Roles Of SIRT1 In Cellular Stress Responses And Underlying Mechanisms

BackgroundThe nucleolus is a non-membrane organelle located in the nucleus. It is the site of ribosome biosynthesis. It plays an important role in the synthesis of cellular proteins and cell proliferation, differentiation and aging. Mounting evidence has suggested that the nucleolus is also a signaling hub involved in mediating cellular stress responses (which is termed nucleolar stress). Nucleolar stress is characterized by disorganization of the normal nucleolar structure and translocation of some nucleolar proteins to the nucleoplasm, nucleophosmin/B23 (NPM) is a nucleolar specific protein, only positioning in the nucleolus under normal condition, while translocating from the nucleolus to the nucleoplasm during nucleolar stress. NPM protein translocation to nucleoplasm is considered to be a marker event of nucleolar stress.Functionally, induction of nucleolar stress major results in up-regulating the abundance of the tumor suppressor P53, likely by disruption of the P53 binding to the E3 ubiquitin ligase MDM2, leading to compromised P53 ubiquitination and degradation. Emerging evidence suggests that occurrence of the nucleolar stress response may be implicated in the pathogenesis of neurodegenerative and cardiovascular diseases. Interestingly, there is evidence showing that micropore irradiation-induced DNA damage per se is not able to trigger P53 stabilization, unless the nucleolus is disrupted; moreover, forced nucleoli disruption in the absence of genotoxic stress also causes P53 stabilization, indicating that nucleolar stress response is both sufficient and necessary for P53 regulation.Mammalian SIRT1 is the orthologue of the yeast SIR2 gene product, and is an NAD+-dependent deacetylase. SIRT1 plays a protective role in regulating lipid metabolism, cell cycle regulation, cell cycle regulation, cell senescence inhibition, inhibition of inflammation and oxidative stress injury by participating in the regulation of cellular stress response. It is thought that the subcellular localization of SIRT1 may have significant impacts on the role of SIRT1 in cell biology. SIRT1 is generally recognized as a nuclear protein, although it is also present in the cytosol. Intriguingly, some evidence has suggested that SIRT1 may also have a role in the nucleoli. For instance, Murayama and colleagues have demonstrated that SIRT1 is a partner of a nucleolar protein complex termed eNoSC (energy-dependent nucleolar silencing complex), which senses intracellular energy status and controls rRNA transcription. These data indicate a critical role of SIRT1 in the nucleoli in modulating cellular stress response. Nonetheless, whether SIRTl has any relationship with the nucleolar stress response is not clear.The biological effects of SIRT1 are mediated by both of transcription-dependent and -independent mechanisms. Among the numerous SIRT1 targets, the tumor suppressor P53 has been extensively studied and shown to have a critical role in mediating SIRT1 effects. SIRT1 is a negative regulator of P53. SIRT1 catalyzes deacetylation of P53 at K382. It is thought that deacetylation of the key lysine residues at the C-terminal suppresses P53 functions. Inhibition of P53 function may be a major mechanism that is responsible for the anti-senescence and pro-survival effects of SIRT1. In addition to directly regulating P53 function, SIRT1 also affects the intracellular accumulation of P53 protein. For example, cells in which the SIRT1 gene was deleted or SIRT1 expression was knocked down with siRNA exhibited increased protein levels of P53. Moreover, it was shown that microRNA-34a could inhibit SIRT1 expression and this effect resulted in accumulation of acetylated P53.Currently, the mechanism of SIRT1-regulated P53 protein accumulation is still poorly understood. Although initial evidence suggests that deacetylation of P53 may accelerate P53 degradation by increasing its ubiquitination level, later studies shows that P53 still can be accumulated after mutations of all the acetylation sites of P53. These data show both in vitro and in vivo do not support a exclusive role of acetylation/deacetylation in the stabilization of P53.The aim of our study was to (1) clarify whether the phenomenon of nucleolar stress was present in vascular cells and whether nucleolar stress was stimuli-specific; (2) investigate the effect and mechanism of SIRT1 on nucleolar stress, which might provide a plausible mechanism to explain SIRT1-mediated, acetylation-independent regulation of P53 stabilization.The whole thesis includes 5 parts:Part1:Nucleolar stress in vascular cells.Part2:The effects of SIRT1 on