The Effect And Mechanism Of DKK3 Suppresses Lesions Of Atherosclerosis And Myocardial Fibrosis

1 BackgroundAtherosclerosis(AS)is a progressive inflammatory disease of the arterial wall that can be clinically silent for many years before triggering acute episodes such as myocardial infarctions or strokes.During the development of AS,following endothelial cell injury,fat and cholesterol gradually deposited on the arterial wall,forming the lipid core.A fibrous cap,formed by smooth muscle cells(SMC),fibroblasts and collagenous tissue is covered outside the lipid core.There are monocytes,T cells,and neutrophils infiltrating in the lipid core during the development of AS.As with the decrease of SMC,the increased degradation of extracellular matrix or decreased synthesis of extracellular matrix in the fibrous cap,the plaque is prone to rupture,leading to the formation of thrombosis and the initiation of acute coronary events.In this way,looking for ways and drugs to stabilize the plaque is of important clinical significance.Currently,stem cell research in the AS field indicates that mesenchymal stem cells(MSC)can migrate to damaged tissue,differentiate into functional cells and repair damaged tissue.The transplanted MSC could localize to a ruptured plaque,differentiated into endothelial cells and secreted collagen fibers.In addition,bone marrow,blood circulation,and smooth muscle progenitor cells in the adventitia can differentiate into SMC and migrate to the damaged area.In some conditions,embryo fibroblasts in the adventitia can also migrate to the intima and differentiate into SMC.Stem cell antigen 1(Stem cell antigen 1,Seal)is a common marker of adventitia smooth muscle progenitor cells.Considering the adventitia smooth muscle progenitor cells and embryo fibroblasts are closer to the atherosclerosis intima and the source of adventitia smooth muscle progenitor cells are more widely,how to regulate Scal+ cells and fibroblast cells to SMC precisely becomes clinical important.Accumulating studies have shown that DKK3 plays an essential role in the regulation of embryonic development,including development of the neural epithelium,limb bud,and bone,DKK3 has been shown to be down-regulated in several types of human cancer,and its overexpression can inhibit cell proliferation.DKK3 is particularly important in regions of epithelial-mesenchymal transformation.DKK3 has been shown to be involved in the differentiation of partially reprogrammed cells to smooth muscle cells,so DKK3 may has be seen a kind of stem cells inducer.To study the mechanism in which DKK3 induced Scal+ cells and embryo fibroblasts is of great clinical meanings.2 ObjectivesThere are four purpose of this study.Firstly,we investigate the mechanism of DKK3 induced stem cell antigen l(Scal+)cell and embryo fibroblasts differentiating into SMC.Secondly we study the influence of two kinds of SMC derived from different cells induced by DKK3 on the disease of atherosclerotic lesions in mice.Also we study the effects of DKK3 on inflammation,the number of smooth muscle cells and extracellular matrix content of AS plaques.Thirdly we study the effect of DKK3 on atherosclerotic plaque in rabbits.Fourthly we study the relationship between the human atherosclerotic plaque stability and serum DKK3 level.3 Methods1.Tandem stenosis surgeryDKK3-/-ApoE-/-and DKK3+/+/ApoE-/-mice(C57BL/6J background)at 8 weeks of age were fed of high fat diet for 6 weeks and assigned randomly to a surgery or control group.Mice were anaesthetized and then an incision was made in the neck and the right common carotid artery was dissected from circumferential connective tissues.A tandem stenosis with 150 ?m outer diameter was introduced with the distal point 1 mm from the carotid artery bifurcation and the proximal point 3 mm from the distal stenosis.Mice were kept for another 4 weeks until the atherosclerotic model was built.2.Cell application on adventitialFor cell application,the artery was seeded with Sca-1+ progenitors derived from GFP transgenic mice(1x1O6 cells)within 25 ?l Matrigel?Basement