| BackgroundVascular calcification(VC)is associated with an increased cardiovascular mortality in aging,chronic kidney disease(CKD),type II diabetes mellitus,and atherosclerosis.In the cardiovascular system,VC is characterized by deposition of calcium and phosphate.Calcified arteries are present in the intima and media of the vessel wall as well as in the cardiac valves.Calcification inducers and inhibitors are both involved in the regulation of VC balance.Increased calcium and phosphate deposits,inflammation,and vascular injuries can promote transformation of endothelial cells into osteoblasts,and express osteoblast related factors like Runx2,Alp,BMP2,and BMP4.Recent evidence indicates that VC can serve as an accurate predictor of cardiovascular events including myocardial infarction and stroke.The pathogenesis of VC is characterized by transformation of vascular smooth muscle cells(VSMCs)and endothelial cells into chondrocytes,apoptosis,matrix vesicle release,and matrix remolding.Convincing evidence suggests that VC is an active process,resembling osteogenesis.However,the precise mechanisms involved in this process require further exploration.Interestingly,parathyroid hormone(PTH)has been reported to play a role in osteogenesis;moreover,PTH can promote osteoblast matrix mineralization and induce VC.In addition,vascular cell apoptosis and apoptotic bodies are present during the process of calcification,including formation of hydroxyapatite.In vitro experiment have that,apoptosis regulates vascular smooth muscle calcification and,thus,inhibition of apoptosis alleviates this process.However,the role of interaction between increased PTH and apoptosis in the process of VC needs to be further elucidated.Parathyroid hormone(PTH)is secreted by the chief cells of the parathyroid glands and eosinophilic granulocytes as a polypeptide containing 84 amino acids with a relative molecular weight of 9500.It mainly acts on skeleton and kidney to maintain the balance of calcium and phosphate,by increasing serum calcium and decreasing serum phosphate.Recent studies have shown that PTH also acts on the cardiovascular system to increase heart rate and coronary blood flow,and to induce left ventricular hypertrophy,arrhythmia,and valve calcification.An extended large number of clinical trials have shown that patients with hyperparathyroidism are at high risk for developing CVD and have a high mortality rate.Meanwhile,PTH affects other organs,tissues and cells(e.g.endothelial cell)via its interaction with membrane PTH receptor.In hemodialysis patients,PTH correlates with valve calcification and inflammatory markers.In addition to activating osteoblasts and osteoclasts,PTH also plays a role in immune regulation and inflammation,by inducing cytokine secretion and inflammatory cell recruitment.Moreover,elevated PTH levels also induce endothelial-to-chondrogenic transition leading to calcification of endothelial cells.PTH can activate Jagged1/Notch1 signaling in osteoblasts,but its role in calcification is not yet described.Recently,elevated Notch1 has been observed in human calcified aortic valves.In this study,we investigated the role of PTH in vascular calcification using an in vitro calcification-induced model by PTH.We found that PTH in a dose dependent manner,elevated the calcification of HUVECs,and increased the expression of osteogenesis-related factors including BMP2 and BMP4.Moreover,flow cytometric analysis showed that PTH significantly increased HUVECs apoptosis,suggesting that PTH had an effect in vascular calcification.Furthermore,we found that PTH elevated the expression of pro-apoptotic proteins(Cleaved caspase3 and Bax),while decreasing the expression of anti-apoptotic Bcl-2 protein in HUVECs.In addition,PTH increased the expression of Jagged1 and Notch1 in HUVECs.In summary,these findings suggest that PTH can act on Jagged1/Notch1 pathway to induce endothelial cell apoptosis and promote vascular calcification.Aims1.To investigate whether parathyroid hormone promotes osteogenic differentiation and calcification of HUVECs.2.To elucidate the mechanism by which parathyroid hormone promotes osteogenic differentiation,calcification and apoptosis of HUVECs is through regulation of Jagged1/Notch1 signaling pathway.Methods1.The expression of BMP2 and BMP4 were detected by western blot and q PCR.HUVECs were used for the experiments between 4 and 8 passages.HUVECs were incubated with PTH(10-11mmol/l、10-10mmol/l、10-9mmol/l、10-8mmol/l)at different doses for 5 days.The m RNA of BMP2 and BMP4 were detected by RT-PCR,and the protein expression of BMP2 and BMP4 were detected by western blot.2.The expression of PTH1R、BMP2 and BMP4 were detected by western blot.PTH receptor(PTH1R)-si RNA-3 treatment showed the most effective inhibition and was chosen for further experiments.PTH1 R expression in HUVECs was detected via Western blot analysis before inhibiting its expression with PTH1R-si RNA.HUVECs were transfected with PTH1 R si RNA(50u M)or Scrambled si RNA(50u M).48 h later,cells were incubated with PTH(10-8mmol/l)for 72 h.Western blot analysis were used to examine the protein expression of BMP2 and BMP4.3.The expression of Jagged1 、 Notch1 、 BMP2 and BMP4 were detected by western blot and q PCR.Notch1 specific ligand Jagged1 was applied to activate Notch1.The levels of BMP2 and BMP4 were analyzed after treatments.HUVECs were incubated with PTH(10-11mmol/l,10-10mmol/l,10-9mmol/l,10-8mmol/l)for 