The Relationship And Mechanism Of PDGF-BB Induced Autophagy And Calcification In Vascular Smooth Muscle Cells

Objective: Recent studies have showed that autophagy as a novel adaptive mechanism that protects against phosphate induced vascular calcification by reducing matrix vesicle release.Furthermore,atorvastatin protect vascular smooth muscle cells(VSMCs)from TGF-?1 stimulated calcification through suppression of the ?-catenin pathway.Previous studies suggested that platelet-derived growth factor(PDGF)-BB-mediated autophagy is essential for the conversion of VSMCs from a contractile to a synthetic phenotype and that this increase in autophagy in synthetic cells could also prevent cell death due to oxidative stress.However,the relationship between autophagy and calcification in VSMCs has not been identified.In this study,we elucidated the relationship autophagy and calcification in PDGF-BB-stimulated VSMCs and explored its mechanism.Methods:1 VSMC cultureVSMCs were isolated from the thoracic aorta of 80-100 g male Sprague Dawley rats.VSMCs were grown in low glucose Dulbecco's-modified Eagle's medium(DMEM)with 10% fetal bovine serum(FBS),100 U/mL penicillin and 100?g/mL streptomycin.The VSMCs were maintained at 37? in a humidified atmosphere containing 5% CO2,and only passages 3 to 5 cells at 70-80% confluence were used in the experiments.2 Western blot analysisEqual 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 overnight,washed with TBST,and then with the HRP-conjugated secondary antibody for 2 h.The blots were evaluated with the ECL(enhanced chemiluminescence)detection system.3 Immunofluorescent assayThe VSMCs were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 15 min.Thereafter,cells were blocked with 10% goat serum for 2 h at room temperature,incubated with anti-Beclin1 and anti-LC3 antibodies and further stained with Alexa Fluor 555 secondary antibodies.Confocal microscopy was performed with the Confocal Laser Scanning Microscope Systems.4 Co-immunoprecipitation assayCells stimulated with PDGF-BB were lysed in a buffer composed of 50 mM Tris-HCl,pH 7.6,150 mM NaCl,1% NP-40,10 mM sodium phosphate,10 mM NaF,2 mM PMSF.After centrifugation,clarified cell lysate was incubated with protein A and anti-Beclin1 or anti-PI3KC3 antibodies.After 12 h incubation,the immune complexes were centrifuged,and washed with ice-cold lysis buffer.The immunoprecipitated protein was further analyzed by Western blot as described above.Results1 The effect of PDGF-BB on the expression of calcification markers in VSMCsCultured VSMCs were stimulated with PDGF-BB(10 ng/mL)for different time(0-72 h),the expression of vascular calcification related proteins BMP2 and ALP was observed by western blotting analysis.The expression of BMP2 and ALP showed a trend from decline to rise,ALP slumped at 12 h,and BMP2 slumped at 6 h.2 The effect of PDGF-BB on the expression of autophagy markers in VSMCsCultured VSMCs were stimulated with PDGF-BB for different time(0-72 h),the expression of autophagy related proteins Beclin1 showed a trend from rise to decline,and peaked at 12 h.The conversion of LC3-?to ? was considered a reliable marker of increased autophagic activity and autophagosome formation.The conversion of LC3-?to ? increased in a time-dependent manner,and peaked at 48 h.Furthermore,the expression of Beclin1 and LC3 was markedly enhanced in VSMCs stimulated with PDGF-BB for 12 h,using immunofluorescence staining.3 The effect of autophagic inhibitor 3-MA on PDGF-BB stimulated VSMC calcificationCultured VSMCs were incubated with 3-MA(10 m M)for 2 h and then treated with PDGF-BB for additional 12 h.The result showed that the expression of Beclin1 and LC3 was reduced in VSMCs incubated with 3-MA and PDGF-BB,compared with PDGF-BB-stimulated group.The similar results were observed using immunofluorescence staining.Furthermore,the expression of BMP2 and ALP was increased in VSMCs incubated with 3-MA and PDGF-BB,compared with PDGF-BB-stimulated VSMCs.4 The effect on Beclin1 phosphorylation and interaction between Beclin1 and PI3KC3 in PDGF-BB-stimulated VSMCsCultured VSMCs were stimulated with PDGF-BB for different time(0-24 h),The interaction between Beclin1 and PI3KC3 was detected using co-immunoprecipitation.The results showed the interaction between Beclin1 and PI3KC3 was enhanced at 6 h after PDGF-BB stimulated,peaked at 12 h,and kept in high level at 24 h.Moreover,Beclin1 phosphorylation was detected by immunoprecipitation.The results showed the phosphorylation level of Beclin1 was enhanced by PDGF-BB stimulation,and peaked at 6 h.Conclusions:1 Calcification was inhibited at the early stage,and promoted at the last stage in PDGF-BB stimulated VSMCs.2 PDGF-BB-induced VSMC autophagy was abolished by 3-MA,followed VSMC calcification accelerated.3 PDGF-BB stimulated VSMC autophagy by enhancing Beclin1 phosphorylation and interaction between Beclin1 and PI3KC3.