The Role And Mechanism Of Smooth Muscle Dickkopf1 In Vascular Calcification By Phospholipase D1

BackgroundVascular calcification(VC)is a pathological change associated with cardiovascular mortality and morbidity.The characteristics of VC is the deposits of calcium and phosphorus in the membrane of the blood vessel,manifesting as vascular stiffness and reduced compliance.However,the mechanism of VC is not yet fully understanded.At present,vascular calcification was thought to be a positive and controllable process,involving the osteogenic transdifferentiation of vascular smooth muscle cells(VSMCs)and extracellular matrix remodeling.Defining the mechanism of vascular calcification is of great significance for the prevention and treatment of vascular calcification and related cardiovascular diseasesThe Dickkopf(DKK)family is composed of four secretory glycoproteins.Dickkopf1(DKK1)is the first and most characteristic recognized molecule,which can antagonize the downstream Wnt pathway by binding to LRP6,and can also recruit PI3K by binding to type Ⅱtransmembrane cytoskeleton related protein 4(CKAP4),activate Akt and promote proliferation.DKK1 participates in many pathological processes,which not only promotes the occurrence and development of various tumors,but also plays a more and more important role in cardiovascular diseases.Drug development targeting DKK1 has been progressed to Phase II clinical trials.Previous studies of our group found that DKK1 plays an important regulatory role in vascular endothelial cells and smooth muscle cells,which can promote vascular remodeling,promote plaque occurrence and increase plaque instability.In addition,DKK1 also plays a crucial role in osteogenesis.Overexpression of DKK1 in osteoblasts can lead to osteoporosis and pathological bone loss.However,the role of DKK1 in vascular calcification is not clear.Although some studies have shown that DKK1 is involved in smooth muscle cells calcification,most of the previous studies have been carried out as a typical inhibitor of Wnt signal pathway.The exact role and mechanism of DKK1 in vascular calcification need to be further studied.In this study,we constructed smooth muscle specific DKK1 knockout mice to study the role and potential mechanism of DKK1 in regulating vascular calcification.Objective1.To study the effect of DKK1 on vascular calcification;2.To explore the molecular mechanism of vascular calcification regulated by DKK1 in smooth muscle cells.Materials and methods1.Construction of smooth muscle specific DKK1 knockout miceThe mouse model of DKK1flox/flox SM22-Cre+(DKK1SMKO)was established by CRISPR/Cas9 method.2.Establishment of vascular calcification model in mice.Male DKK1SMKO mice and littermate control mice were injected subcutaneously with vitamin D3(VD3)(500000 IU/kg/day)for 4 days,and the samples were harvested 10 days later.3.Animal sampling and vascular tissue staining.The mice were anesthetized by 0.8%pentobarbital sodium.The serum was taken after the apical blood was taken,and the DKK1 level,calcium concentration and ALP activity were detected.The mouse aorta was preserved in 4%paraformaldehyde or liquid nitrogen.After G fixation,the paraffin-embedded sections were stained with alizarin red and Von-kossa to show the area of calcification.The expression of DKK1 and PLD1 was detected by immunohistochemistry.4.Smooth muscle cells culture and calcification model.Human primary vascular smooth muscle cells were purchased from ScienCell and cultured with DMEM of 10%FBS.In order to induce calcification,(HASMCs)were stimulated with 2.6mM Na2HPO4/NaH2PO4 solution for 4-12 days.The culture medium is changed every other day.5.Western Blot(WB).The total protein in HASMCs and tissue samples was extracted with RIPA buffer containing PMSF(RIPA:PMSF=100:1).Detection of protein concentration by BCA method,after boiling denaturation,electrophoresis,membrane transfer,sealing,incubation of the first antibody overnight at 4℃,washing the membrane,chemiluminescence after the second antibody was incubated,and the target protein level was determined by ImageJ software.6.RNA interference and plasmid transfection.The target or control genes siRNA to HASMCs were transfected with Lipofectamine 2000,and the PLD1-overexpression plasmid was transfected with Lipofectamine 3000.Cells were used for further experiment after transfection for 48 hours.7.Calcification analysis.The levels of calcium in aortic tissue,HASMCs and serum were measured by calcium determination kit.Alkaline phosphatase assay kit was used to determine the activity of HASMCs and ALP in serum.The BCA protein concentration kit was used to standardize the test results with the total protein concentration.8.Alizarin red staining.After PBS washed the cells,the cells were fixed for 15 min in 4%paraformaldehyde and stained for 8 min with alizarin red staining solution.The tissue sections were stained for 4 min,then rinsed with deionized water and placed in the oven for 4 hours.9.Von-kossa staining.The tissue sections were washed with deionized water for at least 5 times and exposed to ultraviolet light for 2 hours after dripping with Von Kossa dye solution.The excess solution is washed with deionized water,the sample is dehydrated and imaged under a microscope.10.Statistical analysis.All data were analyzed by GraphPad Prism 6.0 software and expressed as mean±standard error(SEM).One-way analysis of variance(One-way ANOVA)was used to compare the significant differences between the two groups.T-test