The Role Of Notch1—RBP-Jκ Signaling Pathway On The Medial Artery Calcification

Part Ⅰ The relationship between Notchl signaling pathway and medial artery calcificationObjective:To investigate the role of Notchl-RBP-Jκ signaling pathway in the medial calcification.Methods:A vascular smooth muscle cell calcification model was set up. Modles were seperated into three groups according to their calcification intervention time:0day group,7days group and14days group. The degree of local calcium deposition and calcium-regulating markers, that includes von kossa staining, calcium content, alkaline phosphatase (ALP) activity, Msx2content, were tested to reflect the osteogenic transformation. Then the expression of moleculars in Notchl-RBP-Jκ signaling pathway were analysed by RT-PCR, western blot detection, and immunohistochemical detection.Results:VSMCs calcification model was successfully established.von kossa staining showed that positive correlation existed betweencalcium deposition and calcification intervention time:few scattered calcium deposits were displayed in the7days group, while obvious black calcium deposition wereobserved in14days group samples. Results of calcium content test proved that expression of calciumcontent were also increased along with the increase of intervention time, and reached its peak in14days group (P<0.05). The mRNA and protein level of Notchl and RBP-Jic,as well as the expression of Msx2, were found increased with the degree of calcification and reached its peak after14days intervention (P<0.05). Immunohistochemical results showed that activation levels of N1-ICD were also increased with theseverityof calcification.Conclusion:Notchl-RBP-Jκ signaling pathway is activated during the arterial medial calcification process. Its expression was positively correlated with the degree of VSMCs calcification. Part Ⅱ The effect of Notchl signaling pathway on medial artery calcificationObjective:To investigate the effect of Notchl signaling pathway on VSMCs calcification by inhibiting the signaling pathway.Methods:A blocker for Notchl signaling pathway--DAPT--was employed to inhibit the VSMCs calcification. It was also divided into6groups based onintervention time:0day group,7day group,14day group,0day group with DAPT,7day group with DAPT, and14day group with DAPT. The protein and mRNA expression of Notchl, RBP-Jκ and N1-ICD activation were tested to clearifythe effect of the Notchl signaling pathway.After inhibition of Notchl signaling pathway,von kossa staining, calcium content, ALP activity, MSX2content were adopted to clearifytheextent of osteogenic transformation.Results:After treated with DAPT, the transcription and translation level of Notchl, RBP-Jκ were down-regulated significantly when compared with calcification group at same intervention time point (P<0.05), Nl-ICD content was also decreased. All these proved that the Notchl signal pathway was inhibited, von kossa staining also revealed that the calcium deposition, the degree of calcium content and ALP activity of14-day with DAPT group was significantly decreased compared with14-day group(P<0.05).The expression of Msx2, an transcriptional factorof osteogenic transformation, was decreased at the same time (P<0.05).Conclusion:Inhibition of the expression and activation of Notchl signaling pathway can suppress the medial calcification. Part Ⅲ The effect of osteoprotegerinon the Notch1-RBP-Jκ signaling pathway in the medial artery calcificationObjective:To investigate the osteoprotegerin in the development of calcification of rat aortic vascular smooth muscle cells.Methods:The cells were divided into6groups based on intervention time:control group, calcified group, DAPT group, OPG group1with O.lng/ml OPG, OPG group2with lng/ml OPQand OPG group3with10ng/ml OPQ. The protein and mRNA expression of Notchl, RBP-Jk and N1-ICD activation were detected to clearify the effect of the Notchl signaling pathway. After inhibition of Notchl signaling pathway, von Kossa staining, calcium content, ALP activity, Msx2content were determined to clearify the extent of osteoblast transformation.Results:Based on the results of real-time qPCE and western blotting, we indentified that expression of Notchl and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both mRNA and protein levels, these effects were abolished by the treatment of OPG dose-dependently. Furthermore, we found that Msx2, a downstream target of Notchl/RBP-JK, was markedly down-regulated by the treatment of OPGConclusion:OPG could inhibit vascular calcification through the down regulation of Notchl-RBP-Jκ signaling pathway. Part Ⅳ The effect of alendronate on the Notchl-RBP-JK signaling pathway in the medial artery calcificationObjective:To investigate the effect of alendronate (ALN) on the Notch1-RBP-Jκ signaling pathway in the artery medial calcification.Methods:The cells were also divided into7groups based on intervention time:control group, calcified group, DAPT group, ALN group1with1×10-8mmol/1ALN, ALN group1with1×10-6mmol/1ALN, ALN group1with1×10-4mmol/l ALN, and ALN group1with1×10-3mmol/1ALN. The protein and mRNA expression of Notchl, RBP-Jk and N1-ICD activation were tested expression of the Notch1-RBP-Jκ signaling pathway. Von kossa staining, calcium content, ALP activity, Msx2content were determined to clearify the extent of osteoblast transformation.Result:Compared with calcification group, von kossa staining showed that calcium deposit was inhibited in the ALN groups in a does-dependent way. And calcium content, ALP activity and MSX2were inhibited in the ALN groups in a does-dependent way. The expressions of Notch1, RBP-Jκand N1-ICD were inhibited by alendronate does-dependently.Conclusion:alendronate could inhibit vascular calcification through the down regulation of Notchl-RBP-Jκ signal pathway.