The Role And Mechanism Of Autophagy On Endothelial Progenitor Cells Function

Background and objective:Atherosclerosis,the leading cause of cardiovascular diseases,is initiated by the hypercholesterolemia-induced disruption of vascular endothelium integrity.Endothelial progenitor cells(EPCs)play a major role in the maintenance of endothelial integrity and contribute to re-endothelialization of injured vessels as well as revascularization of ischemic tissues.Originated from bone marrow and spleen,EPCs were first described and isolated by Asahara and his colleagues nearly 2 decades ago.Recently,EPC-based therapy has demonstrated that increasing the number or improving function of circulating EPCs may be promising in the treatment of atherosclerotic diseases.However,the poor viability of EPCs after transplantation limits the large-scale use of cell-based therapy.Therefore,exploring new strategies in improving the survival post-transplantation is crucial to enhancing the success of EPC therapy.Autophagy is an evolutionarily conserved process that turns over long-lived proteins or impaired organelles through a lysosomal-mediated pathway and acts as a survival mechanism under stress conditions to maintain cellular homeostasis.Recently,the variety of intracellular calcium homeostasis was related to autophagy activation,but the downstream calcium signal and the effect of autophagy were still in debate.Store-operated calcium channel(SOCC)was the major calcium influx pathway,as EPC was non-excited cell.Our previous study had revealed that EPCs expressed STIM1,TRPC1 and ORAI1,which were the component protein of SOCC.We further confirmed that SOCC contributed to EPCs proliferation and migration.Therefore,we speculated that calcium influx through SOCC may associate with EPCs autophagy,moreover,autophagy activation may contribute to EPCs survival under stress condition.This will be beneficial to EPCs transplantation and re-endothelialization of injured vessels.In view of those mentioned above,we will apply the atherosclerosis model mice and mimic the micro-environment of atherosclerosis,ox-LDL,to explore the mechanism and effect of store-operated calcium entry(SOCE)mediated-autopahgy on EPCs bio-function,and reveal the impact of autophagy in pitavastatin-improved EPCs function.Our study may be beneficial to EPCs-based therapy and provide novel target of the treatment of anti-atherosclerosis.Methods:1.The change of EPCs proliferation,migration and autophagy level in atherosclerosis model mice1.1 Replication of atherosclerosis mice model,culturing and identification of EPCsApoE-/-mice were fed with high fat diet to 12 weeks and 16 weeks respectively.We evaluated the atherosclerosis burden through oil O staining of the aorta.We took the C57BL/6 mice as the control to compared with Apo E-/-mice.Density-gradient centrifugation was used to harvest the EPCs from mice bone marrow.Then the EPCs were cultured with endothelial basal medium in the plates which were covered by fibronectin.1.2 EPCs identificationTo confirm the phenotype of EPCs,we incubated the cells with Di I-ac LDL and FITC-UEA-I.Both emergence red and green cells were EPCs in laser scanning confocal microscope.In addition,we approached the anti-body of CD133,CD34 and VEGFR-2 to mark the cells and evaluated the expression of CD133,CD34 and VEGFR-2 in fluorescence-activated cell sorting.1.3 The change of EPCs proliferation and migration in atherosclerosis mice modelWe applied cell number counting and CCK-8 dyeing to analyze EPCs proliferation.Transwell was used to measure EPCs migration.1.4 The change of EPCs autophagy in atherosclerosis mice modelRT-PCR and Western Blot were used to detect the transcription and expression of autophagy protein of EPCs in atherosclerosis mice model.EPCs were infected by adenoviral vectors containing GFP-mRFP-LC3 to mark LC3 protein.After 24 h,EPCs were observed under laser scanning confocal microscope to been counted the number of autophagosomes and autolysosomes.2.1 The effect of ox-LDL on EPCs proliferation and migrationCell counting and CCK-8 dyeing were applied to evaluate EPCs proliferation after different concentrations of ox-LDL treatment with different hours.In addition,Transwell was approached to detect the migration of EPCs as well.2.2 The impact of ox-LDL on EPCs SOCE2.2.1 Intracellular calcium ion was firstly marked by Fluo3-AM,a special calcium fluorescent probe.The fluorescence intensity represented the concentration of intracellular calcium.EPCs were cultured in calcium free medium.Then different concentrations of ox-LDL were injected to the medium,respectively.A few minutes later,we recovered the extracellular calcium concentration to normal.The change of fluorescence intensity was recorded by laser scanning confocal microscope.2.2.2 Intracellular calcium ion was marked by Fluo3-AM.The fluorescence intensity represented the concentration of intracellular calcium.2-APB,a special