Role Of EZH2 In Vascular Calcification And The Molecular Mechanisms

BackgroundVascular calcification,a phenomenon with calcium-phosphate complexes deposit in the vessel wall aberrantly,leading to arterial stiffness,is the common pathological manifestation of many cardiovascular diseases.Phenotypic switch of vascular smooth muscle cells(VSMCs)is the crucial part of vascular calcification,which involves calcium-phosphate imbalance and extracellular matrix remodeling.Enhancer of zeste homolog 2(EZH2),a histone methyltransferase,modulates gene transcription through methylation of histone,non-histone,or protein-protein interaction.It has been proved that EZH2 plays a key role in cardiovascular development,atherosclerosis and osteogenic differentiation of mesenchymal stem cells.However,the role of EZH2 in vascular calcification has not been reported.ObjectiveTo investigate the role of EZH2 in vascular calcification and the molecular mechanisms.MethodsIn vitro,calcification of VSMCs and arterial rings was induced by high phosphate and high calcium medium.The expression of EZH2 was altered by adenovirus,small interfering RNA and inhibitor.In vivo,high phosphate and calcium diet was used to induce vascular calcification in CKD rats and vitamin D3 was used to induce mouse vascular calcification.Alizarin red staining,formic acid quantification and calcium assay were used to assess the extent of calcification.The expression levels of osteogenic differentiation biomarkers were detected by western blot.ResultsWestern blot and immunofluorescence staining showed that EZH2 expression was decreased in calcified human VSMCs and arterial rings,rats VSMCs,and aortas from CKD rats and VitD3-overloaded mice.Overexpression of EZH2 promoted rat VSMC calcification and osteogenic differentiation.Conversely,inhibition of EZH2 reduced calcification and osteogenic differentiation of rat VSMCs and arterial rings.Similar results were obtained in cells transfected with EZH2 siRNA.Mechanistically,RNAsequencing showed that MMP9 expression was changed obviously by EZH2 overexpression in rat VSMCs,which was verified by western blot.Under calcification condition,inhibition of MMP9 eliminated the aggravation effect of EZH2 overexpression on rat VSMCs calcification.Conclusions1.EZH2 expression levels were decreased during calcification.2.EZH2 promoted calcification of VSMCs and arterial rings.3.EZH2 regulated VSMC calcification through MMP9.