogen according to subsequent experiments.After fixed for 24 hours,it was washed under running water for 6-8 hours.After dehydration,paraffin embedding was performed,and then continuous sections were made.4.Cell and tissue protein extractionAdd 80-120 μL cell and tissue lysate(RIPA)to each well according to cell density,and add 200 μL RIPA containing PMSF(1:100)to each 20mg tissue.The tissue was fully cut and crushed,standing on ice for 10min,and then centrifuged at 14000rpm at 4℃ for 5min.The supernatant was added with 5×Loading Buffer,boiled at 99℃ for 10min to denature them,and then stored in a refrigerator at-20℃.5.Western Blot(WB)Protein sample 10 μL/well,electrophoresis,film transfer,incubate primary antibody at 4℃ overnight,wash the film,incubate secondary antibody after chemiluminescence,and analyze strip gray value after developing.6.Staining tissue sectionsCarotid artery sections were stained with hematoxylin-eosin(H&E),immunohistochemical staining and tissue immunofluorescence staining,respectively.Endometrial area of neovascularization,DKK1 expression level,PCNA,Ki67 and other value-added indicators were measured.7.Extraction and culture of mouse primary endothelial cellsDKK1ECKO gene knockout mice of 4-5 weeks old and DKK1fl/fl male mice of the same lair were used to extract aortic endothelial cells and pulmonary artery endothelial cells.8.Culture of human primary smooth muscle cells HASMC and umbilical vein endothelial HUVECsThe cells were purchased from Sciencell,USA,and cultured in complete medium of SMCM or ECM.9.Cell co-cultureThe flow cavity system used for shearstress loading in this experiment mainly consists of three parts:(1)the parallel plate flow cavity used for the joint culture of endothelial cells and smooth muscle cells;(2)liquid perfusion system;(3)Temperature and pH value control device(cell incubator).Endothelial cells were loaded with physiological shear stress(12dyne/cm2)and pathological low shear stress(4dyne/cm2).Transwell chamber was used for co-culture under static state.Endothelial cells transfected with Lenti-GFP-DKK1 lentivirus overexpressing DKK1 were inoculated in the upper compartment,and smooth muscle cells were inoculated in the lower compartment.10.Detection of VSMC cell migrationThe effect of rDKK1 on smooth muscle cell migration was detected by cell scratch assay.11.VSMC cell proliferation was detectedEdU kit was used to detect the effect of RDKK1 on the proliferation of smooth muscle cells.12.Statistical analysisData are presented as mean±SD and analyzed by SPSS 17.0.Unpaired t test was used for comparison of two samples.ANOVA analysis was used for the comparison among many samples.P<0.05 means statistical sense.Results1.Pathological shear stress up-regulated the expression of DKK1 in mouse vascular wall cellsThe vascular endothelium at the lesser curvature of the aortic arch of mice was mainly subjected to pathological low shear stress and oscillating shear stress,while the endothelium at the straight thoracic aorta was subjected to physiological pulsatile shear stress.Western Blot results showed that the expression of DKK1 in the vessels of the lesser curvature of the aortic arch was significantly higher than that at the straight thoracic aorta(P<0.05).In order to further observe the effect of pathological shear stress on the expression of DKK1 in vascular wall cells,the common carotid artery ligation model of mice was used to artificially induce pathological low shear stress.48 hours after ligation,the expression of DKK1 of the carotid artery was detected by Western Blot and immunofluorescence.The results showed that the expression of DKK1 of the common carotid artery in the ligation group was higher than that in the sham group.These results indicate that in vivo,the pathological low shear stress at the prone site of AS can up-regulate the expression of DKK1 in vascular cells.2.Endothelial cell DKK1 has paracrine effect under pathological shear stressIn the state of co-culture,the endothelial cells were stimulated by shear stress for 12 hours.Western blot was used to detect the expression of DKK1 protein in endothelial cells and smooth muscle cells.In conclusion,the expression of DKK1 in both cells was up-regulated after co-cultured ECs were subjected to pathological shear stress(P<0.05).The content of DKK1 in endothelial cells and smooth muscle cell medium was detected by ELISA.Compared with physiological shear stress(12dyne/cm2,NSS),pathological shear stress(4dyne/cm2,LSS)promoted the secretion of DKK1 by endothelial cells,and increased the content of DKK1 in the supernatant of smooth muscle cells on the opposite side of the PET membrane(P<0.05).Endothelial cells(upper