GDF11 Inhibits Calcification Of Human Endothelial Cells And Aortic Smooth Muscle Cells And Its Mechanism

Background Vascular calcification is a common problem among the elderly and those with chronic kidney disease(CKD)and diabetes.Vascular calcification can cause severe consequences such as blood pressure fluctuations,plaque rupture,and thrombosis.As the mechanism of arterial calcification is complex and diverse,and it is not clear,there is currently no effective targeted treatment.However,the vascular calcification process includes passive and active factors,and is closely related to osteogenic transdifferentiation,inflammation,apoptosis or exosomes release from vascular smooth muscle cells(VSMCs)or endothelial cells(ECs).Research on vascular calcification has focused on Transforming growth factor-?(TGF-?)family molecules.In recent years,growth differentiation factor 11(GDF11)has shown a positive role as a new member of the TGF-? family.Studies have shown that exogenous GDF11 can inhibit osteogenic differentiation of bone marrow mesenchymal stem cells and islet ?-cell apoptosis through the smad2 / 3 signaling pathway.According to the current research of GDF11,it is speculated that GDF11 may affect vascular calcification,but there's no related reports.Since the arterial wall mainly includes endothelial cells and smooth muscle cells,this study intends to use ?-glyceryl phosphate,dexamethasone,and L-ascorbic acid to induce calcification of human aortic endothelial cells(HAEC)and human aortic smooth muscle cells(HASMC),and explore the effects of exogenous GDF11 on HAEC osteogenic differentiation,endothelial-to-mesenchymal transition differentiation,and calcification.And explore the effects of exogenous GDF11 on HASMC osteogenic differentiation,phenotypic transformation,apoptosis,calcification for revealing the effect and mechanism of GDF11 on HAEC and HASMC calcification.Provide more possibilities for the treatment or prevention of vascular calcification.Aim 1.To explore the effect of exogenous recombinant GDF11 on HAEC calcification and its effect on osteogenic differentiation and endothelial-to-mesenchymal differentiation during calcification 2.To investigate the effect of exogenous recombinant GDF11 on the calcification of HASMC and osteogenic differentiation,smooth muscle cell contraction to synthetic conversion and apoptosis during calcificationMethods 1.Detection of calcification-related proteins,genes and GDF11 expression levels in HAEC samples: ?-glyceryl phosphate as a variable is divided into 3 groups:(1)blank group;(2)Low concentration of ?-glycerophosphate group(10mmol / L ?-glyceryl phosphate + 100 nmol / L dexamethasone + 50 ug / ml L-ascorbic acid);(3)High concentration of ?-glycerophosphate group(30mmol / L ?-glyceryl phosphate + 100 nmol / L dexamethasone + 50 ug / ml L-ascorbic acid).After 10 days of calcification induction in HAEC,the protein expression levels of BMP2,BMP4,and GDF11 were detected by western blot,and the gene expression levels of Runx2,Osterix,and GDF11 of HAEC were detected by RT-PCR.2.Cellular immunofluorescence detection of GDF11 expression in HAEC: After stimulating HAEC for 10 days with 30 mmol / L ?-glyceryl phosphate + 100 nmol / L dexamethasone + 50 ug / ml L-ascorbic acid,the expression of intracellular GDF11 was detected by cellular immunofluorescence;3.Using GDF11 intervention at different concentrations and durations as variables,explore the effective concentration and time of exogenous recombinant GDF11 to interfere with HAEC calcification.HAEC:(1)Pretreatment with GDF11 for 48 h,and calcification was induced for another 10 days,then divided into 5 groups according to concentration gradient:(1)blank group,(2)calcification group,(3)30 ng / ml group(30 ng / ml GDF11),(4)50 ng / ml group(50 ng / ml GDF11),(5)100 ng / ml GDF11,(2)Pretreatment with 100 ng / ml GDF11,,and calcification was induced for another 10 days,then divided into 5 groups according to time gradient:(1)blank group,(2)calcification group,(3)GDF11 treated 8 h group,(4)GDF11 treated 24 h group,(5)GDF11 treated 48 h group;Western blot was used to detect the protein expression levels of BMP2 and BMP4 in each group.4.After screening the effective intervention concentration and time of GDF11,HAEC was divided into 4 groups for follow-up experiments:(1)blank group(12 days of conventional culture),(2)GDF11 group(pretreated with 100 ng / ml GDF11 for 48 hours,and then cultured for 10 days)(3)Calcification group(2 days after conventional culture,and then calcification induction for 10 d),(4)GDF11 + calcification group(pre-treatment with 100 ng / ml GDF11 for 48 h,and then calcification induction for 10 d).Western blot was used to detect BMP2,BMP4,?