Membrane Matrix.After 4 weeks mice were sacrificed for the subsequent experiment.3.DKK3 release experimentFor the treatment with DKK3,30%of Pluronic gel-127 mixed with recombinant DKK3(200ng/mL)was applied to the adventitial site to envelope the vessel.Animals were sacrificed 4 weeks after surgery.4.Vein Graft Procedure.3-month-old DKK1-/-/ApoE-/-and DKK3+/+/ApoE-/-mice were anesthetized.The right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal and cut for 2 cm.The vena cava vein was harvested from an isogenic donor and grafted between the two ends of the carotid artery by ligating them together with an 8-0 suture.Vigorous pulsation in the grafted vein confirmed successful engraftment.5.Femoral artery injuryMice were anesthetized.Both of the femoral arteries of each mouse were injured by inserting a 0.25 mm guide wire 4-mm in length from one of the distal muscle branches to the femoral artery.One artery was seeded with Sca-1+ progenitors and fibroblasts derived from SM22-LacZ mice(1x106 cells)within 25 ?1 Matrigel?Basement Membrane Matrix,and the other injured artery served as a control.Arteries were harvested 72 hours after the operation.6.In vivo matrigel plug assay.HUVECs combined with control cells or induced SMCs labelled with Qtracker?605 were mixed with 50 ?l of Matrigel and injected subcutaneously into the back or flank of SCID mice.Six injections were conducted for each group.12 days later,the mice were sacrificed and the plugs were harvested,frozen in liquid nitrogen and then cryosectioned.Samples were then fixed with 4%paraformaldehyde in PBS at 4?overnight after which histological staining was applied.7.Histological stainingParaffin sections or frozen sections were stained with H&E staining to observe the morphological feture;immunohistochemical staining was used to detect RAM-11,?-SMA,MMP-9;immunofluorescence staining was used to detect SM22,SMMHC,SMA,CD68,Col1a1,DKK3,elastin,CD31,CD144,Calponin and Sca1;sirius red staining was used to detect the elastic fibers and collagen.8.The beta-gal stainingTake 5 mm femoral artery after injury surgery,remove the adipose tissue and adventitia,and then cut the artery longitudinally.The artery tissue was then fixed with fixing solution,beta-gal dyeing liquid was added and incubated until cells turned to blue.Then artery tissue was observed and photographed under normal light microscope.9.Cell culturePrenatal human embryonic lung fibroblasts from ATCC were cultured on gelatin in ATCC F-12K Medium supplemented with 10%ATCC Fetal Bovine Serum and 100 U/mL penicillin/streptomycin in a humidified incubator with 5%CO2.Cells were passaged every 3 days at a ratio of 1:3 or 1:7 and the medium was refreshed every 2 days.Sca-1+ mouse adult progenitor cells and fibroblasts were isolated from outgrowth of aortic adventitial tissues from ApoE-/-,transgenic(GFP-Sca-1;SM22-LacZ)or wild-type mice and purified using an Anti-Sca-1 MicroBead Kit.Cells were then maintained in DMEM supplemented with 10%EmbryoMax(?)ES Cell Qualified Fetal Bovine Serum,10ng/ml leukaemia inhibitory factor(LIF)and 0.1mM 2-mercaptoethanol on a 0.04%gelatin substrate.After isolation of Sca-1+ cells,the rest of cells were cultivated in the medium for fibroblasts.After 5 passages,the cells were pure fibroblasts.10.Cell transfection and drug stimulation.Human embryonic lung fibroblasts and Scal+ cells were transfected with adenovirus,and 24 hours later,special drug was added into the culture medium.Cells were collected at the planned time point.Human embryonic lung fibroblasts and Scal+ cells were transfected with silencing lentivirus and polybrene,and 24 hours later,special drug was added into the culture medium.Cells were collected at the planned time point.11.Nucleofection of fibroblasts with ATF6Human embryonic lung fibroblasts were transfected with the ATF6 transcription factor plasmid by electroporation using an NHDF nucleofector kit.A PCMV5 empty vector was used as a control.Each transfection required 2x106 cells and 2?g of control or ATF6 plasmid.Cells were then seeded on gelatin and