compartment)transfected with DKK1 overexpressing lentivirus with GFP label were co-cultured with smooth muscle cells(lower compartment),and fluorescence expression of lower compartment cells was observed 24h later.GFP fluorescence was found in co-cultured smooth muscle cells,suggesting that DKK1 secreted by endothelial cells could be taken up by co-cultured smooth muscle cells.3.Effect of endothelial DKK1 on smooth muscle cell function through paracrine action under pathological shear stressIn the time-effect experiment,co-cultured endothelial cells were subjected to pathological low shear stress for 0,1,3,6,12,and 24h.The expression of PCNA in smooth muscle cells was detected by Western blot.The results showed that compared with Oh,the pathological low shear stress promoted the expression of PCNA in smooth muscle cells,which began to increase at 3h,reached the peak at 12h,and continued to 24h(p<0.05).In the strength-effect experiment,the co-cultured endothelial cells were subjected to Normal shear stress(NSS)of 12dyne/cm2 and Low shear stress(LSS)of 4dyne/cm2 for 12 hours.The results showed that compared with the NSS group,the expression of PCNA in smooth muscle cells was upregulated by the pathological low shear stress in co-cultured systhem(P<0.05).The DKK1 interfered stable transgenic endothelial cells and smooth muscle cells were cultured together,and then endothelial cells were stimulated by pathological low shear stress for 12h.Western blot was used to detect the expression of PCNA protein in smooth muscle cells.The results indicated that the inhibition of DKK1 in endothelial cells could abrogate the proliferation of smooth muscle cells induced by pathological low shear stress(P<0.05).Neutralizing antibody blocking experiment:10μg/mL neutralizing antibody of DKK1 was added into the culture medium of smooth muscle cells 3h before shear stress.Then the co-cultured endothelial cells were subjected to low shear stress for 12 hours.The results showed that the promotion effect of pathological low shear stress on the proliferation of smooth muscle cells was inhibited by using DKK1 neutralizing antibody(P<0.05).The recombinant protein stimulation experiment:smooth muscle cells were stimulated by different concentrations of rDKKl(0,25,50,100,150,200 ng/mL)for different time(0,1,3,6,12,24h),and the expression of PCNA was up-regulated(P<0.05).Immunofluorescence showed that the expression of PCNA and Ki67 were increased in rDKK1 group.EdU results:compared with Con group,the number of EdU positive cells in rDKK1 group was increased.The results suggested that exogenous DKK1 could promote the proliferation of smooth muscle cells.4.General condition of DKK1 endothelial conditional knockout mouse modelThe serum levels of TC,TG,LDL-C and HDL-C of DKK1ECKO group and DKK1fl/fl group showed no statistical difference,indicating that DKK1 did not affect blood lipid.5.DKK1 protein does not participate in the maintenance of vascular morphology in miceDKK1 did not affect the vascular morphology of mice.Mice at age of 5,6 and 7 weeks in the DKK1ECKO group and the control group were selected for H&E staining of carotid artery.The results showed that the vascular morphology of both groups were normal and there was no significant difference in vascular inner diameter and vascular media thickness.6.DKK1 protein deficiency in endothelial cells inhibited neointima formation of ligated vessels in miceH&E staining showed that endothelial conditional knockout of DKK1 significantly inhibited the neointima formation at the pathologically low shear stress region caused by carotid artery ligation in mice compared with control group(P<0.05).7.DKK1 protein deficiency inhibits the proliferation of vascular smooth muscle cellsImmunofluorescence staining of PCNA and Ki67 and EdU in vivo showed that DKK1 protein deficiency significantly inhibited the proliferation of smooth muscle cells(P<0.05).8.Complete Genome Sequencing Analysis of Vascular Tissue(RNA-Seq)In order to determine the molecular characteristics and biological processes involved in the effect of DKK1 on vascular function,6 homologous DKK1ECKO male mice and 6 homologous DKK1fl/fl male mice were selected.The full length of aorta and carotid artery were collected at 8 weeks of age,and the blood vessels of 2 homologous mice were used as one sample for whole genome sequencing analysis of vascular tissue.Sequencing results showed that COX2 and transcription factor MYB showed significant difference.9.DKK1 promotes the proliferation of smooth muscle cells through COX2Sequencing results showed that the deletion of DKK1 gene could down-regulate the expression of COX2.Real-time RT-PCR and Western Blot were used to verify the