nucleolar stress.Part3:The possible mechanism of SIRT1-mediated inhibit of nucleolar stress response.Part4:Role of SIRT1 in acetylation-independent regulation of P53 stabilization.Part5:Nucleolar stress phenomenon in myocardial hypertrophy and myocardial infarction.Part1:Nucleolar stress in vascular cells.Objectives1. To explore whether and how nucleolus of Hela cell react to multiple stress stimuli (ACTD, H2O2, serum starvation, amino acid starvation and heat shock).2. To examine whether nucleolar stress could occur in vascular cells.Materials and Methods1. Culture of cell linesHela cells were maintained in DMEM medium containing 10% fetal bovine serum (FBS),1% penicillin/streptomycin. HUVECs were maintained in ECM medium containing 10% fetal bovine serum(FBS),1% penicillin/streptomycin and 1% growing factors. HSMCs were maintained SMCM medium containing 10% fetal bovine serum(FBS),1% penicillin/streptomycin and 1% growing factors.2. Nucleolar stress analysisHela cells, HUVECs and HSMCs were stimulated by ACTD (5nM) for 6,12 and 24 hours. Hela cells, HUVECs and HSMCs were stimulated with H2O2 of increasing concentrations and at different time points. We cultured Hela cells, HUVEC and HSMC for 6,12,24 hours using serum-free culture medium to produce serum starvation model, using balanced salt solution in stead of culture medium to produce amino acid starvation model, and at 42℃ or 45℃ to produce heat shock model. The NPM and NS localization in these cells were detected by immunofluorescence. According to these immunofluorescence staining images, we calculated relative fluorescence intensity (nucleoplasm/nucleolus)-P/L of NPM and the percentage of intact nucleolus as indice of nucleolar stress. Detection of other markers of nucleolar stress (pre-rRNA by RT-PCR and P53 accumulation by Western blot) was performed after stimulation of ACTD and H2O2.3. Statistical analysisData were expressed as mean ± standard error of the mean (SEM). For statistical analysis, unpaired t-test or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparisons as appropriate were performed using SPSS 13.0 software. A value of P< 0.05 was considered statistically significant.Results1. ACTD induced nucleolar stress in Hela cells, HUVEC cells and HSMC cells.2. H2O2 induced nucleolar stress in Hela cells, HUVEC cells and HSMC cells.3. Serum starvation had no effect on the nucleolus morphology in Hela cell, but could induce nucleolar stress in HUVEC and HSMC cells.4. Amino acid starvation could induce nucleolar stress in Hela cell, HUVEC cell and HSMC cell.5. Heat shock could induce nucleolar stress in Hela cell, HUVEC cell and HSMC cell.6. ACTD and H2O2 could down-regulate the expression of pre-rRNA in Hela cells.7. ACTD could enhance P53 protein accumulation in Hela cells.Conclusion1. The phenomenon of nucleolar stress could be observed in vascular cells.2. Nucleolar stress is a ubiquitous response of cells reacting to multiple stress stimuli.3. Different cells may have different sensitivity of stress stimuli-induced nucleolar stress.Part2:The effects of SIRT1 on nucleolar stress.ObjectivesTo clarify whether SIRT1 can affect nucleolar stress caused by multiple stimuli.Materials and Methods1, Four methods were used to change the SIRT1 level in Hela cells:(1) Over-expression of SIRT1 by transfection of SIRTl-Flag plasmid (pEGFP plasmid as control); (2) SIRT1 gene silencing by transfection of si-SIRT1(si-nc as control); (3) isolation of SIRT1-/-mouse aortic smooth muscle cells (mouse SMC line as control); (4) SIRT1 activator Resveratrol was used to increase the activity of SIRT1 protein (DMSO as control). NPM was measured by immunofluorescence after stimulation of ACTD and H2O2.2, Western blot:SIRT1 protein expression level was detected.3, Statistical analysis:Data were expressed as mean ± standard error of the mean (SEM). For statistical analysis, unpaired t-test or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparisons as appropriate were performed using SPSS 13.0 software. A value of P< 0.05 was considered statistically significant.Results1. Deregulation of SIRT1 aggravates the occurrence of nucleolar stress.Silencing of SIRT1 did not induce Nucleolar stress. But additional of ACTD or H2O2, Nucleolar stress occurred earlier and more severe compared to the si-nc control group. The nucleolus of SIRT1-/-mouse aortic smooth muscle cells are intact; hoever, NPM diffusion to the nuleoplasm can be observed under resting conditions. After treatment with H2O2, Nucleolar stress occurred more severe in SIRT1-/-clles as compared to wild type mouse SMCs.2. Upregulation of SIRT1 attenuated the nucleolar stress response.Over-expression of wild type SIRT1 improved