maintained in DM for 48 hours after which samples were harvested and subjected to Q-PCR.12.RNA extraction,reverse transcription and Q-PCR.Total RNA was extracted using the RNeasy Mini Kit according to the manufacturer's protocol.The QuantiTech Reverse Transcription kit was used to reverse transcribe the RNA to cDNA and then QPCR was performed.13.Immunoblotting.20-50?g of protein was boiled in lxSDS loading buffer for 10 minutes before loading onto a,4-12%Bis-Tris gel at 90V for 2 hours while immersed in running buffer.The gel was then transferred onto a PVDF membrane at 200mA for 2 hours while immersed in transfer buffer.The membrane was then blocked with 5%milk in PBS-Tween containing 0.02%sodium azide for 1 hour at room temperature before incubation in the primary antibody solutions overnight at 4?.The next day,the PVDF membrane was incubated with corresponding horseradish peroxidase marked secondary antibody,incubated for 2 hours at room temperature,and then detected by ECL chemiluminescence.14.Immunofluorescent staining of cells.Cells were fixed with 4%paraformaldehyde and blocked with 5%swine serum in PBS for 30 min at room temperature.They were then incubated with primary antibodies.The bound primary antibodies were revealed by incubation with the secondary antibodies for 30 minutes at 37 ?.Cells were counterstained with DAPI,mounted in Fluoromount G,and examined with a fluorescence microscope or confocal microscope.15.Enzyme-linked immunosorbent assay(ELISA)The concentration of the DKK3 released glycoprotein in the supernatant of fibroblasts or mouse,rabbit and human serum was detected by a DKK3 ELISA kit according to the protocol provided.Similarly,the concentration of collagenlal and TGF?1 in the supernatant was detected by an ELISA kit according to the protocol provided.16.Luciferase activity assay.For the human fibroblast experiments,cells were seeded in 12 well gelatin coated plates and infected with null or human DKK3 adenovirus.24h later cells were transfected using FuGene?6 reagent with a myocardin or a SMMHC promoter reporter vector(0.33?g/well).pGL3-Luc-Renilla(0.1 ?g/well)was also included in all transfections as an internal control.The luciferase and Renilla enzymatic activities were detected 48 hours after transfection using the Multiscan Spectrum.Relative Luciferase Unit(RLU)was defined as the ratio of luciferase activity to Renilla activity with that of control set as 1.0.17.Rabbit modelNew Zealand rabbits weighing from 1.5 to 2.0 kg(8-10 weeks)were randomly divided into two groups(n=10/group).After they had been anesthetized with an intravenous injection of pentobarbital sodium(30 mg/kg),balloon-induced aortic wall injury was perforIed with a 4-Fr balloon catheter(3.5x15 mm2)introduced through the right femoral artery to the thoracic aorta.The balloon was inflated with saline to obtain 8 atm,and the catheter was retracted down to the iliofemoral artery.This process was repeated three times in each rabbit to ensure denudation of the endothelium of the abdominal aorta.Then rabbits were fed on a diet of 1%cholesterol and 3%lard for 12 weeks.The Rabbits received a suspension of adenovirus expressing DKK3(2.5×109 pfu)while rabbits in the control group received a suspension of adenovirus-vector(2.5 x 109pfu)at the end of week 12,14,16 and 18 respectively.All the rabbits were sacrificed at week 20 and the artery from arcus aortae to arteria iliaca communis was isolated for analysis.18.Aortic tissue preparation and oil Red 0 stainingThe entire artery was separated and then opened longitudinally to expose the arterial intima.After that image was captured on digital camera,the entire artery was incubated in oil red 0 solution for 2-4 hours and then in 70%ethyl alcohol until the artery turned red.The artery was rinsed in distilled water and preserved using 4%formaldehyde.Images were captured again after the staining.The quantification of atherosclerosis was measured in a region extending from the aortic arch to the bifurcation of the iliac arteries.19.Blood serum lipid measurementMouse blood