The Role Of TWIST1in Regulating Human Aortic Valve Interstitial Cells Transdifferentiating Into Osteoblasts And The Underlying Mechanisms

ObjectiveCalcific aortic valve disease (CAVD) is one of the most commen valvular heartdiseases. The exact mechanisims underlying the disease process is still not fullyunderstdood. Previous studies have shown the existence of osteoblastic markers in thecalcified aortic valves and suggested that the transifferentiation of valve interstitial cellsinto osteoblasts is a core step during the pathological process of valve calcification. In thisstudy, we aimed to:(A) study the expression changes of TWIST1and the osteoblasticmarkers, such as RUNX2et al.(B) Successfully isolate and culture primary human aorticvalve interstitial cells in vitro and establish a valve interstitial cell osteoblastictransdifferentiation model, laying a foundation for future studies on the cellular andmolecular mechanisms of valve interstitial cells phenotype trandifferentiation and valvecalcification.(C) Construct a TWIST1over-expression plasmid and a TWIST1-specificinterfering RNA, and up-and down-regulate the expression of TWIST1in the establishedvalve interstitial cell osteoblastic transdifferentiation model, to study the role of TWIST1in the progress of valve interstitial cells transdifferentiating into osteoblasts.(D) Constructa dual luciferase reporter system, to study the role of TWIST1in regulating RUNX2P2promoter, and illuminate the mechanisms of TWIST1function in regulating valveinterstitial cell osteoblastic transdifferentiation.Methods(A) The expression of TWIST1and the osteoblastic marker RUNX2in calcifiedaortic valvesCollect28calcified aortic valves excised during aortic valve replacement due toCAVD as the experiment group, and and12non-calcified aortic valves from patientsundergoing heart transplantation or autopsy as control group. Compare the mRNAexpression differences of TWIST1, RUNX2and osteocalcin between two groups throughtotal RNA extraction and quantitative real-time PCR. Compare the protein expressiondifference of TWIST1between two groups through total protein extraction and westernblot. Detect the expression differences and the cell location of TWIST1, RUNX2andPCNA through tissue section and immunohistochemistry.(B) Isolation and culture of primary human aortic valve interstitial cells in vitroand establishment of valve interstitial cells osteoblastic transdifferentiation model Valve leaflets were digested in collagenase type II solution (2.0mg/ml) for30min at37°C. After vigorous vortexing to remove the endothelial cells，the leaflets were furtherdigested in fresh collagenase type II solution (2.0mg/ml) for2h at37°C. Collect theinterstitial cells and resuspend them in Dulbecco’s modified Eagle medium (DMEM) with10%fetal bovine serum. The cells were cultured with5%CO2at37°C. Subculture thecells when they reached80–90%confluence. Vimentin immunofluorescence and flowcytometry for CD31positive rating detection were applied for valve interstitial cellidentification. The cells were treated with osteogenisis-inducing medium (OIM), whichcontains50ng/mL BMP-2,50ug/mL ascorbate-2-phosphate,10nmol/L dexamethasoneand10mmol/L β-glycerol phosphate, to establish the valve interstitial cells osteoblastictransdifferentiation model. Quantitative real-time PCR, wetern blot and alkalinephosphatase activity analysis were applied to detect the expression of osteoblastic markers,such as RUNX2et al, for identification of the cell model.(C) The role TWIST1in human valve interstitial cells transdifferentiating intoosteoblastsThe TWIST1protein coding sequence was connected into the carrier plasmid pCMVafter PCR amplification and restriction endonuclease digestion, to build TWIST1expression plasmid pTWIST1. DNA sequencing was applied for identification of theconstructed plasmid. The transfection efficiency of Lipofectamine2000in primary aorticvalve interstitial cells was tested by a FAM-tagged RNA. The valvular interstitial cellswere transfected by pTWIST1. PCR and western blot were applied for detecting TWIST1expression. TWIST1-specific small interfering RNAs were designed and synthesized and asmall interfering RNA with high interfering efficiency was selected. Valve interstitial cellsosteoblastic transdifferentiation model were respectively transfected with TWIST1expression plasmid or TWIST1-specific small interfering RNA, using Lipofectamine2000.The expression of osteoblast markers, such as RUNX2et al, was detected throughfluorescence quantitative PCR, western blot and alkaline phosphatase activity analysis, tostudy the role of TWIST1on human aortic valve interstitial cells transdifferentiating intoosteoblasts. Flow cytometry was applied to detect the effect of TWIST1over-expressionon human aortic valve interstitial cells apoptosis.(D) The molecular mechanism of TWIST1in regulating human aortic valveinterstitial cells osteoblastic transdifferentiation Human RUNX2gene P2promoter region was searched for E-box sequences in theGenebank, and primers for each E-box were designed and synthesized. Through chromatinimmunocoprecipitation, the E-boxes that TWIST1may combine were detected. TheRUNX2P2promoter region that contains the above E-box sequences was cloned into aluciferase reporter gene vector plasmid pGL3-Basic. DNA sequencing was applied to makesure the recombinant plasmid sequences was correct and the plasmid was buildsuccessfully. TWIST1expression plasmid and luciferase reporter gene plasmid wereco-transfected into human aortic valve interstitial cells. Through the dual luciferasereporter gene system, the role of TWIST1in regulating RUNX2P2promoter was studied.By directed mutagenesis technology, luciferase reporter gene plasmids containing mutatedE-box were constructed. The effects of TWIST1on RUNX2P2promoter region withmutated E-box were detected, to identify the specific targeting E-box of TWIST1.Results(A) The expression of TWIST1and the osteoblastic marker RUNX2in calcifiedaortic valvesCompared with the control group of non-calcified valves, RUNX2, OCN and PCNAexpression level increased significantly in the experimental group of degenerative calcifiedaortic valves, while TWIST1mRNA and protein expression levels significantly decreased.TWIST1expression was detected in the nuclear of valve interstitial cells throughimmunohistochemical staining.