Nucleolar stress induced by ACTD or H2O2.ConclusionSIRT1 is a negative regulator of nucleolar stress.Part3:The possible mechanisms of SIRTl-mediated inhibition of nucleolar stress responseObjective:1, To investigate the biological mechanism of SIRTl-mediated inhibition of nucleolar stress response.2, To clarify the relationship between SIRT1 and nucleolus, whether there is co-localization of SERT1 and the nucleolar protein NPM.3, To explore the relationship between SIRT1 and NPM, whether SIRT1 participates in deacetylation of NPM.Methods:1, Isolation and characterization of nucleolus from Hela cell:preparation of Hela cells without treatment, and Hela cells which were treated with EX-527 (inhibitor of SIRT1) for 24hrs. All cells were disrupted by hypo-osmolality and density gradient centrifugation was performed to isolate nucleolus with monitoring under a phase contrast microscope. To verify the purity of nucleolus, western blot were conducted to detect the NPM (nucleolus marker), NS, Fibrillarin, Histone H3 (nucleoplasm marker), and GAPDH (cytoplasm marker).2, SILAC-based proteomics analysis:one group Hela cells were cultured for 6 passages in SILAC DMEM medium supplemented with 15N labeled arginine, the other group was cultured in normal DMEM medium. The two groups of cells were transfected with SIRT1-Flag plasmid and then mixed in equal proportion. The protein was extracted and immunoprecipitatio was conducted using anti-Flag antibody, then the mass spectrometry was performed to screen all the protein which was combined with exogenous SIRT1 in Hela cells.3, Co-IP:Hela cells were transfected with SIRT1-Flag plasmid, protein was extracted and Co-IP was performed to detect whether NPM was combined with exogenous SIRT1, and whether endogenous NPM was combined with SIRT1. The Hela cells were transfected with pEGFP vector (control), H363Y vector (inactivating SIRT1) and SIRT1 vector (wild type) respectively,24hrs after adding ACTD into the medium, protein was extracted and Co-IP was performed to determine whether acetylization of NMP was changed.4, Western Blot:Hela cells were transfected with pEGFP vector (control), SIRT1-H363Y vector (acetyltransferase inactive SIRT1) and SIRT1 vector (wild type) respectively,24hrs after ACTD was added into the medium, protein was extracted and western blot was performed to detect NPM, Flag, AC-lysine and P-Actin:Cells were treated with EX-527 for 24hrs and then western blot was conducted to detect NPM, Flag, AC-lysine and P-Actin.5, Immunofluorescence double labeling was carried out to determine the distribution of SIRT1 and NPM, and to clarify whether SIRT1 and NPM is co-localized.6, Three experiments were performed to change the activity of SIRT1: ① pretreatment with Resveratrol for 2 hrs (DMSO was used as control) to increase the activity of SIRT1; ②retreatment using SIRT1 inhibitor EX-527 for 2 hrs (DMSO was used as control) to decrease the activity of SIRT1;③cells were transfected with SIRT1-H363Y (pEGFP vector was used as control); all of the groups of cells were stimulated with ACTD, then immunofluorescence was carried out to detect nucleolar stress.7, Construction and transformation of NPM-pGEX:The NPM and the expression vector pGEX-4T-1 were digested with BamH I, ligated by T4 DNA Ligase and then transformed into BL21 Competent Cells. Single clones were recovered and the NPM-pGEX vector was constructed and verified by sequencing.8, E Coli expression and purification of fusion protein NPM-GST:bacteria were recovered and cultured for 4hrs, IPTG was added for lhrs and fusion protein NPM-GST was extracted and purified by resin-beads. SDS-PAGE electrophoresis was performed and Coomassie Blue Staining was used to determine the expression of GST-NPM.9, Acetylation in vitro:CBP and Acetyl-CoA were added in fusion protein NPM-GST, acetylization of NPM need to be sufficient in the acetylation reaction mixture, then the SIRT1 and NAD+ were added and the mixture was cultured in deacetylation reaction mix, fusion protein NPM-GST was collected and mass spectrum was performed.10, SIRT1 truncate experiment:three pairs of specific primers were designed according to complete genome sequence of SIRT1 in GenBank. The SIRT1-flag was used as substrate, reverse PCR was performed to truncate SIRT1 and the left part would intra-loop and form three recombinant plasmids (SIRTI104, SIRT1265, SIRT1519). Hela cells were transfected with the above plasmids, IP using anti-Flag antibody and western blot was performed to detect NPM and then to determine the molecular parts which SIRT1 combined with NPM. Immunofluorescence was carried out to clarify whether SIRT1-NPM binding is indispensable for SIRT1 inhibiting