The Role Of Autophagy In Aortic Valve Calcification And ROS-induced Valvular Interstitial Cell Damage And The Underlying Mechanisms

BackgroundCalcific aortic valve disease(CAVS)mainly affects aged people and is the third leading cardiovascular disease.Its pathological characteristics involve fibrotic thickening and calcification of aortic valve leaflets and commissural fusion,which lead to the stenosis of aortic valve area.Since the molecular mechanisms are not well understood,there is no medical therapies for effective prevention and the only available treatment is aortic valve replacement.A growing body of literatures show that aortic valve calcification is not only a dystrophic process,which involves cellular apoptosis and passive calcium deposition,but also relies on the active phenotypic transdifferentiation of valvular interstitial cells(VICs)into osteoblast-like cells.VICs are the main component of the heterogeneous cellular population in aortic valve and essential for maintaining the structure and physiological function of the valvular leaflets.In vitro osteogenesis model of VICs is a powerful tool which is widely utilized to investigate molecular mechanisms and potential medical therapies of CAVS.Oxidative stress,which results from accumulation of reactive oxygen species(ROS)raised by imbalance of the oxidation-reduction system,can cause cell damage or even death.Recent progress indicated an elevation of ROS level in specimens of CAVS patients,which is associated with the reduction of antioxidant enzymes and can lead to DNA damage and early activation of VICs.Increasing evidence showed a tight and complicate relationship between autophagy and ROS/oxidative stress.Autophagy is a highly conserved phenomenon that self-digesting cellular constituents for removal of hazardous substances or recycling of nutrients,which is crucial for cellular survival and homeostasis.ROS is considered to possess the capability of positively or negatively regulating autophagy,while autophagy can also affect ROS by degrading oxidized proteins and damaged organelles.However,whether autophagy is involved in the pathological process of CAVS has not been comprehensively investigated,nor the interaction between oxidative stress and autophagy in the context of VICs been reported.Objectives1.To observe the occurrence and level of autophagy in aortic valve calcification and in vitro osteogenesis process of VICs.2.To explore the influences of autophagy on aortic valve calcification and ROS induced cellular damage and apoptosis.3.To investigate the interaction between oxidative stress and autophagy in the pathological progress of CAVS.Methods(1)Tissue specimens and VIC culture.1.Procurement and pretreatment of tissues: fibrotic and calcific aortic valves were respectively explanted from CAVS and aortic valve regurgitation patients who underwent aortic valve replacement in our department.Controlled normal aortic valves were obtained during heart transplantation or from autopsy.2.Isolation of porcine aortic valve interstitial cells(PAVICs): immerse the explanted porcine aortic valves in type I collagenase solution at 37℃ for 10 mins;scratch the surface of the leaflets with sterile cotton swabs to eliminate valvular endothelial cells(VECs);digest the valves in type Ⅱ collagenase at 37℃ with shaking for 2 hours,followed by infiltration,centrifugation and resuspension.3.Confirmation of VICs: using immunofluorescence to detect the expression of Vimentin(marker of interstitial cells),α-SMA(marker of fibroblasts)and CD31(marker of endothelial cells).4.Culture of PAVICs: cells were maintained in Dulbecco’s Modified Eagle’s medium(DMEM)supplemented with 10% fetal bovine serum(FBS)and 1% penicillin and streptomycin;the medium were changed every 2-4 days and passage 3-6 of PAVICs were used in the following experiments.