(B) Isolation and culture of primary human aortic valve interstitial cells in vitroand establishment of valve interstitial cells osteoblastic transdifferentiation modelThe primary valve interstitial cells present a circular shape in the suspention state justafter isoaltion. The cells grew with adherence, presenting fusiform or spindle-shaped,polygon morphology. Cell density increased after culturing, and cell clusters formed.Somecells appeared as the paving stone shape. Vimentin cell immunofluorescence stainingresults showed that cell positive rate was almost100%, suggesting of high mesenchymalcell purity. Flow cytometry results showed that cell CD31positive rate is less than2%,suggesting little pollution of endothelial cells. The primary aortic valve interstitial cellswere collected after treated with OIM for2d and4d, respectively, to detect the expressionchanges of osteoblast markers. Fluorescence quantitative PCR results showed that RUNX2mRNA expression level increased to1.80times and2.55times respectively. OCN mRNAexpression level increased to1.41times and2.44times respectively. ALP mRNA expression level increased to3.03times and8.66times respectively. RUNX2, OPN, OCNprotein expression levels were also elevated according to the results of western blot. ALPactivity test results showed that ALP activity increased to2.53times and5.91timesrespectively. The differences were all statistically significant.(C) The role TWIST1in human valve interstitial cells transdifferentiating intoosteoblastsThe transfection efficiency of Lipofectamine2000in primary aortic valve interstitialcells can reach more than90%. According to DNA sequencing results, the insertedsequence in TWIST1expression plasmid pTWIST1is correct, without errors or missingbases. The recombinate plasmid was constructed successfully. Fluorescence quantitativePCR results showed that in cells with pTWIST transfection, TWIST1mRNA expressionlevel increased to460times. Western blot results also confirmed that TWIST1proteinexpression level in cells transfected with pTWIST1was significantly higher than that incontrol group. In human aortic valve interstitial cells osteoblastic transdifferentiationmodel, RUNX2, OCN, COL1, ALP mRNA expression level were reduced to0.72,0.74,0.64,0.74times of the control group respectively, after transfected pTWIST1. Accordingto the results of western blot, RUNX2, OPN and OCN protein expression level alsodecreased after pTWIST1transfection. ALP activity test results showed that in cells withpTWIST1transfection, ALP activity was about60%of that in the control group.Interfering RNA selection showed TWIST1interfering efficiency reached near70%. Incells with transfection of TWIST1-specific interfering RNA, the expression levels ofRUNX2, OCN, COL1and ALP mRNA were1.49,1.53,1.93and1.53times of the controlgroup, respectively. There was no statistically significant difference in cell apoptosis ratebetween pTWIST1transfection group and the control group.(D) The molecular mechanism of TWIST1in regulating human aortic valveinterstitial cells osteoblastic transdifferentiationWe found10E-box sequences in the RUNX2P2promoter region. According to therelative position of first base pair to the TSS (+1), they were E-box-2651, E-box-2426,E-box-2333, E-box-2056, E-box-1604, E-box-1556, E-box-1333, E-box-820, E-box-100and E-box+6, respectively. Through chromatin immunocoprecipitation, we found twoE-boxes that TWIST1may be directly combined with, E-box-1333and E-box-820. The1.5kbp DNA fragment containing E-box-1333and E-box-820was cloned into a vectorplasmid pGL3-Basic, constructing a luciferase reporter gene plasmid pGL3-RUNX2-P2, which contains the human RUNX2gene P2promoter region. According to the results ofDNA sequencing results, the inserted sequence is correct, without errors or missing basepairs. In valve interstitial cells with transfection of pGL3-RUNX2-P2, the fluorescenceintensity was significantly higher than that in cells with transfection of the empty vectorplasmid pGL3-Basic. The pGL3-RUNX2-P2fluorescence intensity was reduced in cellswith transfection of pTWIST1. TWIST1still had inhibitory effect on pGL3-P2-1333M andreduce its fluorescence intensity, but had little or no obvious effect on pGL3-P2-820Mwithout reducing its fluorescence intensity.Conclusions(A) The expression of TWIST1and the osteoblastic marker RUNX2in calcifiedaortic valvesTWIST1expression was decreased in calcified aortic valves. It may play a role invalve calcification process by affecting the function of aortic valve interstitial cells.(B) Isolation and culture of primary human aortic valve interstitial cells in vitroand establishment of valve interstitial cells osteoblastic transdifferentiation modelIn vitro isolated and cultured primary aortic valvular interstitial cells are of highpurity, with little pollution, and can be used in the following cytological experiments. Thein vitro valve interstitial cells osteoblastic transdifferentiation model was successfullyestablished, laying a solid foundation for the study of the role of valvular interstitial cellsphenotype transdifferentiation in valvular calcification.(C) The role TWIST1in human valve interstitial cells transdifferentiating intoosteoblastsWe successfully constructed a TWIST1expression plasmid, which successfullyexpressed TWIST1protein in human aortic valve interstitial cells. TWIST1-specificinterfering RNA was successfully synthesized. TWIST1overexpression inhibits, whileTWIST1interfering promotes, valve interstitial cells transdifferentiation into osteoblasts.(D) The molecular mechanism of TWIST1in regulating human aortic valveinterstitial cells osteoblastic transdifferentiationA luciferase reporter gene plasmid containing human RUNX2gene P2promoterregion was successfully constructed. TWIST1can directly combine to the E-box-820inRUNX2P2promoter region, inhibiting its activity, thus inhibiting the expression ofRUNX2, and further inhibiting valve interstitial cells osteoblastic transdifferentiation.