The Roles Of DKK1 In Mechanical Stretch Mediated Vascular Smooth Muscle Cell Function And Vascular Remodeling And Underlying Mechanism

1 BackgroundPathological mechanical stretch can lead to cardiac and vascular remodeling,including myocardial hypertrophy,myocardial fibrosis,heart failure,thickening and stiffening of the vessel wall,and atherosclerosis.Vascular smooth muscle cells(VSMCs)in the media of the arterial wall,which are essential for maintaining vessel homeostasis,are exposed to mechanical cyclic stretch in vivo.In response to pathologically increased stretch,the VSMCs phenotype switch from a contractile to a synthetic phenotype characterized by increased proliferative and migratory activities,loss of contractility,and abnormal extracellular matrix production.However,the molecular mechanisms by which mechanical stretch regulates the function of VSMCs remain to be further elucidated.Mechanical stretch regulation of smooth muscle cell gene expression and cell function requires the conversion of mechanical forces into intracellular signals,a process that is mediated by a number of structures and components on the cell membrane surface.Previous studies have shown that primary cilia,integrins,ion channels,calmodulin,and recently reported nuclear membrane proteins are involved in the process.Integrin subunit ?3 mediates the regulation of pathological stretch to smooth muscle cell proliferation and phenotypic switch.Dickkopf-1(DKK1)is a member of the Dickkopf family.DKK1 competitively binds to low-density lipoprotein receptor-related protein(LRP)5/6 and Kremen-1 on the cell membrane to block the Wnt pathway.In addition,DKK1 can also bind cytoskeleton-associated protein 4(CKAP4)on the cell membrane and activate the PI3K/AKT pathway.An increasing number of studies have revealed that DKK1 exert exerts tumor-promoting effects.To date,multiple DKK1-neutralizing antibodies have been developed as antitumor agents and are being evaluated in clinical trials.Recent studies have also shown that DKK1 has an important function in the development of cardiovascular disease,but the molecular mechanisms involved are not clear.Therefore,revealing the role of DKK1 in the development of cardiovascular disease is important to guide the application of antitumor agents targeting DKK1 in tumors within the population of patients with cardiovascular disease.Recently,the role of DKK1 in cardiovascular disease is attracting increasing attention.The function of DKK1 in vascular remodeling is remain poorly understood.The implication of DKK1 in the development of atherosclerosis has become progressively more evident.But the evidence of its involvement in other types of vascular remodeling and the potential mechanisms remain elusive.Previous studies confirmed that DKK1 expression in endothelial cells was upregulated in response to disturbed flow in vivo and oscillatory shear stress(OSS)treatment in vitro.Knockdown of DKK1 attenuated OSS-induced monocyte adhesion and endothelial impairment.It is unknown whether DKK1 plays a role in the regulation of smooth muscle cell function by mechanical stretch.2 Objectives(1)To clarify the role of mechanical stretch in the regulation of DKK1 expression.(2)To explore the role of DKK1 in arterial remodeling in mice caused by pathological stretch stimulation.3 Materials and methods3.1 Construction of a mouse abdominal aortic constriction(AAC)modelAn animal model of pathological distraction of the proximal segment of the stenotic abdominal aorta was constructed by partially ligating the mouse abdominal aorta above the level of the renal artery using surgical sutures.3.2 Generation of smooth muscle-specific DKK1 knockout miceMating Dkklflox/flox mice with SM22-Cre mice to ablate DKK1 specifically in smooth muscle.The mice were named DkklSMKO.3.3 Heart rate and blood pressure measurement The baseline heart rate(HR)and blood pressure(BP)were measured by the mouse-tail cuff method using the automated Blood Pressure Analysis System.The HR and BP of mice in the aorta-constriction and sham-operated groups were measured with a Millar catheter placed in the left carotid artery.3.4 EchocardiographyEchocardiography was performed using a Vevo 2100 Imaging System.Mice were anesthetized with isoflurane.The echocardiography system automatically calculates left ventricular fractional shortening(FS,%)and ejection fraction(EF,%).Diastolic function was evaluated by tissue Doppler echocardiography presented as the E/E' ratio.3.5 Histopathology and immunohistochemistryParaffin sections were deparaffinized and rehydrated.Tissue sections were stained with hematoxylin and eosin to examine the morphology of the artery.The expression levels of DKK1,PCNA and ?