SOCC inhibitor,was used to inhibit SOCE after exposure to 60 ?g/ml ox-LDL under laser scanning confocal microscope.The change of fluorescence intensity was recorded by laser scanning confocal microscope.2.2.3 Intracellular calcium ion was marked by Fluo3-AM.The fluorescence intensity represented the concentration of intracellular calcium.Lentiviral vector carrying STIM1 sh RNA was constructed to knock down STIM1.STIM1-knockdown EPCs were exposure to 60 ?g/ml under laser scanning confocal microscope.The change of fluorescence intensity was recorded by laser scanning confocal microscope.2.3 The effect of ox-LDL on EPCs autophagyRT-PCR and Western Blot were used to detect the transcription and expression of autophagy protein of EPCs after different concentrations of ox-LDL exposure respectively.EPCs were infected by adenoviral vectors containing GFP-mRFP-LC3 to mark LC3 protein.After 24 h infection,EPCs were exposed to 60 ?g/ml ox-LDL.Then the cells were observed under laser scanning confocal microscope to been counted the number of autophagosomes and autolysosomes.2.The effect of ox-LDL on EPCs proliferation,migration,SOCE and autophagy3.The mechanism and relationship between EPCs autophagy and EPCs bio-function3.1 SOCE-CAMKK2-MTOR signaling pathway contributes to autophagy induction under ox-LDL exposure3.1.1 20 ?M BAPTA-AM(intracellular calcium chelator)and 10 m M EGTA(extracellular calcium chelator)pretreated EPCs 20 min respectively before exposure to 60 ?g/ml ox-LDL 12 h.Then we used Western Blot to measure the LC3 II and LC3 I protein expression.3.1.2 20 ?M BAPTA-AM(intracellular calcium chelator)and 10 m M EGTA(extracellular calcium chelator)pretreated EPCs 20 min respectively before exposure to 60 ?g/ml ox-LDL 12 h.Then EPCs were infected by adenoviral vectors containing GFP-m RFP-LC3 to mark LC3 protein.We observed the autophagosomes and autolysosomes under laser scanning confocal microscope to evaluate the autophagy level.3.1.3 After EPCs exposure to 60 ?g/ml ox-LDL 12 h,Western Blot was used to detect the CAMKK2,AMPK,MTOR,P70S6 K protein expression as well as their phosphorylation levels.3.1.4 EPCs were treated with STO-609,a special inhibitor of CAMKK,before exposure to 60 ?g/ml ox-LDL 12 h.Western Blot was used to measure the protein expression of LC3 II,LC3I,CAMKK2,AMPK,MTOR,P70S6 K and their phosphorylation levels.3.1.5 EPCs were transfected with lentiviral vector carrying sh STIM1 to knock down STIM1 expression.Then EPCs were exposure to 60 ?g/ml ox-LDL 12 h.We detected the protein expression of LC3 II,LC3I,CAMKK2 and the phosphorylation of CAMKK2.3.2 The relationship of autophagy and EPCs bio-function3.2.1 EPCs were transfected with lentiviral vector carrying sh ATG7 to knock down ATG7 expression.Then EPCs were exposure to 60 ?g/ml ox-LDL 12 h.Real-time cell analyzer(RTCA)was used to measure the cell proliferation continuously.In addition,CCK-8 analysis was used to detect EPCs proliferation as well.3.2.2 Besides the lentiviral vector silencing,we applied pharmacologic inhibitor of autophagy,3-MA,to block autophagy before EPCs exposure to 60 ?g/ml ox-LDL 12 h.RTCA and CCK-8 were both used to measure the cell proliferation.4.Pitavastatin improved EPCs bio-function through autophagy activation4.1 The effect of pitavastatin on EPCs proliferation and migration4.1.1 CCK-8 and Transwell were used to measure the proliferation and migration respectively after EPCs exposure to different concentrations of pitavastatin.4.2 The impact of pitavastatin on EPCs autophagy4.2.1 We approached pitavastatin to treat EPCs for 6 h,then detected the protein expression of LC3 II,LC3I and p62.4.2.2 EPCs were exposed to pitavastatin for 6 h,then marked by GFP-RFP-LC3 adenovirus for 4 h.We observed and counted the autophagosomes and autolysosomes under laser scanning confocal microscope.4.3 The relationship of pitavastatin-induced autophagy and EPCs bio-function.4.3.1 3-MA was applied to inhibit EPCs autophagy before exposure to pitavastatin.CCK-8 and Transwell were used to measure cell proliferation and migration respectively.4.3.2 Lentiviral vector carrying sh ATG7 was applied to silence EPCs autophagy before exposure to pitavastatin.CCK-8 and Transwell were used to measure cell proliferation and migration respectively.Results:1.The change of EPCs bio-function and autophagy level from atherosclerosis model mice1.1 The construction of atherosclerosis model mice and EPCs culture and identificationApoE-/-mice were fed with high fat diet for 12 and 16 weeks.We found that the lipid plaque in aorta was more significant in high fat diet mice compared with control.EPCs were successfully isolated from bone marrow and cultured in endothelial basic medium.After seeding,EPCs attached the plates and exhibited spindly,irregular and fusiform shape.Flow cytometry indicated that most of the cells were positive to CD133,CD34 and VEGFR-2.Immunofluorescent staining showed that the majority of the