nucleolar stress.11, Statistics:Continuous variables were summarized with descriptive statistics (mean +SEM), Data was analyzed by student t-test and one-way ANOVA technique. Differences in the values were considered significant when P<0.05. All analyses were two-tailed.Results1, NPM interacted with SIRT1.In our study, by using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics approach, we identified a physical interaction between SIRT1 and NPM. Co-IP and Western blot showed that both exogenous and endogenous SIRT1 was combined with NPM. Immunofluorescence showed that SIRT1 and NPM were partially overlapped.2, SIRT1 was expressed in the nucleolus of Hela cells.We purified nucleoli and analyzed the cellular fractions obtained during nucleoli purification. Fibrillarin and NS were only expressed in the nucleolar fraction and Histone H3 was expressed in the nucleoplasmic fraction, indicating there was not contaminations by nucleoplasmic proteins. GAPDH was not expressed in the nucleolar fraction, indicating that the isolated nucleoli were without contaminant derived from cytoplasmic proteins. These data showed that SERT1 was mostly expressed in nucleoplasm, but was also present in nucleolus.3, SIRT1 truncate experimentCo-IP and Western blot showed that both SIRT1104 and SIRT1519, but not SIRT1265, were combined with NPM. Immunofluorescence showed that SIRT1265 can not protect nucleolus from stress induced by ACTD.4, The inhibitory effect of SIRT1 on nucleolar stress response is only partly dependent on the deacetylase activity.Over-expression of SIRT1-H363Y could also reduce Nucleolar stress induced by ACTD, although over-expression of wild type SIRT1 showed a larger nucleolus-protective effect. After pretreatment with SIRT1 inhibitor EX-527, over-expression of H363Y and wild type SIRT1 both inhibited nucleolar stress induced by ACTD similarly.5, SIRT1 did not significantly deacetylate NPM both in vivo or in vitro.Over-expression of SIRT1 did not change the acetylation level of NPM. After treatment with EX-527, changed levels of certain acelylated nucleolar proteins can be detected using the same antibody, indicating the lack of change in NPM acetylation was not due to a low sensitivity of the antibody used.Based on the results of acetylation assay in vitro, there is only one lysine site(K223) of the C-terminal at which SIRT1 catalyzes deacetylation of NPM. These data indicate that SIRT1-mediated NPM deacetylation may not play a decisive role in the inhibition of nucleolar stress.6, SIRTl increased NPM protein expression.Over-expression both SIRT1 and H363Y increased NPM protein expression in normal condition or after adding ACTD. NPM expression decreased by treating cells with EX-527.Conclusion1, SIRT1 was expressed in the nucleolus and interacted with NPM.2, The protein region combinding to NPM was located to the central part of SIRT1. Without SIRT1-NPM binding, SIRT1 lost its nucleolus-protective effect.3, The inhibitory effect of SIRT1 on nucleolar stress response is only partly dependent on the deacetylase activity.4, SIRTl-mediated NPM deacetylation may not play a decisive role in the inhibition of nucleolar stress.Part4:SIRT1 could regulate P53 stabilization via deacetylation-independent mechanismsObjective:To clarify whethwe SIRT1 can modulate P53 accumulation under nucleolar stress condition via acetylation/deacetylation-independent mechanisms.Methods:1, Immunofluorescence and Western blot were performed to detect P53 expression and localization.2, P53(8KR) plasmid in which all of the potential 8 lysine acetylation sites were mutated to arginine was created by synthetic DNA construction.3, Statistics:Continuous data were expressed as mean±SEM, and were analyzed by student t-test or one-way ANOVA. Differences were considered significant when the P value was <0.05. All analyses were two-tailed.Results1. Nucleolar stress occurs earlier than the accumulation of P53.Treatment of cells with ACTD for 2,4,6,8,10,12,14,16,18,20,22 and 24 hours showed that Nucleolar stress started from 4 hours while accumulation of P53 started around 22 hours post ACTD stimulation.2. SIRTl inhibits nucleolar stress-induced P53 protein accumulation.Immunofluoresence and western blot results showed that ACTD could increase the protein levels of P53, whereas overexpression of SIRT1 could inhibit P53 protein accumulation.3. SIRT1 inhibit P53 protein accumulation independent of its deacetylase activity.Immunofluoresence and western blot results showed that over-expression of non-functional SIRT1-H363Y could also inhibit P53 protein accumulation. Cells were then pre-treated with SIRT1 inhibitor EX-527 before overexpressing SIRT1-H363Y, thus