Mechanisms Of SIRT1and SIRT2on Regulating Phenotypic Differentiation And Extralcelluar Matrix Expression Of Vascular Smooth Muscle Cells

Being one of the most important cells in vascular wall, vascularsmooth muscle cells (VSMCs) could switch their phenotype fromcontractile state to synthetic state in response to pathiological stimuli,which consists the essential event in vascular remodeling.Extracellular matrix (ECM) synthesised by VSMCs play an importantrole in cell proliferation and migration, and it is critical for maintenancethe elasticity and function of vascular wall. Hemodynamics and growthfactor are key elements modulate VSMC differentiation and ECMdeposition. VSMCs are permanently exposed to the cyclic tensile strainfor pulsatile blood flow in vivo and transforming growth factor-β1(TGF-β1) is one of the key factors that regulate VSMC differentiationand ECM deposition. The sduty on the effect and mechanism of cyclic strain and TGF-β1on VSMC phenotypic differentiation and ECMdeposition will contribute a lot to illustrate the mechanism in vascularremodeling.The cultured rat VSMCs were subjected to10%-1.25Hz-cyclicstrain using FX-4000T Flexercell system for24hours. VSMCs withoutstrain were used as a static control. The expressions of histonedeaceytlase SIRT1and SIRT2and contractile phenotypic markers,α-actin, calponin and SM22α in VSMCs were examined by Westernblotting. The inhibitor of SIRT1and SIRT2, Salermide was used toreduce the expression of SIRT1and SIRT2. Differentially expressedgenes between VSMCs subjected to cyclic strain with or withoutSalermide were detected by gene chip, which would provide helpfulinformation for understanding the regulatory role and mechanism ofSIRT1and SIRT2in the VSMC phenotypic differentiation induced byphysiological cyclic strain. TGF-β1recombinant protein was used totreat rat VSMCs at concentration of10ng/mL for24h. VSMCs withoutTGF-β1treatment were used as a control. The expression of SIRT1,SIRT2; contractile phenotypic markers, calponin, SM22α and ECMproteins, collagen I, elastin, collagen III in VSMCs was examined byWestern blotting. Salermide was used, as well as the methods of RNAinterference were applied to block the expression of SIRT1and SIRT2 in order to investage the regulatory role and mechanism of SIRT1andSIRT2in the VSMC phenotypic differentiation and ECM depositioninduced by TGF-β1.The results were as follows:(1) After loading10%-1.25Hz-cyclicstrain, the expressions of contractile phenotypic markers, α-actin,calponin and SM22α, were enhanced in VSMCs.(2) Cyclic straininduced the SIRT1and SIRT2expression in VSMCs.(3) SIRT1andSIRT2inhibitor Salermide inhibited the expression of SIRT1, SIRT2and contractile phenotypic markers induced by cyclic strain.(4) Genechip detected121differentially expressed genes which might beregulated by SIRT1and SIRT2. Function classification analysissuggested that these genes might participate in several cellular processesincluding differentiation.(5) Treatment VSMCs with TGF-β1atconcentration of10ng/mL for24hours induced contractile phenotypicmarkers, calponin, SM22α and ECM proteins, collagen I, elastin,collagen III expression.(6) TGF-β1could increase the SIRT1andSIRT2expression in VSMCs.(7)50μM Salermide decreased theexpression of SIRT1and SIRT2induced by TGF-β1in VSMCs, andinhibited calponin, SM22α and ECM proteins, collagen I, elastinexpression, while it had no effect on collagen III expression.(8)SIRT1and SIRT2siRNA decreased the expression of SIRT1and SIRT2, and inhibited the VSMC contractile phenotypic markers, calponin andSM22α.(9) Salermide inhibited Smad5, Akt and ERK phosphorylationinduced by TGF-β1, but had no effect on CTGF expression.Our data indicated that physiological cyclic strain and TGF-β1could promote the phenotypic differentiation of VSMCs, and TGF-β1could induce ECM deposition. These processes were mediated at leastpartly by the activation of SIRT1and SIRT2pathway. It suggested thatphysiological cyclic strain and TGF-β1treatment protected thephenotype of VSMCs switching from contractile state to synthesis stateand TGF-β1treatment promoted ECM deposition, which exerts aprotective role in maintaining the vascular structure and function at ahomeostaic state.