-SMA,and VE-Cadherin protein expressions,RT-PCR was used to detect Runx2,Osterix gene expression levels,cellular immunofluorescence was used to detect ?-SMA,VE-Cadherin expression,and alizarin red was used to detect calcified deposits; 5.Detection of calcification-related proteins,genes and GDF11 expression levels in HASMC samples: HASMC was divided into two groups:(1)blank group;(2)10mmol / L ?-glyceryl phosphate + 100 nmol / L dexamethasone + 50 ug / ml L-ascorbic acid group;Western blot was used to detect the expression levels of Runx2,BMP2,BMP4,and GDF11 in the samples after 14 days of calcification induction in HASMC.RT-PCR was used to detect the expression levels of Runx2,Osterix,GDF11,ALP,BMP2,and BMP4 in the samples;6.Using GDF11 intervention at different concentrations and durations as variables,explore the effective concentration and time of exogenous recombinant GDF11 to interfere with HASMC calcification.HASMC:(1)Pretreatment with GDF11 for 48 h,and calcification was induced for another 10 days,then divided into 6 groups according to concentration gradient: blank control group,calcification group,30 ng / ml group(30 ng / ml GDF11),50 ng / ml group(50 ng / ml)GDF11),100 ng / ml group(100 ng / ml GDF11),(6)200 ng / ml group(200 ng / ml GDF11);(2)Pretreatment with 100 ng / ml GDF11,and calcification was induced for another 10 days,then divided into 6 groups according to time gradient:(1)blank Group,(2)calcification group,(3)GDF11 treatment 4h group,(4)GDF11 treatment 8h group,(5)GDF11 treatment 24 h group,(6)GDF11 treatment 48 h group;Western blot was used to detect Runx2,BMP2,BMP4 protein expression levels in each group;7.After screening the effective intervention concentration and time of GDF11,HASMC was divided into 4 groups for follow-up experiments:(1)blank group(16 days of conventional culture),(2)GDF11 group(pretreated with 100 ng / ml GDF11 for 48 hours,and then cultured for 10 days)),(3)calcification group(2 days after conventional culture,pre-calcification induction for 14 d),(4)GDF11 + calcification group(pretreatment with 100 ng / ml GDF11 for 48 h,and then calcification induction for 14 d).Western blot detected Runx2,BMP2,BMP4,?-SMA,OPN,Bcl-2,Bax,Caspase3,cleaved-Caspase3 protein expression levels,and cellular immunofluorescence detected ?-SMA,OPN protein expression,q-RT-PCR was used to detect gene expression levels of ALP,BMP2,BMP4,Runx2,and Osterix.Alizarin Red S staining was used to detect calcified deposits;Results 1.The establishment of HAEC calcification model and the effect of calcification on the expression of endogenous GDF11: Western blot results showed that the expression levels of BMP2,BMP4,GDF11 protein in the high-concentration ?-glycerol phosphate group were higher than those in the blank group(all P <0.05);RT-PCR results showed that the gene expression levels of Runx2,Osterix,and GDF11 in the high-concentration ?-glyceryl phosphate group were higher than those in the blank group(all P <0.05).The protein expression was stronger than that in the blank group and the low-concentration ?-glycerol phosphate group.In the subsequent experiments,the induction protocol of the calcification group,that is,the high-concentration ?-glycerol phosphate group;2.Effective concentration and time of exogenous GDF11 on HAEC calcification: Western blot results showed that 100 ng / ml GDF11 was pretreated for 48 h,and the expressions of BMP2 and BMP4 proteins in HAEC were down-regulated(both P <0.05).100 ng / ml GDF11 was pretreated for 48 h as effective GDF11 intervention concentration and time;3.Effects of exogenous GDF11 on HAEC calcification-related proteins,calcification,and endothelial-to-mesenchymal transition differentiation: Western blot results showed that BMP2 and BMP4 protein expression levels in the GDF11 + calcification group were lower than those in the calcification group(all P <0.05)and GDF11 The expression levels of BMP2 and BMP4 proteins in the group were not statistically different from those in the blank group.Western blot results showed that the expression level of ?-SMA protein in the GDF11 + calcification group was lower than that in the calcification group(P <0.05).The expression level of ?