(2)In vitro osteogenesis of PAVICs and calcification related detection.1.In vitro calcification models: PAVICs were treated with osteogenesis-induced medium-1(OIM-1)consisted of DMEM with 5% FBS,2m M Na H2PO4,50μg/ml ascorbic acid and 107 M insulin and OIM-2 consisted of DMEM with 10% FBS,10 m M betaglycerol phosphate,50μg/ml ascorbic acid and 100 n M dexamethasone to induce calcification of PAVICs for up to 7 and 21 days,respectively.2.Detection of the phenotypic switching of VICs into osteoblast-like VICs(ob VICs): the protein levels of α-SMA and osteogenic markers RUNX2 and OPN were examined by western blot(WB)analysis;BCIP/NBT alkaline phosphatase(ALP)staining was used to detect expression of ALP.3.Detection of calcium deposits: 1% Alizarin Red S(ARS)(p H=4.3)was applied to cultured PAVICs to stain calcific nodules.(3)Assays of autophagic measurement and intervention of autophagy.1.Morphological detection: autophagic structures(e.g.autophagic vacuoles and autophagosomes)in aortic valve specimens were directly observed by transmission electron microscopy(TEM).2.Analysis of autophagic level in specimens: immunohistochemistry(IHC)and WB were employed to evaluate the expression levels of autophagy related proteins(e.g.LC3,BECN1,ATG5,ATG7);co-localization of vimentin and BECN1 was examined by dual immunofluorescence.3.Formation of autophagosomes and pathways: turnover of LC3-Ⅱ/LC3-Ⅰ,other autophagic proteins,p-AKT(ser473)and p-P70S6K(Thr389)was detected by WB with intervention of bafilomycin A1.4.Occurrence of autophagic flux: WB was conducted to assess the degradation of P62;transfection of PAVICs with GFP-m RFP-LC3 adenovirus was used to monitor formation of autophagosomes and autolysosomes.5.Intervention of autophagy: rapamycin was used to augment autophagic activity while wortmannin and chloroquine(CQ)for inhibition of autophagy;gene silencing of BECN1 by adenovirus-delivered si RNA was transfected into PAVICs to block autophagy specifically.(4)Induction and intervention of oxidative stress and cellular damage detection.1.In vitro model of oxidative stress: using 100-1000μM hydrogen peroxide(H2O2)to stimulate PAVICs for 0.5 hour-14 days.2.Detection of ROS: evaluate the level of ROS through fluorescence microscope and flow cytometry(FCM)by labeling cellular ROS with 2’,7’-DCFH.3.Intervention of oxidative stress: applicate N-Acetylcysteine(NAC)to scavenge excessive cellular ROS of PAVICs.4.Assessment of cytotoxicity: analyze cell viability through Cell Counting-8(CCK-8)assay;Cytotoxic Lactic Dehydrogenase(LDH)Releasing Assay was used to detect membranous damage of PAVICs under oxidative stress.5.Intervention and detection of apoptosis: pan-caspase inhibitor Z-Vad-FMK was applied to suppress apoptosis;FCM was conducted to assess early/late apoptosis of Annexin V/PI labeled PAVICs;the activation of Caspase pathway was detected by WB focusing on expression level of cleaved Caspase3 and PARP.Results(1)Evaluation of autophagy in calcific aortic valve disease.In contrast to rarely found autophagic vacuoles in controlled normal aortic valves,moderate formation of autophagosomes and large amount of autophagosomes and autolysosomes could be observed in fibrotic and calcific aortic valves,respectively,which provided a direct evidence for increasing autophagic activity in CAVS.Abundant positive immunostaining of LC3,BECN1,ATG5 and ATG7,which is essential for autophagosome formation,was found in diseased aortic valve specimens by IHC.WB analysis showed a elevated turnover of LC3-Ⅰ to LC3-Ⅱ and expression of BECN1 and ATG5 in aortic valve sclerosis(AVSc)and CAVS compared to normal aortic valves.Double immunofluorescence showed that prominent detection of BECN1 was located in vimentin positive cells,which demonstrated elevated autophagic activity occurred in valvular interstitial cells of CAVS.