results,which were consistent with the RNA-Seq.Human recombinant DKK1 protein(rDKK1)(100ng/ml)was stimulated for different time(0,1,3,6,12,24h)of HASMCs in vitro,and the expression of COX2 was upregulated.siRNA transfection interferes with COX2,and then rDKK1 stimulation was given for 12h.Western Blot analysis showed that compared with the negative control group,the expression of PCNA in HAMCs under the intervention of rDKK1 is up-regulated and inhibited,indicating that COX2 is involved in the regulation of DKK1 on the proliferation of smooth muscle cells.10.COX2 is the target gene of c-MYB,and DKK1 promotes the proliferation of smooth muscle cells through c-MYB/COX2Sequencing results showed that the deletion of DKK1 gene could down-regulate the expression of c-myb.Real-time RT-PCR and Western Blot were used to verify the results,which were consistent with the RNA-Seq.Human recombinant DKK1 protein(rDKK1)(100ng/ml)was given to HASMCs for different time(0,1,3,6,12,24h)to stimulate the increase of c-myb expression.C-myb was transfected with siRNA and then stimulated by rDKK1 for 6h.Western Blot analysis showed that compared with the negative control group,COX2 expression in HASMCs was up-regulated by rDKK1,indicating that DKK1 could regulate the expression of COX2 through c-myb.11.DKK1 mainly promotes the proliferation of smooth muscle cells through PI3K/Akt pathways by binding to CKAP4 receptors on the surface of smooth muscle cellssiRNA transfection was used to interfere with the common receptors of DKK1,LRP6 and CKAP4,and then human recombinant protein DKK1 was given for 12h.CKAP4 knockout of the receptor could significantly inhibit DKK1’s promotion of smooth muscle cell proliferation(P<0.05).Akt is the downstream pathway of receptor CKAP4,and the inhibitor of Akt pathway can block the promotion effect of exogenous DKK1 on smooth muscle cell proliferation,as well as the promotion effect of DKK1 on c-myb expression(P<0.05).Conclusions1.Pathological low shear stress could increase the secretion of DKK1 by endothelial cells and promoted the proliferation of smooth muscle cells;2.DKK1 protein deficiency in endothelial cells inhibited the formation of new intima in the low shear stress area caused by vascular ligation in mice;3.DKK1 can promote the proliferation of smooth muscle cells through c-myb/COX2.IntroductionCardiovascular disease is the main cause of death in human beings,and hypertension is the main risk factor of cardiovascular disease.Hypertension is often accompanied by mechanical abnormalities in the vascular walls,including stretch and shear stress,with increased tension being the main mechanical change.Abnormal tension can cause morphological and functional changes of vascular wall cells,resulting in abnormal vascular structure and function,promoting atherosclerotic plaque formation,vascular hyperplasia,aneurysm,and aggravating the damage of hypertensive target organs.Therefore,further research on the occurrence and pathogenesis of hypertension is of great significance for reducing cardiovascular events.Renin-angiotensin system(RAS)is closely related to cardiovascular disease.Angiotensin-converting enzyme 2(ACE2),a new member of the RAS system,puts a premium on the protection of vascular function.ACE2 is homologue of ACE,but is unlike the ACE on substrate specificity.ACE2 can crack Ang Ⅱ and produce peptide vasodilation Ang(1-7).ACE2 can improve cardiac and vascular remodeling in hypertensive rats,diabetic cardiomyopathy and nephropathy,and myocardial infarction.Our laboratory studies have proved that ACE2 overexpression in atherosclerosis models can delay the occurrence and development of plaques,reduce plaque vulnerability index and stabilize plaques.Our previous study found that,compared with physiological stretch,pathological stretch down-regulated the expression of ACE2 in vascular smooth muscle cells,and ACE2 was involved in the regulation of stretch on the proliferation and migration of smooth muscle cells.These studies suggest that mechanical stretch can affect smooth muscle cell function and vascular wall remodeling via ACE2,but its exact effect and mechanism still need to be further studied.Dickkopf-1(DKK1)is a multifunctional secreted protein and inhibitor of Wnt signaling pathway.It was initially found in oncology studies that DKK1 plays an important role in the occurrence of lung cancer,colon cancer and other tumors,and was used as a drug development target in Phase Ⅱ clinical studies.Recent clinical studies have shown that DKK1 is closely related to cardiovascular disease.Our previous study found that peripheral blood DKK1 level is an independent predictor of recurrent adverse cardiovascular events.Further