-SMA in the thoracic aorta and coronary arteries of mice were detected by immunofluorescence staining.3.6 Western blotHASMCs and tissue sample proteins were extracted using RIPA buffer containing protease inhibitors.BCA protein detection reagent was used to determine protein concentration.Lysates were denatured by boiling with SDS-PAGE loading buffer(Reducing,5x).The proteins were resolved on 10%SDS-PAGE gels and transferred to PVDF membranes.Membranes were incubated with primary antibodies before incubated with HRP-conjugated secondary antibody.Detection was performed with Immobilon ECL substrate.3.7 Cell cultureHASMCs were cultured in smooth muscle cell medium(SMCM).Mouse primary aortic smooth muscle cells were extracted using tissue-block culture method and cultured using DMEM medium containing 10%serum.All cells were cultured at 37?in 5%CO2.Cells from passages 4 to 6 were used for experiments.3.8 In vitro mechanical stretchCyclic stretch was performed using a FX-5000T FlexCell Tension Plus System.HASMCs were plated on type I collagen coating Bioflex plates,and cyclic stretch was performed using a frequency of 1.0 Hz(60 cycles/min)and an elongation of 5%or 18%for the indicated time.3.9 SiRNA and RNA interferenceHASMCs were transfected with DKK1-siRNA or scrambled control siRNA using Lipofectamine 2000 in Opti-MEM.3.10 EdU assaysCell proliferation in vitro was analyzed using an EdU kit.HASMCs were incubated for 6 hours with 10?M EdU-labeling solution in prewarmed tissue culture medium.The cells were fixed with 4%paraformaldehyde.After treated with 0.5%Triton X-100 in PBS,the cells were incubated with Apollo staining reaction solution in the dark for 30 minutes.Cell nuclei were counterstained with Hoechst 33342.Finally,fluorescence images were taken.3.11 Cell Counting Kit-8 assaysCell Counting Kit-8(CCK-8)assays were performed according to the manufacturer's instructions.The absorbance at 450 nm was measured in a microplate reader.3.12 Flow cytometry assay of cell cycleCell cycle assay was performed using a cell cycle phase determination kit.HASMCs were centrifuged and washed with PBS.70%ethanol was used to fix the cells at 4? overnight.After washing twice,cells were incubated with 0.25 mg/mL RNase A and PI at 37? for 30 min in the dark.Cells were filtered and analyzed in the FL2 channel of flow cytometer.3.13 Transwell assaysTranswell assays were performed using 24-well inserts with an 8 ?m pore size.Briefly,600 ?l SMCM medium supplemented with 10%FBS was loaded into the lower side of the Transwell compartment,whereas 2 × 104 VSMCs in 200 ?l FBS-free SMCM medium were loaded into the upper side compartment after the application of cyclic stretching.After 6 h,cells penetrating to the bottom of the membrane were fixed,stained with crystal violet,and analyzed under a microscope.3.14 ELISADKK1 in supernatants and serum from animal experiments and HASMCs was analyzed with ELISA kits.All kits were used according to the manufacturer's protocols.3.15 Statistical analysisData were tested for normal distribution.Normally distributed data are presented as means±SEM,and non-normally distributed data are presented as medians and interquartile ranges(IQR).For data fitting a normal distribution,unpaired Student's t-test,one-way analysis of variance(ANOVA)and two-way ANOVA followed by Turkey's post hoc test was used to analysis the data.For data not fitting a normal distribution,non-parametric tests were used.P<0.05 was considered statistically significant.Prism 6 software was used for statistical analysis.4 Results4.1 AAC increased DKK1 expression in mouse arterial smooth muscle cell.The systolic blood pressure(SBP)and diastolic blood pressure(DBP)of AAC mice were significantly higher than those of sham-operated controls.A significant increase in the DKK1 protein level was detected in the media of the thoracic aorta and coronary artery in the AAC mice compared with the sham-operated control mice at 1 week after the operation.Thoracic aortas were harvested and analyzed by Western blotting,and the relative protein expression levels of DKK1 were significantly increased in the AAC mice.4.2 Generation and analysis of Dkk1SMKO miceImmunofluorescence,Western blot results showed that DKK1 expression in Dkk1SMKO aorta decreased significantly compared with control group.There were no significant differences between Dkk1SMKO or control mice regarding body weight,blood pressure,or heart rate.4.3 Smooth muscle(SM)-specific deletion of DKK1 in mice resulted in improved vascular remodelingDkklSMKO and control mice were randomly divided into the sham groups and the AAC groups.Mice were monitored for 3 weeks after the operation and then sacrificed,and hearts and aortas were harvested.HE staining showed that the thickening of the media layer of thoracic aorta and coronary artery caused by AAC was reduced in the DkklSMKO mice compared with the control mice(P<0.05).Immunohistochemistry and Western blot analysis showed AAC increased expression of PCNA but decreased expression of ?