(2)Measurement and functional analysis of autophagy in in vitro calcification model of PAVICs Immunocytofluorescence confirmed little contamination of VECs in cultured PAVICs isolated by our modified methods involved two-step collagenase digestion.During the processes of inducing calcification of PVAICs using OIM-1 and 2,we observed increasing expression of OPN and RUNX2 with declined α-SMA and positive ALP staining,which indicates a phenotypic transdifferentiation of VICs from fibroblast-like cells to osteoblastlike cells in both calcification models.ARS staining showed extensive calcific deposition of PAVICs cultured in OIM-1 and 2 at day 7 and 21,respectively.When OIM-1/2 was applied to PAVICs,the ratio of LC3-Ⅱ/LC3-Ⅰ increased and expression of P62 declined in a short period(<24 hours).Blocking the fusion of autophagosomes and lysosomes with bafilomycin A1,we could still detect elevated LC3-Ⅱ expression,indicating formation of autophagosomes in calcification model of PAVICs.A direct evidence of autophagolysosome formation was provided using GFP-m RFP-LC3 monitoring system.Increasing levels of BECN1,ATG5 and ATG7 were also observed in prolonged culture of PAVICs in OIM-1/2(4-21 days).ARS staining revealed that the autophagy inducer,rapamycin,significantly alleviated calcium deposition in OIM-1 and OIM-2 while the autophagy inhibitor wortmannin and transfection of sh BECN1 exacerbated calcification of PAVICs.As shown in WB analysis,OPN and RUNX2 decreased along with a recovery of α-SMA after treating PAVICs with rapamycin in OIM-1/2 while wortmannin and sh BECN1 led to a opposite results,suggesting autophagy can relieve calcification of VICs by retarding transdifferentiation into osteoblast-like cells.Besides,application of rapamycin in the halfway of osteogenesis induction can also mitigate the extension of calcification in the terminal point.(3)Evaluation of the interaction between autophagy and ROS in the oxidative stress model of PAVICs.Elevated ROS level was observed in PAVICs cultured in OIM-1 and OIM-2 by labeling 2’,7’-DCFH-DA probe within 6 hours.WB analysis shown occurrence of caspasedependent apoptosis in calcification model of PAVICs.Elimination of excessive ROS by NAC alleviated apoptosis of PAVICs in OIM,as proved by FCM.Furthermore,NAC and pan-caspase inhibitor Z-Vad-FMK could decrease calcium deposition,demonstrating that oxidative stress induced cellular apoptosis is a crucial mechanism involved in the pathological process of CAVS.Immunostaining of LC3-Ⅱ/LC3-Ⅰ and P62 indicated that autophagic activity depended on the concentration of hydrogen peroxide while NAC can inhibit autophagy in the oxidative stress model of PAVICs.Time-course analysis revealed that autophagy was induced by ROS in PAVICs through PI3K-AKT-m TOR pathway.Intervention by bafilomycin A1 and GFP-m RFP-LC3 monitoring system confirmed the formation of autophagosomes and occurrence of intact autophagic flux,respectively,in the oxidative stress model of PAVICs.NAC can inhibit autophagy in calcification models,which suggests that activated autophagy during aortic valve calcification is at least partially induced by oxidative stress.After prolonged stimulation with H2O2,protein levels of BECN1,ATG5 and ATG7 in PAVICs increased at day 7,but declined at day 14.Z-Vad-FMK significantly improved cell viability of PAVICs treated with 250-750μM H2O2,indicating cellular apoptosis is a major type of cell death of PAVICs under oxidative stress.CCK-8 and LDH release assay showed that treatment of rapamycin made PAVICs more susceptible to oxidative stress at different periods induced by 500-750μM H2O2 while wortmannin and CQ exacerbated cell death and membranous damage.Specifically,augmentation of autophagic activity could alleviate caspase-dependent apoptosis of PAVICs,as proved by FCM and WB.Mechanisms contributing to the protective effects of autophagy was deconvoluted by the quantitative FCM analysis of 2’,7’-DCF positive PAVICs,which indicated that autophagy scavenged the excessive ROS accumulated in PAVICs.Conclusions Oxidative stress are responsible for augmented autophagic activity of valvular interstitial cells in pathological process of CAVS.Autophagy provides a protective function through alleviating VIC damage and apoptosis induced by oxidative stress via scavenging excessive cellular ROS.The phenotypic transdifferentiation of VICs into osteoblast-like cells is inhibited by autophagy,which can retard aortic valve calcification.