samples(0.5-1 ml)were taken by cardiac puncture at the time of sacrifice and placed at room temperature for one hour to clot before being centrifuged at 2000 g for 15 min.Rabbit blood samples were obtained by puncturing the marginal ear vein at the end of week 12,13,14,15,17,20.The serum concentrations of total cholesterol,triglycerides,low-density lipoprotein(LDL)and high-density lipoprotein(HDL)were determined using an automated biochemical analyser.20.Measurement of patients' specimens.Collecting Qilu Hospital of Shandong University between July 2015 and December 2015.The patients were divided into stable angina(SA)group and unstable angina pectoris(UA)group by medical history and clinical symptoms according to the Braunwald classification.The baseline characteristics of the 2 groups were collected from patients just before their treatment.The general clinical date including age,gender,height,weight,blood pressure,diabetes,smoking and alcohol consumption were recorded.Leukocyte count in the blood and serum levels of creatinine,fibrinogen,homocystein,fast glucose,total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)were measured using a commercial method(company).The serum DKK3 of the patients was measured with an ELISA kit purchased by Abacam.4 Results1.DKK3 Knockout Effects Plaque CompositionTandem stenosis of the carotid artery of DKK3+/+ApoE-/-and DKK3-/-ApoE-/-mice was performed to construct murine atherosclerotic plaque model,and samples harvested after 4 weeks were analysed for atherosclerotic plaque development and plaque phenotype.Hematoxylin/eosin(H&E)staining of sections obtained revealed that fewer plaques from DKK3+/+ ApoE-/-animals revealed the presence of red blood cells.Immunofluorescent staining revealed that fewer SMCs resided but more CD68+cells infiltration within the DKK3-/-plaques when compared to those of DKK3?/?mice.Miller and Picosirius staining of sections revealed that there was significantly less extracellular matrix(ECM)deposition in the form of elastin and collagen in the plaques from DKK3-/-ApoE-/-mice when compared to DKK3+/+ApoE-/-plaques.2.DKK3 Knockout Increases Plaque Rupture Incidence of Vein Graft ModelA mice vein graft model was used.Vein graft rupture complications were obseverd in the total 10 mice in DKK3-/-ApoE-/-group.Dissection,necrosis and calcification could be seen within intramural thrombosis.However,only 2 of 10 mice developed vein graft rupture complications in DLK3+/+ApoE-/-group.Moreover,cell composition analysis of the plaques exhibited decreased SMCs and increased CD68+macrophages in the DKK3-/-ApoE-/-group,and Miller and Picosirius staining indicated a decrease of matrix proteins in lesions of DKK3-/-ApoE-/-group.3.DKK3 Promotes Vascular Progenitors Migration.GFP-Scal+-progenitors isolated from the vessel wall were seeded on the adventitia of an ApoE-/-mouse artery after tandem ligation.Fluorescence microscopy revealed that Scal+ cells migrated to the lesion of the vessel within 4 weeks.No GFP+cells were detected in the control vessel.Administration of Sca-1+ cells to the adventitia of tandem ligated vessels resulted in a significant reduction of intraplaque haemorrhage,while no red blood cells were observed within the plaque in 10/10 animals.4.Scal+ Progenitors and Fibroblasts Promote Wire Injury RepairUsing the wire injury model,Scali progenitors and fibroblasts were isolated from SM22-LacZ transgenic mice and seeded on the adventitia of a wild type mouse after femoral artery injury.Beta-gal staining revealed that both SM22-LacZ-progenitors and SM22-LacZ-fibroblasts migrated to the intima of the injured vessel and differentiated into SMCs after 72 hours.5.DKK3 Can Induce Differentiation of Fibroblasts to SMCs in vitro.Human fibroblasts were seeded on a gelatin substrate and infected with null or DKK3 adenovirus under differentiation conditions.Initial observations of cells overexpressing DKK3 after 4 days in DM revealed a morphological SMC-like change.This correlated with upregulation of SMC markers as seen by Q-PCR,Western blot and