-SMA in the media layer of the aorta and coronary artery,and this change was significantly attenuated in DkklSMKO mice(P<0.05).We calculated the ratios of heart weight to body weight(HW/BW)and heart weight to tibia length(HW/TL)in all groups.The AAC mice had significantly higher HW/BW and HW/TL ratios than the sham mice.However,there was no significant difference in HW/BW or HW/TL between the Ctr mice and the Dkk1SMKO mice.We also evaluated cardiac function using echocardiography in all groups.No difference was observed in EF%,FS%,E/E' or LVPW between the CTR mice and the Dkk1SMKO mice.4.4 Mechanical overstretch increased DKK1 expression in VSMCs in vitroWhen HASMCs were treated with high-level stretch(18%),both HASMCs and the culture supernatant showed an increase in the time dependence of DKK1 protein and mRNA level.No such change was observed when the cells were under 5%cyclic stretch.HASMCs were treated for 6 hr with no stretch,normal stretch(5%)or high-level stretch(18%),and then cell lysates were harvested for Western blotting analysis.DKK1 expression was significantly higher in cells treated with high-level stretch.4.5 Integrin subunit beta 3(ITGB3)mediates the regulation of DKK1 by mechanical stretchWestern blot,ELISA and RT-qPCR analysis indicated that DKK1 expression in HASMCs was significantly downregulated after knocking down ITGB3 by siRNA under high-level stretch.4.6 DKK1 contributes to the regulation of VSMC function under mechanical stretchHASMCs were stimulated with 5%or 18%stretch for 24 hr after transfection with DKK1-siRNA or scrambled siRNA.The EdU and CCK-8 assays indicated that 18%cyclic stretch significantly promoted the proliferation of HASMCs,and knocking down DKK1 suppressed this effect(P<0.05).Flow cytometric analysis of cell cycle distribution indicated that cells in G0/G1 phase increased significantly,while cells in S phase decreased significantly,after DKK1 knockdown under high-level stretch(P<0.05).Similarly,the Transwell assay results consistently indicated that the number of cells that migrated into the lower chambers was significantly lower in the DKK1-siRNA group(P<0.05).Whole-cell lysates were examined for the protein levels of PCNA and ?-SMA by WB analysis.Compared with the normal(5%)treatment,the high-level stretch(18%)treatment upregulated the protein levels of PCNA and downregulated the protein levels of ?-SMA(P<0.05).These changes were partially reversed by knocking down the expression of DKK1.The addition of exogenous rhDKK1 promoted cell proliferation and migration,facilitated the expression of PCNA and inhibited the expression of ?-SMA.5 Conclusions(1)DKK1 expression in HASMCs is induced by pathological stretch.(2)DKK1 is involved in the process of HASMCs response to mechanical stretch via the promotion of abnormal proliferation and migration.(3)Knockout of DKK1 in smooth muscle cells attenuates arterial vascular remodeling caused by pathological stretch.1 BackgroundDickkopf-1(DKK1)protein is a secreted glycoprotein and a member of the DKK family.It is best known for its role in antagonizing the classical Wnt/?-catenin pathway by competing with Wnt for the receptor LRP5/6 and promoting endocytosis and degradation of LRP5/6.DKK1 also affects cell proliferation,migration,apoptosis and inflammatory responses by regulating ?