Effect Of T3 On Calcium And Phosphorus Induced Calcification In Rat Vascular Smooth Muscle Cells And Its Molecular Mechanism

ObjectiveThis paper intends to study the roles of T3(3,5,3'-Triiodothyronine)in the process of vascular calcification and vascular smooth muscle cells(VSMCs)changes into osteoblast like phenotype related proteins and transcription factors in the cell level by calcification model,and to further clarify the main signal transduction pathway of vascular calcification and the phenotypic transformation of VSMCs to osteoblast like cells.Thus demonstrates that T3 is an important endogenous protective substance to inhibit vascular calcification,which provides an effective experimental basis for clinical regulation and reversal of vascular calcification caused by arterial hypertension related diseases.MethodRat thoracic aortic smooth muscle cells(A7r5)were cultured in vitro and randomly assigned into normal control group(cells were maintained in DMEM without other treatments),calcification group(Cells were treated with 5 mmol/L?-glycerophosphate and 2.5 mmol/L CaCl2),T3 group(In the presence of calcium and phosphorus,cells were treated with T3 at different concentrations 10-7,10-8 and 10-9mol/L)and inhibitor group.Alizarin red staining was used to assess Ca deposition in VSMC cell layers,by which Alizarin red S dye binds with Ca ions in cell layer matrix.The content of osteocalcin(BGP)and the ALP activity were measued with commercially available kits.The mRNA of Runx2 and Osterix were detected by RT-PCR,and the protein expression of OPN,SM22 alpha-actin,SM alpha-actin,ERK1/2,P-ERK,Akt,P-Akt were detected by Western blot.The mRNA content of Runx2 and Osterix and OPN,SM22 alpha-actin and SM alpha protein content were detected again after the addition of signal pathway inhibitors.Data are expressed as mean ± standard deviation(SD).Means were compared with t test between two groups and with one way analysis of variance(ANOVA)among groups followed by Student2Newman2Keuls test.A value of P<0.05 was considered statistically significant.ResultsWhen compared with normal control group,the osteocalcin content,ALP activity,Osterix and Runx2 mRNA expression and OPN protein expression increased significantly(P<0.01),and the protein expression of SMa and SM22a reduced dramatically in A7r5 cells of calcification group(P<0.01).After T3 treatment,the osteocalcin content and ALP activity reduced markedly,mRNA expression of Osterix and Runx2 and OPN protein expression reduced significantly.However,MMI(inhibitor of T3)was able to block the above effects of T3.When compared with calcification group,Osterix and Runx2 mRNA expression and OPN protein expression increased markedly(P<0.01).In addition,the protein expression of ERK1/2,p-ERK,Akt and p-Akt increased significantly in calcification group.In the presence of integrin ?v?3/ERK blocker(PD98059)and/or PI3K/Akt antagonist(LY294002),T3 was still able to inhibit the calcification,and this effect was similar to that after treatment with inhibitors alone.Moreover,LY294002 had a better inhibitory effect as compared to PD98059.1.Effect of T3 on calcium and phosphorus induced calcification of A7r5After induction with ?-glycerophosphate and CaC12 for 12 days,Alizarin red S staining showed orange calcified nodules in calcification group;when compared with calcification group,the orange calcified nodules reduced significantly in T3 group,and the higher the T3 concentration,the smaller the number of orange calcified nodules was.In addition,cells were treated with T3 for 3 h,6 h,12 h,1 d and 3 d,but the calcified cells had a low adherence.Thus,after induction for 3 days,a majority of cells was suspended in the medium.On the basis of osteocalcin content and ALP activity,the optimal concentration of T3 was 10-7 mol/L and the optimal duration of T3 treatment was 6 h.Thus,in following experiments,cells were treated with 10-7 mol/L T3 for 6 h.2.Effects of T3 on the osteocalcin content and ALP activity of calcium and phosphorus treated A7r5 cellsThe BGP content and ALP activity increased significantly in calcified cells.After treatment with T3 at different concentration,the BGP content and ALP activity reduced markedly,and the inhibition was the most evident in Ca+ T3(10-7 mol/L)group.However,this inhibition did not further become more obvious over time,which was ascribed to the low adherence of calcified cells.These findings were consistent with those observed after Alizarin red S staining.3.Effects of MMI on the T3 induced inhibition of calcification in A7r5 cells after calcium and phosphorus treatmentWhen compared with normal control group,the orange calcified nodules increased significantly after calcification induction,accompanied by significant increases in BGP content and ALP activity(P<0.01).After treatment with 10-7 mol/L T3 for 6 h,the number of calcified