immunofluorescence.Luciferase promoter assays revealed an increase in promoter activity of both myocardin and SMMHC upon overexpression of DKK3 in DM conditions.6.Fibroblast Differentiation to SMCs upon DKK3 Overexpression is Mediated by TGF?1 Induction.Fibroblasts were stimulated with human recombinant TGF?1 with or without DKK3 overexpression and compared to untreated cells.Initial observations in cell morphology revealed that the cells treated with a combination of TGF?1 and DKK3 were distinctly different from untreated fibroblasts and fibroblasts treated with TGF?1 alone.Additionally,Q-PCR and Western blot analysis revealed that upon TGF?1 stimulation,cells overexpressing DKK3 expressed SMC markers and components of the ECM at higher levels.A specific inhibitor of the receptor for TGF?1(ALK5)was used in DKK3 overexpressing fibroblasts.Upon inhibition of ALK5,there was a striking ablation of SMC marker expression.Furthermore,a neutralizing antibody specifically inhibiting active TGF?1 was utilized in the culture medium,which revealed an inhibition of SMC marker expression.Fibroblasts were stimulated by TGF?1 in the presence(NT lentivirus)or absence(shDKK3)of DKK3.It was revealed that upon silencing of DKK3 there was a dramatic abrogation of the expression of SMC markers and ECM components.Finally,the endogenous expression of TGF?1 was silenced using a lentivirus.The result showed that the expression of SMC markers as well as matrix components was inhibited.7.DKK3 Regulates TGF?1 via Activation of ATF6After utilizing the Qiagen Champion ChiP Transcription Factor Search Portal,we found that the transcription factor ATF6 was upregulated upon DKK3 overexpression.Fibroblasts were infected with NT or shDKK3 during differentiation.We found that upon DKK3 silencing,ATF6 expression was also reduced.Moreover,siATF6 led to a decrease in TGF?1 at both RNA and protein levels while ATF6 overexpression not only upregulated TGF?1 but also led to an increased expression of a full panel of SMC markers and ECM components8.DKK3 Mediated Fibroblast Differentiation to Functional SMCsA matrigel plug assay was employed to examine the ability of differentiated SMCs to generate functional vessels in vivo when mixed with human umbilical vein endothelial cells.DKK3 induced SMCs were labeled with a red cell tracker(QTracker red),mixed with HUVECs in matrigel and then injected subcutaneously into SCID mice.Samples were obtained after 2 weeks and sections were stained with H&E.It was observed that these cells had the ability to form complete vessels characteristic of the vessels formed when mixing functional SMCs and endothelial cells(ECs)in vivo.Immunofluorescent staining of frozen sections for EC markers revealed that while ECs formed tubular structures comprising the inner lining of vessels,the induced SMCs correctly positioned on the outside layer,enveloping the EC tubes and forming robust 2 layer vessels.We thus concluded that DKK3 can induce the differentiation of functional SMCs capable of forming complete vessels in vivo.9.Scal + Progenitors Can Differentiate to SMCs via DKK3.Scal+cells were seeded on a collagen IV substrate and maintained in DM for 6 days.Analysis of the samples harvested displayed an upregulation of SMC markers accompanied by an upregulation of DKK3.Additionally,ELISA revealed that there was an increasing release of DKK3 in the supernatant during differentiation towards SMCs.Immunofluorescent staining of differentiated cells revealed characteristic SMC marker staining parallel to an increase in DKK3 staining.Silencing of DKK3 using shDKK3 led to a downregulation of SMC markers.Neutralization of secreted DKK3 by utilization of a DKK3 antibody also revealed a downregulation of SMC marker expression.Cells were stimulated with recombinant DKK3 or adenoviral overexpression of DKK3.Western blot analysis indicated a further induction of SMCs markers upon stimulation with exogenous DKK3.10.DKK3 Promots Sca1+ Progenitors Differentiate to SMCs via Activation of the Wnt Signalling PathwayInitially,an anti-DKK3 antibody was used to