The Role And Mechanism Of Parathyroid Hormone In Promoting Vascular Endothelial Cell Calcification

BACKGROUNDCoronary artery calcification has become one of the key factors and restricting factors that affect the treatment effect of coronary heart disease.Clinically,vascular calcification in patients with chronic renal failure mostly involves smooth muscle and is mainly manifested as media calcification.For patients with coronary heart disease without renal failure,vascular calcification is more common in intimal calcification due to the presence of atherosclerotic plaques.In recent years,the effect of serum parathyroid hormone(PTH)on vascular calcification has become a research hotspot.However,most of these studies were focused on patients with chronic kidney disease,and there are few studies on the correlation between PTH and vascular calcification in non-renal failure patients.Previous basic studies have shown that PTH can promote vascular calcification.NOTCH protein is abundant in vascular endothelial cells,and its intracellular segment can interact with IKK to activate NF-κB,thereby promoting inflammation.On the other hand,NF-κB activation can also cause the expression of Caspase3(CASP3),which leads to the occurrence of apoptosis.Based on the above findings,we have reason to believe that the NOTCH 1/IKK/CASP3 pathway plays an important regulatory role in the process of vascular endothelial cell calcification.However,whether PTH can promote vascular endothelial apoptosis and calcification through NOTCH1/IKK/CASP3 signaling pathway has not been reported so far,which deserves further study.OBJECTIVE1.To evaluate the correlation between serum PTH level and coronary artery calcification in patients without renal failure.2.To explore the specific mechanism of PTH regulating HUVECs apoptosis and calcification through NOTCH1/IKK/CASP3 signaling pathway.METHODS(一)PatientsA total of 157 cases of non-renal failure patients receiving CTA were retrospectively analyzed.All patients were divided into calcification group(CACS>0)and control group(CACS=0)according to the coronary artery caicification score(CACS).The t test of two independent samples was used for the comparison between the two groups.ROC curve was drawn to determine the predictive value of PTH for CAC.Logistic regression analysis model of reaction variables for binary classification was used in order to determine independent factors correlated with CAC.(二)Quantitative real time polymerase chain reaction(qRT-PCR)analysisRNA was extracted from HUVECs by using specific primers according to Trizol routine method.The qRT-PCR was performed in accordance with the standard steps of Takara TB Green Premix,and LightCycler(?)480 System was used for amplification.Select GAPDH as reference and calculate Δ Ct values.Using the 2-ΔΔCt method to calculate mRNA expression levels relatively.(三)Western blot analysisHUVECs sample protein was extracted with RIPA lysate,and 4×NuPAGETM LDS Sample Buffer was added after quantification.Using a prefabricated adhesive with a SurePAGETM 4-12%gradient,trarsmembran were performed using conventional electrophoresis at 80V-100V and wet rotation method at 100V for 2 h.After sealing with 5%skim milk,the primary antibody was incubated at 4℃ overnight,and after washing with TBST for 3 times,the secondary antibody was incubated in darkness for 1 h at room temperature.ImageQuant LAS 4000 series rapid chemical optical imaging system was used to detect and analyze the protein bands.(四)Flow cytometry(FCM)5-10×104 cells were centrifuged and added with 200 μl Annexin V-FITC binding solution to resuscitate the cells.The cells were incubated at room temperature in dark for 10 min.After centrifugation at 200g for 5 min,190 μl Annexin V-FITC binding solution was added to resuscitate the cells,and 10 μl propidium iodide staining solution was added and mixed for flow cytometry detection.(五)Cell transfection125μl Opti-MEM culture medium was added to Lipofectamine(?)3000 reagent 3.75μl for dilution.At the same time,125μl Opti-MEM culture medium was added to 2500ng plasmid DNA and 5μl P3000TM reagent or 50pmol siRNA(excluding P3000TM)for dilution.The two parts of liquid were mixed and incubated at room temperature for 10-15min to prepare DNA-liposome complex,and the final complex was added to the cell culture solution and incubated at 37℃ for 2 days.