studies have shown that DKK1 can promote plaque formation and increase plaque instability.Further research is needed on the mechanism of mechanical stretch regulating ACE2 expression in vascular smooth muscle cells.It has not been reported whether stretch affects the expression of ACE2 by regulating smooth muscle cell DKK1,influencing the function of smooth muscle cells.Objectives:1.To observe the effect of periodic tension on the expression of ACE2 and DKK1 in vascular smooth muscle cells;2.To investigate whether ACE2 is involved in the effects of pathological stretch on the proliferation and migration of smooth muscle cells;3.To investigate the role of DKK1 in stretching regulation of ACE2 expression in smooth muscle cells.Methods1.Culture and treatment of human aortic smooth muscle cells(HASMCs)1.1.Smooth muscle cell cultureCells were purchased from Sciencell,USA,and cultured in complete SMCM medium.1.2.Smooth muscle cell interventionThe 6-9 generations of HASMCs cultured in vitro were used in the experiment.Smooth muscle cells were inoculated into Flexcell 6-well plate coated with type Ⅰ rat tail collagen.When the density of smooth muscle cells reached 80%-90%,the serum-free SMCM medium was replaced and cultured for another 24 hours.Before loading the tension stimulation,the medium was replaced with a new complete medium containing serum.Smooth muscle cells were stimulated using the Flexcell 5000-Tension Instrument in a 37℃,5%CO2 cell incubator.2.Establishment and sampling of animal modelsThe rats were randomly divided into two groups:the abdominal aortic coarctation group and the sham operation control group.The rats were sacrificed 3,5 and 7 days after operation.The rats were anesthetized by intraperitoneal injection of 1%pentobarbital sodium.After full perfusion with normal saline,the hearts,liver,spleen,lungs,kidneys and aorta were retained and stored in 4%paraformaldehyde or liquid nitrogen.The aortic tissue was fixed with formaldehyde for 24 h,then rinsed with water,dehydrated with gradient alcohol and embedding with paraffin.Paraffin slicer was used for 5 um continuous sectioning.3.Cell and tissue protein extractionAdd 80-120 μL cell and tissue lysate(RIPA)to each well according to cell density,and add 200 μL RIPA containing PMSF(1:100)to each 20mg tissue.The tissue was fully cut and crushed,standing on ice for 10min,and then centrifuged at 14000rpm at 4℃ for 5min.The supernatant was added with 5 X Loading Buffer,boiled at 99 ℃ for 10min to denature them,and then stored in a refrigerator at-20℃.4.Western BlotAdd protein sample 10 μL/well,electrophoresis,film transfer,incubate primary antibody at 4℃ overnight,wash the film,incubate secondary antibody after chemiluminescence,and analyze strip gray value after developing.6.Enzyme-linked immunosorbent assay(ELISA)After the stimulation was completed,the supernatant of smooth muscle cells was collected,and the contents of DKK1,angiotensin 1-7 and angiotensin Ⅱ in the supernatant were detected by ELISA.7.EdU was used to detect smooth muscle cell proliferationThe EDU kit was used to detect the role of ACE2 in the regulation of pathological stretch and smooth muscle cell proliferation.8.Cell scratch testThe role of ACE2 in the pathological tension regulation of smooth muscle migration was examined by cell scratch test.9.Apoptosis was detected by flow cytometryAnnexin V/PI staining was used to detect the role of ACE2 in regulating the proliferation of smooth muscle cells by flow cytometry.10.Cell transfectionDKK1 siRNA,ATF3 siRNA and Negtive control siRNA were respectively transfected with Invitrogen’s LIPO3000 to detect the changes in ACE2 expression and verify whether DKK1 and ATF3 are involved in the regulation of ACE2 expression by pathological tension.11.Double luciferase report assayACE2 promoter sequences of different lengths were constructed respectively.Human Embryonic Kidney cells(HEK293)were cotransfected with ATF3 overexpressed plasmid and ACE2 promoter sequence by Lipofectamine 2000 to detect fluorescence expression intensity.The binding of ATF3 to the promoter of ACE2 gene was identified.12.Histochemical stainingThe expression of ACE2,ACE and DKK1 in vascular tissues was observed by immunohistochemical staining.At the same time,the expression of ACE2 and DKK1 in blood vessels of human tissues were observed and compared between hypertensive and non-hypertensive vessels.13.Statistical analysisData are presented as mean±SD and analyzed by SPSS 17.0.Unpaired t test was used for comparison of two samples.ANOVA analysis was used for the comparison among many samples.P<0.05 means statistical sense.Results1.In vivo abdominal aortic coarctation of rats affected the expression