nodules reduced significantly,and BGP content and ALP activity decreased markedly when compared with calcification group(P<0.01).When compared with T3 group,the number of orange calcified nodules increased markedly,and BGP content and ALP activity elevated dramatically in MMI+T3 group(P<0.01).These indicate that MMI may antagonize the effects of T3.However,the BGP content and ALP activity in MMI+T3 group were still higher than in calcification group,suggesting that MMI partially antagonizes the effects of T3.4.Effects of T3 on phenotype markers of A7r5 cells after calcium and phosphorus treatmentWhen compared with normal control group,the mRNA expression of Osterix and Runx2(two phenotype specific marker)increased significantly in calcification(P<0.01),the protein expression of OPN(a marker of osteogenesis)elevated markedly(P<0.01),but the protein expression of SMa and SM22a(two markers of contractile phenotype)reduced dramatically(P<0.01).When compared with calcification group,the mRNA expression of Osterix and Runx2 reduced significantly in T3 group(P<0.01),accompanied by marked reduction in OPN protein expression(P<0.01)and increase in the protein expression of SMa and SM22a(P<0.01).The mRNA expression of Osterix and Runx2 reduced significantly,the protein expression of SMa and SM22a increased markedly,but OPN protein expression remained unchanged in T3+MMI group when compared with calcification group(P<0.05).These findings were consistent with above finding that MMI partially antagonizes the effects of T3.In addition,MMI alone failed to significantly alter the expression of above molecules.5.Effects of PD98059 and/or LY294002 on phenotype markers in calcium and phosphorus treated A7r5 cellsWhen compared with normal control group,the protein expression of ERK1/2,p-ERK,Akt and p-Akt increased significantly(P<0.01)in calcification group.When compared with calcification group,the protein expression of these molecules reduced markedly after T3 treatment(P<0.05),and the reduction in p-Akt protein expression was the most obvious(P<0.01).This suggests that the phenotype switch of calcium and phosphorus induced A7r5 cells is related to the activation of ?v?3/ERK and PI3K/Akt pathways.The ?v?3/ERK blocker PD98059 and/or PI3K/Akt inhibitor LY294002 was used to treat A7r5 cells.Results showed,when compared with calcification group,the mRNA expression of Osterix and Runx2 reduced,and significant difference was observed in Runx2 expression(P<0.01)after inhibitor treatment.In addition,with the increase in inhibitor concentration,OPN protein expression reduced gradually,and protein expression of SMa and SM22a restored progressively.The optimal concentration was 40 mg/L for PD98059 and 30 ?mol/L for LY294002.After treatment with inhibitors at optimal concentration,the calcification was still inhibited although calcification induction and T3 treatment were also administered,but the effects were similar to those after treatment with inhibitors alone.This suggests that T3 induced calcification inhibition is not overlapped with that of PD98059 and LY294002.Moreover,the inhibitory effects of LY294002 were more obvious than those of PD98059(P<0.01),suggesting that calcium and phosphorus induced calcification of A7r5 cells is more related to the activation of PI3K/Akt signaling pathway.Conclusions1.Calcium phosphate induced A7r5 cells can be successfully constructed in 12 days.2.T3 can reduce the calcium deposition and alkaline phosphatase activity of A7r5 calcification cells,this effect is concentration dependent,indicating that T3 is an endogenous protective substance that regulates vascular calcification.3.T3 up regulation of smooth muscle cell marker SM22 and alpha SM alpha,the expression of bone related protein OPN increased,at the same time to detect important transcription factors Runx2 and Osterix differentiation level of mRNA decreased,while adding T3 antagonist MMI,the T3 effect was partially antagonized the inhibition of calcification,that by inhibiting the T3 phenotype vascular smooth muscle cell transformation is a key link to the regulation of vascular calcification.4.Calcium phosphate induced detected ERK1/2,Akt activation of A7r5 cells in calcification,add the corresponding expression of inhibitor phenotype specific transcription factors Osterix and Runx2 of mRNA were decreased,with the increase of bone differentiation protein OPN protein inhibitor concentration gradually decreased,the protein level of SM contraction phenotype and.SM22 alpha gradually recovered,adding liquid with T3 calcification intervention using the inhibitor concentration were screened,results showed that the inhibitory effect of LY294002 is more obvious than PD98059,there are more obvious relationship between the activation of A7r5 cells induced by calcium phosphate calcification may be related to PI3K/Akt signal pathway.