Regulation Of Vascular Calcification By Proprotein Convertase Subtilisin/Kexin Type 9 And Its Mechanism

Objectives:Vascular calcification(VC),characterized by deposition in the vessel wall as calcium phosphate complexes,is an independent risk factor for cardiovascular disease morbidity and mortality.The proprotein convertase bacillus subtilis proteinase 9(PCSK9)was detected to be upregulated in human atherosclerotic plaques(including endothelial cells),and alirocumab(PCSK9 inhibitor)was found to inhibit atherosclerosis formation and improve plaque morphology in the clinic.Endothelial cells first respond to flow shear stress and various signaling molecules in the blood,and then they transmit messages to induce vascular cells and inflammatory cells to participate in VC.Endothelial mesenchymal transdifferentiation(End MT)converts the endothelium to a pluripotent state allowing cells to differentiate to poorly(e.g.,cardiac fibrosis and VC).In this study,we intend to observe the biological role of PCSK9 in the development and progression of vascular calcification through in vivo and ex vivo experiments.Methods:We first constructed a C57BL/6 mouse model of vascular calcification and gave subcutaneous injections of alirocumab weekly starting from week 2.Plasma,aorta and liver tissues were preserved after uniform execution at week 12 to detect the effect of downregulation of PCSK9 after intervention,and experimental methods such as aortic pathology staining and protein blotting(Western blot)were performed.To further investigate and verify whether PCSK9 would reduce calcification by inhibiting End MT and thus,we constructed a human aortic endothelial cell(HAEC)calcification model and observed PCSK9 expression and calcification and End MT phenomena by real-time fluorescence quantitative polynucleotide chain reaction and Western blot;knocked down PCSK9 by small interfering RNA(si RNA).PCSK9 was knocked down by small interfering RNA(si RNA)to observe the effect of PCSK9 downregulation on End MT and calcification in endothelial cells.Results:It was found that the level of PCSK9 was down-regulated by Western blot in plasma and liver.HE and Von Kossa calcium salt staining showed that compared with the control group,the elastic fibers of intima and media in the aorta of the model group were obviously broken,and a large number of black particles were deposited in extracellular matrix and smooth muscle cells,and the normal structure of aortic blood vessels was destroyed.At the same time,Western blot method found that the calcification-related marker protein 4(BMP4)was up-regulated.At the same time,we detected by Western blot that the mesenchymal related expression protein(α-smooth muscle actin)was upregulated,and the endothelial marker protein(vascular endothelial cadherin)was downregulated,indicating that calcification occurred and End MT occurred at the same time.However,after the intervention of alirocumab,the calcification and End MT phenomenon in the aorta of mice were alleviated.In vitro experiments,we found that PCSK9 was upregulated and End MT occurred while inducing calcification of HAEC.Then we knock down PCSK9 in HAEC by si RNA,which can significantly reduce endothelial cell calcification and End MT.Conclusion:In this study,we found that PCSK9 expression levels increased and End MT occurred after induction of calcification in vitro and in vivo,while downregulation of PCSK9 inhibited the progression of calcification and alleviated End MT,suggesting that we downregulated PCSK9 may alleviate VC progression by inhibiting the pathway of End MT,providing a new idea for understanding the mechanism of VC and discovering new therapeutic approaches.