neutralize the effect of DKK3 in the supernatant of differentiating Sca1+ cells.There was a downregulation of Wnt signalling targets upon DKK3 blocking,suggesting inhibition of Wnt signalling.Similar results were obtained when the endogenous expression of DKK3 was silenced by shDKK3.In addition,a mouse Wnt signalling pathway RT2 Profiler PCR Array was utilized.Upon overexpression of DKK3,the majority of the genes associated with Wnt signalling were significantly upregulated.Conversely,upon shDKK3 treatment the majority of wnt associated genes were downregulated,suggesting suppression of the signalling pathway.Utilization of a specific a wnt inhibitor revealed that inhibition of wnt signalling led to ablation of SMC marker induction during Scali differentiation.Western blot analysis indicated that overexpression of DKK3 in mouse Sca-1+ cells resulted in an increase of active-?-catenin,while inhibition of Wnt signalling in Sca-1+ cells overexpressing DKK3 inhibited SMC marker protein production.Taking these results into consideration,we concluded that DKK3 can induce Sca1+ differentiation to SMCs via activation of the Wnt signalling pathway.11.Exogenous DKK3 Can Rescue Atherosclerotic Plaques in DKK3-/-ApoE-/-Mice.The tandem stenosis model in DKK3-/-ApoE-/-mice was used,in which 30%pluronic gel containing 200?g/ml of recombinant DKK3 was applied to the adventitial side of the carotid artery to envelope the treated vessels.DKK3++ApoE-/-mice treated with pluronic gel containing mouse serum proteins(200?g/ml)were used as the control group.Cross sections obtained from the vessel 4 weeks after surgery were initially stained with H&E.This revealed that very few red blood cells were present within the plaques of DKK3-/-ApoE-/-mice treated with DKK3 protein,which was comparable to the plaques of the control group.Additionally,sections were immunostained with anti-SMA antibody.Interestingly,DKK3 treatment resulted in increased numbers of SMCs within the plaque,comparable to those found within the control animals.Similarly,sections were immunostained for CD68 and exhibited a similar macrophage infiltration between DKK3 treated and untreated animals.Finally,Miller and Picosirus staining exhibited a similar deposition of ECM proteins between the 2 groups.Hence,these results indicate that exogenous DKK3 can successfully change the composition of atherosclerotic plaques by reducing intraplaque haemorrhage,increasing SMC and ECM deposition and reduced macrophage infiltration.12.Administration of DKK3 Adenovirus Alters Atherosclerotic Plaque Phenotypes in Rabbits.New Zealand White rabbits were subjected to a balloon-induced aortic wall injury and fed on a high fat diet for 10 weeks to trigger development of atherosclerotic plaques.They then received a suspension of either adeno-vector(control)or DKK3 adenovirus.ELISA detection of DKK3 in the serum of both groups revealed that levels of released DKK3 in the serum were consistently higher in the adeno-DKK3 infected rabbits when compared to the controls.Immunostaining of sections with anti-SMA antibody indicated a significant increase of SMCs numbers and decrease of macrophages within the plaques of subjects injected with DKK3 adenovirus when compared to control.The MMP9 immunostaining results showed sections obtained from the adeno-DKK3 rabbits presented with fewer MMP9+ cells compared to the control group.Finally,Sirius red staining of sections for elastin and collagen showed that there was an increased accumulation of ECM in rabbit plaques treated with DKK3 versus the control group.However,Oil Red O staining indicated no significant difference between the adeno-DKK3 and control groups13.Decreased DKK3 Levels in Patients with Unstable AnginaThe serum of patients with stable angina pectoris and unstable angina was collected.The DKK3 level in serum was detected by ELISA which showed a relative lower level in patients with unstable angina pectoris.5 Conclusions1.We provide the first evidence that DKK3 is a potent SMC differentiation factor.In fibroblasts,DKK3 may bind to its "receptor" or indirectly to a Wnt binging site and vascular progenitors.Consequently,DKK3 activates ATF6 to induce TGF-?1,which results in SMC differentiation from fibroblasts.2.In Sca1+ vascular progenitor cells,DKK3 was indirectly combined with the Wnt binding site ATF6,and then activated the Wnt/beta-catenin pathway leading to the differentiation of Sca1+ cells into smooth muscle cells.3.DKK3 increases smooth muscle cells and collagen,elastic fiber content in mice and rabbits atherosclerosis vulnerable plaques,reduces the incidence of macrophage infiltration and intraplaque neovasculature depending on its differentiation function.DKK3 might have a therapeutic effect in atherosclerotic plaque.4.Serum DKK3 concentration decreased in patients with unstable angina,suggesting that DKK3 may be used as a molecular marker to predict the occurrence of unstable angina.1.BackgroundMyocardial fibrosis refers to abnormal collagen proliferation and different types of collagens deposition in cardiac tissues.Factors that increase the pression on the heart or affect the neuro-humoral can lead to hypertrophy and cardiac fibrosis.The cardiac fibrosis can be characterized by stiffness,contraction,and decreased perfusion.The activation of renin-angiotensin-aldosterone system is the most important cause of myocardial fibrosis.Angiotensin ?(Ang?),one of the most important component in RAAS system,can directly affect the cardiac muscle cells and fibroblasts,causing cardiomyocyte hypertrophy,fibroblast proliferation,and excessive secretion of extracellular matrix.Therefore,the method that can block the biological effects of Ang? will have a protective effect on the damaged heart.The current study found that Angiotensin converting enzyme 2(ACE2)can pyrolysis Angiotensin ? into Angiotensin 1-7(Ang1-7),the latter has a protective effect for myocardial hypertrophy and fibrosis,reflecting the ACE2 potential therapeutic value.Recent studies have found that metalloproteinase 17(A disintegrin and metalloproteinase 17,ADAM 17)has the function of degrading ACE2,so we speculate that it may be related to myocardial injury caused by angiotensin.?-catenin is the key effector of the Wnt pathway.When ?-catenin increased in the cytoplasm,the nucleus translocation increased,which resulted in the myocardial hypertrophy gene and fibrosis gene transcription.The transcription resulted in the myocardial fibrosis and myocardial hypertrophy eventually.Glycogen synthesis kinase 3?(GSK-3?)is a kind of anti-myocardial hypertrophy kinase.Free state of GSK-3? can be recruited to Frizzled receptor,where combined with ?-catenin,causing the degradation of P-catenin,and thus blocked the nuclear transcription of?-catenin.The biological functions of transformation growth factor(TGF-?1)are very extensive,which mainly include enhancing endothelial synthesis function,promoting collagen synthesis from fibroblasts,and inhibiting matrix metalloproteinase activity.In the process of myocardial fibrosis,TGF-?1 not only regulates various signal transduction pathways,but also directly stimulates CFs.The proliferation of CFs is the most important factor leading to myocardial fibrosis.Therefore,we examined the relationship between TGF-?1 and Angll in this experiment.At present,it has been reported that dickkopf-3(DKK3)has certain protective effect on the myocardium,however,its role in Ang?-induced myocardial fibrosis is not clear,and there is no research on the expression changes of GSK-3?,?