-SMA protein in the GDF11 + calcification group was not statistically different from that in the blank group.Both were higher than the calcification group(P <0.05).The expression level of VE-cadherin protein in the GDF11 group was not statistically different from that in the blank group.The cellular immunofluorescence results of ?-SMA and VE-cadherin proteins were consistent with Western blot results.Alizarin Red S staining showed that the calcification deposits in the GDF11 + calcification group were less than those in the calcification group.Decreased(all P <0.05),there was no statistical difference in the expression levels of Runx2 and Osterix genes in the GDF11 group with the blank group;4.The establishment of HASMC calcification model and the effect of calcification on the expression of endogenous GDF11: Western blot results showed that 10 mmol / L ?-glyceryl phosphate + 100 nmol / L dexamethasone + 50 ug / ml L-ascorbic acid stimulated HASMC for 14 d could induce calcification.Western blot results showed that Runx2,BMP2,BMP4,and GDF11 protein expression levels in the calcification group were higher than those in the blank control group(all P <0.05);RT-PCR results showed that BMP2,BMP4,Runx2,Osterix,ALP,GDF11 in the calcification group The gene expression level was higher than that in the blank group(all P <0.05);5.Effective concentration and time of exogenous GDF11 on calcification of HASMC:Western blot results showed that pretreatment with 100 ng / ml GDF11 for 24 h resulted in the most significant down-regulation of BMP2 and BMP4 protein expressions in HASMC(all P <0.05).Because the solution was changed every 48 h during HASMC culture,the subsequent experiments were pretreated with 100 ng / ml GDF11 for 48 h as the effective intervention concentration and time of GDF11;6.Effects of exogenous GDF11 on HASMC calcification-related proteins / genes,calcification deposition,apoptosis,and smooth muscle cell phenotypic transformation: Western blot results showed that Runx2,BMP2,and BMP4 protein expression levels in the GDF11 + calcification group were lower than those in the calcification group(all P <0.05),there was no statistical difference in the expression levels of Runx2,BMP2,and BMP4 protein in the GDF11 group compared to the blank group;Decreased(all P <0.05),the corresponding gene expression levels in the GDF11 group were not statistically different from those in the blank group;alizarin red S staining showed that the calcium deposition in the GDF11 + calcification group was less than that in the calcification group,but there was no statistical difference in the GDF11 group compared with the blank group;Western blot results showed that the expression levels of pro-apoptotic proteins Bax,Caspase3,and cleaved-Caspase3 in the GDF11 + calcification group were lower than those in the calcification group(all P <0.05).But Bax,Caspase3,cleaved-Caspase3,and Bcl-2 protein expression levels in the GDF11 group were not statistically different from the blank group;Western blot results suggested that The expression level of PN protein was lower than that of calcification group(all P <0.05),while the expression level of ?-SMA protein in GDF11 + calcification group was higher than that of calcification group(all P <0.05),but the expression levels of ?-SMA and OPN protein in GDF11 group were blank.There was no statistical difference in the group;the results of cellular immunofluorescence showed that the ?-SMA fluorescence intensity of the GDF11 + calcification group was higher than that of the calcification group,and the OPN fluorescence intensity was weaker than the calcification group,but there was no significant difference in fluorescence intensity between the GDF11 group and the blank group;The fluorescence intensity was lower than the calcification group,but there was no significant difference in fluorescence intensity between the GDF11 group and the blank group;Conclusion 1.A mixed calcification induction solution containing ?-glycerol phosphate,dexamethasone,L-ascorbic acid can stimulate osteogenic differentiation and calcification of HAEC and HASMC;2.During the calcification of HAEC and HASMC,the protein and gene levels of GDF11 are up-regulated;3.Exogenous recombinant GDF11 can inhibit osteogenic differentiation and calcification of HAEC,and at the same time inhibit endothelial-to-mesenchymal transition during calcification;4.Exogenous recombinant GDF11 can inhibit the osteogenic differentiation and calcification of HASMC,and at the same time inhibit the smooth muscle cell contraction to synthetic conversion and apoptosis that occur during calcification.