The Effect And Mechanism Of Autophagy On Vascular Smooth Muscle Cell Osteogenesis

Objective: Vascular calcification is a common pathological feature of vascular remodeling diseases such as atherosclerosis,hypertension and restenosis.Vascular smooth muscle cells(VSMCs)have been shown to differentiate to osteoblast like phenotype and upregulate the expression of bone-related factors,including bone morphogenetic protein(BMP2)and alkaline phosphatase(ALP).Recent studies implicated that autophagy involved in the inhibition of vascular calcification.In the calcified VSMCs,the autophagy inducer valproic acid reduces calcification,and the autophagy inhibitor 3-MA promotes calcification.However,whether autophagy is involved in VSMC osteogenesis remains unclear.In this study,in response to platelet-derived growth factor(PDGF)-BB or vascular intimal injury stimulation,autophagy may acts a protection mechanism in inhibiting VSMC osteogenesis.Methods:1 Carotid artery ligation modelsTwenty-five male LDLR-/-mice(8 to 12 weeks old)were randomly divided into 3,7,14,28 d and sham group and underwent the left carotid artery ligation to induce neointimal formation.After different days of surgery,the carotid arteries were harvested.Some arteries embedded in OCT compound for frozen sections,and the others preserved at-80 ℃ for Western Blot.2 VSMC cultureVSMCs were prepared from medial explants of thoracic aorta from male Sprague Dawley rats,and cultured in DMEM with 10% FBS.VSMCs were maintained at 37 ℃ in a humidified atmosphere containing 5% CO2 and only passages 3 to 5 cells were used in the experiments.VSMCs were made quiescent by serum-starvation for 24 h and then treated with PDGF-BB(10 ng/ml)for different time(6,12,24,48,72 h).3 Western Blot analysisVascular samples or VSMCs were prepared with lysis buffer.Equal amounts of protein were separated by 10% SDS-PAGE,and electrotransfered to a PVDF membrane.Membranes were blocked with 5% BSA(bovine serum albumin)for 2 h at room temperature,and incubated with specific antibodies against ALP,BMP2 and AMP-activated protein kinase(AMPK)overnight,and then with the HRP-conjugated secondary antibody(1:20000)for 2 h.The blots were evaluated with the ECL(enhanced chemiluminescence)detection system.4 Immunofluorescent stainingThe samples of ligated artery were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 20 min.Thereafter,incubated with anti-Beclin1 and anti-LC3 antibody and further stained with appropriate secondary antibodies.Confocal microscopy was performed with the Confocal Laser Scanning Microscope Systems(Leica).Results1 The effects of intimal injury on VSMC osteogenesis and autophagyTo determine the relationship between VSMC osteogenesis and autophagy during neointimal formation,a mouse carotid artery ligation model was prepared.The results showed as ligation time increased,the lumen stenosis,intimal gradually thickening,the ratio of intimal/Media increased,reached a peak at 28 d(Fig.1A).Then we examined the effects of ligation on VSMC osteogenesis and autophagy.The results showed the expression of ALP and BMP2 increased at 7 d and peaked at 14 d(Fig.1B),while the expression of Beclin1 and LC3 peaked at 7 d and then declined,recovering to the normal level at 28 d(Fig.1C).2 Inhibition autophagy promotes VSMC osteogenesis and intimal hyperplasiaUsing an autophagy inhibitor 3-MA,we observed that PDGF-BB-induced expression of Beclin1 and LC3 was blocked by 3-MA(Fig.2A).However,the expression of ALP and BMP2 increased after incubation with 3-MA(Fig.2B).Next we determined the effect of 3-MA on intimal hyperplasia.After incubation with 3-MA,the I/M ratio was significantly higher than that of the ligation group without 3-MA(Fig.2C).3 Autophagy inhibits VSMC osteogenesis via reducing AMPK expressionUnder PDGF-BB stimulation,the expression of AMPK increased at 12 h,peaked at 24 h,and remained high level at 72 h(Fig.3A).We next incubated VSMC with 3-MA and rapamycin respectively,and then treated with PDGF-BB.The PDGF-BB-induced AMPK high expression was blocked by 3-MA and promoted by rapamycin(Fig.3B and 3C).Furthermore,3-MA promoted the ALP activity induced by PDGF-BB(Fig.3D),implying that autophagy may inhibit VSMC osteogenesis by inducing the expression of AMPK.Conclusions:1 When vascular intimal injured,cell autophagy is induced first,and then vascular cells differentiate to osteoblast like phenotype,and eventually intimal hyperplasia is promoted;2 Inhibition autophagy promotes vascular cell osteogenesis and intimal hyperplasia;3 Autophagy may inhibit VSMC osteogenesis by inducing the expression of AMPK.