the deacetylase activity was completely inhibited. However, P53 accumulation could still be inhibited in the presence of EX-527. Cells were transfect with P53 (8KR)-GFP mutant (so that P53 protein was unable to be deacetylated and insensitive to SIRT1 deacetylase activity), then SIRT1 was overexpressed. We found that P53 (8KR)-GFP protein accumulation was inhibited by SIRT1, further indicating that SIRTl could suppress P53 accumulation via deacetylation independent pathways.Conclusion1. SIRT1 inhibits nucleolar stress-induced P53 protein accumulation.2. SIRT1 can mediate an deacetylation-independent regulation of P53 stabilization.Part5:The nucleolar stress phenomenon in cardiovascular diseaseObjective:To clarify whether the nucleolar stress phenomenon is present in myocardial cells under pathological conditions such as hypertrophy and myocardial infarction.Methods:1. Myocardial hypertrophy animal model.Male C57BL/6 mice at the age of 8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). Male C57BL/6 mice at the age of 12-wk were randomly divided into 3 groups:sham group; TAC-2 week group; TAC-4 week group. All animal studies were complied with the Animal Management Rules of the Chinese Ministry of Health and approved by the Ethical and Use Committee of the Qilu Hospital of the Shandong University. The myocardial hypertrophy model was created by transverse aortic constriction (TAC).2. Echocardiographic measurementsUltrasound echocardiography was performed before and at 2 and 4 weeks after TAC. Using a high resolution echocardiography system (Vevo 770, Visual Sonics, Canada) with a 35-MHz transducer, two-dimensional parasternal long-and short-axis images of the LV were recorded. The following index were measured and calculated by the echocardiographic system:(1) the LV end-diastolic diameter (LVDd); (2) LV end-systolic diameter (LVDs); (3) inter-ventricular septum end-diastolic thickness (IVSTd); (4) LV posterior wall end-diastolic thickness (PWTd); (5) LV fractional shortening (FS) ejection fraction (FS); (6) corrected LV mass.3. Immunofluorescence:Hearts were embedded in paraffin. Sections were stained with specific NPM antibody for immunofluorescence labeling.4. Statistics:Continuous variables were summarized with descriptive statistics (mean ±SEM), Data was analyzed by student t-test or one-way ANOVA. Differences were considered significant when P<0.05. All analyses were two-tailed.ResultsNucleolar stress could be observed in the myocardium of mice with decompensatory myocardial hypertrophy induced by chronic pressure overload.ConclusionThe pheniomenon of nucleolar stress is present in cardiac cells under cardiovascular disease conditions.BackgroundVascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the mechanical forces imposed by blood flow, of which the shear stress acting on the surface of ECs is recognized to be one of the most important vasoactive factors in EC. It is well documented that a relatively high level of laminar shear stress is protective, whereas abnormal (too low or oscillatory) shear stress caused by turbulent blood flow is a detrimental cellular stress to ECs, which is implicated in endothelial dysfunction and various vascular diseases. Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions such as gene expression, proliferation, migration, morphogenesis, permeability and inflammation.Autophagy is an evolutionarily conserved cellular stress response. Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12 for example. Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment. Autophagy is induced under metabolic stress conditions, such as nutrient starvation, and the Akt-mTOR (mammalian target of rapamycin) pathway plays a central role in mediating the autophagic response. Inhibition of mTOR triggers cell autophagy.In endothelial cells, an autophagic response can be initiated by different stress stimuli. However, the cellular outcome following induction of autophagy in endothelial cells, either cell survival or cell death, varies depending on the nature of stimuli and specific experimental settings. Moreover, there is evidence showing that autophagy may also be involved in modulating other endothelial cell functions such as angiogenesis and cellular senescence. Therefore, understanding the mechanisms of regulation of autophagy in endothelial cells will be important in the discovery of strategies for protecting normal endothelial functions under stress conditions. Currently, however, little is known about the relationship between shear stress and autophagy in endothelial cells.Mammalian SIRT1 is the orthologue of the yeast SIR2 gene product, and is an NAD+-dependent deacetylase. SIRT1 has a