cells were both expressing UEA and banding LDL.CCK-8 analysis results showed that the proliferation of EPCs from ApoE-/-mice with high fat diet 12 weeks decreased significantly compared with control.Furthermore,EPCs from ApoE-/-mice fed with high fat diet 16 weeks decreased more than 12 weeks'.Transwell results revealed that EPCs migration decreased from Apo E-/-mice with high fat diet 12 weeks,and further decreased in high fat diet 16 weeks group.1.3 The change of EPCs autophagy in atherosclerosis model miceWestern Blot showed that the ratio of LC3II/LC3 I decreased and p62 expression increased significantly in EPCs from atherosclerosis model mice compared with control.The number of autophagosomes in EPCs from atherosclerosis model mice increased significantly compared with control under laser scanning confocal microscope.All the results demonstrated that autophagy level decreased in atherosclerosis model mice EPCs.2.The effect of ox-LDL on EPCs bio-function,SOCE and autophagy level2.1 CCK-8 analysis results revealed that ox-LDL dose-and time-dependent decreased EPCs proliferation.Transwell results showed that ox-LDL decreased EPCs migration as well.2.2 We detected that ox-LDL elicited EPCs calcium release from calcium store under laser scanning confocal microscope.Furthermore,ox-LDL dose-dependently induced calcium influx through SOCC.This results indicated that ox-LDL activated SOCE in dose-dependent.Calcium concentration was derived after in situ calibration according to the following equation.[Ca2+]i(n M)= Kd ×(F-Fmin)/(Fmax-F)Kd is the dissociation constant of Fluo3-AM for calcium at room temperature(400 n M).2.3 Both silencing STIM1 and pharmacologic inhibitor of SOCC,2-APB,reduced the calcium influx amplitude elicited by ox-LDL under laser scanning confocal microscope.This further confirmed that ox-LDL induced SOCE significantly.2.4 Given the effect of ox-LDL on EPCs bio-function and SOCE,we further detected the autophagy level after ox-LDL exposure.Western Blot results indicated that ox-LDL increased the ratio of LC3II/LC3 I and decreased the expression of p62.In addition,we observed and counted the autophagosomes as well as autolysosomes combined with the 1.2 The change of proliferation and migration of atherosclerosis model mice EPCs autophagy inhibitor bafilomycin A1(BAFA1)and chloroquine(CQ)under laser scanning confocal microscope.Both the results of Western Blot and counting confirmed that ox-LDL indcued autophagy flux significantly.3.The relationship of SOCE-mediated autophagy and EPCs bio-function.3.1 We pretreat EPCs with BAPTA-AM(intracellular calcium chelator)or EGTA(extracellular calcium chelator)before ox-LDL exposure,then we detected a reduce of the ratio of LC3II/LC3 I and a decrease of autophagosomes number,which indicated that BAPTA-AM or EGTA reversed ox-LDL-indcued autophagy.3.2 Given that calcium contributed to ox-LDL-activated autophagy,we further studied the downstream mechanism of calcium signal in autophagy induction.We detected the according protein expression,the results showed that ox-LDL activated the phosphorylation of CAMKK2 and AMPK,inactivated the phosphorylation of MTOR and P70S6 K.Ox-LDL-induced CAMKK2 and AMPK phosphorylation could be blocked by STO-609.Additionally,STO-609 reversed the ratio of LC3II/LC3 I and increased the phosphorylation of MTOR and P70S6 KB.Silencing STIM1 as well reversed the ratio of LC3II/LC3 I and CAMPKK2 phosphorylation.All the results indicated that ox-LDL activated autophagy through SOCE-CAMKK2-MTOR signal pathway in EPCs.3.3 To further reveal the relationship of autophagy and EPCs bio-function,we detected EPCs proliferation both in RTCA and CCK-8 after autophagy inhibition in ox-LDL exposure.The results indicated that both 3-MA and silencing ATG7 further decreased EPCs proliferation after ox-LDL exposure,which confirmed that autophagy served as a protective role in ox-LDL exposure.4.Pitavastatin improved EPCs bio-function through autophagy regulationPitavastatin was confirmed to improve EPCs bio-function before.To further reveal the mechanism and whether autophagy associated with the process,we detected an increase of proliferation,migration and autophagy level after pitavastatin exposure.Next,we applied 3-MA or lentiviral vector carrying ATG7 to knock down EPCs autophagy before exposure to pitavastatin.Western Blot results indicated that pitavastatin-increased proliferation and migration were reversed after autophagy inhibition.This showed that pitavastatin improved EPCs bio-function through autophagy regulation.Conclusion:1.EPCs proliferation,migration and autophagy level decreased in atherosclerosis model mice2.Ox-LDL reduced EPCs proliferation and migration,but activated SOCE and autophagy3.Ox-LDL activated autophagy through SOCE-CAMKK2-MTOR signal pathway,which served as a protective role in EPCs bio-function4.Pitavastatin improved EPCs proliferation and migration through autophagy regulation...