5days.Western blot and RT-PCR were used to detect the protein and m RNA expression of Jagged1 and Notch1.HUVECs were incubated with Jagged1 for 24 h and switched to PTH treatment for 5 days.Western blot and RT-PCR were used to detect the protein and m RNA expression of BMP2 and BMP4.HUVECs were stimulated in the presence of γ-secretase inhibitor DAPT(50u M)for 24 hours,and then treated with PTH for 5 days.The expression of Notch1 in HUVECs was determined by western blot and RT-PCR.4.Flow cvtometry detected the apoptosis of HUVECs and western blot detected the expression of Cleaved caspase3、Bax and Bcl-2.We tested the effect of PTH on HUVECs apoptosis.Through flow cvtometry were applied to test cell apoptosis in HUVECs.Western blot analysis detected pro-apoptotic proteins in HUVECs when cells were treated with of PTH(10-11-10-8mmol/l).HUVECs were incubated with Jagged1 for 24 h and switched to PTH treatment for 5 days.Western blot was used to detect the pro-apoptotic protein expression including Cleaved caspase3 and Bax.HUVECs were exposed to different concentrations of PTH for 5 days,western blot was used to detected apoptotic related protein expression.5.Western blot detected the expression of BMP2、BMP4、Cleaved caspase3 and Bax and q PCR detected the expression of BMP2、BMP4、ALP and Runx2.HUVECs were pretreated with DAPT for 48 hours,and then treated PTH for 3 days.Western blot was used to detect pro-apoptotic protein and calcified realted protein expression.Use RT-PCR to detect bone realted prote in BMP2、BMP4、ALP and Runx2 expression.Results1.PTH promotes osteogenic differentiation and calcification of Human umbilical vein endothelial cells.HUVECs were exposed to different consentration of PTH for5 days.PTH(10-11mmol/l,10-10mmol/l,10-9mmol/l,10-8mmol/l)dose-dependently increased the protein and m RNA expression of BMP2 and BMP4 were obviously higher compared with control group,particularly at 10-8mmol/l of PTH(P<0.05).2.Effects of PTH1 R on Human umbilical vein endothelial cells calcification.PTH receptor(PTH1R)-si RNA-3 treatment showed the most effective inhibition and was chosen for further experiments(A).HUVECs were transfected with PTH1 R si RNA(50u M)or Scrambled si RNA(50u M).48 h later,HUVECs were incubated with PTH (10-8mmol/l)for 72 h.Western blot showed that PTH1 R silencing decreased PTH-induced BMP2 and BMP4 expression(P<0.05).3.PTH promotes expression of Jagged1 and Notch1 in Human umbilical vein endothelial cells.HUVECs were incubated with PTH(10-11mmol/l,10-10mmol/l,10-9mmol/l,10-8mmol/l)for 5 days.PTH in a dose-dependent manner increased the protein expression of Jagged1 and Notch1,particularly at 10-8mmol/l(P<0.05).Western blot and RT-PCR were used to detect the protein and m RNA expression of Jagged1 and Notch1,respectively.HUVECs were incubated with Jagged1 for 24 h and switched to PTH treatment for 5 days.Western blot and RT-PCR were used to detect the protein and m RNA expression of BMP2 and BMP4.Western blot showed that DAPT significantly inhibited PTH-induced Notch1 expression.We applied DAPT aγ-secretase inhibitor,to inhibit Notch1 protein expression.Western blot showed that the Notch1 protein was higher than compared with control group(P<0.05).RT-PCR showed that Notch1 m RNA was higher than compared with control group(P<0.05).4.PTH promotes apoptosis of Human umbilical vein endothelial cells.The effect of PTH on HUVECs apoptosis was tested using Annexin V/PI double staining.Flow cytometry instrument testing showed that apoptotic rate of control group was 14.45%(14.45 ± 0.30%),the 10-11mmol/l group was 16.76%(16.76 ± 0.56%),the10-10mmol/l group was 20.52%(20.52±0.98%),the 10-9mmol/l group was 22.90%(22.90±0.60%),the 10-8mmol/l group was 26.75%(26.75±0.45%).Western blot showed that PTH in a dose-dependent increased the protein levels of Cleaved caspase3 and Bax,particularly at 10-8mmol/l(P<0.05).HUVECs were incubated with Jagged1 for 24 h and switched to PTH treatment for 5 days.Western blot was used to detect the pro-apoptotic protein expression including cleaved caspase3 and Bax.The level of Cleaved caspase3 and Bax was higher compared with control group(P<0.05).HUVECs were incubated with Jagged1 for 24 h and switched to PTH treatment for 5days.Western blot showed that the pro-apoptotic protein expression was higher than control group(P<0.05).5.Effects of inhibition of Jagged/Notch1 signaling on Human umbilical vein endothelial cells,calcification and apoptosis.Notch1 specific γ-secretase inhibitor(DAPT)significantly decreased the protein expression of PTH-induced BMP2 、BMP4、Cleaved Caspase3、and Bax.Western blot showed that DAPT reduced the levels of BMP2 and BMP4 of PTH trestment(P<0.05).The effect of apoptotic related protein(Caspase3 and Bax)inhibition was greater compared with control group.RT-PCR showed that the m RNA expression of BMP2、BMP4,、Runx2、and ALP was higher than control group(P<0.05).Conclusion Parathyroid hormone promotes osteogenic differentiation and calcification of HUVECs,as well as cell apoptosis.Additionally,parathyroid hormone promotes the protein of Jagged1 and Notch1 expression.PTH could inhibit HUVECs apoptosis and calcification and the Jagged1/Notch1 signaling pathway plays an important role in these processes.Parathyroid hormone promotes apoptosis and calcification of HUVECs,presumably via inhibition of Jagged1/Notch1 signaling pathway. |
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