-catenin-independent pathways,such as the non-canonical Wnt pathway.DKK 1 has also been found to bind to type ?cytoskeleton-associated protein 4(CKAP4),activate PI3K/AKT,and promote tumor cell proliferation.Due to its important role in a variety of pathological processes,DKK1 has been identified as a potential drug target.DKK1 also plays a role in cardiovascular diseases,and its role in endothelial cell dysfunction and atherosclerosis has been documented.However,the effect of DKK1 in smooth muscle cell dysfunction and other types of vascular remodeling remains to be investigated.It is of importance to elucidate the role of DKK1 in cardiovascular diseases and its mechanism in detail.In previous,we found that knockout of DKK1 in mouse smooth muscle inhibited vascular remodeling caused by pathological stretch in vivo.In vitro,the silence of DKK1 attenuated smooth muscle cell dysfunction caused by pathological stretch,including increased cell proliferation and migration and reduced contractile phenotypic markers.However,the mechanisms by which DKK1 is involved in the regulation of smooth muscle cell function by mechanical stretch need to be further investigated.2 Objectives(1)To identify target molecular involved in the regulation of vascular smooth muscle function by DKK1.(2)To uncover the signaling pathways and molecular mechanisms by which DKK1 regulates this target.3 Methods3.1 Cell CultureHASMCs were cultured in smooth muscle cell medium(SMCM)at 37? in 5%CO2-Cells from passages 4 to 6 were used for experiments.293T cells and primary murine aortic smooth muscle cells were cultured in DMEM medium.3.2 Plasmid and transfectionWild-type and mutant UHRF1-promoters were cloned downstream of the Renilla reporter gene into the PGL-3 plasmid(UHRF1-WT&UHRF1-MUT).PcDNA3.1 YAP plasmid and pcDNA3.1 empty plasmid were constructed.Transient transfections were performed with Lipofectamine 3000 Transfection Reagent.3.3 SiRNA and RNA interferenceHASMCs or 239T cells were transfected with specific siRNA or control siRNA using Lipofectamine 2000 according to the manufacturer's recommendations.3.4 Real-Time Quantitative PCR(RT-qPCR)Total RNA was extracted from cells.After cDNA was synthesized,RT-qPCR was performed on a CFX96 TouchTM Real-Time PCR System using SYBR Green PCR Kit.Relative gene expression was determined using the 2-??CT method.CT values were normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase(GAPDH).3.5 In vitro mechanical stretchCyclic stretch was performed using a FX-5000T FlexCell Tension Plus System.HASMCs were plated on type I collagen coating Bioflex plates,and cyclic stretch was performed using a frequency of 1.0 Hz(60 cycles/min)and an elongation of 5%or 18%for the indicated time.3.6 EdU assaysCell proliferation in vitro was analyzed using an EdU kit.HASMCs were incubated for 6 hours with 10?M EdU-labeling solution in prewarmed tissue culture medium.The cells were fixed with 4%paraformaldehyde.After treating with 0.5%Triton X-100,the cells were incubated with Apollo staining reaction solution in the dark for 30 minutes.Cell nuclei were counterstained with Hoechst 33342.Finally,fluorescence images were taken.37 Cell Counting Kit-8 assaysCell Counting Kit-8(CCK-8)assays were performed according to the manufacturer's instructions.3.8 Transwell assaysTranswell assays were performed using 24-well inserts with an 8 ?m pore size.Briefly,600 ?l SMCM medium supplemented with 10%FBS was loaded into the lower side of the Transwell compartment,whereas 2 × 104 VSMCs in 200 ?l FBS-free SMCM medium were loaded into the upper side compartment After 6 h,cells moving to the bottom of the membrane were stained with crystal violet,and analyzed under a microscope.3.9 HistologyFresh tissues were fixed,embedded in paraffin,and sectioned transversely.Paraffin sections were deparaffinized and rehydrated.The expression levels of YAP in the thoracic aorta and coronary arteries of mice were detected by immunofluorescence staining,and UHRF1 expression levels were detected by immunohistochemical staining.3.10 Western blotHASMCs and tissue sample proteins were extracted using RIPA buffer containing protease inhibitors.Lysates were denatured by boiling with SDS-PAGE loading buffer(Reducing,5x).The proteins and prestained protein ladder were resolved on SDS-PAGE gels and transferred to methanol-activated polyvinylidene fluoride membranes.Membranes were incubated with primary antibodies and HRP-conjugated secondary antibody.Detection was performed with Immobilon ECL substrate.3.11 Chromatin ImmunoprecipitationHASMCs were crosslinked with 1%formaldehyde.After adding 125 mM glycine and washing,the cells were sonicated and immunoprecipitated with normal IgG,anti-Histone H3,anti-TEAD1 and anti-TEAD4 antibodies at 4? overnight.After elution and reverse crosslinking the antibody/DNA complexes,DNA were purified by a DNA purification kit.DNA was analyzed by PCR using primer pairs covering a specific region of the UHRF1 promoter.3.12 Luciferase Reporter AssayWild-type and mutant UHRF1-promoters were cloned downstream of the Renilla reporter gene into the PGL-3 plasmid and transfected into YAP1-overexpressing 293T cells and