-catenin signaling pathway and ADAM17/ACE2 in myocardial tissues.In addition,we also studied whether DKK3 affected TGF-?1/Smad3 signaling pathway to regulate Angll effect.2.ObjectivesThere are four purposes of this study:(1)to study whether DKK3 overexpression alleviates Angll induced myocardial hypertrophy and fibrosis;(2)to study whether DKK3 could affect GSK-3 beta/beta-catenin signaling pathway in the model of angll-induced myocardial fibrosis mice;(3)to study the relationship between DKK3 and ADAM17/ACE2 signaling pathway and TGF-beta 1 signaling pathway;(4)to study the effect of DKK3 on inflammation reaction and fibroblast proliferation induced by Angll.3.Methods1.Primary cultures of cardiac fibroblastsPrimary cardiac fibroblasts(CFs)were prepared from the hearts of neonatal C57BL/6 mice.The heart was isolated and cut into pieces in DMEM,digested for 3 h at 37?.Then,the liquid supernatant was centrifuged,and cells were cultured in DMEM containing fetal bovine serum(FBS)and penicillin-streptomycin.Two hours later,the culture medium was replaced to remove all cells except the CFs.The cells were subcultured in subsequent experiments.2.Cell transduction and drug administrationThe DKK3-overexpressing adenovirus was purchased from Abm and amplified by the Chinese National Human Genome Center,Beijing.CFs were pretreated with DKK3 overexpression adenovirus or adenovirus vehicle(MOI=100)for 24 h before Ang?(1 ?g/ml)stimulation and were cultured for an additional 48 h to evaluate the effect of DKK3 overexpression.The CFs were divided into six groups as follows:Group 1(OV+Ang?):DKK3-overexpressing adenovirus+ Angiotensin ?;Group 2(AD+Ang?):adenovirus vehicle+ Angiotensin ?;Group 3(Ang?):Angiotensin only;Group 4(OV):DKK3-overexpressing adenovirus;Group 5(AD):adenovirus vehicle;Group 6(NC):no additions3.DKK3-siRNA transfection experiment and groupingCFs were extracted and plated the day before transfection.When the cells grew to 30%confluence,NC-siRNA or DKK3-siRNA mixed with delivery reagent was added into the plate following the manufacturer's instructions.After four hours,the cell culture medium was replaced,and after 24 hours,Ang ?(1 ?g/ml)was added into the plate.The cells were cultured for an additional 48 hours until proteins were extracted for western blot analysis.The CFs were divided into four groups as follows:Group 1(NC-siRNA):negative control-siRNA only;Group 2(NC-siRNA+ Ang?):negative control-siRNA+Ang?;Group 3(DKK3-siRNA+Ang?):DKK3-siRNA+AngIl;Group 4(DKK3-siRNA):DKK3-siRNA only4.Animal modelBased on the results of the preliminary in vitro experiment,the Ang? group presented similar results as the AD+Ang? group,and the results observed in the NC group were similar to the AD group.We removed the Ang? and NC groups to conduct our in vivo experiment with fewer animals.Sixty male wild-type C57BL/6 mice(10-12 weeks of age)were chronically infused with saline or Ang? at a rate of 1000 ng/kg/min using osmotic mini-pumps(Alzet,Cupertino,CA)for 28 days as described previously.The DKK3-overexpressing adenovirus or vehicle(2×109 pfu)was administered by a caudal vein injection 3 days before the operation.On day 15,mice were injected again.Mice were divided into four groups randomly and treated as follows:Group 1(OV+Ang?):DKK3-overexpressing adenovirus injection ?Angiotensin ? pumping;Group 2(AD+Ang?):adenovirus vehicle injection ?Angiotensin ? pumping;Group 3(OV):DKK3-overexpressing adenovirus injection+saline pumping;Group 4(AD):adenovirus vehicle injection +saline pumping5.Quantitative real-time RT-PCRTotal RNA was extracted from the left ventricle of mice or CFs using TRIzol reagent(Invitrogen,CA).A real-time PCR thermocycler(IQ5 real-time PCR cycler;Bio-Rad)was utilized to perform the quantitative real-time PCR.The hypertrophic genes atrial natriuretic peptide(ANP)and beta-myosin heavy chain(?-MHC)were detected.PCR was performed at 95? for 30 s,followed by 40 cycles of 95? for 5 s and 56? for 10 s.The transcript GAPDH content was quantified as an internal control.The results were calculated using the 2 ??CT method.6.Western blot(WB)analysesTotal proteins and nuclear proteins were extracted from CFs or mouse cardiac tissues,and equal amounts of protein were electrophoretically separated and then transferred to PVDF membranes.The membranes were incubated overnight at 4?with a primary antibody.The antibodies used in this study were anti-DKK3,anti-collagen ?,anti-collagen ?,anti-MMP2,anti-MMP9,anti-phosphor-ADAM17(T735),anti-ACE2 and anti-TGF-?1,anti-ADAM17,anti-GSK-3?,anti-?-catenin,anti-phospho-GSK-3?(Ser9),anti-active-?-catenin(Ser33/37/Thr41),anti-bcl-2,anti-bax,anti-Smad3,anti-phospho-Smad3(Ser...