of ACE2 and DKK1Both immunohistochemical results and Western Blot showed that the expression of ACE2 decreased after abdominal aorta coarsis in rats compared with the sham group(P<0.01),while the expression of DKK1 and ACE increased(P<0.01),2.The expression of ACE2 decreased and the expression of DKK1 increased in patients with hypertensionWe collected paracancerous tissue from patients with rectal cancer with or without hypertension.Immunohistochemical staining showed that the arterial wall was significantly thickened in patients with hypertension.The expression of ACE2 in smooth muscle cells significantly decreased,while the expression of DKK1 significantly increased.3.Pathological stretch regulates the expression of DKK1,ACE2/Ang-(1-7)and ACE/AngⅡ in vascular smooth muscle cells18%mechanical stretch inhibited the expression of ACE2(P<0.05),while it promoted the expression of ACE and DKK1(P<0.05).The level of Ang Ⅱ and DKK1 increased significantly after 18%mechanical tension(P<0.01),and the level of Ang(1-7)was significantly decreased(P<0.01).4.Effect of ACE2 on the function of pathologically stretch-induced human aortic smooth muscle cellsThe effect of ACE2 overexpression on the proliferation of smooth muscle cells under pathological stretch was detected by EdU.The results showed that compared with the static group,pathological stretch promoted the proliferation of smooth muscle cells by 18%(P<0.01);ACE2 overexpression inhibited the proliferation of smooth muscle cells(P<0.05);The effect of ACE2 overexpression on the migration ability of smooth muscle cells under pathological stretch was detected by cell scratch test.The results showed that,compared with the static group,pathological stretch promoted the migration of smooth muscle cells by 18%(P<0.01);ACE2 overexpression inhibited smooth muscle cell migration(P<0.05);Flow cytometry was used to detect the effect of ACE2 overexpression on the apoptosis of smooth muscle cells under the pathological stretch.The results showed that compared with the static group,pathological tension promoted the apoptosis of smooth muscle cells by 18%(P<0.01);ACE2 overexpression can inhibit apoptosis of smooth muscle cells(P<0.05);The influence of ACE2 overexpression on collagen synthesis of smooth muscle cells under the pathological stretch was detected by Western Blot.The results showed that compared with the static group,the expression of collagen Ⅰ and Ⅲ in smooth muscle cells was promoted by 18%pathological stretch tension(P<0.01);ACE2 overexpression can inhibit the synthesis of collagen Ⅰ and collagen Ⅲ in smooth muscle cells(P<0.05).5.Pathological stretch is involved in the regulation of ACE2 expression through P38/DKK1/ATF3Pathological stretch(18%elongation,1Hz)promoted phosphorylation of ERK,JNK and P38(P<0.05).MAPKs signaling inhibitors PD98059(ERK1/2 inhibitor),SP600125(JNK inhibitor)and SB203580(P38 inhibitor)were used to treat pathologic stretch stimulation(18%elongation,1Hz).It was found that SB203580 could block the promoting effect of pathological stretching on DKK1(P<0.05).These results suggest that p38 is involved in the regulation of pathological stretch on DKK1 expression.By interfering with the expression of DKK1 in HASMCs with siRNA or using anti-DKK1 neutralizing antibody in advance,the down-regulation of ACE2 by pathological tension can be blocked(P<0.01),indicating that DKK1 is involved in mediating the regulation effect of pathological tension on ACE2.The siRNA of ATF3 was transfected into HASMCs to silence it.Western Blot results showed that compared with the negative control group,the interference of ATF3 could block the downregulation of ACE2 by exogenous DKK1(P<0.01),indicating that ATF3 is involved in the down-regulation of ACE2 by DKK1.5.ACE2 is direct target of ATF3The siRNA of ATF3 was transfected into HASMCs and silenced.Western Blot results showed that the interference of ATF3 could block the down-regulation of ACE2 by pathological tension compared with the negative control group(P<0.01),suggesting that ATF3 is involved in the downregulation of ACE2 by pathological stretch(18%elongation,1Hz).By dual luciferase and ChIP assay,the transcription factor ATF3 can directly bind to ACE2 promoter.Conclusions1.Pathological stretch promoted the expression of DKK1 and inhibited the expression of ACE2 in vascular smooth muscle cells;2.ACE2 is involved in the effects of pathological stretch on the proliferation and migration of smooth muscle cells;3.Traction tension can inhibit the expression of ACE2 by promoting the secretion of smooth muscle cell DKK1;4.Pathological tension regulates the expression of ACE2 through p38/ATF3,which is the target gene of transcription factor ATF3.