| Objective: Vascular calcification often occurs with advancing age,atherosclerosis and metabolic disorders such as diabetes mellitus andend-stage renal disease. TGF-β1is capable of promoting vascular smoothmuscle cell (VSMC) differentiation and regulating vascular calcification. The3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins)exert proven beneficial effects on cardiovascular diseases. β-catenintranslocates to the nucleus involved in vascular calcification promote specificgene expression. Autophagy is a highly conserved cellular process regulatingturnover of cytoplasmic proteins via a lysosome-dependent pathway. However,there are no studies about the effect of statins on autophagy andTGF-β1/β-catenin pathway in osteogenic differentiation in vascular smoothmuscle cells. In the present study, we elucidated the roles of autophagy andβ-catenin in TGF-β1-induced osteogenic differentiation in vascular smoothmuscle cells.Methods and Results: TGF-β1increased osteogenic differentiation inVSMCs. TGF-β1induced rapid β-catenin nuclear translocation of VSMCs invitro through TGF-β1receptor I (TGF-βRI). Degrading β-cateninpharmacologically significantly decreased osteogenic differentiation withoutaltering Smad2/3activity. Meanwhile, increasing β-catenin availability withWnt3A markedly increased β-catenin expression. The enhanced effect ofatorvastatin on TGF-β1induced autophagy was reversed by Wnt3A.Evenmore, the inhibition effect of atorvastatin on TGF-β1-induced osteogenicdifferentiation was reversed by Wnt3A.These data support a critical role ofβ-catenin in the effect of atorvastatin on autophagy and calcification. Similarly,our results showed that atorvastatin induced autophagy and provided protection from TGF–β1induced cell osteogenic differentiation via β-catenindependent manner.1Atorvastatin Inhibit TGF-β1-Induced VSMC Calcification in VSMCsTo induce VSMC calcification, cells were incubated with TGF-β1for6,12,24,48and72h. We found that TGF-β1(2ng/mL) induced Ca depositionin a time-dependent manner. Moreover, we confirmed that TGF-β1treatmentincreased the expression of osteogenic factors such as ALP, typeⅠcollagen,BMP-2and osteocalcin in a time-dependent manner.To investigate the effect of atorvastatin on TGF-β1-induced calcification,VSMC were incubated with atorvastatin in the presence of TGF-β1(2ng/mL).Ca deposition was significantly suppressed by atorvastatin treated for24hours.An inhibitory effect of the atorvastatin on VSMCs calcification was also foundby the detection of ALP, typeⅠcollagen, BMP-2and osteocalcin expression.2Inhibitory Effect of Atorvastatin on Calcification is caused by inducingautophagyIt is demonstrated that autophagy was activated to regulate cellCalcification. We examined the effect of atorvastatin on autophagy in VSMCscalcification. Autophagic vacuolization was rarely detected in normal VSMCsby TEM. During calcification, little autophagic vacuolization were formed inthe cytoplasm after TGF-β1(2ng/mL) treatment for24h. Atorvastatin, atconcentrations exerting inhibition of calcification, induced a large number ofautophagic vacuolization distributed throughout the whole cytoplasm.Moreover, the autophagy induced by atorvastatin in VSMCs was alsoobserved using fluorescence microscopy. Furthermore, atorvastatin treatmentincreased expression of Beclin-1and Atg5in VSMCs in a time-dependentmanner. LC3II conversion was also observed after atorvastatin treatment a24h, and the ratio of LC3Ⅱ/LC3Ⅰwas enhanced after atorvastatin treatment.To investigate the relationship between autophagy and calcification, weused3-MA, a specific autophagic inhibitor, and we found that3-MA (5mmol/L) significantly inhibited TGF-β1-induced autophagyas well ascalcification.In addition,atorvastatin-induced LC3II conversion was also inhibi ted by3-MA, indicating that the protective effect of atorvastatin on VSMCscalcification was attributable to activating autophagy.3Downregulation of β-catenin Is Associated With atorvastatin-InducedautophagyIt is demonstrated that β-catenin was involved in vascular calcification bypromoting the related gene expression, so we investigated the role of β-cateninin the effects of atorvastatin in TGF-β1-induced VSMCs osteogenicdifferentiation. As shown in Fig.3A, TGF-βRI and β-catenin expression weremarkedly increased in a time-dependent manner after2ng/mL TGF-β1treatment. Moreover, we found that TGF-β1stimulated β-catenin translocationinto the nucles in VSMCs. To further investigate the role of β-catenin signal inthe process of autophagy and calcification, the TGF-βRI inhibitor SD208orthe β-catenin inhibitor JW74were supplemented in TGF-β1-treated VSMC.The addition of SD208(5μgl/mL) or JW74(10μgl/mL) clearly abrogatedboth TGF-β1-induced autophagy and calcification. These results indicate thatTGF-β1-induced autophagy and calcification are associated with upregulationof TGF-βRI and β-catenin.To determine whether the autophagic effect of atorvastatin is dependenton downregulation of the β-catenin, we assessed the effect of atorvastatin onβ-catenin expression. Atorvastatin treatment significantly decreased β-cateninexpression. Furthermore, we overexpressed β-catenin and determined theeffects of atorvastatin on TGF-β1-induced autophagy and calcification. Wnt3Awas added to cells at a concentration of150ng/mL for24hours beforeperforming functional studies to increase the availability of β-catenin inducenuclear accumulation of β-catenin. Wnt3A markedly increased β-cateninexpression. The enhanced effect of atorvastatin on TGF-β1-induced autophagywas reversed by Wnt3A. Similarly, the beneficial effect of atorvastatin onTGF-β1-induced calcification was also abolished by Wnt3A. These datasupport a critical role of β-catenin in the effect of atorvastatin on autophagyand calcification. Conclusion:1Atorvastatin inhibited TGF-β1induced osteogenic differentiation inVSMCs.2Atorvastatin inhibited osteogenic differentiation via inducingautophagy.3Atorvastatin induced autophagy via β-catenin dependent manner. |
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