the relevant controls,using Lipofectamine 3000.Cells were harvested 48 h after transfection and analyzed with a Dual-Luciferase Reporter Assay Kit.3.13 Statistical analysisData were tested for normal distribution.Normally distributed data are presented as means ± SEM,and non-normally distributed data are presented as medians and interquartile ranges(IQR).For data fitting a normal distribution,unpaired Student's t-test,one-way analysis of variance(ANOVA)and two-way ANOVA followed by Turkey's post hoc test was used to analysis the data.For data not fitting a normal distribution,non-parametric tests were used.P<0.05 was considered statistically significant.Prism 6 software was used for statistical analysis.4 Results4.1 The canonical Wnt pathway is not involved in the roles of DKK1 in VSMCsPretreatment of HASMCs with FH535 failed to reverse the downward trend in PCNA and the upward trend in ?-SMA protein expression in HASMCs transfected with DKK1-siRNA.EdU assays,CCK-8 assays and Transwell assays also indicated that pretreatment of HASMCs with FH535 failed to reverse the downward trend in cell proliferation and cell migration in HASMCs transfected with DKK1-siRNA.4.2 UHRF1 expression is regulated by DKK1We verified that the protein level of UHRF1 significantly increased under high-level stretch,and no change was detected under normal conditions.When DKK1 was knocked down by siRNA under high-level stretch,UHRF 1 expression was significantly downregulated at the mRNA and protein levels.Moreover,rhDKK1 stimulation significantly increased UHRF1 expression in HASMCs.Immunohistochemistry and Western blot analysis showed AAC increased expression of UHRF 1 in the media layer of the aorta and coronary artery,and this change was significantly attenuated in Dkk1SMKO mice(P<0.05).4.3 UHRF1 mediates the effects of DKK1 on HASMCsWe knocked down UHRF1 with siRNA for 48 hr and treated the cells with normal-(5%)or high-level stretch(18%)in the presence of rhDKK1 stimulation for 24 h.Knocking down UHRF1 significantly attenuated the increase in cell proliferation and migration observed in rhDKK1-stimulated HASMCs(P<0.05).Western blotting analysis demonstrated that the protein level of PCNA significantly decreased and the protein level of ?-SMA increased after UHRF 1 knockdown.4.4 YAP/TEAD pathway regulates UHRF1 expressionBy using the JASPAR database,we predicted that members of the TEAD protein family could directly bind to the UHRF1 promoter region.Interference with TEAD1 and TEAD4 expression showed marked downregulation of UHRF 1 mRNA(P<0.05).When we knocked down both TEAD1 and TEAD4 simultaneously,the decrease in UHRF1 protein became more pronounced than with the individual siRNA treatments(P<0.05).Furthermore,chromatin immunoprecipitation(ChIP)experiments demonstrated that there was an enrichment of TEAD 1 and TEAD4 binding between 275 bp and 229 bp upstream of ATG of the UHRF1 gene.When we used verteporfin(VP)to disrupt YAP-TEAD interactions or transduced HASMCs with small interfering RNA against YAP,the UHRF1 mRNA and protein levels dropped significantly(P<0.05,).The data from the dual-luciferase assays confirmed that YAP overexpression significantly activated WT promoter luciferase activity and that this activation was inhibited via knockdown of TEAD1 and TEAD4(P<0.05),YAP overexpression significantly activated the WT but not MUT promoter luciferase activity(P<0.05).4.5 DKK1 regulates UHRF1 through the YAP/TEAD pathwayWestern blotting analysis demonstrated that high-level stretching reduced the phosphorylation of YAP at Ser1 27,which was enhanced after DKK1 knockdown(P<0.05).Phosphorylation of YAP at Ser127 was also reduced in HASMCs after treatment with rhDKK1(P<0.05).Moreover,the immunofluorescence results confirmed that DKK1 knockdown markedly reduced nuclear YAP localization.VP treatment significantly reversed the effect of rhDKK1 in HASMCs,leading to a decrease in UHRF1 and PCNA expression and an increase in ?-SMA expression in HASMCs.Western blotting analysis showed that compared with the sham mouse aortas,AAC mouse aortas exhibited higher YAP protein levels,and DKK1 deletion could reduce the AAC-induced upregulation of YAP(P<0.05).Immunofluorescence showed AAC increased the proportion of YAP-positive nuclei in vascular smooth muscle cells.In contrast,this proportion was significantly decreased in the DkklSMKO mice.5 Conclusions(1)UHRF1 is involved in the regulation of HASMCs function by DKK1.(2)DKK1 is involved in the regulation of UHRF1 via YAP/TEAD pathway during cyclic stretch application.