(六)Statistical analysisAll data were statistically analyzed using SPSS 18.0 software.The measurement data were expressed as mean ± standard deviation,and the experimental data of each group were repeated at least 3 times.P<0.05 was used as the criterion of statistical significance.RESULTS(一)Serum PTH levels were correlated with CAC in patients without renal failure1.Serum PTH level in the calcification group was significantly higher than that in the control group,P<0.05,indicating that the serum PTH level was significantly correlated with the presence of CAC.2.Partial correlation analysis showed a nonlinear correlation between PTH level and CACS(P>0.05).3.The ROC curve indicated that serum PTH level>31.05pg/ml was the optimal cut-off point for predicting CAC.4.The result of Logistic regression analysis showed that serum PTH levels remained an independent predictor of coronary calcification(OR=1.050,95%CI:1.027-1.074,P<0.001).(二)PTH significantly promoted HUVECs calcification and apoptosis in vitroHuman recombinant PTH was added to HUVECs culture medium at concentrations of 10-8mmol/L and 10-7mmol/L to induce cell calcification,and cells were collected for detection 0-5 days after treatment.The results of qRT-PCR showed that the mRNA expression levels of BMP2 and BMP4 at the concentration of 10-7mmol/L were significantly higher than those at the concentration of 10-8mmol/L(P<0.05).From the perspective of time,the mRNA expression of BMP2 and BMP4 gradually reached its peak on the 3rd day after PTH-induced cells,and then declined again on the 4th and 5th day.Further verification by qRT-PCR and Western blot showed that the mRNA and protein expression levels of BMP2,BMP4 and RUNX2 in HUVECs were significantly higher than those in the control group after 3 days of 10-7mmol/L PTH treatment(P<0.05),confirming that in vitro PTH intervention could significantly promote calcification of HUVECs.Flow cytometry results showed that HUVECs underwent significant apoptosis 3 days after PTH treatment,suggesting that cell calcification was associated with apoptosis.(三)PTH up-regulated the expression of NOTCH1/IKK/CASP3 in HUVECsPTH was used to induce HUVECs with 10-7mmol/1 concentration for 3 days.qRT-PCR results showed that mRNA expressions of NOTCH1,IKK-α,IKK-β and CASP3 in the downstream pathways of calcification were significantly higher than those in the control group(P<0.05).Western blot analysis showed that protein expressions of NOTCH 1,IKK-α,IKK-β and CASP3 were also significantly increased compared with the control group.(四)Overexpression of NOTCH1 promoted PTH-induced HUVECs calcificationWe transfected HUVECs with NOTCH1 overexpressed plasmid.The results of quantitative PCR showed that,in HUVECs induced by PTH under the same conditions,the NOTCH1 overexpression plasmid significantly increased the mRNA level of NOTCH1 in the cells(P<0.05).Correspondingly,mRNA expression levels of calcification indicators BMP4,BMP2,RUNX2,and the signaling molecules IKK-α,IKK-β and CASP3 in the downstream pathway of calcification were significantly increased compared with the control group(P<0.05).Meanwhile,Western blot results also showed that the protein expression levels of BMP4,BMP2,RUNX2,IKK-α,IKK-β and CASP3 were also significantly increased after the overexpression of NOTCH1 compared with the control group(P<0.05).(五)Down-regulated NOTCH1 expression inhibited PTH-induced HUVECs calcificationWe further transfected HUVECs with NOTCH1-knocked down small interfering RNA(siRNA).Quantitative PCR showed a significant decrease in mRNA expression of NOTCH1 compared with the control group(P<0.05).Further quantitative PCR and Western blot results showed that,in PTH-induced calcification HUVECs under the same conditions,the mRNA and protein expressions of BMP-4,BMP-2,RUNX2,IKK-α,IKK-βand CASP3 were also significantly decreased compared with the control group(P<0.05).It was further demonstrated that PTH promoted the occurrence of HUVECs calcification through NOTCH1/IKK/CASP3 signaling pathway.CONCLUSIONS1.Serum PTH levels are correlated with the occurrence of CAC in patients without renal failure,which can be used as a reliable predictor of CAC.2.Recombinant PTH could induce calcification and apoptosis of HUVECs in vitro,and showed time and dose correlations.3.PTH promotes calcification of HUVECs through NOTCH1/IKK/CAPS3 signaling pathway.