pivotal role in modulating stress responses in mammalian cells. Several lines of evidence have shown that SIRT1 exerts diverse beneficial effects in maintaining normal endothelial cell functions. For example, SIRT1 physically interacts with and deacetylates endothelial nitric oxide synthase, leading to enhanced nitric oxide production and vasodilatation. Activation of SIRT1 also led to decreased inflammatory reactions and cellular senescence in endothelial cells. Moreover, accumulating evidence has suggested that SIRT1 may have an important role in modulating cell autophagy. Based on these observations, in the present study we tested the hypothesis that shear stress may modulate endothelial cell autophagy through SIRT1.Objectives1, To investigate the effect of shear stress on autophagy in endothelial cells2, To explore the role of SIRT1 in regulating autophagy in endothelial cellsMethods1. In vitro flow simulation:human umbilical vein ECs (HUVECs) were seeded on glass slides coated with collagen, cultured in a Streamer parallel-plate flow chamber (FlexCell, Burlington, NC, USA) system and divided into Flow group (shear) and the Static group (no shear). M199 medium was used as a fluid buffer, we treated HUVECs with laminar flow (20 dyn/cm2) to mimics physiological blood flow shear status, laminar flow (4 dyn/cm2) to mimics low-magnitude flow shear status, and oscillatory flow (±5 dyn/cm2 at 1 Hz) to mimics oscillatory flow shear model.2. Following methods were applied to evaluate autophagy:(1) Immunofluorescence to detect the cellular localization of LC3 and count LC3 puncta aggregation (two indicators:LC3 puncta gathered cell percentage of total cells, LC3 puncta per cell).(2) RT-PCR to detect autophagy-related gene P62 expression. (3) Western blots to detect autophagy related proteins including LC3、P62、ATG5、ATG7% FoxO1、mTOR expression.3. Co-immunoprecipitation method to detect acetylation level of autophagy-related proteins(ATG5、ATG7、FoxO1) after laminar flow.4. Amplex Red Hydrogen Peroxide Assay to detect the effect of flow-induced ROS in HUVECs treated with NADPH oxidase inhibitor.5. Caspase3/7 activity assay to detect apoptosis of HUVECs after laminar flow or with various autophagy inhibitors.6. Construct a plasmid with SIRT1 promoter sequence, transfect it into HUVECs, then detect activity of SIRT1 gene promoter after laminar flow with luciferase reporter gene system.Results1. Immunofluorescence found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy in physiological conditions.2. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action.3. The flow-induced autophagic response was comparable to that induced by amino-acid starvation, which was used to mimic a positive control response.4. Shear stress induced by laminar flow can promote P62 transcription but unchanged P62 level.5. The autophagy inhibitor chloroquine induced significant accumulation of LC3 in HUVECs.6. Bafilomycin Al,an inhibitor of the late phase of autophagy, alone increased LC3 puncta, whereas application of laminar flow also exhibited an increasing effect on this response in the presence of bafilomycin.7. Laminar flow had no significant impacts on the phosphorylation levels of mTOR in HUVECs.8. Laminar flow significantly enhanced the gene promoter activity in HUVECs.9. Deacetylase SIRT1 may have an important role in modulating autophagy.10. Flow-induced autophagy is redox dependent. Shear stress regulates ROS production in EC, which can be blocked by the NADPH oxidase inhibitors. EUK-134,a synthetic superoxide dismutase,and antioxidant NAC significantly blocked the upregulating effects of flow on SIRT1 and LC3-Ⅱ.11. SIRT1-dependent activation of FoxO1 is critical in mediating shear-induced autophagy. Flow significantly decreased the acetylation level of FoxO1.Constitutively active FoxO1 increased the expression levels of LC3 in HUVECs.12. SIRT1 decreased the acetylation levels of autophagic proteins Atg5 and Atg7.13. Autophagy was cytoprotective in EC. Autophagy inhibitor 3-methyladenine (3-MA) aggravated serum deprivation-induced cell death. Flow-induced autophagy was blocked by 3-MA. Atg5 siRNAs partially attenuated the cytoprotective effect of resveratrol on cell apoptosis induced by serum deprivation. Atg5 gene silencing also partially reversed the cytoprotective effect of shear stress using staurosporine as an apoptosis inducer.Conclusion:In summary, we have presented evidence showing that laminar flow-induced shear stress promotes autophagic responses in ECs via a redox-and SIRT1-dependent mechanism. Shear stress-induced autophagy in ECs may represent a novel mechanism by which laminar blood flow produces its vascular-protective actions.