was used to compare the significant difference between the two groups.P<0.05 was considered to be statistically significant.Results1.DKK1 expression was decreased under calcifying conditions.When HASMCs was given different calcification stimuli(Na2HPO4/NaH2PO4,glucose and dexamethasone),the level of DKK1 protein decreased significantly compared with the control group(P<0.05).When HASMCs was stimulated by high Pi at different time points(0,3,6,9,12 days),with the increase of time the expression of Runx2 and BMP2 were gradually increased,while DKK1 expression was decreased(P<0.05).Meanwhile,the protein expression level of DKK1 in the aorta of vitamin D3-induced calcified mice was lower than that of control mice(P<0.01).Immunohistochemistry showed the same results(P<0.01).2.DKK1 overexpression alleviated high phosphate-induced vascular calcification.First of all,the lentivirus overexpressed by DKK1/GFP was transfected into the cells.After stimulation of high phosphate for 4-6 days in HASMCs,the expression levels of Runx2,BMP2 and OPN in no-load virus group were significantly up-regulated.After overexpression of DKK1,the expression levels of Runx2,BMP2 and OPN were decreased significantly(P<0.05).Alizarin red staining showed that the deposition of calcification was significantly increased under the induction of phosphate,and it was restored by the overexpression of DKK1(P<0.01).Similarly,compared with the high phosphate stimulation group,the calcium concentration and alkaline phosphatase(ALP)activity in the cell lysate supernatant were significantly down-regulated in the DKK1 overexpression group(P<0.05).3.DKK1 affected the protein expression of PLD1.Proteomic sequencing of smooth muscle cells transfected with DKK1 small interference or control group showed that PLD1 increased significantly after DKK1 knockdown.Western blot showed that the level of PLD1 protein was significantly increased after DKK1 interference(P<0.01).At the same time,the level of PLD1 protein in DKK1 overexpression group was significantly lower than that in GFP group(P<0.01).It is suggested that PLD1 may be the downstream target of DKK1.4.PLD1 mediated DKK1-induced inhibition of smooth muscle calcification.Firstly,the overexpression plasmid of PLD1 was constructed and transfected into HASMCs.Under the condition of calcification induced by high phosphorus,the overexpression of PLD1 could significantly increase the decrease of calcification caused by overexpression of DKK1(P<0.05).Alizarin red staining,calcium concentrations and ALP activity in cells also showed the same results(P<0.01).5.DKK1 promoted the degradation of PLD1 protein through regulating autophagosome formation and maturation.RT-PCR results showed that knockdown of DKK1 had no significant difference in mRNA level of PLD1,suggesting that DKK1 may promote the protein degradation of PLD1.When Cycloheximide(CHX),an inhibitor of protein synthesis,was used in DKK1 overexpression group and GFP group,it was found that the degradation rate of PLD1 protein in DKK1 overexpression group was significantly faster than that in GFP group(P<0.05).Proteasome pathway inhibitor MG132,autophagy pathway inhibitor 3-MA and lysosomal pathway inhibitor CQ were used to determine which protein degradation pathway DKK1 affected PLD1 degradation(P<0.05).The results showed that DKK1 affected the protein degradation of PLD1 mainly through autophagy lysosome pathway rather than proteasome pathway.After the knockdown of DKK1 in HASMCs,the ratio of p-Akt/Akt to p-mTOR/mTOR,the protein expression of autophagosome maturation inhibitor Rubicon and P62 were increased,while the autophagy flux ratio LC3BII/I was decreased(P<0.05).In contrast,in DKK1 overexpression group,HASMCs autophagy was activated(P<0.05).The results suggest that DKK1 may regulate autophagy and then regulate PLD1 by affecting autophagy inhibitory pathway Akt/mTOR.MK-2206 2HCl,an inhibitor of Akt,could reverse the increase of PLD1 protein expression induced by DKK1 interference(P<0.05).On the other hand,Rubicon,as a key protein that inhibits autophagy maturation,is also regulated by DKK1.Interestingly,the expression of PLD1 protein decreased significantly after Rubicon was blocked by siRNA interference(P<0.05).These results suggest that DKK1 activates autophagy and further affects the degradation of PLD1 protein by regulating the formation and maturation of autophagosomes.6.Smooth muscle DKK1-specific knockout aggravated VD-induced vascular calcification in mice.Animal model of VD-induced vascular calcification was constructed.Alizarin red and Von-Kossa staining showed that the model was successful,and the calcification area at the aortic arch in DKK1SMKO mice was significantly larger than that in control mice(P<0.01).Western bolt showed that in mouse aorta the Runx2 was increased in the calcification group,and was more obvious in DKK1SMKO mice(P<0.001).Meanwhile,the serum calcium concentration,ALP activity and vascular calcium concentration in the calcified DKK1SMKO group were higher than those in the simple calcification group(P<0.05).In addition,the expression level of PLD1 in DKK1SMKO group was significantly higher than that in control group(P<0.05),which indicated that the specific knockout of smooth muscle DKK1 aggravated VD3-induced vascular calcification in mice.Conclusions1.Smooth muscle specific DKK1 knockout aggravates VD-induced vascular calcification;2.DKK1 inhibits vascular smooth muscle calcification by promoting the protein degradation of PLD1.